Targeted Deletion of Kcne2 Causes Gastritis Cystica Profunda and Gastric Neoplasia

Gastric cancer is the second leading cause of cancer death worldwide. Predisposing factors include achlorhydria, Helicobacter pylori infection, oxyntic atrophy and TFF2-expressing metaplasia. In parietal cells, apical potassium channels comprising the KCNQ1 α subunit and the KCNE2 β subunit provide a K+ efflux current to facilitate gastric acid secretion by the apical H+K+ATPase. Accordingly, genetic deletion of murine Kcnq1 or Kcne2 impairs gastric acid secretion. Other evidence has suggested a role for KCNE2 in human gastric cancer cell proliferation, independent of its role in gastric acidification. Here, we demonstrate that 1-year-old Kcne2−/− mice in a pathogen-free environment all exhibit a severe gastric preneoplastic phenotype comprising gastritis cystica profunda, 6-fold increased stomach mass, increased Ki67 and nuclear Cyclin D1 expression, and TFF2- and cytokeratin 7-expressing metaplasia. Some Kcne2−/−mice also exhibited pyloric polypoid adenomas extending into the duodenum, and neoplastic invasion of thin walled vessels in the sub-mucosa. Finally, analysis of human gastric cancer tissue indicated reduced parietal cell KCNE2 expression. Together with previous findings, the results suggest KCNE2 disruption as a possible risk factor for gastric neoplasia

Visualization of extracellular matrix components within sectioned Salmonella biofilms on the surface of human gallstones.

Chronic carriage of Salmonella Typhi is mediated primarily through the formation of bacterial biofilms on the surface of cholesterol gallstones. Biofilms, by definition, involve the formation of a bacterial community encased within a protective macromolecular matrix. Previous work has demonstrated the composition of the biofilm matrix to be complex and highly variable in response to altered environmental conditions. Although known to play an important role in bacterial persistence in a variety of contexts, the Salmonella biofilm matrix remains largely uncharacterized under physiological conditions. Initial attempts to study matrix components and architecture of the biofilm matrix on gallstone surfaces were hindered by the auto-fluorescence of cholesterol. In this work we describe a method for sectioning and direct visualization of extracellular matrix components of the Salmonella biofilm on the surface of human cholesterol gallstones and provide a description of the major matrix components observed therein. Confocal micrographs revealed robust biofilm formation, characterized by abundant but highly heterogeneous expression of polysaccharides such as LPS, Vi and O-antigen capsule. CsgA was not observed in the biofilm matrix and flagellar expression was tightly restricted to the biofilm-cholesterol interface. Images also revealed the presence of preexisting Enterobacteriaceae encased within the structure of the gallstone. These results demonstrate the use and feasibility of this method while highlighting the importance of studying the native architecture of the gallstone biofilm. A better understanding of the contribution of individual matrix components to the overall biofilm structure will facilitate the development of more effective and specific methods to disrupt these bacterial communities

Histopathological Analysis of <i>Salmonella</i> Chronic Carriage in the Mouse Hepatopancreatobiliary System

<div><p><i>Salmonella</i> Typhi asymptomatic chronic carriage represents a challenge for the diagnosis and prevention of typhoid fever in endemic areas. Such carriers are thought to be reservoirs for further spread of the disease. Gallbladder carriage has been demonstrated to be mediated by biofilm formation on gallstones and by intracellular persistence in the gallbladder epithelium of mice. In addition, both gallstones and chronic carriage have been associated with chronic inflammation and the development of gallbladder carcinoma. However, the pathogenic relationship between typhoid carriage and the development of pre-malignant and/or malignant lesions in the hepatopancreatobiliary system as well as the host-pathogen interactions occurring during chronic carriage remains unclear. In this study, we monitored the histopathological features of chronic carriage up to 1 year post-infection. Chronic cholecystitis and hepatitis ranging from mild to severe were present in infected mice regardless of the presence of gallstones. Biliary epithelial hyperplasia was observed more commonly in the gallbladder of mice with gallstones (uninfected or infected). However, pre-malignant lesions, atypical hyperplasia and metaplasia of the gallbladder and exocrine pancreas, respectively, were only associated with chronic <i>Salmonella</i> carriage. This study has implications regarding the role of <i>Salmonella</i> chronic infection and inflammation in the development of pre-malignant lesions in the epithelium of the gallbladder and pancreas that could lead to oncogenesis.</p> </div

Chronic hepatitis is exacerbated in infected mice at 3 and 6 months post-infection.

<p>(A) Inflammation grades in the liver at all time points post-infection (± bacteria, ± gallstones). Means of each time point values were compared by a Student’s <i>t</i> test (*, <i>p</i><0.05; ***, <i>p</i><0.001). (B) Representative HE images of each group at 3 and 6 months post-infection. Inflammation scores are as follows: B1 (0); B2 (1); B3 and B6 (2); B4 and B5 (4). Infected mice have scattered inflammatory cells distributed randomly within the lobule (B3-B5) and/or in portal areas (B6) composed of neutrophils, lymphocytes, plasma cells and histiocytes. Inflammation was often associated with necrosis of individual hepatocytes (B3) or large confluent areas of hepatocellular necrosis (B4-B5) (noted as N). 20x. </p

Chronic cholecystitis occurs as a result of both gallstone disease and <i>Salmonella</i> carriage.

<p>(A) Inflammation grades in the gallbladder at all time points post-infection (± bacteria, ± gallstones). Means of each time point values were compared by a Student’s <i>t</i> test. No statistical significant difference was observed between values of each time point (<i>p</i><0.5). (B) Representative HE images of each group at 3 and 9 months post-infection. B1 has an inflammation score of 0; B2, B3, B5 and B6 have a inflammation score of 2; whereas B4 has a score of 3. Hyperplasia was only evident in mice harboring gallstones and with concurrent epithelial hyalinosis (*) (B2, B5 and B6). Black arrows indicate scattered inflammatory cells within the lamina propria and muscularis, they were mainly composed of lymphocytes, plasma cells and fewer neutrophils. 40x.</p

<i>Salmonella</i> LPS staining was detected in the liver regardless of the presence of gallstones.

<p>Representative IHC images of mouse liver sections at 3 and 9 months post-infection using an anti-<i>Salmonella</i> LPS antibody. Panel C shows a typhoid nodule. Black arrows indicate the presence of <i>Salmonella</i> inside inflammatory macrophages (Panel C) or Kupffer cells (Panel F) or intracellularly in hepatocytes (Panels D, E). DAB and hematoxylin counterstain. 40x.</p

<i>Salmonella</i> detection in organs and bodily fluids of chronically infected mice up to 1 year post-infection.

<p>Bacterial CFU enumeration of <i>Salmonella</i> at various times post-infection in the absence and presence of gallstones. nd, not detected. Limit of detection was 100 CFU /mL. No statistically significant difference was observed between infected mice with or without gallstones at any time point (Student’s <i>t</i> test). nd, not detected.</p

<i>Salmonella</i> LPS staining was detected in the exocrine pancreas regardless of the presence of gallstones.

<p>Representative IHC images of mouse pancreas sections at 3 and 9 months post-infection using an anti-<i>Salmonella</i> LPS antibody. <i>Salmonella</i> was detected in the interstitium around acini or ductular cells (Panels D, E and F). DAB and hematoxylin counterstain. 40x.</p

Pancreatic mucinous metaplasia was only evident in infected mice regardless of the presence of gallstones.

<p>Representative HE images of pancreas 6 months post-infection. Black arrows indicate mucinous metaplasia associated with exocrine pancreatic acini at the periphery of marked chronic necrosuppurative inflammation. 20x. </p