Relationship of hydrolase activities to epithelial cell turnover in distal colonic mucosa of normal rats

BACKGROUND: The relationships between changes induced by diet in colonic epithelial kinetics and in the activities of brush border hydrolases are poorly defined. The aims of this study are to define these relationships, as changes in kinetics would be expected to influence differentiation, and to determine whether the type of ingested dietary indigestible carbohydrates influences hydrolase activities. METHODS: Groups of eight rats were fed a low fibre diet +/- supplements of different types of indigestible carbohydrates for 4 weeks. Alkaline phosphatase (ALP) and dipeptidyl peptidase IV (DPPIV) activities and epithelial kinetics were measured in distal colonic mucosa. RESULTS: Median ALP activities correlated positively and DPPIV activity negatively with the median proportion of cells entering metaphase (r = 0.58 and -0.58, respectively; P < 0.05) and number of metaphase arrests per crypt column across the diets (r = 0.59 and 0.58, respectively; P < 0.05). Stepwise regression analysis showed that both hydrolases independently predicted these kinetic indices (R2 > 63% for each). Mucosal ALP activities were markedly elevated during consumption of raw potato starch, guar gum and methylcellulose, while only potato starch caused a significant elevation of DPPIV activities. CONCLUSIONS: The type of indigestible carbohydrate in the diet influences colonic mucosal hydrolase activities. The opposite relationship between kinetics and each of the two hydrolases indicates that these hydrolases do not reflect the same event; dipeptidyl peptidase IV might relate to differentiation status while ALP could also be influenced by epithelial irritation due to changes in luminal conditions.     

Lactobacillus reuteri maintains intestinal epithelial regeneration and repairs damaged intestinal mucosa

ABSTRACT

Little is known about the regulatory effect of microbiota on the proliferation and regeneration of ISCs. Here, we found that L. reuteri stimulated the proliferation of intestinal epithelia by increasing the expression of R-spondins and thus activating the Wnt/β-catenin pathway. The proliferation-stimulating effect of Lactobacillus on repair is further enhanced under TNF -induced intestinal mucosal damage, and the number of Lgr5⁺ cells is maintained. Moreover, compared to the effects of C. rodentium on the induction of intestinal inflammation and crypt hyperplasia in mice, L. reuteri protected the intestinal mucosal barrier integrity by moderately modulating the Wnt/β-catenin signaling pathway to avoid overactivation. L. reuteri had the ability to maintain the number of Lgr5⁺ cells and stimulate intestinal epithelial proliferation to repair epithelial damage and reduce proinflammatory cytokine secretion in the intestine and the LPS concentration in serum. Moreover, activation of the Wnt/β-catenin pathway also induced differentiation toward Paneth cells and increased antimicrobial peptide expression to inhibit C. rodentium colonization. The protective effect of Lactobacillus against C. rodentium infection disappeared upon application of the Wnt antagonist Wnt-C59 in both mice and intestinal organoids. This study demonstrates that Lactobacillus is effective at maintaining intestinal epithelial regeneration and homeostasis as well as at repairing intestinal damage after pathological injury and is thus a promising alternative therapeutic method for intestinal inflammation.     

Crosstalk between intestinal epithelial cell and adaptive immune cell in intestinal mucosal immunity

Constantly challenged by luminal bacteria, intestinal epithelium forms both a physical and biochemical defense against pathogens. Besides, intestinal epithelium senses dynamic and continuous changes in luminal environment and transmits signals to subjacent immune cells accordingly. It has been long accepted that adaptive immune cells fulfill their roles partly by modulating function of intestinal epithelial cells. Recent studies have brought up the proposal that intestinal epithelial cells also actively participate in the regulation of adaptive immunity, especially CD4+ adaptive T cells, which indicates that there is reciprocal crosstalk between intestinal epithelial cells and adaptive immune cells, and the crosstalk may play important role in intestinal mucosal immunity. This Review makes a comprehensive summary about crosstalk between intestinal epithelial cells and CD4+ adaptive T cells in intestinal immunity. Special attention would be given to their implications in inflammatory bowel disease pathogenesis and potential therapeutic targets.     

The influence of conditioned medium from mouse intestinal epithelial cells on the proliferative activity of crypt cells: role of granulocyte-macrophage colony-stimulating factor

Background: The aim of this work was to study the influence of soluble factors produced by native mouse intestinal epithelial cells (IECs) on the proliferative activity of freshly isolated intestinal crypt cells. Methods: The crypt cells were cultured with either conditioned medium and its ultrafiltrates or recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence or absence of neutralizing anti-GM-CSF antibodies. GM-CSF in culture medium was identified by the electrochemiluminescence method. Results: It was demonstrated that the IEC conditioned medium contained GM-CSF. This cytokine led to both the upregulation and downregulation of crypt cell proliferative activity, depending on its concentration in the culture medium. The effect of native GM-CSF was reproduced with recombinant mouse GM-CSF: 25 and 5 ng/ml inhibited the proliferative activity, whereas 1 ng/ml led to its significant stimulation. Conclusions: Freshly isolated murine IECs produce GM-CSF, which plays a critical role in crypt cell proliferative activity in vitro. These results suggest the involvement of this factor in the regulation of the crypt proliferative zone, in an autocrine and/or paracrine manner. Received: February 25, 2002 / Accepted: July 26, 2002     

Crosstalk between Wnt and Notch signaling in intestinal epithelial cell fate decision

Continuous renewal of the intestinal epithelium requires coordinated regulation to maintain the balance between proliferation and differentiation of the epithelial stem cells and immature progenitor cells. Canonical Wnt signaling has long been regarded as the signaling pathway playing a central role in this epithelial cell fate determination; however, recent studies have shown that Notch signaling is also indispensable for this process. Here, we review the current concepts of how the Wnt and Notch pathways control intestinal epithelial cell fate decisions, particularly focusing on their crosstalk at both tissue and cellular levels. As several features are shared between stem cell renewal and cancer cell renewal, comprehensive understanding of how the Wnt and Notch signaling pathways cooperate and integrate in the gut epithelium has significant implications for the development of novel therapeutic modalities for intestinal neoplasia.     

The intestinal barrier: a fundamental role in health and disease

Introduction: The gastrointestinal mucosa constitutes a critical barrier where millions of microbes and environmental antigens come in close contact with the host immune system. Intestinal barrier defects have been associated with a broad range of diseases and therefore denote a new therapeutic target.

Areas covered: This review is based on an extensive literature search in PubMed of how the intestinal barrier contributes to health and as a trigger for disease. It discusses the anatomy of the intestinal barrier and explains the available methods to evaluate its function. Also reviewed is the importance of diet and lifestyle factors on intestinal barrier function, and three prototypes of chronic diseases (inflammatory bowel disease, celiac disease and nonalcoholic fatty liver disease) that have been linked to barrier defects are discussed.

Expert commentary: The intestinal barrier has been investigated by various methods, but correlation of results across studies is difficult, representing a major shortcoming in the field. New upcoming techniques and research on the effect of barrier-restoring therapeutics may improve our current understanding of the gut barrier, and provide a step forward towards personalised medicine.     

Heterotopic gastric mucosa with focal intestinal metaplasia and squamous epithelium in the rectum

Heterotopic gastric mucosa has been described in all levels of the gastrointestinal tract. However, gastric heterotopia of the rectum is a rare finding. It is usually reported along with polyp located in the rectum between 5 and 8 cm from the anal verge. The most common symptom is painless rectal bleeding, and non-specific gastrointestinal symptoms may also be presented. We report an incidentally found case of a 46-year-old man without any gastrointestinal symptoms. The pathology showed gastric mucosa and squamous epithelium and focal intestinal metaplasia. This finding could be a clue as to the origins of the heterotopic gastric mucosa. Although there are no guidelines for treatment or the follow-up period, regular endoscopic surveillance is necessary for gastric cancer screening.     

Effect of butyrate on paracellular permeability in rat distal colonic mucosa ex vivo

BACKGROUND AND AIMS: The effects of butyrate on colonic epithelial barrier function are poorly understood. The aim of this study was to examine the short-term effects of butyrate on paracellular permeability of rat distal colonic epithelium. METHODS: Mucosa mounted in Ussing chambers was treated with butyrate (1-10 mmol/L) for 4 h. Transepithelial conductance, [51Cr]-EDTA flux, mucosal brush border hydrolase activity and epithelial kinetics, using proliferating cell nuclear antigen (PCNA) staining, were measured. RESULTS: On exposure to butyrate (10 mmol/L, but not 1 or 5 mmol/L), transepithelial conductance was 65 +/- 2% higher (mean +/- SEM; n = 8, P < 0.05, paired t-test) and the rate coefficient for [51Cr]-EDTA flux was 65 +/- 25% higher (P = 0.03) than those of control tissue. Histologically, the epithelium exhibited no signs of injury, but butyrate-treated tissue exhibited interstitial oedema consistent with water uptake in association with butyrate absorption. Butyrate caused a reduction in crypt column height to 30.6 +/- 1.6 cells from 33.4 +/- 1.8 cells in controls (n = 10, P < 0.03), but the number of cells per crypt column staining with PCNA was unchanged. Butyrate significantly reduced the mucosal activities of alkaline phosphatase by 40 +/- 16%, maltase by 54 +/- 12% and dipeptidyl peptidase IV by 41 +/- 14%. CONCLUSIONS: Acute exposure to butyrate increased paracellular permeability in rat distal colon. The mechanism involved may relate to the loss of differentiated surface epithelial cells, or as a physiological response to Na+-coupled butyrate uptake.     

Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions

Apoptosis is now recognized as an important process responsible for maintenance of the cellular balance between proliferation and death. Apoptosis is distinct from necrosis in that it is a programmed form of cell death and occurs without any accompanying inflammation. This form of cell death can be induced by a wide range of cellular signals, which leads to activation of cell death machinery within the cell and is characterized by distinct morphological changes. Apoptosis is especially relevant in the gastrointestinal tract, as the mammalian intestinal mucosa undergoes a process of continual cell turnover that is essential for maintenance of normal function. Cell proliferation is confined to the crypts, while differentiation occurs during a rapid, orderly migration up to the villus. The differentiated enterocytes, which make up the majority of the cells, then undergo a process of programmed cell death (apoptosis). Although apoptosis is essential for the maintenance of normal gut epithelial function, dysregulated apoptosis is seen in a number of pathological conditions in the gastrointestinal tract. The cellular mechanisms regulating this tightly regimented process have not been clearly defined and this topic represents an area of active investigation as delineation of this process will lead to a better understanding of normal gut mucosal growth.     

Sustained epithelial proliferation in a functionally irreversible fundic mucosa after Helicobacter pylori eradication

Background  We recently reported the expansion of the acid-secreting mucosa following Helicobacter pylori eradication with Congo red chromoendoscopy for a short-term follow-up of up to 7 months. We aimed to extend the observation period and to clarify the characteristic features of acid-secreting and non-acid-secreting mucosa. Methods  In 24 H. pylori-positive patients with fundic atrophy, Congo red chromoendoscopy was performed prior to, 1 month, 7 months, and finally more than 2 years after the eradication. The areas of the acidsecreting mucosa were evaluated semiquantitatively. Two gastric biopsy specimens were taken from the acidsecreting and non-acid-secreting areas at the final chromoendoscopy and were subjected to histologic evaluation and immunohistochemistry for Ki-67 as a proliferation index. Results  After a gradual increase in acidsecreting areas for up to 7 months after eradication, they further increased in 79% subjects between 7 months and the final observation at a mean follow-up of 62 months. However, there still existed non-acid-secreting mucosa in the fundic area in all subjects, indicating that the expansion of acid-secreting mucosa remained partial. Compared with the neighboring acid-secreting area, the non-acid-secreting area was characterized histologically by higher degrees of residual inflammation, mucosal atrophy, and intestinal metaplasia, and by sustained hyperproliferation as well. Conclusions  Functionally irreversible (non-acid-secreting) gastric mucosa after eradication was associated with extensive intestinal metaplasia and sustained hyperproliferation, suggesting that such mucosa still possesses malignant potential. Congo red chromoendoscopy may be useful for estimating the risk of subsequent development of gastric cancer following successful H. pylori eradication by determining the distribution of functionally irreversible mucosa.     

Environmental stress causes mast cell degranulation, endothelial and epithelial changes, and edema in the rat intestinal mucosa.

OBJECTIVE: Mental stress has been shown to produce intestinal disease, but the effects of a mild environmental stress on intestinal physiology have not been elucidated. This study was performed to determine the effects of environmental stress on the ultrastructure of the intestinal mucosa, using the rat as an experimental model. METHODS: One group of rats (group A, n = 3) was examined immediately upon arrival at the animal care facility. Groups B (n = 6) and C (n = 6) were housed in rooms with high and low personnel activity, respectively, for up to 4 wk. Group D (n = 8) was housed in the high activity room for 3 to 4 wk followed by 1, 2, or 3 in the low activity room. RESULTS: Rats in group B had the greatest number of degranulated intestinal mucosal mast cells, and activated goblet cells. Intestinal villi were edematous and epithelial cells were detaching from the basement membrane at villus tips. Changes were observed in capillary endothelial ultrastructure. In group B there were greater numbers of vesicles and multilamellar fenestral diaphragms compared to group C. Rats in groups A and C had the lowest numbers of degranulated mast cells and activated goblet cells. Intestinal villi showed normal ultrastructure. Group D was in a recovery phase and the condition of the intestinal mucosa was improved relative to group B, but the number of degranulated mast cells was not significantly reduced. CONCLUSIONS: This study demonstrates that environmentally induced stress causes pathological changes in the rat intestinal mucosa that compromise the epithelial-endothelial exchange barrier. These results emphasize the importance of closely monitoring the environment of experimental animals and provide evidence to stimulate further research into the mechanisms linking mental stress to gastrointestinal dysfunction in humans.     

Role of urokinase and its receptor in basal and stimulated colonic epithelial cell migration in vitro

BACKGROUND: Migration of colonic epithelial cells is important for mucosal repair following injury. The urokinase (u-PA) system regulates migration in other cell types. AIM: To examine the role of u-PA and its receptor (u-PAR) in colonic epithelial cell migration. METHODS: Migration was assessed over 24 hours in circular wounds made in confluent monolayers of LIM1215 and Caco-2 human colon cancer cells. The function of u-PA and u-PAR was ablated with antisense oligonucleotides to block expression, with synthetic u-PA peptides to block interaction, and with aprotinin to block u-PA mediated proteolysis. RESULTS: Migration was stimulated two to threefold by exogenous u-PA, an effect dependent on u-PAR binding but independent of u-PA mediated mitogenesis and proteolysis. Expression of u-PA and u-PAR was inhibited by 80% by the appropriate antisense oligonucleotide. Basal migration and the motogenic effects of butyrate, epidermal growth factor, and phorbol-12-myristate-13-acetate were suppressed by the u-PAR antisense oligonucleotide (40-60%) but were at best minimally affected following inhibition of u-PA expression and binding. CONCLUSIONS: In an in vitro model of wounded colonic epithelium, u-PAR promotes cell migration through mechanisms that are not exclusively dependent on u-PA binding. Therefore, u-PA and u-PAR may contribute to colonic mucosal repair in vivo.     

Immunopathogenesis of primary sclerosing cholangitis: possible role of a shared colonic and biliary epithelial antigen

Abstract   Primary sclerosing cholangitis (PSC) is a chronic fibrosing disease of both extrahepatic as well as intrahepatic bile ducts that is strongly associated with inflammatory bowel disease, particularly ulcerative colitis. It is characterized by progressive destruction of the bile ducts leading to widespread biliary obstructions and biliary cirrhosis. PSC is currently one of the more common indications for liver transplantation. It is an immune mediated disorder associated with autoantibodies against both colon epithelium as well as biliary epithelium, infiltration of the portal tract with functional T cells, abnormal expression of HLA molecules on biliary epithelial cells and a restricted T-cell receptor repertoire. Activation of the complement system, following the deposition of antigen-specific autoantibody with biliary epithelial cells, may also contribute to the pathogenesis of PSC. Four different HLA haplotypes have been associated with PSC: three with increased risk of disease and one with reduced risk. Bacterial products entering the biliary epithelium from colon may be a triggering factor strongly associated with ulcerative colitis; however, a further immune mediated chronic inflammation may be associated with cellular antigen(s) which is shown to be shared by human colon and biliary epithelium by molecular mimicry.     

Modulation of distal colonic epithelial barrier function by dietary fibre in normal rats

BACKGROUND: Dietary fibre influences the turnover and differentiation of the colonic epithelium, but its effects on barrier function are unknown. AIMS: To determine whether altering the type and amount of fibre in the diet affects paracellular permeability of intestinal epithelium, and to identify the mechanisms of action. METHODS: Rats were fed isoenergetic low fibre diets with or without supplements of wheat bran (10%) or methylcellulose (10%), for four weeks. Paracellular permeability was determined by measurement of conductance and 51Cr-EDTA flux across tissue mounted in Ussing chambers. Faecal short chain fatty acid (SCFA) concentrations were assessed by gas chromatography, epithelial kinetics stathmokinetically, and mucosal brush border hydrolase activities spectrophotometrically. RESULTS: Body weight was similar across the dietary groups. Conductance and 51Cr-EDTA flux were approximately 25% higher in animals fed no fibre, compared with those fed wheat bran or methylcellulose in the distal colon, but not in the caecum or jejunum. Histologically, there was no evidence of epithelial injury or erosion associated with any diet. The fibres exerted different spectra of effects on luminal SCFA concentrations and pH, and on mucosal indexes, but both bulked the faeces, were trophic to the epithelium, and stimulated expression of a marker of epithelial differentiation. CONCLUSIONS: Both a fermentable and a non-fermentable fibre reduce paracellular permeability specifically in the distal colon, possibly by promoting epithelial cell differentiation. The mechanisms by which the two fibres exert their effects are likely to be different.     

双歧杆菌分泌型黏附素对肠上皮细胞形态学的影响

目的:观察双歧杆菌分泌型黏附素对肠上皮细胞形态学变化的影响.方法:肠上皮细胞经LPS.H2O2和双歧杆菌分泌型黏附素预孵处理后,采用光镜、透射电镜和丫啶橙染色分别观察其形态学的变化,结果:光镜下观察见LPS或H2O2处理组细胞变小、变圆,胞质颗粒明显增多,部分细胞脱落漂浮于培养基中;先经黏附素处理后,变小、变圆,胞质颗...     

Epithelial cell turnover in relation to ongoing damage of the gastric mucosa in patients with early gastric cancer: increase of cell proliferation in paramalignant lesions

Background Gastric cancer is typically an end result of Helicobacter pylori-associated chronic gastritis. The pathogenesis is thought to involve effects on gastric mucosal epithelial cell turnover. In this study, we aimed to compare apoptosis and proliferation in the noncancer-containing mucosa of H. pylori-positive patients with early gastric cancer with these phenomena in H. pylori-positive controls.Methods Two specimens each were obtained from the greater and lesser curvatures of the corpus and from the greater curvature of the antrum. The histopathological grading used was the updated Sydney System. Apoptotic epithelial cells were detected using the terminal deoxy nucleotidyl transferase-mediated deoxy-uridine triphosphate (dUTP) biotin nick-end labeling (TUNEL) method. The expression of Ki 67 was evaluated by immunostaining.Results Forty-five H. pylori-positive patients with endoscopic mucosal resection for early gastric cancer and 52 H. pylori-positive controls were studied. Gastric cancer was associated with a higher frequency of incomplete intestinal metaplasia (IM; odds ratio [OR], 19.1; 95% confidence interval [CI], 6.9–53.2; P < 0.001). The apoptotic index (AI) in the greater curvature of the corpus and the proliferation index (PI) in each part were significantly higher in cancer patients than in the control group. The median PI in the antrum was significantly higher in the incomplete IM group than that in the complete IM group (17.6 vs 12.6; P = 0.009). The PI and the AI in the greater curvature of the corpus correlated with the activity score, and the PI correlated with the IM score.Conclusions In the cancer patients, H. pylori-induced gastritis was associated with increased cell proliferation and apoptosis compared with mucosal findings in the controls. IM seems to be one of the most important factors affecting cell proliferation and may be one of the components of carcinogenesis that results in proliferation-dominant cell kinetics.     

Localization of immunoreactive secretory leukocyte protease inhibitor (SLPI) in intestinal mucosa

Secretory leukocyte protease inhibitor (SLPI) is the dominant protease inhibitor in the mucus secretions of the repiratory and genital tracts, and local production seems likely, as immunoreactive SLPI has been found in the corresponding mucosa. To our knowledge, SLPI has not been previously demonstrated in intestinal epithelia or secretions. In an earlier study, however, we found surprisingly high levels of SLPI in peritonitis exudate from patients with gastrointestinal perforations. This study extends these observations by demonstrating the presence of immunoreactive SLPI in intestinal mucosa. In the small intestine, SLPI was present in Paneth cells and in scattered mucosa cells of goblet-type. In normal mucosa of the large bowel, SLPI was also found in scattered cells of goblet-type in the epithelium. In addition, immunoreactive SLPI was frequently found in colonic adenomas. The findings in this study raise several interesting questions on the possible role of SLPI in the gut epithelial defense against inflammatory assaults.