Regulation of expression of the intestinal actin-binding protein, villin, a marker of intestinal epithelial differentiation, is poorly understood. Activation of the extracellular calcium-sensing receptor (CaSR) on sub-epithelial myofibroblasts stimulated the secretion of Wnt5a, while activation of the CaSR on intestinal epithelia increased expression of Ror2, a Wnt-family co-receptor. Immunocytochemistry has localized Ror2 expression in the epithelia lining the small intestine from the crypt base to the villus tip. The aim of this study was to determine whether Wnt5a binding Ror2 in intestinal epithelia stimulated transient increases in phospho-ERK1/2 (pERK1/2) which lead to increased expression of villin transcript and protein. To examine Wnt5a-Ror2 regulation of villin expression, we transgenically overexpressed wild-type, truncated, or mutant Ror2 constructs in HT-29 adenocarcinoma cells and nontransformed fetally-derived human intestinal epithelial cells (HIECs), added conditioned media containing Wnt5a and measured changes in ERK1/2 phosphorylation, villin amplicons and protein expression by RT-PCR and Western blot techniques. Wnt5a addition caused a transient increase in pERK1/2, which was maximal at 10 min but diminished by 30 min. Transient transfection with a siRNA duplex against Ror2 diminished Ror2 amplicons and protein and reduced the extent of pERK1/2 activation. Structure-function analysis revealed that deletion of the cysteine-rich, kringle, or tyrosine kinase domain or substitution mutations of tyrosine residues in the intracellular Ser/Thr-1 region of Ror2 prevented the Wnt5a-stimulation of pERK1/2. Deletion of the intracellular proline and serine/threonine rich regions of Ror2 had no effect on Wnt5a-stimulation of pERK1/2 in HT29 cells. Western blot analysis demonstrated that villin protein was increased by over-expression of wild-type Ror2 in HT-29 cells and HIECs in the presence of Wnt5a. The increase in villin expression was blocked by pharmacological inhibition of MEK1&2 and casein kinase 1, but not by PKC and p38 inhibitors. Neither Wnt3a nor EGF addition increased villin protein. This work suggested that stromal Wnt5a will stimulate pERK1/2 via the Ror2 tyrosine kinase domain to generate increased villin protein. These findings suggested that Ror2 homeostasis and Wnt5a interaction with Ror2 are important determinants of the regulation of villin expression in the intestine. / Thesis (Master, Physiology) -- Queen's University, 2009-07-14 23:34:39.397

No description available.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Since the discovery of the p120ctn binding partner, Kaiso, a BTB/POZ transcription factor, several studies have implicated the protein in both development and tumourigenesis. Most information about Kaiso’s function in vertebrate development has been gleaned from studies in Xenopus laevis embryos where Kaiso negatively regulates the Wnt signalling pathway. Since the Wnt signalling pathway is crucial in intestinal development, intestinal-specific Kaiso overexpressing mice were generated and characterized to elucidate Kaiso’s role in a mammalian context. Kaiso transgenic (KaisoTg/+) mice were viable and fertile but developed gross histopathological changes in the small intestine. The KaisoTg/+ mice exhibited enlarged crypts accompanied by increased secretory cell differentiation reminiscent of inhibition of the Notch pathway. Indeed, the Notch effector protein, HES1, is decreased in KaisoTg/+ mice. Additionally, KaisoTg/+ mice display a neutrophil-specific intestinal inflammation reminiscent of the knockdown of p120ctn. Interestingly, the KaisoTg/+ mice display decreased p120ctn localization at the membranes and an increase in the neutrophil adhesion molecule, ICAM-1, both of which induce neutrophilia. Notably, the KaisoTg/+ mice developed multiple crypt abscesses over time due to massive neutrophil infiltration of the epithelial cell layers. This is the first study to examine the in vivo roles of Kaiso in a mammalian context and our findings suggest a regulatory role for Kaiso in the inflammatory and Notch signalling pathways. / Thesis / Candidate in Philosophy

Autophagy, an intracellular degradation, and recycling process is essential in maintaining cellular homeostasis. Dysregulated autophagy is linked to the pathogenesis of various diseases, including inflammatory bowel disease (IBD) which consists of Crohn’s disease and ulcerative colitis. In IBD, enterochromaffin cell numbers and one of its main product serotonin (5-hydroxytryptamine; 5-HT) levels are elevated. Previously, we had shown that tryptophan hydroxylase 1 deficient (Tph1-/-) mice, with reduced gut 5-HT had decreased severity of colitis. Here, we showed that gut 5-HT plays a vital role in modulating autophagy and thus regulating gut microbial composition and susceptibility to intestinal inflammation. Tph1-/- mice, had upregulated colonic autophagy via the mammalian target of rapamycin pathway (mTOR), and decreased colitis severity. Tph1-/- mice after 5-HT replenishment, and serotonin reuptake transporter deficient (SERT-/-) mice, which have increased 5-HT levels, showed converse results. Deletion of intestinal epithelial cell-specific autophagy gene, Atg7, in Tph1-/- mice (DKO mice) abolished the protective effect of Tph1 deficiency in colitis, decreased the production of antimicrobial peptide, β-defensin 1 and promoted colitogenic microbiota. Furthermore, using cecal microbial transplantation, we found that the colitic microbiota of the DKO mice contributed to the increased severity of colitis. Supporting this pathway's translational importance, we uncovered that 5-HT treatment of peripheral blood mononuclear cells from both healthy volunteers and patients with Crohn’s disease inhibited autophagy via the mTOR pathway. Our results in this thesis emphasize the role of 5-HT-autophagymicrobiota axis in intestinal inflammation. Moreover, these findings suggest 5-HT as a novel therapeutic target in intestinal inflammatory disorders such as IBD that exhibit dysregulated autophagy. / Thesis / Doctor of Philosophy (PhD) / Approximately 0.7% of Canadians are currently affected with inflammatory bowel disease (IBD). The gut hormone serotonin, which regulates many normal functions, is elevated in gut inflammation. Reduced serotonin levels decrease the severity of inflammation. IBD pathology has been linked to a unique cell self-eating process called autophagy. Using cell lines, mice, and samples from IBD patients, we assessed the interactions between serotonin signaling and autophagy during gut inflammation. I found that an increase in serotonin levels enhances the severity of gut inflammation by inhibiting autophagy. We also established the connection between serotonin and autophagy in the intestinal epithelial cells, and how this modulates epithelial cell function. Furthermore, we demonstrated the establishment of an altered gut microbiota upon disruption of the serotonin-autophagy axis in the epithelial cells, which subsequently influenced gut inflammation severity. Thus, we identified one of the key triggers related to the pathogenesis and severity of IBD.

No description available.

Since the removal of in feed antibiotics in the past few years commercial poultry production is especially sensitive to the health of the small intestine. Healthy small intestines balance nutrient absorption and defensive barrier functions to ensure the chicken is able to meet the whole-body nutritional needs and is able to help prevent internalization of pathogens or potentially toxic components. This balance can only be maintained under stable conditions. When a disturbance event occurs the intestine imbalances until a new, and less efficient, balance can be achieved. The objective of this dissertation is to propose a novel model to understanding intestinal homeostasis in the face of various disturbance events. Chapter 2 investigated the effects of Runting Stunting Syndrome on broiler chickens in four different groups of chicks displaying clinical symptoms. The major finding in this study was that in two of the four groups the expression of stem cell gene Olfactomedin 4 was absent from the crypt though other functional genes were found to still be expressed there. Chapter 3 characterized intestinal gene expression following a single challenge of Eimeria acervulina in broiler chickens. During Eimeria infection gene expression of multiple host defense peptide genes were decreased compared to uninfected chickens. Further, Eimeria infected chickens increased cell proliferation within the crypt and post-peak infection showed signs of intestinal recovery. Additionally, chapter 3 developed a novel method for visualizing Eimeria as it infects the intestine. In chapters 4 and 5 cell type population changes during the peri-hatch intestinal maturation process were evaluated. Peri-hatch intestinal maturation is critical for the successful transition from embryonic to post-hatch life. Chapter 4 profiled changes in proliferative cells and gene expression of various stem cell marker genes during the peri-hatch period: the last three days of embryogenesis and the first week post-hatch. The stem cell marker gene Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) decreased during the post-hatch period while Olfactomedin 4 increased post-hatch. Both stem cell genes were expressed within the intestinal crypt, though prior to hatch Lgr5 was expressed in the lamina propria and villi as well. Additionally, the marker of proliferation Ki67 gene was expressed in cells throughout the intestine prior to hatch but became restricted to the crypts and along the center of the villi. Chapter 5 assessed the effect of providing probiotics to late term embryos via in ovo feeding (IOF). The effects of IOF were primarily observed on embryonic day 20 (e20), roughly 48 hours after IOF. On e20 the embryos in ovo fed probiotics in saline had increased expression in the ileum of Peptide Transporter 1 (PepT1) a marker gene for enterocytes and Mucin-2 (Muc2) a marker gene for goblet cells compared to non-injected control embryos. Also, on e20 the embryos in ovo fed saline only had numerically increased PepT1 and Muc2 compared to non-injected control embryos. The difference in responses between the probiotic and saline fed embryos on e20 suggests different routes of stimulation. These investigations illustrate various possible scenarios and means of investigating intestinal homeostasis during disturbance events. / Doctor of Philosophy / In healthy birds, the small intestine absorbs nutrients while preventing the free passage of microbes or toxic chemicals into the body. The two functions: absorption and barrier exclusion seem contradictory, but a balance is struck to ensure both functions continue. This balance-point, homeostasis, persists until an event disturbs it. Once disturbed the balance-point is changed and the intestine is unable to maintain both functions, until a new balance is found following recovery. The objective of the dissertation is to better understand intestinal homeostasis, through four different research projects. Experiment 1 characterized the intestinal cell population changes in broiler chickens during Runting Stunting Syndrome, a viral infection. The major finding of this chapter was that a stem cell gene, that is normally robustly expressed was not expressed in some groups of infected chicks but not all. Experiment 2 investigated the intestinal response of broiler chickens to Eimeria acervulina, an intestinal parasitic infection. Eimeria, which infects intestinal enterocytes, caused a decrease in defensive genes during the peak of infection. Then after the peak the intestine began to recover, as indicated by increased cell proliferation. Experiment 3 profiled changes in the expression patterns of stem cell and proliferation genes in the small intestine during the last days before hatch and the first week post-hatch. Pre-hatch stem and proliferative gene expression occurred in the crypt and villus, but became restricted to the crypt early during the post-hatch period. Experiment 4 assessed the effect of feeding probiotics to embryos before hatching on intestinal gene expression. Embryos fed probiotics had increased Mucin-2 and Peptide Transporter 1 gene expression in the last segment of the intestine, the ileum compared to non-fed embryos 48 hours after feeding. Additionally, treatments fed saline also showed increased gene expression, though to a lesser extent. Together these projects illustrate various disturbances to intestinal homeostasis and how intestinal cells change and respond during the disturbance and recovery periods.

A study was performed to identify apoptotic cells in the equine intestine and to determine if the occurrence of apoptosis is affected by gastrointestinal disease and tissue layer of intestine. Samples of intestine were collected from 38 horses that underwent surgery or were humanely destroyed for small or large bowel obstruction, strangulation or distension. Samples were also taken from 9 horses which were humanely euthanized for reasons other than gastrointestinal disease or systemic disease. Specimens were collected at surgery from intestine involved in the primary lesion, distant to the primary lesion, or at necropsy from several sites including the primary lesion. Tissues were fixed, serially sectioned and stained with hematoxylin and eosin (H&E) and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. The number of apoptotic cells per high power field were counted in the mucosa, circular muscle, longitudinal muscle and serosa for each sample of intestine. Apoptotic staining nuclei were seen in all layers of intestine. An increased number of apoptotic cells were found in the circular muscle of the intestine from horses with simple obstruction. Intestine distant from the primary strangulating lesion had higher numbers of apoptotic cells than intestine distant from a simple obstruction lesion or intestine taken at the site of a strangulating or simple obstructive lesion. Intestine from horses with obstructing or strangulating lesions in the small intestine and large colon has increased numbers of apoptotic cells. Further investigation is required to determine whether increased apoptosis affects intestinal function. / Master of Science

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Master of Science / Department of Biology / Sherry D. Fleming / Helicobacter is a common intestinal pathogen of most laboratory mice from both commercial and academic sources worldwide. Not previously thought to have an effect, recent evidence indicates Helicobacter infection alters cytokine, chemokine, and gene expression in the stomach, intestine, and colon. Though the in vivo cell types responsible for these changes are currently unknown, in vitro results suggest macrophages are the likely source. In addition to detection and elimination of pathogens, intestinal macrophages play a role in maintaining homeostasis. By altering gene expression and cytokine production in the microenvironment, we hypothesized that Helicobacter infection altered the phenotype and inflammatory response of submucosal intestinal macrophages. To test this hypothesis, we examined macrophages within whole mounts of intestinal muscle as well as isolated macrophages from Helicobacter-infected or uninfected mouse intestine. Macrophages from the intestinal muscle of Helicobacter-infected mice showed increased expression of F4/80 and CD11b, altered gene expression, and increased phagocytosis when compared to macrophages from uninfected mice. Infection also altered the macrophage response to stimuli. Macrophages from infected mice produced significantly lower concentrations of cytokines, chemokines, and PGE[subscript]2 in response to stimulation with either IFN and LPS or IL-4 and IC. These data support our hypothesis demonstrating that the intestinal muscle macrophage phenotype, function, and response to stimulation are altered by Helicobacter infection both in vivo and in vitro.