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)     

Immunocytochemical characterization of the DNA-polymerase alpha-positive colonic mucosal epithelial cells in patients with ulcerative colitis

Colonic mucosal epithelial cells (EpC) in patients with ulcerative colitis (UC) have been shown to express HLA-DR antigen. In the present study, we observed the characteristics of HLA-DR-positive EpC using immunoperoxidase technique. In the control group, colonic EpC expressed HLA-ABC, but not HLA-DR. Only the EpC at the base of glands revealed positive for DNA-polymerase alpha (DNA-P). On the other hand, in actively inflamed mucosa of UC, about 80% of glands strongly expressed HLA-DR. Furthermore, most of EpC in the HLA-DR-positive glands showed positive nuclear stainings for DNA-P. This indicates that these EpC are not in the resting stage. It is strongly suggested that there are close relationships between the regeneration or proliferation of the EpC and class II MHC (HLA-DR) expression on the EpC in UC.     

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.     

Significance of glucocorticoid receptor expression in colonic mucosal cells of patients with ulcerative colitis

AIM: Glucocorticoid (GC) resistant ulcerative colitis (UC) remains a serious disease and is difficult to manage. Although the molecular basis of GC insensitivity is still unknown, GC receptors (GRAAA and GRp) may play an important role in it. This study was aimed to investigate the relationship between the expression of GRa and GRp in colonic mucosal cells of patients with UC, the efficacy of GC therapy and the intensity of inflammation. METHODS: Twenty-five cases of UC were classified into: GC sensitive (n = 16) and GC resistant (n - 9) cases. Patients consisted of mild (n = 6), moderate (n = 8) and severe (n = 11) cases. GRa and GRp expression in colonic mucosal specimens were investigated by immunohistochemistry, and compared between GC resistant and sensitive groups, and also among various degrees of inflammation. RESULTS: All cases were positive for GRa and GRp expression. Both positive association between GRa expression and the response of UC to GC and strong negative association between GRp expression and the response of UC to GC were identified. There was no significant association between GRa/GRp expression and the degree of inflammation of UC. CONCLUSION: These findings suggest that both GRa and GRp may play an important role in the action of GC, and that GRp functions as a dominant negative inhibitor of GRa. Expression of GRa and GRp in colonic mucosal cells of patients with UC may serve as predictors of glucocorticoid response, but can not function as markers of inflammatory intensity.     

糖尿病结肠动力障碍大鼠结肠Cajal间质细胞凋亡和间隙连接蛋白43的表达

目的 探讨糖尿病结肠动力障碍大鼠结肠Cajal间质细胞(ICC)的凋亡及ICC的间隙连接蛋白43(Cx43)表达的变化在结肠动力障碍发生中的意义.方法 雄性Sprague-Dawley(SD)大鼠36只,根据体质量及血糖按随机数字表法分为正常6周组、正常10周组、糖尿病6周组、糖尿病10周组,每组9只.腹腔注射链脲佐菌素建立糖尿病模型,检测体质量、空腹血糖及胃肠推进率;HE染色观察ICC;TUNEL法检测ICC的凋亡指数;免疫组化检测ICC的c-Kit、Cx43蛋白表达.结果 (1)糖尿病组较同时间点正常组体质量下降,空腹血糖升高,胃肠推进率降低,ICC的c-Kit、Cx43蛋白表达降低(F=76.68,1397.24,18.87,137.65,87.73,P均＜0.05).(2)糖尿病10周组较糖尿病6周组空腹血糖升高,胃肠推进率降低,ICC的c-Kit、Cx43蛋白表达降低(F=76.68,1397.24,18.87,137.65,87.73,P均＜0.05).(3)糖尿病组ICC的凋亡指数与同时间点正常组相比,差异无统计学意义;糖尿病10周组与糖尿病6周组ICC的凋亡指数差异也无统计学意义(P均＞0.05).结论 ICC数量减少、Cx43蛋白表达降低可能是糖尿病结肠动力障碍的发生机制之一,且上述改变随病程发展而加重;ICC数量减少可能与ICC凋亡无关.     

胰岛素对2型糖尿病患者结肠Cajal间质细胞及乙酰胆碱变化的影响

目的 比较接受不同治疗的2型糖尿病患者与非糖尿病对照的结肠cajal间质细胞(ICC)及乙酰胆碱(Ach)的形态、分布及数量,了解糖尿病患者结肠ICC及Ach的变化以及胰岛素治疗对其的影响.方法 取结肠癌手术标本切缘的正常结肠组织共81例,其中非糖尿病对照29例,2型糖尿病52例(胰岛素治疗24例,口服降糖药治疗28例).免疫组化检测c-kit阳性细胞及Ach表达;甲苯胺蓝染色肥大细胞. c-kit阳性细胞与肥大细胞数之差为ICC数目,图像处理软件分析Ach表达及密度.结果 结肠的黏膜下层、环行肌及纵行肌层内(IM)、肌间(MY)神经丛均有c-kit阳性细胞分布.糖尿病患者的ICC细胞突起明显减少、分布松散,较多空泡形成,这些变化在口服降糖药组更明显.ICC数目比较:对照组>胰岛素组>口服降糖药组(ICC-MY:60.12比23.95比16.49, P=0.000; ICC.IM:41.79比33.18比25.88, P=0.000).结肠黏膜下神经丛和肌间神经丛均有Ach表达,糖尿病患者Ach阳性细胞分布松散,较多空泡形成,口服降糖药组更明显. Ach阳性表达分析,面密度:对照组>胰岛素组>口服降糖药组(147.50比103.82比86.38, P=0.000).对非糖尿病对照组的分析表明,ICC数目及Ach表达与年龄、性别无相关性.结论 糖尿病患者结肠ICC及Ach较对照组分布松散,较多空泡形成,ICC数量及Ach表达明显减少.胰岛素对上述变化具有保护作用.结肠ICC数目及Ach表达与年龄及性别无关.     

Induction of experimental ulcerative colitis by Fusobacterium varium isolated from colonic mucosa of patients with ulcerative colitis

BACKGROUND: Bacteria are implicated in certain forms of model chronic colitis but the identity and role of bacteria in human ulcerative colitis (UC) are uncertain. AIMS: To isolate pathogenic bacteria from inflamed mucosa of patients with UC, to examine whether the bacteria have a toxin to Vero cells, and to determine whether the toxin induces UC-like lesions in animals. METHODS: Bacteria were isolated from UC patients and supernatants from cultures were filtered and tested for cytotoxicity to Vero cells. Bacterial cells producing the cytotoxic supernatants were examined by polymerase chain reaction for verotoxin genes. Culture supernatants of cytotoxic strains were examined by high performance liquid chromatography for organic acid concentrations. Mice were given enemas containing organic acid at the mean concentration in the supernatants of cytotoxic strains to ascertain whether colonic lesions appear in UC. RESULTS: Only supernatants from cultures of Fusobacterium varium killed Vero cells. Bacterial cells lacked verotoxin genes. Bacterial culture supernatants contained high concentrations of n-butyric acid and the mean concentration (32 mmol/l) was cytotoxic to Vero cells. Twenty four hours after mice were given enemas containing either butyric acid or F varium culture supernatants, colonic ulcers with crypt abscesses, inflammatory cell infiltration, and apoptotic changes were observed. CONCLUSIONS: Butyric acid in culture supernatants from cultures of F varium caused UC-like lesions in mice. This study indicates that F varium may be one of the elusive pathogenic factors in UC.     

Very low intraluminal colonic pH in patients with active ulcerative colitis

Intraluminal gastrointestinal pH was measured in seven patients with active ulcerative colitis (four male, three female). A radiotelemetry capsule was used, and its location was determined by fluoroscopy. Satisfactory measurements were obtained from six, in all of whom pH levels were normal in the stomach and small intestine. Three patients also had normal pH values in the colon. However, in the remaining three patients very low pH levels (lowest values 2.3, 2.9, and 3.4) were found in the proximal parts of the colon. Five of the seven patients, including the three with low pH in the colon, underwent colectomy. The mechanism behind the low intraluminal pH in some patients with ulcerative colitis is speculative. Increased fecal concentrations of lactate occur in active disease, but some of the pH values measured in our study were below the pK_a value of lactate. The study demonstrates that very low intraluminal pH levels in the colon occur in some patients with active ulcerative colitis. This might be an indicator of severe activity of the disease.     

Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis

OBJECTIVE: Increased lymphocyte activation and production of inflammatory cytokines are implicated in the pathogenesis of ulcerative colitis. Because antigen-presenting dendritic cells play a cardinal role in the activation and survival of activated lymphocytes, the aim of the present study was to characterize dendritic cells in ulcerative colitis. DESIGN: This study was designed to compare the phenotypes and functions of peripheral blood dendritic cells among healthy normal volunteers and patients with ulcerative colitis or Crohn's disease. Activated dendritic cells were also localized at the colonic mucosa. METHODS: Peripheral blood dendritic cells were generated from 15 patients with ulcerative colitis, 10 patients with Crohn's disease and 15 healthy control volunteers. The stimulatory capacities of dendritic cells were analysed in an allogenic mixed lymphocyte reaction. Nitric oxide was detected by the Griess method. Single- and dual-colour flow cytometry was employed to study the levels of maturation of dendritic cells. Activated dendritic cells were localized immunohistochemically in the colonic mucosa. RESULTS: In comparison to normal controls, peripheral blood dendritic cells from patients with ulcerative colitis showed significantly increased stimulatory capacities (P < 0.05) and produced significantly higher levels of nitric oxide (P < 0.05). The numbers of activated dendritic cells were also significantly higher in ulcerative colitis (P < 0.05). Mature and activated dendritic cells expressing the CD83 antigen were detected at the inflamed colonic mucosa in patients with ulcerative colitis and Crohn's disease. CONCLUSIONS: Activated and mature dendritic cells may have a role in the induction of an exacerbated immune response in ulcerative colitis. This study provides the scientific and logical basis for blocking the maturation and activation of dendritic cells in ulcerative colitis as a new therapeutic intervention.     

溃疡性结肠炎患者外周血和肠道黏膜辅助性T细胞表型分析

目的 探讨Th细胞各亚群比例在溃疡性结肠炎(UC)发病中的变化及意义.方法 按照2007年济南标准收集UC患者20例,对照组16例.利用细胞内细胞因子染色和四色荧光抗体流式细胞术对肠黏膜及外周血淋巴细胞作表型分析,比较各组肠黏膜固有层单个核细胞(cLPMC)和外周血单个核细胞(PBMC)中Th1、Th2、Th17细胞比例的改变.结果 UC组cLPMC中Th17细胞比例较对照组升高,分别为3.75%(6.93%)和1.25%(3.70%),在PBMC中为1.40%(2.15%)和0.70%(0.33%),两组间差异均有统计学意义(P值均<0.05).UC患者CLPMC中Th17细胞比例与疾病评分呈正相关(r=0.34,P<0.05).UC组和对照组Th1、Th2细胞在cLPMC和PBMC中的比例均差异无统汁学意义(P>0.05),且不同疾病活动度患者间差异亦无统计学意义(P值均>0.05).结论 在Th细胞各亚群中,Th17细胞是介导UC肠道局部和外周免疫应答的优势细胞,可能成为UC治疗的有效靶点.     

Effect of sulfphasalazine on the colonic mucosal malondialdehyde in patients with ulcerative colitis

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溃疡性结肠炎患者外周血和肠道黏膜辅助性T细胞表型分析

目的 探讨Th细胞各亚群比例在溃疡性结肠炎(UC)发病中的变化及意义.方法 按照2007年济南标准收集UC患者20例,对照组16例.利用细胞内细胞因子染色和四色荧光抗体流式细胞术对肠黏膜及外周血淋巴细胞作表型分析,比较各组肠黏膜固有层单个核细胞(cLPMC)和外周血单个核细胞(PBMC)中Th1、Th2、Th17细胞比例的改变.结果 UC组cLPMC中Th17细胞比例较对照组升高,分别为3.75%(6.93%)和1.25%(3.70%),在PBMC中为1.40%(2.15%)和0.70%(0.33%),两组间差异均有统计学意义(P值均<0.05).UC患者CLPMC中Th17细胞比例与疾病评分呈正相关(r=0.34,P<0.05).UC组和对照组Th1、Th2细胞在cLPMC和PBMC中的比例均差异无统汁学意义(P>0.05),且不同疾病活动度患者间差异亦无统计学意义(P值均>0.05).结论 在Th细胞各亚群中,Th17细胞是介导UC肠道局部和外周免疫应答的优势细胞,可能成为UC治疗的有效靶点.     

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.     

溃疡性结肠炎患者肠神经内分泌细胞的观察

目的：观察溃疡性结肠炎患者肠切除标本中神经内分泌细胞的变化。方法：应用免疫组织化学和形态计量学方法。结果：病变肠组织VIP能神经，S-100蛋白及神经特异性烯醇化酶免疫阳性神经元、神经纤维形态异常，其线密度（Lv％）、体密度（Vv％）增高（分别P＜0．01）。生长抑素、血清素免疫阳性细胞及肠嗜银细胞数量明显减少（分别P＜0．01）。结论：肠神经内分泌细胞的异常可能参与溃疡性结肠炎的发生和发展。     

Cytokine production from colonic T cells in patients with ulcerative colitis with and without primary sclerosing cholangitis

PURPOSE: Only five percent of all patients with ulcerative colitis develop primary sclerosing cholangitis. T cells accumulate at the sites of the colonic and bile duct inflammation in both ulcerative colitis and primary sclerosing cholangitis. T helper cell populations comprise functionally distinct subsets characterized by the cytokines they produce. Several alterations in cytokine production have been described in patients with ulcerative colitis. The aim of this study was to investigate possible differences in T helper subsets and cytokine production in peripheral blood and colonic mucosa among ulcerative colitis patients with and without primary sclerosing cholangitis. METHODS: Eleven patients with primary sclerosing cholangitis and extensive ulcerative colitis, 11 patients with extensive ulcerative colitis and no liver disease, and 5 patients without any history of liver disease who underwent routine colonoscopy because of previous polypectomy were included in the study. Colonoscopy with multiple biopsies was performed on all patients. Lamina propria mononuclear cells and peripheral blood mononuclear cells were isolated. A modified version of solid-phase enzyme-linked immunospot assay was used for the separate counting of cells producing interferon-, interleukin-2 (T helper 1), and interleukin-4 (T helper 2). RESULTS: No differences in spontaneous production of cytokines from peripheral blood mononuclear cells was found among the three groups. Patients with primary sclerosing cholangitis compared with patients with ulcerative colitis without liver disease showed a significant increase in the number of cells secreting interferon- after purified protein derivative stimulation (P<0.02). More cells secreting interferon- were found in the two ulcerative colitis groups than in the cell populations from healthy controls (P<0.03). The number of cells secreting interferon- in the primary sclerosing cholangitis group was significantly lower than in the ulcerative colitis group without liver disease (P<0.04). The number of cells secreting interleukin-4 was lower in the primary sclerosing cholangitis group than among the patients with ulcerative colitis only (P=0.05). CONCLUSION: Isolated lymphocytes from colonic mucosa differ in cytokine production in patients with ulcerative colitis with and without primary sclerosing cholangitis.This study was supported by grants from The Swedish Medical Research Council (7129), foundations of the Karolinska Institute, the Nanna Svartz foundation, the Swedish Society of Medicine, and the Ruth and Richard Juhlin foundation.     

神经激肽-1受体在溃疡性结肠炎黏膜中的表达

目的：了解神经激肽-1受体(NK-1R)在溃疡性结肠炎(UC)病人结肠活检黏膜中的表达，探讨该受体的表达与UC严重程度的关系。方法：38个UC黏膜标本取自因该病而行结肠镜检查的病人，男23例，女15例；对照组结肠黏膜取自15例肠易激综合征(IBS)病人，男8例，女7例。应用逆转录聚合酶链反应(RT-PCR)检测对照组和UC肠黏膜NK-1R的mRNA表达水平，应用Western blot技术检测NK-1R的蛋白水平，以免疫组化方法进行NK-1R的组织学定位。结果：与对照组肠黏膜相比，UC黏膜中NK-1R mRNA和蛋白都过度表达，NK-1R mRNA的表达与疾病的严重程度相关。免疫组化检查显示，NK-1R的表达主要位于UC的肠黏膜表面、黏膜固有层的单核细胞、黏膜下层的动静脉等处。结论：UC黏膜组织中NK-1R的表达水平明显上调，扰乱了神经激肽的作用环节，加剧了肠黏膜的病理改变。     

Deficiency of colonic telomerase in ulcerative colitis

OBJECTIVE: GI epithelial cells express telomerase, a ribonucleoprotein that prevents telomeric shortening in proliferating cells. Telomerase levels are high in cancer, but little is known about telomerase expression in other diseases. We, therefore, designed experiments to determine telomerase expression in different colonic segments and to compare this with corresponding segments in patients with ulcerative colitis. Colorectal cancers and adenomatous polyps were included as disease controls. METHODS: In total, telomerase expression was determined in colonic tissues obtained from 62 patients. Twenty-five patients had ulcerative colitis, 21 had normal colons, 11 had colorectal cancer, and nine had adenomatous polyps. Endoscopic biopsies were collected prospectively at colonoscopy, processed for telomerase assays (Telomeric Repeat Amplification Protocol), hematoxylin and eosin staining, and scored for inflammation. RESULTS: Telomerase activity is expressed in arbitrary units (median 95% confidence interval). In the normal colon, telomerase activity in the cecum, transverse, sigmoid, and rectum was 255 (171-449), 707 (374-895), 561 (468-1426), and 563 (402-846), respectively. Telomerase was higher in the distal three segments when compared with the cecum (p = 0.005). In ulcerative colitis, there was a marked decrease in telomerase activity in the cecum 152 (59-272), p = 0.04, transverse 180 (129-365), p < 0.001, sigmoid 352 (114-464), p = 0.005, and rectum 180 (70-337), p = 0.001 when compared with normals. Telomerase activity correlated negatively with inflammation (r = -0.32, p = 0.001) and was also decreased in microscopically normal areas. Cancers expressed high levels of telomerase. CONCLUSIONS: Colonic mucosal expression of telomerase is reduced in ulcerative colitis. Levels are low even in microscopically normal mucosa, suggesting that telomerase deficiency may contribute to the pathogenesis of the disease.     

In vivo measurement of colonic butyrate metabolism in patients with quiescent ulcerative colitis

BACKGROUND: Butyrate, a short chain fatty acid produced by bacterial fermentation, is a major fuel source for the colonocyte. In vitro work has shown that ulcerative colitis may be characterised by a metabolic defect in colonocyte butyrate oxidation. AIMS: To investigate the rate of metabolism of rectally administered butyrate in patients with quiescent colitis. METHODS: [1-(13)C]-butyrate enemas were administered to 11 patients with long standing quiescent ulcerative colitis and to 10 control patients. The rate of production of (13)CO(2) in exhaled breath over four hours was measured by isotope ratio mass spectrometry combined with indirect calorimetry in order to measure CO(2) production. This allowed calculation of the patients' resting energy expenditure and respiratory quotient. RESULTS: Over a four hour period, 325 (SEM 21) micromol (13)CO(2) was recovered in breath samples from the colitis group compared with 322 (17) micromol from the control group (NS). The respiratory quotient of the colitic group was significantly lower than that of the control group. CONCLUSION: There was no difference in the rate of metabolism of butyrate between the two groups. It is unlikely that there is a primary metabolic defect of butyrate metabolism in patients with quiescent ulcerative colitis.