D. Mark Pritchard

Characterization of epithelial cell shedding from human small intestine

Intestinal epithelial cells migrate from the base of the crypt to the villi where they are shed. However, little is known about the cell shedding process. We have studied the role of apoptosis and wound healing mechanisms in cell shedding from human small intestinal epithelium. A method preparing paraffin sections of human small intestine that preserves cell shedding was developed. A total of 14 417 villus sections were studied. The relationship of cell shedding to leukocytes (CD45), macrophages (CD68) and blood vessels (CD34) were studied by immunohistochemistry. Apoptotic cells were identified using the M30 antibody against cleaved cytokeratin 18 and an antibody against cleaved caspase-3. Potential wound healing mechanisms were studied using antibodies against Zona Occludens-1 (ZO-1) and phosphorylated myosin light chains (MLCs). We found that 5.3% of villus sections contained a shedding cell. An eosin-positive gap was often seen within the epithelial monolayer beneath shedding cells. Shedding was not associated with leukocytes, macrophages or blood vessels. Cells always underwent apoptosis during ejection from the monolayer. Apoptotic bodies were never seen in the monolayer but morphologically normal cells that were positive for M30 or cleaved caspase-3 were often seen. ZO-1 protein was usually (41/42) localized to the apical pole of cells neighboring a shedding event. Phosphorylated MLCs could be identified in 50% of shedding events. In conclusion, cell shedding is associated with apoptosis though it remains unclear whether apoptosis initiates shedding. It is also associated with phosphorylation of MLCs; a process associated previously with wound healing.

The management of gastric polyps

Background Gastric polyps are important as some have malignant potential. If such polyps are left untreated, gastric cancer may result. The malignant potential depends on the histological type of the polyp. The literature base is relatively weak and any recommendations made must be viewed in light of this. Definition Gastric polyps are sessile or pedunculated lesions that originate in the gastric epithelium or submucosa and protrude into the stomach lumen. Malignant potential Depending on histological type, some gastric polyps (adenomas and hyperplastic polyps) have malignant potential and are precursors of early gastric cancer. They may also indicate an increased risk of intestinal or extra-intestinal malignancy.

KLF4 gene expression is inhibited by the notch signaling pathway that controls goblet cell differentiation in mouse gastrointestinal tract

In Kruppel-like factor (KLF)-4-deficient mice, colonic goblet cell numbers are significantly reduced. Goblet cell development is regulated by the Notch signaling pathway. The aim of this study was to examine whether Notch represses KLF4 expression to regulate goblet cell differentiation. We first detected that KLF4 gene expression was upregulated in a human progastrin-overexpressing mouse model where goblet cell hyperplasia has been observed. We then found that mice treated with a gamma-secretase inhibitor (compound E, 10 micromol/kg) for 24 h, which inhibits the Notch signaling pathway, had significantly increased KLF4 mRNA levels in small intestine and colon, accompanied by an increased number of KLF4-expressing cells at the bottom of crypts in small intestine and colon. In a colon cancer cell line (HCT116 cells), KLF4 promoter activity was inhibited by a constitutively active form of Notch1 (ICN1) by transient cotransfection assays. This inhibition was significantly compromised by a dominant-negative RBPjk, a repressive mediator of the Notch signaling pathway. An ICN1-responsive element was then mapped in the human KLF4 promoter between -151 and -122 nucleotides upstream of the transcriptional start site. It was also found that an intact ICN1-responsive element is required for ICN1 to inhibit KLF4 promoter activity by transient cotransfection assays. Our findings thus reveal a possible mechanism by which KLF4 is inhibited by Notch, which controls goblet cell differentiation in mouse gastrointestinal tract.

Regular ArticlesRadiation-Induced p53 and p21WAF–1/CIP1 Expression in the Murine Intestinal Epithelium: Apoptosis and Cell Cycle Arrest

p53-dependent expression of p21WAF-1/CIP1 has been studied in murine intestinal epithelium after exposure to ionizing radiation. In un-irradiated small intestine, neither p53 nor p21WAF-1/CIP1 could be detected by immunohistochemistry. After irradiation (8 Gy), there was a time- and dose-dependent increase in the expression of both proteins. In the small bowel, the positional expression of p53 and p21WAF-1/CIP1 was similar but not coincident. Both proteins could be observed throughout the crypts with greatest frequency of expression over the first 15 cell positions, which includes the stem cell population (approximately positions 3 to 5) and the proliferating, transit cell population (approximately positions 5 to 15). p53-positive cells were primarily distributed toward the base of the crypt relative to p21WAF-1/CIP1. Subdivision of the p53-positive cell population revealed that the cells with strongest p53 immunoreactivity were positioned farther toward the base of the crypt, and their distribution was approximately coincident with the frequency distribution of apoptotic cells. Cells that were either weakly or moderately immunoreactive for p53 were located toward the middle of the crypt and were approximately coincident with the distribution of p21WAF-1/CIP1. The numbers of both p53- and p21WAF-1/CIP1-positive cells declined steadily with time, and by 6 days after irradiation there were very few immunoreactive cells to observe. Radiation-induced increase in p53 and p21WAF-1/CIP1 expression was not detected in mice homozygously null for p53. Expression of p21WAF-1/CIP1 and incorporation of tritiated thymidine were found to be mutually exclusive. In the large bowel, p21WAF-1/CIP1 and p53 expression were observed along the entire length of the colonic crypts after irradiation (8 Gy), and, unlike in the small intestine, this expression was not only maintained but increased over 72 hours. p21WAF-1/CIP1 immunoreactivity was detected in large intestine epithelium up to 6 days after irradiation. The differential expression of p21WAF-1/CIP1, observed between the large and small bowel and within the small intestinal crypts, is discussed.

Helicobacter pylori Induces Plasminogen Activator Inhibitor 2 in Gastric Epithelial Cells through Nuclear Factor-κB and RhoA Implications for Invasion and Apoptosis

The gastric pathogen Helicobacter pylori is associated with a progression to gastric cancer. The specific targets of H. pylori that might influence this progression are still unclear. Previous studies indicated that the gastric hormone gastrin, which may be increased in H. pylori infection, stimulates gastric expression of plasminogen activator inhibitor (PAI)-2, which is an inhibitor of the urokinase plasminogen activator and has previously been shown to be increased in gastric adenocarcinoma. Here, we report that H. pylori also increases PAI-2 expression. In gastric biopsies of H. pylori-positive subjects there was increased PAI-2, including subjects with plasma gastrin concentrations in the normal range. PAI-2 was expressed mainly in chief and mucous cells. In a gastric cancer cell line (AGS), H. pylori increased PAI-2 expression, which was associated with inhibition of H. pylori-stimulated cell invasion and apoptosis. The induction of PAI-2 by H. pylori was mediated by release of interleukin-8 and activation of cyclooxygenase-2, and interestingly, gastrin stimulated PAI-2 expression by similar paracrine pathways. The activation of NFκB was required for interleukin-8 and cyclooxygenase-2 activation but did not occur in cells responding to these paracrine mediators. The data suggest that induction of PAI-2 is a specific target in H. pylori infection, mediated at least partly by paracrine factors; induction of PAI-2 inhibits cell invasion and apoptosis and is a candidate for influencing the progression to gastric cancer.

A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide

SUMMARY The gut barrier, composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS) administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1−/−), Tnfr2 (Tnfr2−/−), nuclear factor kappa B1 (Nfκb1−/−) or Nfĸb2 (Nfĸb2−/−). Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and via NFκB2 favoring apoptosis. This model will enable investigation of the importance and regulation of pathological IEC apoptosis and cell shedding in various diseases.

Gastrin-induced apoptosis contributes to carcinogenesis in the stomach.

Hypergastrinemia in INS-GAS mice leads to accelerated carcinogenesis of the stomach, but the mechanisms have not been well defined. We investigated the possible role of gastrin-induced gastric cell apoptosis in the development of gastric cancer. We examined apoptosis and the expression of Bcl-2 family proteins in INS-GAS mice of different ages, as well as in gastrin-deficient (GAS-KO) mice after gastrin-17 (G-17) infusion. In addition, we studied the effects of the gastrin/cholecystokinin-2 (CCK-2) receptor antagonist YF476 and/or histamine H2 (H-2) receptor antagonist loxtidine on apoptosis and atrophy in INS-GAS mice with or without Helicobacter felis (H. felis) infection. INS-GAS mice had age-associated increases in Bax protein expression and decreases in Bcl-2 protein expression, along with increased glandular and epithelial cell apoptosis. At 8-week gastrin infusions in GAS-KO mice resulted in a similar pattern of altered Bax and Bcl-2 expression, followed by gastric cell apoptosis. H. felis infection of INS-GAS mice led to increased apoptosis and the development of atrophy, whereas treatment with either YF476 and/or loxtidine strongly inhibited both apoptosis and atrophy. In vitro studies with Fas-expressing RGM1 cells showed that gastrin stimulation alone directly induced apoptosis via gastrin/CCK-2 receptor and synergized with FasL stimulation. These results indicate that gastrin can induce apoptosis in gastric epithelial cells and contribute to the development of gastric carcinogenesis.

Helicobacter pylori and gastric cancer.

Purpose of review Although chronic Helicobacter pylori infection is the strongest known risk factor for development of gastric adenocarcinoma, only a small proportion of infected individuals will ever develop tumours. This article discusses various bacterial, host and environmental factors which may influence an individuals susceptibility. Recent findings Recent research on bacterial virulence factors has focussed upon the cag pathogenicity island, particularly its roles in regulating epithelial growth and adhesion. Studies of host genetic factors have included several analyses of polymorphisms in inflammatory cytokines in human cohorts. Animal studies have recently clarified the roles of dysregulated epithelial apoptosis, proliferation and differentiation pathways during gastric carcinogenesis, and novel experiments involving H. felis infection of bone marrow transplanted irradiated mice have suggested that gastric cancer may originate from bone marrow-derived stem cells. Important roles for signalling between epithelial and mesenchymal cells, particularly myofibroblasts, are also emerging. Recent research on the importance of environmental factors has demonstrated how helminth coinfection may protect against atrophic gastritis and T helper type 1 responses. Summary Complex interactions between several bacterial, host genetic and environmental factors determine whether H. pylori infected individuals develop gastric carcinoma. The importance of bone marrow stem cell engraftment during human gastric neoplasia is an area requiring urgent investigation.

Conditional Deletion of IκB-Kinase-β Accelerates Helicobacter-Dependent Gastric Apoptosis, Proliferation, and Preneoplasia

BACKGROUND & AIMS The nuclear factor kappaB (NF-kappaB)/IkappaB-kinase-beta (IKKbeta) pathway has been shown to represent a key link between inflammation and cancer, inducing pro-inflammatory cytokines in myeloid cells and anti-apoptotic pathways in epithelial cells. However, the role of NF-kappaB pathway in gastric carcinogenesis and injury has not been well-defined. We derived mice with a conditional knockout of IKKbeta in gastric epithelial cells (GECs) and myeloid cells, and examined responses to ionizing radiation (IR) and Helicobacter felis infection. METHODS Ikkbeta(Deltastom) mice were generated by crossing Foxa3-Cre mice to Ikkbeta(F/F) mice. Cellular stress was induced with IR and H felis in Ikkbeta(Deltastom), Ikkbeta(F/F), and cis-NF-kappaB-enhanced green fluorescent protein (GFP) reporter mice. Gastric histopathology, apoptosis, proliferation, necrosis, reactive oxygen species, and expression of cytokines, chemokines, and anti-apoptotic genes were assessed. The role of myeloid IKKbeta in these models was studied by crosses with LysM-Cre mice. RESULTS NF-kappaB activity was upregulated in myeloid cells with acute H felis infection, but in GECs by IR or long-term H felis infection during progression to dysplasia. Deletion of IKKbeta in GECs led to increased apoptosis, reactive oxygen species, and cellular necrosis, and resulted in up-regulation of interleukin-1alpha and down-regulation of anti-apoptotic genes. Loss of IKKbeta in GECs resulted in worse inflammation and more rapid progression to gastric preneoplasia, while loss of IKKbeta in myeloid cells inhibited development of gastric atrophy. CONCLUSIONS The loss of IKKbeta/NF-kappaB signaling in GECs results in increased apoptosis and necrosis in response to cellular stress, and accelerated development of dysplasia by Helicobacter infection.

Netazepide, a Gastrin Receptor Antagonist, Normalises Tumour Biomarkers and Causes Regression of Type 1 Gastric Neuroendocrine Tumours in a Nonrandomised Trial of Patients with Chronic Atrophic Gastritis

Introduction Autoimmune chronic atrophic gastritis (CAG) causes hypochlorhydria and hypergastrinaemia, which can lead to enterochromaffin-like (ECL) cell hyperplasia and gastric neuroendocrine tumours (type 1 gastric NETs). Most behave indolently, but some larger tumours metastasise. Antrectomy, which removes the source of the hypergastrinaemia, usually causes tumour regression. Non-clinical and healthy-subject studies have shown that netazepide (YF476) is a potent, highly selective and orally-active gastrin/CCK-2 receptor antagonist. Also, it is effective in animal models of ECL-cell tumours induced by hypergastrinaemia. Aim To assess the effect of netazepide on tumour biomarkers, number and size in patients with type I gastric NETs. Methods We studied 8 patients with multiple tumours and raised circulating gastrin and chromogranin A (CgA) concentrations in an open trial of oral netazepide for 12 weeks, with follow-up 12 weeks later. At 0, 6, 12 and 24 weeks, we carried out gastroscopy, counted and measured tumours, and took biopsies to assess abundances of several ECL-cell constituents. At 0, 3, 6, 9, 12 and 24 weeks, we measured circulating gastrin and CgA and assessed safety and tolerability. Results Netazepide was safe and well tolerated. Abundances of CgA (p<0.05), histidine decarboxylase (p<0.05) and matrix metalloproteinase-7(p<0.10) were reduced at 6 and 12 weeks, but were raised again at follow-up. Likewise, plasma CgA was reduced at 3 weeks (p<0.01), remained so until 12 weeks, but was raised again at follow-up. Tumours were fewer and the size of the largest one was smaller (p<0.05) at 12 weeks, and remained so at follow-up. Serum gastrin was unaffected. Conclusion The reduction in abundances, plasma CgA, and tumour number and size by netazepide show that type 1 NETs are gastrin-dependent tumours. Failure of netazepide to increase serum gastrin further is consistent with achlorhydria. Netazepide is a potential new treatment for type 1 NETs. Longer, controlled trials are justified. Trial Registration European Union EudraCT database 2007-002916-24 https://www.clinicaltrialsregister.eu/ctr-search/search?query=2007-002916-24 ClinicalTrials.gov NCT01339169 http://clinicaltrials.gov/ct2/show/NCT01339169?term=yf476&rank=5