Claudin-18 Loss Alters Transcellular Chloride Flux but not Tight Junction Ion Selectivity in Gastric Epithelial Cells.

BACKGROUND & AIMS: Tight junctions form a barrier to the paracellular passage of luminal antigens. Although most tight junction proteins reside within the apical tight junction complex, claudin-18 localizes mainly to the basolateral membrane where its contribution to paracellular ion transport is undefined. Claudin-18 loss in mice results in gastric neoplasia development and tumorigenesis that may or may not be due to tight junction dysfunction. The aim here was to investigate paracellular permeability defects in stomach mucosa from claudin-18 knockout (Cldn18-KO) mice. METHODS: Stomach tissue from wild-type, heterozygous, or Cldn18-KO mice were stripped of the external muscle layer and mounted in Ussing chambers. Transepithelial resistance, dextran 4 kDa flux, and potential difference (PD) were calculated from the chambered tissues after identifying differences in tissue histopathology that were used to normalize these measurements. Marker expression for claudins and ion transporters were investigated by transcriptomic and immunostaining analysis. RESULTS: No paracellular permeability defects were evident in stomach mucosa from Cldn18-KO mice. RNAseq identified changes in 4 claudins from Cldn18-KO mice, particularly the up-regulation of claudin-2. Although claudin-2 localized to tight junctions in cells at the base of gastric glands, its presence did not contribute overall to mucosal permeability. Stomach tissue from Cldn18-KO mice also had no PD versus a lumen-negative PD in tissues from wild-type mice. This difference resulted from changes in transcellular Cl- permeability with the down-regulation of Cl- loading and Cl- secreting anion transporters. CONCLUSIONS: Our findings suggest that Cldn18-KO has no effect on tight junction permeability in the stomach from adult mice but rather affects anion permeability. The phenotype in these mice may thus be secondary to transcellular anion transporter expression/function in the absence of claudin-18.

Loss of Tight Junction Protein Claudin 18 Promotes Progressive Neoplasia Development in Mouse Stomach.

BACKGROUND & AIMS: Loss of claudin 18 (CLDN18), a membrane-spanning tight junction protein, occurs during early stages of development of gastric cancer and associates with shorter survival times of patients. We investigated whether loss of CLDN18 occurs in mice that develop intraepithelial neoplasia with invasive glands due to infection with Helicobacter pylori, and whether loss is sufficient to promote the development of similar lesions in mice with or without H pylori infection. METHODS: We performed immunohistochemical analyses in levels of CLDN18 in archived tissues from B6:129 mice infected with H pylori for 6 to 15 months. We analyzed gastric tissues from B6:129S5-Cldn18tm1Lex/Mmucd mice, in which the CLDN18 gene was disrupted in gastric tissues (CLDN18-knockout mice), or from control mice with a full-length CLDN18 gene (CLDN18+/+; B6:129S5/SvEvBrd) or heterozygous disruption of CLDN18 (CLDN18+/-; B6:129S5/SvEvBrd) that were infected with H pylori SS1 or PMSS1 at 6 weeks of age and tissues collected for analysis at 20 and 30 weeks after infection. Tissues from CLDN18-knockout mice and control mice with full-length CLDN18 gene expression were also analyzed without infection at 7 weeks and 2 years after birth. Tissues from control and CLDN18-knockout mice were analyzed by electron microscopy, stained by conventional methods and analyzed for histopathology, prepared by laser capture microdissection and analyzed by RNAseq, and immunostained for lineage markers, proliferation markers, and stem cell markers and analyzed by super-resolution or conventional confocal microscopy. RESULTS: CLDN18 had a basolateral rather than apical tight junction localization in gastric epithelial cells. B6:129 mice infected with H pylori, which developed intraepithelial neoplasia with invasive glands, had increasing levels of CLDN18 loss over time compared with uninfected mice. In B6:129 mice infected with H pylori compared with uninfected mice, CLDN18 was first lost from most gastric glands followed by disrupted and reduced expression in the gastric neck and in surface cells. Gastric tissues from CLDN18-knockout mice had low levels of inflammation but increased cell proliferation, expressed markers of intestinalized proliferative spasmolytic polypeptide-expressing metaplasia, and had defects in signal transduction pathways including p53 and STAT signaling by 7 weeks after birth compared with full-length CLDN18 gene control mice. By 20 to 30 weeks after birth, gastric tissues from uninfected CLDN18-knockout mice developed intraepithelial neoplasia that invaded the submucosa; by 2 years, gastric tissues contained large and focally dysplastic polypoid tumors with invasive glands that invaded the serosa. CONCLUSIONS: H pylori infection of B6:129 mice reduced the expression of CLDN18 early in gastric cancer progression, similar to previous observations from human gastric tissues. CLDN18 regulates cell lineage differentiation and cellular signaling in mouse stomach; CLDN18-knockout mice develop intraepithelial neoplasia and then large and focally dysplastic polypoid tumors in the absence of H pylori infection.

Cytotoxic Escherichia coli strains encoding colibactin isolated from immunocompromised mice with urosepsis and meningitis.

Immune-compromised mouse models allow for testing the preclinical efficacy of human cell transplantations and gene therapy strategies before moving forward to clinical trials. However, CRISPR/Cas9 gene editing of the Wsh/Wsh mouse strain to create an immune-compromised model lacking function of Rag2 and Il2rγ led to unexpected morbidity and mortality. This warranted an investigation to ascertain the cause and predisposing factors associated with the outbreak. Postmortem examination was performed on 15 moribund mice. The main lesions observed in these mice consisted of ascending urogenital tract infections, suppurative otitis media, pneumonia, myocarditis, and meningoencephalomyelitis. As Escherichia coli strains harboring polyketide synthase (pks) genomic island were recently isolated from laboratory mice, the tissue sections from the urogenital tract, heart, and middle ear were subjected to E. coli specific PNA-FISH assay that revealed discrete colonies of E. coli associated with the lesions. Microbiological examination and 16S rRNA sequencing confirmed E. coli-induced infection and septicemia in the affected mice. Further characterization by clb gene analysis and colibactin toxicity assays of the pks+ E. coli revealed colibactin-associated cytotoxicity. Rederivation of the transgenic mice using embryo transfer produced mice with an intestinal flora devoid of pks+ E. coli. Importantly, these barrier-maintained rederived mice have produced multiple litters without adverse health effects. This report is the first to describe acute morbidity and mortality associated with pks+ E. coli urosepsis and meningitis in immunocompromised mice, and highlights the importance of monitoring and exclusion of colibactin-producing pks+ E. coli.

Cutaneous Dermatophilosis in a Meadow Jumping Mouse (Zapus hudsonius).

A laboratory-housed, wild-caught, subadult, male meadow jumping mouse (Zapus hudsonius) presented with extensive scaling of the face, limbs, and tail and severe edema of the paws. Postmortem examination revealed marked distal limb edema with focal digital hematomas and white scales, scabs, and crusts affecting the majority of nonhaired skin. Histopathologic analysis revealed severe, multifocal, chronic-active exudative and proliferative dermatitis characterized by multilaminated crusts covering the epidermis. The epidermis was expanded by hyperkeratosis, acanthosis, and hyperplasia. The superficial dermis contained moderate edema, hemorrhage, and pigmentary incontinence, and was infiltrated by granulocytes and mononuclear cells. The laminated crusts contained numerous branching filaments of gram-positive coccoid bodies arranged in parallel rows, consistent with cutaneous Dermatophilus congolensis infection. This diagnosis was confirmed through bacterial culture and 16S rRNA PCR analysis. In the presented case, factors that might have contributed to disease progression include climatic conditions at the capture site and stress associated with trapping and laboratory housing.

Evaluation of Best Practices for the Euthanasia of Zebra Finches (Taeniopygia guttata).

Although zebra finches (Taeniopygia guttata) have been used in biomedical research for many years, no published reports are available about euthanizing these small birds. In this study, we compared 5 methods for zebra finch euthanasia: sodium pentobarbital (NaP) given intracoelomically with physical restraint but no anesthesia; isoflurane anesthesia followed by intracoelomic injection of NaP; and CO2 asphyxiation at 20%, 40%, and 80% chamber displacement rates (percentage of chamber volume per minute). Birds undergoing euthanasia were videorecorded and scored by 2 observers for behaviors potentially related to discomfort or distress. Time to recumbency and time until respiratory arrest (RA) were also assessed. RA was achieved faster by using NaP in a conscious bird compared to using isoflurane anesthesia followed by NaP; however, neither method caused behaviors that might affect animal welfare, such as open-mouth breathing, to any appreciable extent. Among the CO2 treatment groups, there was an inverse correlation between the chamber displacement rate used and the duration of open-mouth breathing, onset of head retroflexion, and time to RA. The results demonstrate that the intracoelomic administration of NaP in an awake, restrained zebra finch is a rapid and effective method of euthanasia. If CO2 is used to euthanize these birds, a high displacement rate (for example, 80%) will minimize the duration of the procedure and associated behaviors.

Tight junction disruption: Helicobacter pylori and dysregulation of the gastric mucosal barrier.

Long-term chronic infection with Helicobacter pylori (H. pylori) is a risk factor for gastric cancer development. In the multi-step process that leads to gastric cancer, tight junction dysfunction is thought to occur and serve as a risk factor by permitting the permeation of luminal contents across an otherwise tight mucosa. Mechanisms that regulate tight junction function and structure in the normal stomach, or dysfunction in the infected stomach, however, are largely unknown. Although conventional tight junction components are expressed in gastric epithelial cells, claudins regulate paracellular permeability and are likely the target of inflammation or H. pylori itself. There are 27 different claudin molecules, each with unique properties that render the mucosa an intact barrier that is permselective in a way that is consistent with cell physiology. Understanding the architecture of tight junctions in the normal stomach and then changes that occur during infection is important but challenging, because most of the reports that catalog claudin expression in gastric cancer pathogenesis are contradictory. Furthermore, the role of H. pylori virulence factors, such as cytotoxin-associated gene A and vacoulating cytotoxin, in regulating tight junction dysfunction during infection is inconsistent in different gastric cell lines and in vivo, likely because non-gastric epithelial cell cultures were initially used to unravel the details of their effects on the stomach. Hampering further study, as well, is the relative lack of cultured cell models that have tight junction claudins that are consistent with native tissues. This summary will review the current state of knowledge about gastric tight junctions, normally and in H. pylori infection, and make predictions about the consequences of claudin reorganization during H. pylori infection.