The Experts below are selected from a list of 16254 Experts worldwide ranked by ideXlab platform

Steffen Backert - One of the best experts on this subject based on the ideXlab platform.

  • campylobacter jejuni serine protease htra cleaves the tight junction component Claudin 8
    Frontiers in Cellular and Infection Microbiology, 2020
    Co-Authors: Irshad Sharafutdinov, Delara Soltan Esmaeili, Aileen Harrer, Nicole Tegtmeyer, Heinrich Sticht, Steffen Backert
    Abstract:

    Campylobacter jejuni express the high temperature requirement protein A (HtrA), a secreted serine protease, which is implicated in virulence properties of the pathogen. Previous studies have shown that C. jejuni HtrA can cleave the epithelial transmembrane proteins occludin and E-cadherin in the tight and adherens junctions, respectively. In the present report, we studied the interaction of HtrA with another human tight junction protein, Claudin-8. Confocal immunofluorescence experiments have shown that C. jejuni infection of the intestinal polarized epithelial cells in vitro leads to a relocation of Claudin-8. Wild-type C. jejuni induced the downregulation of Claudin-8 signals in the tight junctions and an upregulation of signals in the cytoplasm, which was not seen during infection with isogenic ∆htrA knockout deletion or protease-inactive S197A point mutants. Western blotting of protein samples from infected vs. uninfected cells revealed that an 18 kDa carboxy-terminal fragment is cleaved-off from the 26 kDa full-length Claudin-8 protein, but not during infection with the isogenic htrA mutant. These results were confirmed by in vitro cleavage assays using the purified recombinant C. jejuni HtrA and human Claudin-8 proteins. Recombinant HtrA cleaved purified Claudin-8 in vitro giving rise to the same 18 kDa sized carboxy-terminal cleavage product. Mapping studies revealed that HtrA cleavage occurs in the first extracellular loop of Claudin-8. Three-dimensional modelling of the Claudin-8 structure identified an exposed HtrA cleavage site between the amino acids alanine 58 and asparagine 59, which is in well agreement with the mapping studies. Taken together, HtrA operates as a secreted virulence factor targeting multiple proteins both in the tight and adherens junctions. This strategy may help the bacteria to open the cell-to-cell junctions, and to transmigrate across the intestinal epithelium by a paracellular mechanism and establish an acute infection.

  • Importance of two PDZ domains for the proteolytic and chaperone activities of Helicobacter pylori serine protease HtrA.
    Cellular microbiology, 2020
    Co-Authors: Urszula Zarzecka, Steffen Backert, Dorota Matkowska, Joanna Skorko-glonek
    Abstract:

    The Helicobacter pylori HtrA protein (HtrAHp ) is an important virulence factor involved in the infection process by proteolysis of components of the tight (Claudin-8 and occludin) and adherens junctions (E-cadherin) between epithelial cells. As a protease and chaperone, HtrAHp is involved in protein quality control, which is particularly important under stress conditions. HtrAHp contains a protease domain and two C-terminal PDZ domains (PDZ1 and PDZ2). In the HtrA protein family, the PDZ domains are proposed to play important roles, including regulation of proteolytic activity. We therefore mutated the PDZ1 and PDZ2 domains in HtrAHp and studied the maintenance of proteolytic activity, assembly and rearrangement of the corresponding oligomeric forms. Our in vitro experiments demonstrated that at least PDZ1 is important for efficient substrate cleavage, while both PDZ domains are dispensable for the chaperone-like activity. However, in living H. pylori cells, only the mutant containing at least PDZ1, but not PDZ2, ensured bacterial growth under stressful conditions. Moreover, we can demonstrate that PDZ1 is crucial for HtrAHp oligomerization. Interestingly, all truncated proteolytically active HtrAHp variants were functional in the in vitro infection assay and caused damage to the E-cadherin-based adherens junctions. These findings provide valuable new insights into the function of HtrAHp in an important pathogen of humans. This article is protected by copyright. All rights reserved.

  • How many protein molecules are secreted by single Helicobacter pylori cells: Quantification of serine protease HtrA.
    Cellular microbiology, 2019
    Co-Authors: Matthias Neddermann, Steffen Backert
    Abstract:

    Infection with Helicobacter pylori represents a major risk for developing peptic ulcer disease, gastric adenocarcinoma, and various other gastric and nongastric sicknesses. A series of H. pylori virulence factors can be secreted into the cell culture supernatant, and the secretome contains more than 100 different proteins. However, the quantities of proteins secreted by the bacteria over time are unknown. One of these factors is the serine protease high-temperature requirement A (HtrA), encoded by an essential bifunctional gene with crucial intracellular and extracellular activities. We have demonstrated recently that secreted HtrA can cleave off the ectodomains of the tight junction proteins occludin and Claudin-8, as well as of the tumour suppressor and adherens junction protein E-cadherin on polarised gastric epithelial cells. The exact mechanism of secretion and the quantity of secreted HtrA, however, have not been studied in detail. Here, we applied protein purification and quantitative Western blotting to determine the number of HtrA molecules secreted by H. pylori cells in liquid culture during a time course. Over a period of 8 hr, actively dividing bacteria secreted HtrA at a similar rate, on average about 9,600 HtrA molecules per cell. We determined minor variation over time corresponding to 9,931 ± 1,768 at an OD600 of 0.4 after 2 hr, 9,403 ± 2,356 2 hr later, and 9,644 ± 2,067 molecules per cell after 8 hr of culturing, when the culture had reached an OD600 of 0.8. This is the first report on the quantification of a secreted virulence protein from the important gastric pathogen H. pylori. Because HtrA has been considered as a promising new target for antibacterial therapy, knowledge about secreted protein quantities is crucial for optimising corresponding treatment regimes.

  • Extracellular HtrA serine proteases: An emerging new strategy in bacterial pathogenesis.
    Cellular microbiology, 2018
    Co-Authors: Steffen Backert, Sabine Bernegger, Joanna Skorko-glonek, Silja Wessler
    Abstract:

    The HtrA family of chaperones and serine proteases is important for regulating stress responses and controlling protein quality in the periplasm of bacteria. HtrA is also associated with infectious diseases since inactivation of htrA genes results in significantly reduced virulence properties by various bacterial pathogens. These virulence features of HtrA can be attributed to reduced fitness of the bacteria, higher susceptibility to environmental stress and/or diminished secretion of virulence factors. In some Gram-negative and Gram-positive pathogens, HtrA itself can be exposed to the extracellular environment promoting bacterial colonisation and invasion of host tissues. Most of our knowledge on the function of exported HtrAs stems from research on Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, Bacillus anthracis, and Chlamydia species. Here, we discuss recent progress showing that extracellular HtrAs are able to cleave cell-to-cell junction factors including E-cadherin, occludin, and Claudin-8, as well as extracellular matrix proteins such as fibronectin, aggrecan, and proteoglycans, disrupting the epithelial barrier and producing substantial host cell damage. We propose that the export of HtrAs is a newly discovered strategy, also applied by additional bacterial pathogens. Consequently, exported HtrA proteases represent highly attractive targets for antibacterial treatment by inhibiting their proteolytic activity or application in vaccine development.

Mateja Erdani Kreft - One of the best experts on this subject based on the ideXlab platform.

  • Reuse of bladder mucosa explants provides a long lasting source of urothelial cells for the establishment of differentiated urothelia.
    Histochemistry and cell biology, 2018
    Co-Authors: Urška Dragin Jerman, Mateja Erdani Kreft
    Abstract:

    Organ explant cultures are well-established in vitro models that are used to study normal cell biological and regeneration processes as well as carcinogenesis. Primary urothelial cultures from bladder mucosa explants are highly differentiated and are thus broadly used as in vitro experimental equivalents of native urothelial tissue. Since experiments on differentiated urothelial cultures from bladder mucosa explants currently allow only a single use of explants, establishment of sufficient quantities of cultures requires large numbers of sacrificed animals. There is thus a great need for a cheaper approach with less ethical dilemmas. Herein, we demonstrate that mouse bladder mucosa explants can be reused. Reused explants produce outgrowths with highly differentiated urothelia, just like primary explants. Even after being recycled ten times, urothelial outgrowths have the supramolecular and ultrastructural features that are comparable to the native urothelium. Ten times reused explants produce superficial urothelial cells that express uroplakins in the apical plasma membrane, Claudin-8 in the tight junctions, and have a subapical network of cytokeratin 20. Basal urothelial cells in urothelial outgrowths of ten times reused explants express p63 which indicates that these urothelial outgrowths have a persistent proliferative capacity. Using our approach, one can perform experiments that were previously not feasible due to low quantities of donor tissue. The method also offers opportunity for effective use of scarce healthy human urothelial tissue.

  • Co-culturing porcine normal urothelial cells, urinary bladder fibroblasts and smooth muscle cells for tissue engineering research.
    Cell biology international, 2017
    Co-Authors: Daša Zupančič, Katjuša Mrak Poljšak, Mateja Erdani Kreft
    Abstract:

    New strategies for culturing and co-culturing of the main types of urinary bladder cells are essential for successful establishment of biomimetic in vitro models, which could be applied for research into, and management of, diverse urological disorders. Porcine normal urothelial cells are available in nearly unlimited amounts and have many properties equivalent to human urothelial cells. In the present study, we established normal differentiated porcine urothelial cells in co-cultures with porcine urinary bladder normal fibroblasts and/or smooth muscle cells. The optimal culture medium for establishment of differentiated urothelial cells, demonstrated by positive immunofluorescence of uroplakins, cytokeratins (CK 7, CK 20), zonula occludens 1 (ZO-1), Claudin 4, Claudin 8, and E-cadherin, was the medium composed of equal parts of Advanced Dulbecco's modified Eagle's medium (A-DMEM) and MCDB 153 medium with physiological calcium concentration of 2.5 mM and without fetal bovine serum, named UroM (+Ca2+  - S). This medium was also proven to be suitable for culturing of bladder fibroblasts and smooth muscle cells and co-culturing of urothelial cells with these mesenchymal cells. Urothelial cell differentiation was optimal in UroM (+Ca2+  - S) medium in all co-culture conditions and when compared to all conditioned-media combinations. To summarize, these strategies for culturing and co-culturing of urinary bladder urothelial cells with mesenchymal cells could be used as new in vitro models for future basic and applicable research of the urinary bladder and thus potentially also for translational tissue engineering studies.

  • The complete functional recovery of chitosan-treated biomimetic hyperplastic and normoplastic urothelial models
    Histochemistry and Cell Biology, 2015
    Co-Authors: Tanja Višnjar, Mateja Erdani Kreft
    Abstract:

    The urinary tract is exposed to a variety of possible injures that may lead to organ damage or loss, and thus, the establishment of valid in vitro urothelial models to study the mechanism of drug candidates is necessary. This study is the first to investigate the effect of chitosan on urothelia in vitro and to evaluate whether chitosan-treated urothelial models can regenerate in vitro and reestablish a functional urothelium. Biomimetic hyperplastic and normoplastic urothelial models were used to test the effect of chitosan (0.05 %) on partially and highly differentiated urothelial cells (UCs) by monitoring their molecular, ultrastructural, and physiological changes for 3 weeks. Chitosan caused an immediate and complete loss of transepithelial resistance (TER), tight junction disruption, cytopathological changes of UCs, and consequently enhanced the permeability of partially and highly differentiated urothelial models. However, 3 weeks after chitosan treatment, TER was reestablished, tight junctions resealed, permeability decreased, and progressive differentiation stages of newly exposed superficial UCs expressing uroplakins and tight junction protein Claudin-8 were found. The in vitro models regenerated and reestablished urothelia with a tight barrier. The biomimetic urothelial models represent appropriate in vitro models for studying urothelial drug candidates as well as evaluating drug permeabilities and their intracellular function. Understanding the possible intracellular function of chitosan could significantly advance approaches to treating urothelial-specific diseases.

  • Hyperplasia as a mechanism for rapid resealing urothelial injuries and maintaining high transepithelial resistance
    Histochemistry and Cell Biology, 2012
    Co-Authors: Tanja Višnjar, Petra Kocbek, Mateja Erdani Kreft
    Abstract:

    When the urothelial barrier, i.e., the blood−urine barrier, is injured, rapid resealing of the injury is crucial for the normal functioning of the organism. In order to investigate the mechanisms required for rapid resealing of the barrier, we established in vitro models of hyperplastic and normoplastic urothelia. We found that hyperplastic urothelia achieve significantly higher transepithelial resistance (TER) than normoplastic urothelia. However, the expression of cell junctional (Claudin-8, occludin, E-cadherin) and differentiation-related proteins (cytokeratin 20 and uroplakins) is weaker in hyperplastic urothelia. Further investigation of cell differentiation status at the ultrastructural level confirmed that superficial urothelial cells (UCs) in hyperplastic urothelial models achieve a lower differentiation stage than superficial UCs in normoplastic urothelial models. With the establishment of such in vitro models and the aid of TER measurements, flow cytometry, molecular and ultrastructural analysis, we here provide unequivocal evidence that the specific cell-cycle distribution and, consequently, the number of cell layers have a significant influence on the barrier function of urothelia. We demonstrate the importance of hyperplasia for the rapid restoration of the urothelial barrier and the maintenance of high TER until the UCs reach a highly differentiated stage and restoration of the urothelial barrier after injury is complete. The information that this approach provides is unique and we expect that further exploitation of hyperplastic and normoplastic urothelial models in future studies may advance our understanding of blood−urine barrier development and functionality.

  • Distribution of junction- and differentiation-related proteins in urothelial cells at the leading edge of primary explant outgrowths
    Histochemistry and Cell Biology, 2006
    Co-Authors: Mateja Erdani Kreft, Maksimiljan Sterle, Kristijan Jezernik
    Abstract:

    Leading edge cells, which are located at the forefront of a wound margin, play a significant role in coordinating the wound healing process. In this study, leading edge cells of the urothelial explant outgrowth, resembling leading edge cells during urothelial full-thickness wound healing in vivo, were analyzed for expression and distribution of junction and differentiation-related proteins. Ultrastructural and immunofluorescence studies revealed that urothelial cells at the leading edge expressed ZO-1, Claudin-4, occludin, E-cadherin, cytokeratin 7 and cytokeratin 20, while no expression of Claudin-8 was noted. ZO-1, Claudin-4, occludin and E-cadherin were localized along the cell membranes where neighbouring leading edge cells were in contact. Cytokeratin 7 was detected as filaments and cytokeratin 20 as small dots and sparse filaments. In conclusion, we detected early expression of ZO-1, Claudin-4 and occludin at the urothelial leading edge, predicating the later formation of tight junctions as a necessary stage for the differentiation process that subsequently begins. The expression of occludin and cytokeratin 20 in urothelial cells at the leading edge suggests that leading edge cells may develop into fully differentiated superficial cells.

Andrew N. Young - One of the best experts on this subject based on the ideXlab platform.

  • Claudin 7 and Claudin 8 immunohistochemical markers for the differential diagnosis of chromophobe renal cell carcinoma and renal oncocytoma
    Human Pathology, 2009
    Co-Authors: Adeboye O. Osunkoya, Diane Lawson, Maria M Picken, Mahul B. Amin, Cynthia Cohen, Andrew N. Young
    Abstract:

    Summary Claudin-7 and Claudin-8 code for tight junction proteins expressed in distal nephron epithelium. In a recent oligonucleotide microarray study, we identified Claudin-7 and Claudin-8 as candidate markers to distinguish chromophobe renal cell carcinoma from other renal tumors, including oncocytoma. Distinction of these lesions can be difficult by light microscopy but is clinically important because chromophobe renal cell carcinoma has malignant biological potential, whereas renal oncocytoma is benign. Claudin-7 and Claudin-8 expression was studied by immunohistochemistry in 11 chromophobe renal cell carcinomas and 17 oncocytomas using formalin-fixed paraffin-embedded tissue sections of tumor with adjacent nonneoplastic kidney. Steam antigen retrieval was performed before immunohistochemistry. Specificity was verified by negative control reactions without primary antibody and appropriate membranous staining patterns in positive control tissues (colon carcinoma and adjacent nonneoplastic kidney). Claudin-7 protein was expressed in a membranous pattern in 10 of 11 chromophobe renal cell carcinomas and 4 of 17 oncocytomas ( P P

  • Claudin-7 and Claudin-8: immunohistochemical markers for the differential diagnosis of chromophobe renal cell carcinoma and renal oncocytoma.
    Human pathology, 2008
    Co-Authors: Adeboye O. Osunkoya, Diane Lawson, Maria M Picken, Mahul B. Amin, Cynthia Cohen, Andrew N. Young
    Abstract:

    Claudin-7 and Claudin-8 code for tight junction proteins expressed in distal nephron epithelium. In a recent oligonucleotide microarray study, we identified Claudin-7 and Claudin-8 as candidate markers to distinguish chromophobe renal cell carcinoma from other renal tumors, including oncocytoma. Distinction of these lesions can be difficult by light microscopy but is clinically important because chromophobe renal cell carcinoma has malignant biological potential, whereas renal oncocytoma is benign. Claudin-7 and Claudin-8 expression was studied by immunohistochemistry in 11 chromophobe renal cell carcinomas and 17 oncocytomas using formalin-fixed paraffin-embedded tissue sections of tumor with adjacent nonneoplastic kidney. Steam antigen retrieval was performed before immunohistochemistry. Specificity was verified by negative control reactions without primary antibody and appropriate membranous staining patterns in positive control tissues (colon carcinoma and adjacent nonneoplastic kidney). Claudin-7 protein was expressed in a membranous pattern in 10 of 11 chromophobe renal cell carcinomas and 4 of 17 oncocytomas (P < .01). Claudin-8 was expressed in multiple patterns: In oncocytoma, 11 of 17 cases showed cytoplasmic, 4 of 17 membranous, and 2 of 17 negative reactions. In chromophobe renal cell carcinoma, 0 of 11 cases showed cytoplasmic, 3 of 11 membranous, and 8 of 11 negative reactions (P < .01). The immunohistochemical pattern of membranous Claudin-7 and negative Claudin-8 was seen in 7 of 11 chromophobe renal cell carcinomas and 1 of 17 oncocytomas (63% sensitivity, 84% specificity, 88% positive predictive value for chromophobe renal cell carcinoma). Negative Claudin-7 and cytoplasmic Claudin-8 were observed in 10 of 17 oncocytomas and 0 of 11 chromophobe renal cell carcinomas (59% sensitivity, 100% specificity and positive predictive value for oncocytoma). The distal nephron proteins Claudin-7 and Claudin-8 have potential use as immunohistochemical biomarkers in the differential diagnosis of chromophobe renal cell carcinoma and oncocytoma. Expression of Claudin-7 and Claudin-8 may reflect the relationship of chromophobe renal cell carcinoma and oncocytoma to intercalated cells of the cortical collecting duct. It may be necessary to identify additional biomarkers to include with Claudin-7 and Claudin-8 in a larger immunohistochemical panel to improve diagnostic sensitivity and specificity.

  • Claudin-7 immunohistochemistry in renal tumors: a candidate marker for chromophobe renal cell carcinoma identified by gene expression profiling.
    Archives of pathology & laboratory medicine, 2007
    Co-Authors: Christopher D. Hornsby, Diane Lawson, Maria M Picken, Mahul B. Amin, Cynthia Cohen, Qiqin Yin-goen, Andrew N. Young
    Abstract:

    Abstract Context.—The differential diagnosis of eosinophilic renal tumors can be difficult by light microscopy. In particular, chromophobe renal cell carcinoma (RCC) is difficult to distinguish from oncocytoma. This differential diagnosis is important because chromophobe RCC is malignant, whereas oncocytoma is benign. Furthermore, chromophobe RCC has distinct malignant potential and prognosis compared with eosinophilic variants of other RCC subtypes. Immunohistochemistry is useful for distinguishing chromophobe RCC from other subtypes of renal carcinoma, but no expression marker reliably separates chromophobe RCC from oncocytoma. Objective.—In a previous gene expression microarray analysis of renal tumor subtypes, we found the distal nephron markers Claudin-7 and Claudin-8 to be overexpressed in chromophobe RCC versus oncocytoma and other tumor subtypes. We have confirmed similar findings in independent microarray data and validated differential Claudin-7 protein expression by immunohistochemistry. Design...

Anthony W Segal - One of the best experts on this subject based on the ideXlab platform.

  • tu1353 mucosal mrna expression profiling from the terminal ileum and colon reveals under expression of Claudin 8 a tight junction molecule as potentially causal in ulcerative colitis
    Gastroenterology, 2012
    Co-Authors: Philip J Smith, Adam P Levine, Gavin W Sewell, Nuala R Oshea, Roser Vega, Stuart Bloom, Andrew M Smith, Anthony W Segal
    Abstract:

    Introduction Intestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC and controls (HC) to identify genes that might be implicated in the pathogenesis of the disease. Methods Mucosal biopsies were taken from 24 quiescent UC patients (Mayo score <3) and 33 HCs undergoing colonoscopy. Patients were on no treatment or on 5-aminosalicylates ± azathioprine. HCs were patients without organic disease. Parallel biopsies were taken for RNA extraction and histology from macroscopically non-inflamed mucosa in the terminal ileum (TI), ascending, descending and sigmoid colon, and rectum. cRNA was hybridised to Illumina HumanHT v12.0 Expression Beadchips. Expression data were log transformed and normalised. Probes with a detection p value <0.01 were analysed. Comparing 85 biopsies from HCs and 68 biopsies from UC patients across the colon, the data for each bowel location were adjusted to the mean HC rectal expression level. Where multiple biopsies were taken from the same individual, the adjusted data across all biopsies for that individual were averaged. T tests between groups and outlier analysis (p<0.005, fold change (FC) ≥1.5) were performed using proprietary software. Results Of the ∼30K probes analysed, the two most significantly under-expressed in UC in the colon were those of Claudin 8 (CLDN8) with FCs 2.94 (p=1.29×10−5) and 3.45 (p=3.92×10−5). The expression of Claudin 8 increased distally in the colon, whereas Claudins 3, 7 and 23 were highly, and uniformly, expressed throughout the colon and were normal in UC. Outlier analysis between HC and UC showed CLDN8 to be significantly under-expressed in 25%>40% of UC patients at all 4 colonic sites. There were no CLDN8 UC outliers in the TI. Most of these outlier patients demonstrated consistent levels of under-expression throughout the colon, and their histology revealed microscopic inflammation. Other patients with histologically active disease had normal CLDN8 expression. Conclusion CLDN8 is significantly under-expressed in the UC colon. Outlier analysis has also identified a group of patients in whom CLDN8 is grossly under-expressed. Low expression of CLDN8 in UC could be secondary to inflammation, although the evidence presented here is against this. Reduced levels of CLDN8 could lead to a weak and permeable mucosa predisposing to UC by reducing barrier resistance and allowing penetration by microbes. Competing interests None declared.

  • Tu1353 Mucosal mRNA Expression Profiling From the Terminal Ileum and Colon Reveals Under Expression of Claudin 8, a Tight Junction Molecule, as Potentially Causal in Ulcerative Colitis
    Gastroenterology, 2012
    Co-Authors: Philip J Smith, Adam P Levine, Gavin W Sewell, Roser Vega, Stuart Bloom, Andrew M Smith, Nuala R. O'shea, Anthony W Segal
    Abstract:

    Introduction Intestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC and controls (HC) to identify genes that might be implicated in the pathogenesis of the disease. Methods Mucosal biopsies were taken from 24 quiescent UC patients (Mayo score

Philip J Smith - One of the best experts on this subject based on the ideXlab platform.

  • tu1353 mucosal mrna expression profiling from the terminal ileum and colon reveals under expression of Claudin 8 a tight junction molecule as potentially causal in ulcerative colitis
    Gastroenterology, 2012
    Co-Authors: Philip J Smith, Adam P Levine, Gavin W Sewell, Nuala R Oshea, Roser Vega, Stuart Bloom, Andrew M Smith, Anthony W Segal
    Abstract:

    Introduction Intestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC and controls (HC) to identify genes that might be implicated in the pathogenesis of the disease. Methods Mucosal biopsies were taken from 24 quiescent UC patients (Mayo score <3) and 33 HCs undergoing colonoscopy. Patients were on no treatment or on 5-aminosalicylates ± azathioprine. HCs were patients without organic disease. Parallel biopsies were taken for RNA extraction and histology from macroscopically non-inflamed mucosa in the terminal ileum (TI), ascending, descending and sigmoid colon, and rectum. cRNA was hybridised to Illumina HumanHT v12.0 Expression Beadchips. Expression data were log transformed and normalised. Probes with a detection p value <0.01 were analysed. Comparing 85 biopsies from HCs and 68 biopsies from UC patients across the colon, the data for each bowel location were adjusted to the mean HC rectal expression level. Where multiple biopsies were taken from the same individual, the adjusted data across all biopsies for that individual were averaged. T tests between groups and outlier analysis (p<0.005, fold change (FC) ≥1.5) were performed using proprietary software. Results Of the ∼30K probes analysed, the two most significantly under-expressed in UC in the colon were those of Claudin 8 (CLDN8) with FCs 2.94 (p=1.29×10−5) and 3.45 (p=3.92×10−5). The expression of Claudin 8 increased distally in the colon, whereas Claudins 3, 7 and 23 were highly, and uniformly, expressed throughout the colon and were normal in UC. Outlier analysis between HC and UC showed CLDN8 to be significantly under-expressed in 25%>40% of UC patients at all 4 colonic sites. There were no CLDN8 UC outliers in the TI. Most of these outlier patients demonstrated consistent levels of under-expression throughout the colon, and their histology revealed microscopic inflammation. Other patients with histologically active disease had normal CLDN8 expression. Conclusion CLDN8 is significantly under-expressed in the UC colon. Outlier analysis has also identified a group of patients in whom CLDN8 is grossly under-expressed. Low expression of CLDN8 in UC could be secondary to inflammation, although the evidence presented here is against this. Reduced levels of CLDN8 could lead to a weak and permeable mucosa predisposing to UC by reducing barrier resistance and allowing penetration by microbes. Competing interests None declared.

  • Tu1353 Mucosal mRNA Expression Profiling From the Terminal Ileum and Colon Reveals Under Expression of Claudin 8, a Tight Junction Molecule, as Potentially Causal in Ulcerative Colitis
    Gastroenterology, 2012
    Co-Authors: Philip J Smith, Adam P Levine, Gavin W Sewell, Roser Vega, Stuart Bloom, Andrew M Smith, Nuala R. O'shea, Anthony W Segal
    Abstract:

    Introduction Intestinal barrier dysfunction plays an important role in the pathogenesis of ulcerative colitis (UC). We investigated mRNA profiles of mucosa from the colon and terminal ileum, in patients with UC and controls (HC) to identify genes that might be implicated in the pathogenesis of the disease. Methods Mucosal biopsies were taken from 24 quiescent UC patients (Mayo score