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Braga-Neto MB, Qazi T, Fulmer C, Holubar SD, Fiocchi C, Ivanov AI, Rieder F. Cellular and molecular mechanisms in the pathogenesis of pouchitis: more than just the microbiota. Gut 2025:gutjnl-2024-334445. [PMID: 40240062 DOI: 10.1136/gutjnl-2024-334445] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/25/2024] [Accepted: 03/28/2025] [Indexed: 04/18/2025]
Abstract
Pouchitis, defined as inflammation of the ileal pouch, is the most common complication following restorative proctocolectomy for refractory ulcerative colitis. Antibiotics remain the first line of therapy for pouchitis, but the majority of patients develop subsequent episodes and some are refractory to antibiotic therapy. This highlights the need for more effective treatment options and points to a more complex pathophysiology beyond the role of th pouch microbiome, similar to what is seen in inflammatory bowel disease. In this review, we outline the putative mechanisms of pouchitis, including genetic predisposition, microbiome alterations, dysfunction of the intestinal barrier and the immune system and review the available animal models of pouchitis. In addition, we introduce the concept of pouchitis as a possible transmural disease and discuss the potential role of non-immune cells, including stromal cells, in perpetuating inflammation and intestinal barrier dysfunction. We discuss future directions, implications for novel therapies and propose novel multicellular disease models that can better capture the complexity of pouchitis pathogenesis.
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Affiliation(s)
- Manuel B Braga-Neto
- Department of Gastroenterology, Hepatology & Nutrition, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio, USA
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Taha Qazi
- Department of Gastroenterology, Hepatology & Nutrition, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Clifton Fulmer
- Department of Pathology, Robert J. Tomsich Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Stefan D Holubar
- Department of Colon and Rectal Surgery, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Claudio Fiocchi
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Andrei I Ivanov
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
| | - Florian Rieder
- Department of Gastroenterology, Hepatology & Nutrition, Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio, USA
- Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA
- Program for Global Translational Inflammatory Bowel Disease, Cleveland Clinic, Cleveland, Ohio, USA
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Knowles JP, Church J. Normal Ileal Mucus Is Inadequate for Epithelial Protection in Ileal Pouch Mucosa. Dis Colon Rectum 2024; 67:635-644. [PMID: 38276959 DOI: 10.1097/dcr.0000000000003163] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/27/2024]
Abstract
BACKGROUND Clinical, nonspecific pouchitis is common after restorative proctocolectomy for ulcerative colitis, but its cause is unknown. A possible lack of protection for the ileal mucosa in its role as a reservoir for colonic-type bacteria may be the missing piece in defining the causes of pouchitis. OBJECTIVE The study aimed to review the causes of pouchitis and introduce the hypothesis that inadequate mucus protection in the pouch, combined with a predisposition to abnormal inflammation, is the most common cause of nonspecific pouchitis. DATA SOURCES Review of PubMed and MEDLINE for articles discussing pouchitis and intestinal mucus. STUDY SELECTION Studies published from 1960 to 2023. The main search terms were "pouchitis," and "intestinal mucus," whereas Boolean operators were used with multiple other terms to refine the search. Duplicates and case reports were excluded. MAIN OUTCOME MEASURES Current theories about the cause of pouchitis, descriptions of the role of mucus in the physiology of intestinal protection, and evidence of the effects of lack of mucus on mucosal inflammation. RESULTS The crossreference of "intestinal mucus" with "pouchitis" produced 9 references, none of which discussed the role of mucus in the development of pouchitis. Crossing "intestinal mucus" with "pouch" resulted in 32 articles, combining "pouchitis" with "barrier function" yielded 37 articles, and "pouchitis" with "permeability" yielded only 8 articles. No article discussed the mucus coat as a barrier to bacterial invasion of the epithelium or mentioned inadequate mucus as a factor in pouchitis. However, an ileal pouch produces a colonic environment in the small bowel, and the ileum lacks the mucus protection needed for this sort of environment. This predisposes pouch mucosa to bacterial invasion and chronic microscopic inflammation that may promote clinical pouchitis in patients prone to an autoimmune response. LIMITATIONS No prior studies address inadequate mucus protection and the origin of proctitis. There is no objective way of measuring the autoimmune tendency in patients with ulcerative colitis. CONCLUSIONS Studies of intestinal mucus in the ileal pouch and its association with pouchitis are warranted.
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Affiliation(s)
- Jonathan P Knowles
- Division of Colorectal Surgery, Columbia University Medical Center, New York, New York
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Chen Y, Tseng SH, Chen CY, Tsai YH. Application of Intestinal Barrier Molecules in the Diagnosis of Acute Cellular Rejection After Intestinal Transplantation. Transpl Int 2023; 36:11595. [PMID: 37745643 PMCID: PMC10514359 DOI: 10.3389/ti.2023.11595] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Accepted: 08/22/2023] [Indexed: 09/26/2023]
Abstract
Diagnosing acute rejection after intestinal transplantation currently heavily relies on histopathological analysis of graft biopsies. However, the invasive risks associated with ileoscopic examination and the inaccessibility for biopsy after ileostomy closure hinder real-time detection of rejection responses. Molecules comprising the intestinal barrier have been identified as physiological and molecular biomarkers for various bowel conditions and systemic diseases. To investigate the potential of barrier function-related molecules in diagnosing rejection after intestinal transplantation, plasma samples were collected longitudinally from transplant recipients. The samples were categorized into "indeterminate for rejection (IND)" and "acute rejection (AR)" groups based on clinical diagnoses at each time point. The longitudinal association between plasma levels of these barrier function-related molecules and acute rejection was analyzed using the generalized estimating equations (GEE) method. Logistic GEE models revealed that plasma levels of claudin-3, occludin, sIgA, and zonulin were independent variables correlated with the clinical diagnosis of acute rejection. The subsequent prediction model demonstrated moderate ability in discriminating between IND and AR samples, with a sensitivity of 76.0%, specificity of 89.2%, and accuracy of 84.6%. In conclusion, monitoring plasma levels of claudin-3, occludin, sIgA, and zonulin shows great potential in aiding the diagnosis of acute rejection after intestinal transplantation.
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Affiliation(s)
- Yun Chen
- Department of Surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan
- Graduate Institute of Medicine, Yuan Ze University, Taoyuan, Taiwan
| | - Sheng-Hong Tseng
- Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan
| | - Chih-Yen Chen
- Medicine and Institute of Emergency and Critical Medicine, School of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
- Division of Gastroenterology and Hepatology, Taipei Veterans General Hospital, Taipei, Taiwan
- Association for the Study of Small Intestinal Diseases, Taoyuan, Taiwan
| | - Ya-Hui Tsai
- Department of Surgery, Far Eastern Memorial Hospital, New Taipei City, Taiwan
- Graduate Institute of Medicine, Yuan Ze University, Taoyuan, Taiwan
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Zhang Y, Jiang Y, Li H, Wang J, Li C, Zhang D. Effect of STING signaling on intestinal barrier damage in severe acute pancreatitis. Exp Cell Res 2023; 428:113630. [PMID: 37196844 DOI: 10.1016/j.yexcr.2023.113630] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 05/02/2023] [Accepted: 05/04/2023] [Indexed: 05/19/2023]
Abstract
BACKGROUND Patients with severe acute pancreatitis (SAP) have a compromised intestinal barrier with decreased barrier function and increased cell death. Intestinal epithelial cells (IECs) create a physicochemical barrier that anchors bacteria in the intestine. Recent studies have shown that the stimulator of interferons genes (STING) signaling pathway plays an important function in a number of inflammatory conditions. METHODS The rat SAP model was established by retrograde injection of freshly prepared sodium taurocholate into the biliopancreatic duct. Serum amylase (AMY), lipase (LIPA), interleukin (IL)-6, interferon (IFN)-β, tumor necrosis factor (TNF)-α, intestinal-type fatty acid binding protein (FABP2), diamine oxidase (DAO) and endotoxin (ET) levels were measured in rats. H&E staining was used to assess histological changes in the intestine and pancreas. The expression of intestinal epithelial cell tight junction (TJ) proteins and STING signaling pathway proteins and genes were measured by RT- PCR, Western blot and immunofluorescence staining were used to analyze. The expression of STING signaling pathway proteins in pancreas were measured by Western blot were used to analyze. TUNEL was used to detect IECs death. RESULTS Upregulation of STING pathway-related proteins and genes occurred after sap-induced IECs. In addition, C-176 reduced serum AMY, LIPA, TNF-α, IL-6, INF-β, FABP2, DAO and endotoxin levels and decreased pancreatic and intestinal histopathological injury in SAP rats; DMXAA aggravated serum AMY, LIPA, TNF-α, IL-6, INF-β, FABP2, DAO and endotoxin levels and increased pancreatic and intestinal histopathological injury in SAP rats. CONLUSIONS The results suggest that inhibition of STING signaling can alleviate IECs after SAP, and activation of STING signaling can aggravate IECs after SAP.
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Affiliation(s)
- Yongkang Zhang
- Department of the First General Surgery, Qingdao Municipal Hospital, Dalian Medical University, No. 1 Jiaozhou Road, Qingdao, 266011, Shandong, China
| | - Yingjian Jiang
- Department of the First General Surgery, Qingdao Municipal Hospital, Dalian Medical University, No. 1 Jiaozhou Road, Qingdao, 266011, Shandong, China
| | - Hongbo Li
- Department of the First General Surgery, Qingdao Municipal Hospital, Dalian Medical University, No. 1 Jiaozhou Road, Qingdao, 266011, Shandong, China
| | - Jiang Wang
- Department of the First General Surgery, Qingdao Municipal Hospital, Dalian Medical University, No. 1 Jiaozhou Road, Qingdao, 266011, Shandong, China
| | - Chang Li
- Department of the First General Surgery, Qingdao Municipal Hospital, Dalian Medical University, No. 1 Jiaozhou Road, Qingdao, 266011, Shandong, China
| | - Dianliang Zhang
- Department of the First General Surgery, Qingdao Municipal Hospital, Dalian Medical University, No. 1 Jiaozhou Road, Qingdao, 266011, Shandong, China.
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Zhang J, Jiang Y, Li H, Wang J, Li C, Zhang D. Elevation of HO-1 expression protects the intestinal mucosal barrier in severe acute pancreatitis via inhibition of the MLCK/p-MLC signaling pathway. Exp Cell Res 2023; 424:113508. [PMID: 36764591 DOI: 10.1016/j.yexcr.2023.113508] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2023] [Revised: 02/05/2023] [Accepted: 02/07/2023] [Indexed: 02/11/2023]
Abstract
In severe acute pancreatitis (SAP), intestinal mucosal barrier damage can cause intestinal bacterial translocation and induce or aggravate systemic infections. Heme oxygenase-1 (HO-1) is a validated antioxidant and cytoprotective agent. This research aimed to investigate the effect and mechanism of HO-1 on SAP-induced intestinal barrier damage in SAP rats. Healthy adult male Sprague-Dawley rats were randomly separated into the sham-operated group, SAP group, SAP + Hemin group, and SAP + Znpp group. The rat model of SAP was established by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Hemin (a potent HO-1 activator) and Znpp (a competitive inhibitor of HO-1) were injected intraperitoneally in the selected groups 24 h before SAP. Serum and intestinal tissue samples were collected for analysis after 24 h in each group. Hemin pretreatment significantly reduced systemic inflammation, intestinal oxidative stress, and intestinal epithelial apoptosis in SAP by increasing HO-1 expression. Meanwhile, pretreatment with Hemin abolished the inhibitory effect on the expression of the tight junction proteins and significantly inhibited the activation of the MLCK/P-MLC signaling pathway. Conversely, ZnPP completely reversed these effects. Our study indicates that upregulation of HO-1 expression attenuates the intestinal mucosal barrier damage in SAP. The protective effect of HO-1 on the intestine is attributed to MLCK/p-MLC signaling pathway inhibition.
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Affiliation(s)
- Jingyin Zhang
- Qingdao University, Shandong Province, China; Department of The First General Surgery, Qingdao Municipal Hospital, Shandong Province, China
| | - Yingjian Jiang
- Department of The First General Surgery, Qingdao Municipal Hospital, Shandong Province, China
| | - Hongbo Li
- Department of The First General Surgery, Qingdao Municipal Hospital, Shandong Province, China
| | - Jiang Wang
- Department of The First General Surgery, Qingdao Municipal Hospital, Shandong Province, China
| | - Chang Li
- Department of The First General Surgery, Qingdao Municipal Hospital, Shandong Province, China
| | - Dianliang Zhang
- Qingdao University, Shandong Province, China; Department of The First General Surgery, Qingdao Municipal Hospital, Shandong Province, China.
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Droessler L, Cornelius V, Boehm E, Stein L, Brunner N, Amasheh S. Barrier Perturbation in Porcine Peyer’s Patches by Tumor Necrosis Factor is Associated With a Dysregulation of Claudins. Front Physiol 2022; 13:889552. [PMID: 35707009 PMCID: PMC9189282 DOI: 10.3389/fphys.2022.889552] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Accepted: 04/26/2022] [Indexed: 12/22/2022] Open
Abstract
The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer’s patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer’s patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer’s patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer’s patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer’s patches, in agreement with the major role of this receptor in Peyer’s patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer’s patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer’s patches as a primary mucosal immune defense.
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Bekusova V, Droessler L, Amasheh S, Markov AG. Effects of 1,2-Dimethylhydrazine on Barrier Properties of Rat Large Intestine and IPEC-J2 Cells. Int J Mol Sci 2021; 22:10278. [PMID: 34638619 PMCID: PMC8508681 DOI: 10.3390/ijms221910278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2021] [Revised: 09/19/2021] [Accepted: 09/20/2021] [Indexed: 11/26/2022] Open
Abstract
Colon cancer is accompanied by a decrease of epithelial barrier properties, which are determined by tight junction (TJ) proteins between adjacent epithelial cells. The aim of the current study was to analyze the expression of TJ proteins in a rat model of 1,2-dimethylhydrazine (DMH)-induced colorectal cancer, as well as the barrier properties and TJ protein expression of IPEC-J2 cell monolayers after incubation with DMH. Transepithelial electrical resistance and paracellular permeability for sodium fluorescein of IPEC-J2 were examined by an epithelial volt/ohm meter and spectrophotometry. The expression and localization of TJ proteins were analyzed by immunoblotting and immunohistochemistry. In the colonic tumors of rats with DMH-induced carcinogenesis, the expression of claudin-3 and -4 was significantly increased compared to controls. The transepithelial electrical resistance of IPEC-J2 cells increased, while paracellular permeability for sodium fluorescein decreased, accompanied by an increased expression of claudin-4. The increase of claudin-4 in rat colon after chronic DMH exposure was consistent with the acute effect of DMH on IPEC-J2 cells, which may indicate an essential role of this protein in colorectal cancer development.
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Affiliation(s)
- Viktoria Bekusova
- Department of General Physiology, Faculty of Biology, Saint Petersburg State University, Universitetskaya nab., 7–9, 199034 Saint Petersburg, Russia;
| | - Linda Droessler
- Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany; (L.D.); (S.A.)
| | - Salah Amasheh
- Institute of Veterinary Physiology, Department of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany; (L.D.); (S.A.)
| | - Alexander G. Markov
- Department of General Physiology, Faculty of Biology, Saint Petersburg State University, Universitetskaya nab., 7–9, 199034 Saint Petersburg, Russia;
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Effects of Lipid Peroxidation-Mediated Ferroptosis on Severe Acute Pancreatitis-Induced Intestinal Barrier Injury and Bacterial Translocation. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2021; 2021:6644576. [PMID: 34257815 PMCID: PMC8245223 DOI: 10.1155/2021/6644576] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Revised: 05/12/2021] [Accepted: 06/08/2021] [Indexed: 12/14/2022]
Abstract
Ferroptosis is a recently recognized type of regulated cell death characterized by iron- and lipid peroxidation-mediated nonapoptotic cell death. However, whether ferroptosis is involved in severe acute pancreatitis- (SAP-) induced intestinal barrier injury is unknown. The aim of this study was to investigate whether ferroptosis is involved in SAP-induced intestinal barrier injury, particularly intestinal epithelial cell (IEC) death, and determine whether the inhibition of ferroptosis would ameliorate intestinal barrier injury and prevent bacterial translocation (BT). Sodium taurocholate (5%) was retrogradely perfused into the biliopancreatic duct to establish a rat model of SAP. The rats were divided into three groups: sham operation (SO), SAP-induced intestinal barrier injury (SAP), and ferroptosis inhibitor liproxstatin-1 (SAP + Lip). Serum indexes were measured in the rats. In addition, the biochemical and morphological changes associated with ferroptosis were observed, including iron accumulation in intestinal tissue, lipid peroxidation levels, and mitochondrial shrinkage. Hematoxylin staining and eosin staining were used to assess histological tissue changes. Western blot, RT-PCR, and immunofluorescent staining were performed to analyze the expression of ferroptosis-related proteins and genes as well as tight junction. BT was detected by 16S rDNA sequencing analysis. The results indicated that ferroptosis was significantly induced in the IECs from rats with SAP and ferroptosis was mediated by lipid peroxidation. The specific lipid peroxidation of IECs clearly upregulated ferroptosis and exacerbated intestinal barrier injury. Furthermore, treatment with liproxstatin-1 lowered the levels of serum damage markers, decreased lipid peroxidation, and alleviated intestinal and acute remote organ injury in SAP rats. In addition, inhibition of ferroptosis reduced BT. Our findings are the first to demonstrate that ferroptosis contributes to SAP-induced intestinal barrier injury via lipid peroxidation-mediated IEC death. These results suggest that ferroptosis is a potential therapeutic target for SAP-induced intestinal barrier injury.
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Li KY, Wang X, Liu G, He AQ, Zheng ZC, Zhao XY, Liu T. A New Rat Model of Pouchitis After Proctocolectomy and Ileal Pouch-Anal Anastomosis Using 2,4,6-Trinitrobenzene Sulfonic Acid. J Gastrointest Surg 2021; 25:1524-1533. [PMID: 32424688 DOI: 10.1007/s11605-020-04642-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Accepted: 05/03/2020] [Indexed: 01/31/2023]
Abstract
BACKGROUND Pouchitis is a common complication after ileal pouch-anal anastomosis (IPAA) in patients with ulcerative colitis. However, an ideal model remains lacking. Therefore, we aimed to establish an appropriate model resembling human pouchitis. METHODS Sprague-Dawley rats were randomly assigned to five groups: TNBS group, DSS group, NS group (following IPAA procedure, administrated with TNBS enema, DSS orally, normal saline enema, respectively), NI group (underwent IPAA), and sham group (underwent switch abdominal surgery). General status, weight change, hematochezia, and fecal scores were recorded. Fecal microbiota were counted under a microscope and analyzed by 16S rRNA gene high-throughput sequencing. Specimens of ileal pouch and small intestine (proximal, mid, distal) were collected to evaluate myeloperoxidase and occludin expression by immunohistochemistry and mRNA expression of pro-inflammatory markers by PCR. RESULTS General status, hematochezia, fecal score, and increased mRNA expression of interleukin-6 and TNF-α in the TNBS group were similar to those in the DSS group, whereas the TNBS-induced model displayed a more stable weight change and more serious dysbacteriosis, not only was fecal bacterial diversity reduced, the dominant microbiota was altered. Histopathology scores of the distal small intestine in the TNBS group were lower compared with those in the DSS group (P < 0.05). A significant difference in myeloperoxidase and occludin expression in the small intestine was also detected between the TNBS and DSS groups. CONCLUSIONS Our model mimicked the characteristics of human pouchitis and avoided potential side effects in the small intestine, and thus could be employed for further research.
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Affiliation(s)
- Kai-Yu Li
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China
| | - Xin Wang
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China
| | - Gang Liu
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China.
| | - An-Qi He
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China
| | - Zi-Cheng Zheng
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China
| | - Xin-Yu Zhao
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China
| | - Tong Liu
- Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, 300052, China
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Protective Role of Natural and Semi-Synthetic Tocopherols on TNFα-Induced ROS Production and ICAM-1 and Cl-2 Expression in HT29 Intestinal Epithelial Cells. Antioxidants (Basel) 2021; 10:antiox10020160. [PMID: 33499140 PMCID: PMC7911239 DOI: 10.3390/antiox10020160] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Revised: 12/31/2020] [Accepted: 01/19/2021] [Indexed: 12/12/2022] Open
Abstract
Vitamin E, a fat-soluble compound, possesses both antioxidant and non-antioxidant properties. In this study we evaluated, in intestinal HT29 cells, the role of natural tocopherols, α-Toc and δ-Toc, and two semi-synthetic derivatives, namely bis-δ-Toc sulfide (δ-Toc)2S and bis-δ-Toc disulfide (δ-Toc)2S2, on TNFα-induced oxidative stress, and intercellular adhesion molecule-1 (ICAM-1) and claudin-2 (Cl-2) expression. The role of tocopherols was compared to that of N-acetylcysteine (NAC), an antioxidant precursor of glutathione synthesis. The results show that all tocopherol containing derivatives used, prevented TNFα-induced oxidative stress and the increase of ICAM-1 and Cl-2 expression, and that (δ-Toc)2S and (δ-Toc)2S2 are more effective than δ-Toc and α-Toc. The beneficial effects demonstrated were due to tocopherol antioxidant properties, but suppression of TNFα-induced Cl-2 expression seems not only to be related with antioxidant ability. Indeed, while ICAM-1 expression is strongly related to the intracellular redox state, Cl-2 expression is TNFα-up-regulated by both redox and non-redox dependent mechanisms. Since ICAM-1 and Cl-2 increase intestinal bowel diseases, and cause excessive recruitment of immune cells and alteration of the intestinal barrier, natural and, above all, semi-synthetic tocopherols may have a potential role as a therapeutic support against intestinal chronic inflammation, in which TNFα represents an important proinflammatory mediator.
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Wang H, Jiang Y, Li H, Wang J, Li C, Zhang D. Carbachol protects the intestinal barrier in severe acute pancreatitis by regulating Cdc42/F-actin cytoskeleton. Exp Ther Med 2020; 20:2828-2837. [PMID: 32765779 PMCID: PMC7401956 DOI: 10.3892/etm.2020.8985] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2019] [Accepted: 12/06/2019] [Indexed: 12/17/2022] Open
Abstract
The present study aimed to investigate the effect of carbachol on the intestinal tight-junction barrier in a rat model of severe acute pancreatitis (SAP) without aggravating pancreatic injury, and to determine whether cell division cycle 42 (Cdc42)/F-actin could have a regulatory role. Rats were separated into a sham-operation (SO) group (n=10), SO + carbachol group (n=10), SAP group (n=60) and SAP + carbachol group (n=60). Sodium taurocholate (5%) was retrogradely injected into the biliopancreatic duct of rats to induce SAP. Subsequently, 16S rRNA sequencing was used to detect bacterial translocation (BT) in the gut of surviving animals. Hematoxylin and eosin staining was used to detect morphological changes in the pancreas and intestine. The expression of F-actin and tight junction proteins was analyzed by western blotting and immunofluorescence, and Cdc42 expression was analyzed by immunohistochemistry and western blotting. The results demonstrated that the intestinal injury in SO and SO + carbachol groups was lower than that in the SAP + carbachol group (P<0.05); however, the intestinal injury was similar in the SO and SO + carbachol groups (P>0.05), and was significantly more severe in the SAP group compared with the SAP + carbachol group (P<0.05). Similarly, pancreatic injury in the SAP and SAP + carbachol groups was significantly higher compared with the SO and SO + carbachol groups (P<0.05); however, pancreatic injury was similar in the SAP and SAP + carbachol groups (P>0.05), and in the SO and SO + carbachol groups (P>0.05). Furthermore, the mortality rate and BT in the SAP group were significantly higher compared with the SAP + carbachol group (mortality rate, 50% vs. 30%, P<0.05; BT, 60% vs. 33.3%, P<0.05). In addition, the expression of Cdc42, F-actin and claudin-2 was significantly higher in the SAP and SAP + carbachol groups compared with the SO and SO + carbachol groups (P<0.05), and the expression of occludin and zonula occludens-1 were significantly higher in the SO and SO + carbachol groups compared with the SAP and SAP + carbachol groups (P<0.05). In conclusion, these findings demonstrated that carbachol may protect the intestinal barrier in the SAP rat model without aggravating pancreatic injury via regulation of Cdc42/F-actin expression.
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Affiliation(s)
- Hanlin Wang
- Center of Colon and Rectum, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, P.R. China
| | - Yingjian Jiang
- Center of Colon and Rectum, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, P.R. China
| | - Hongbo Li
- Center of Colon and Rectum, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, P.R. China
| | - Jiang Wang
- Center of Colon and Rectum, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, P.R. China
| | - Chang Li
- Center of Colon and Rectum, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, P.R. China
| | - Dianliang Zhang
- Center of Colon and Rectum, Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong 266011, P.R. China
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Brunner N, Stein L, Cornelius V, Knittel R, Fallier-Becker P, Amasheh S. Blood-Brain Barrier Protein Claudin-5 Expressed in Paired Xenopus laevis Oocytes Mediates Cell-Cell Interaction. Front Physiol 2020; 11:857. [PMID: 32848831 PMCID: PMC7396581 DOI: 10.3389/fphys.2020.00857] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2020] [Accepted: 06/26/2020] [Indexed: 01/08/2023] Open
Abstract
Claudin-5 determines the sealing properties of blood-brain barrier tight junctions and its function is impaired in neurodegenerative and neuroinflammatory disorders. Focusing on the contribution of claudin-5 to the trans-interaction within the tight junction seal, we used Xenopus laevis oocytes as an expression system. Cells were clustered and challenged in a novel approach for the analysis of claudin interaction. We evaluated the strengthening effect of claudin-5 to cell-cell-connection in comparison to claudin-3. Application of a hydrostatic pressure impulse on clustered control oocyte pairs revealed a reduction of contact areas. In contrast, combinations with both oocytes expressing claudins maintained an enhanced connection between the cells (cldn5-cldn5, cldn3-cldn3). Strength of interaction was increased by both claudin-3 and claudin-5. This novel approach allowed an analysis of single claudins contributing to tight junction integrity, characterizing homophilic and hetrophilic trans-interaction of claudins. To test a new screening approach for barrier effectors, exemplarily, this 2-cell model of oocytes was used to analyze the effect of the absorption enhancer sodium caprate on the oocyte pairs.
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Affiliation(s)
- Nora Brunner
- Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
| | - Laura Stein
- Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
| | - Valeria Cornelius
- Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
| | - Ria Knittel
- Institute of Pathology and Neuropathology, University Hospital of Tuebingen, Eberhard Karls University of Tuebingen, Tuebingen, Germany
| | - Petra Fallier-Becker
- Institute of Pathology and Neuropathology, University Hospital of Tuebingen, Eberhard Karls University of Tuebingen, Tuebingen, Germany
| | - Salah Amasheh
- Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
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Harnoss JM, Gebhardt JM, Radhakrishnan P, Leowardi C, Burmeister J, Halligan DN, Yuan S, Kennel KB, Strowitzki MJ, Schaible A, Lasitschka F, Taylor CT, Schneider M. Prolyl Hydroxylase Inhibition Mitigates Pouchitis. Inflamm Bowel Dis 2020; 26:192-205. [PMID: 31618435 DOI: 10.1093/ibd/izz218] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Indexed: 12/12/2022]
Abstract
BACKGROUND Pouchitis is the most common long-term complication after restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) or familial adenomatous polyposis (FAP), which can eventually progress to pouch failure, necessitating permanent stoma construction. Hypoxia-inducible transcription factor prolyl hydroxylase-containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors that control adaptive gene expression through hypoxia-inducible factor (HIF). Emerging evidence supports PHDs as being therapeutic targets in intestinal inflammation. However, pharmacological inhibition of PHDs has not been validated as a treatment strategy in pouchitis. METHODS PHD1-3 mRNA and protein expression were analyzed in mucosal pouch and prepouch ileal patient biopsies. After establishment of a preclinical IPAA model in rats, the impact of the pan-PHD small-molecule inhibitor dimethyloxalylglycine (DMOG) on dextran sulfate sodium (DSS)-induced pouchitis was studied. Clinical and molecular parameters were investigated. RESULTS PHD1, but not PHD2 or PHD3, was overexpressed in pouchitis in biopsies of patients with IPAA for UC but not FAP. In addition, PHD1 expression correlated with disease activity. DMOG treatment profoundly mitigated DSS-induced pouchitis in a rodent IPAA model. Mechanistically, DMOG restored intestinal epithelial barrier function by induction of tight junction proteins zona occludens-1 and claudin-1 and alleviation of intestinal epithelial cell apoptosis, thus attenuating pouch inflammation. CONCLUSIONS Together, these results establish a strong therapeutic rationale for targeting PHD1 with small-molecule inhibitors in pouchitis after IPAA for UC.
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Affiliation(s)
- Jonathan M Harnoss
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Jasper M Gebhardt
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Praveen Radhakrishnan
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Christine Leowardi
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Julius Burmeister
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Doug N Halligan
- School of Medicine, Systems Biology Ireland and the Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
| | - Shuai Yuan
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Kilian B Kennel
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Moritz J Strowitzki
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany.,School of Medicine, Systems Biology Ireland and the Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
| | - Anja Schaible
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
| | - Felix Lasitschka
- Institute of Pathology, University of Heidelberg, Heidelberg, Germany
| | - Cormac T Taylor
- School of Medicine, Systems Biology Ireland and the Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland
| | - Martin Schneider
- Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany
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Vitzthum C, Stein L, Brunner N, Knittel R, Fallier-Becker P, Amasheh S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins. FASEB J 2019; 33:5312-5319. [PMID: 30645152 DOI: 10.1096/fj.201801451rr] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ-specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn-1, -2, or -3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including "kissing-point"-like structures representing homophilic trans-interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultrastructural analysis revealed a self-assembly of TJ strands and a cldn-specific strand morphology.-Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier-Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.
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Affiliation(s)
- Constanze Vitzthum
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany; and
| | - Laura Stein
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany; and
| | - Nora Brunner
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany; and
| | - Ria Knittel
- Institute of Pathology and Neuropathology, University of Tuebingen, Tuebingen, Germany
| | - Petra Fallier-Becker
- Institute of Pathology and Neuropathology, University of Tuebingen, Tuebingen, Germany
| | - Salah Amasheh
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany; and
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15
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Shen B. Pathogenesis of Pouchitis. POUCHITIS AND ILEAL POUCH DISORDERS 2019:129-146. [DOI: 10.1016/b978-0-12-809402-0.00011-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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Nam HH, Nan L, Choo BK. Dichloromethane Extracts of Geranium Koreanum Kom. Alleviates Esophagus Damage in Acute Reflux Esophagitis-Induced Rats by Anti-Inflammatory Activities. Int J Mol Sci 2018; 19:ijms19113622. [PMID: 30453554 PMCID: PMC6274961 DOI: 10.3390/ijms19113622] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2018] [Revised: 11/05/2018] [Accepted: 11/14/2018] [Indexed: 12/14/2022] Open
Abstract
Reflux esophagitis (RE) is a gastrointestinal disease caused by the reflux of gastric acid and stomach contents, and it leads to esophageal damage. Therefore, it is necessary to study the improvement of esophageal damage on a RE-induced model. The present study was accomplished to demonstrate the protective effects of a dichloromethane fraction of Geranium koreanum (DGK) plant on esophageal damage in an acute RE rat model. First, we examined the potential of anti-inflammatory effects of various fractions measured by cell cytotoxicity, morphological changes and nitric oxide (NO) production on lipopolysaccharide (LPS)-induced Raw 264.7 macrophage cells. Then, to evaluate the protective effects on RE, rats were partitioned into the following groups: normal control, RE-induced control and RE rats pre-treated with DGK 100 and 200 mg/kg body weight. The esophageal mucosal ulcer ratio was measured by the Image J program and histological changes were examined using a hematoxylin and eosin staining of the esophageal mucosa. The expression of pro-inflammatory proteins, cytokines and tight junction proteins involved in the esophageal mucosal damage were investigated using Western blotting and an enzyme-linked immunosorbent assay (ELISA) kit with esophagus tissue. DGK chemical profile and phenolic contents were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The results showed that DGK exhibited anti-inflammatory effects against LPS-stimulated cells by significantly inhibiting NO production. Additionally, the results in vivo showed that improvement effects of DGK on esophageal mucosal damage. The expression of inflammatory proteins involved in nuclear factor κB (NF-κB) signaling pathways and tight junction protein (claudin-4 and -5) were significantly decreased in esophageal mucosa. We found the potential of DGK as source of replacement therapy products for inflammatory and RE disease.
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Affiliation(s)
- Hyeon Hwa Nam
- Department of Crop Science & Biotechnology, Chonbuk National University, Jeonju 54896, Korea.
| | - Li Nan
- Department of Crop Science & Biotechnology, Chonbuk National University, Jeonju 54896, Korea.
| | - Byung Kil Choo
- Department of Crop Science & Biotechnology, Chonbuk National University, Jeonju 54896, Korea.
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Autophagy Strengthens Intestinal Mucosal Barrier by Attenuating Oxidative Stress in Severe Acute Pancreatitis. Dig Dis Sci 2018; 63:910-919. [PMID: 29427225 DOI: 10.1007/s10620-018-4962-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Accepted: 02/01/2018] [Indexed: 12/15/2022]
Abstract
BACKGROUND Intestinal mucosal barrier dysfunction can be caused by severe acute pancreatitis (SAP). It is normally associated with changes to mucosal autophagy and oxidative stress. OBJECTIVE The aim of this study was to investigate the correlation between autophagy and oxidative stress on the intestinal mucosal barrier of SAP rat model. METHODS SAP was induced by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Bacterial translocation (BT) was detected by 16S rDNA sequencing analysis. Morphological alterations in the pancreas and gut were determined by hematoxylin-eosin staining. Oxidative stress status was determined by measuring the level of intestinal malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Western blot, RT-PCR, and immunofluorescent staining were preformed to analyze the expression of tight junction and autophagy proteins. RESULTS According to the sequencing analysis, rats in SAP group were divided into BT (+) group (n = 9) and BT (-) group (n = 8). Pancreatic and intestinal injuries in SAP group were significantly higher than sham operation group. The content of MDA was clearly elevated, and SOD as well as GPx activities were decreased in BT (+) group as compared with BT (-) group. The expression of LC3II and Beclin1 in BT (-) group was higher than that observed in BT (+). In contrast, BT (+) group had a higher level of claudin-2 and a lower level of zonula occluden-1, occludin, and claudin-1. CONCLUSION These results suggest that activated autophagy may attenuate intestinal mucosal barrier dysfunction by preventing and reducing the oxidative stress in SAP.
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Radloff J, Falchuk EL, Markov AG, Amasheh S. Molecular Characterization of Barrier Properties in Follicle-Associated Epithelium of Porcine Peyer's Patches Reveals Major Sealing Function of Claudin-4. Front Physiol 2017; 8:579. [PMID: 28855873 PMCID: PMC5557736 DOI: 10.3389/fphys.2017.00579] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2017] [Accepted: 07/26/2017] [Indexed: 12/22/2022] Open
Abstract
The pig represents a preferred model for the analysis of intestinal immunology. However, the barrier of the follicle-associated epithelium (FAE) covering porcine Peyer's patches (PP) has not yet been characterized in detail. This study aimed to perform this characterization in order to pave the way toward an understanding of the functional contribution of epithelial barrier properties in gut immunology. Porcine tissue specimens were taken from the distal small intestine in order to obtain electrophysiological data of PP FAE and neighboring villous epithelium (VE), employing the Ussing chamber technique. Transepithelial resistance (TER) and paracellular fluorescein flux were measured, and tissues were morphometrically compared. In selfsame tissues, expression and localization of major tight junction (TJ) proteins (claudin-1, -2, -3, -4, -5, and -8) were analyzed. PP FAE specimens showed a higher TER and a lower apparent permeability for sodium fluorescein than VE. Immunoblotting revealed an expression of all claudins within both epithelia, with markedly stronger expression of the sealing TJ protein claudin-4 in PP FAE compared with the neighboring VE. Immunohistochemistry confirmed the expression and localization of all claudins in both PP FAE and VE, with stronger claudin-4 abundance in PP FAE. The results are in accordance with the physiological function of the FAE, which strongly regulates and limits antigen uptake determining a mandatory transcellular route for antigen presentation, highlighting the importance of this structure for the first steps of the intestinal immune response. Thus, this study provides detailed insights into the specific barrier properties of the porcine FAE covering intestinal PP, at the interface of intestinal immunology and barriology.
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Affiliation(s)
- Judith Radloff
- Institute of Veterinary Physiology, Freie Universität BerlinBerlin, Germany
| | - Evgeny L Falchuk
- Department of General Physiology, Saint Petersburg State UniversitySt. Petersburg, Russia
| | - Alexander G Markov
- Department of General Physiology, Saint Petersburg State UniversitySt. Petersburg, Russia
| | - Salah Amasheh
- Institute of Veterinary Physiology, Freie Universität BerlinBerlin, Germany
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Radloff J, Zakrzewski SS, Pieper R, Markov AG, Amasheh S. Porcine milk induces a strengthening of barrier function in porcine jejunal epithelium in vitro. Ann N Y Acad Sci 2017; 1397:110-118. [PMID: 28445594 DOI: 10.1111/nyas.13340] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2016] [Revised: 02/26/2017] [Accepted: 02/28/2017] [Indexed: 12/13/2022]
Abstract
Milk contains a variety of components that have been shown to affect the expression and localization of epithelial tight junction proteins and therefore the intestinal barrier. Thus, we hypothesized that milk would have an effect on intestinal barrier properties, owing to effects on the tight junction in an intraspecies porcine intestinal in vitro model. Jejunal samples of piglets derived from different age groups were analyzed. Transepithelial electrical resistance was recorded employing the Ussing chamber technique. Porcine milk or predigested milk in buffer solution was added to the apical side, and effects were compared to untreated controls. Unidirectional paracellular flux measurements were performed using sodium fluorescein. Tight junction protein expression and localization were analyzed by immunoblotting and immunofluorescence microscopy. Incubation with milk or predigested milk led to an increase in transepithelial electrical resistance, while paracellular permeability for sodium fluorescein did not result in significant changes. Densitometric analysis of immunoblot signals did not show significant alterations in claudin expression, but a reduction of claudin signals in apicolateral membrane compartments in both approaches became apparent via immunohistology. The functional effect might reflect a physiological protective mechanism, when offspring exclusively rely on their mother's milk and are exposed to a plethora of potentially barrier-perturbing factors.
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Affiliation(s)
- Judith Radloff
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
| | - Silke S Zakrzewski
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
| | - Robert Pieper
- Institute of Animal Nutrition, Freie Universität Berlin, Berlin, Germany
| | - Alexander G Markov
- Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia
| | - Salah Amasheh
- Institute of Veterinary Physiology, Freie Universität Berlin, Berlin, Germany
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20
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Markov AG, Aschenbach JR, Amasheh S. The epithelial barrier and beyond: Claudins as amplifiers of physiological organ functions. IUBMB Life 2017; 69:290-296. [PMID: 28371008 DOI: 10.1002/iub.1622] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2016] [Accepted: 03/03/2017] [Indexed: 12/22/2022]
Abstract
Epithelial cell layers are interconnected by a meshwork of tight junction (TJ) protein strands, which are localized within apicolateral membranes. The proteins that form TJs are regarded to provide a static barrier, determining epithelial properties. However, recent findings in the field of barriology suggest that TJs contribute to more physiological aspects than indicated by the sum of the qualities of the single TJ proteins. Generally, TJs exhibit four major functions: (i) a "gate function," defining transepithelial permeability (i.e., barrier) properties, (ii) a "fence function" determining epithelial cell polarity, (iii) a "signaling function," affecting regulatory pathways, and (iv) a "stabilizing function," maintaining the integrity of the epithelium. This review presents a critical view on how the efficacy of physiological processes in epithelia and thus organ function might be improved by changes in the expression of claudins, the latter representing the largest and most variable family of TJ proteins. Major focus is set on (i) the coordinated regulation of transport and barrier in the intestine, (ii) the role of TJs in defining the route for antigen uptake and presentation in intestinal Peyer's patches, and (iii) the TJ function in mammary glands in response to milk accumulation, which represent impressive examples to highlight the amplification of epithelial functions by TJ proteins. © 2017 IUBMB Life, 69(5):290-296, 2017.
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Affiliation(s)
- Alexander G Markov
- Department of General Physiology, St. Petersburg State University, Russia
| | - Jörg R Aschenbach
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Veterinary Physiology, Berlin, Germany
| | - Salah Amasheh
- Department of Veterinary Medicine, Freie Universität Berlin, Institute of Veterinary Physiology, Berlin, Germany
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Schieffer KM, Williams ED, Yochum GS, Koltun WA. Review article: the pathogenesis of pouchitis. Aliment Pharmacol Ther 2016; 44:817-35. [PMID: 27554912 PMCID: PMC5785099 DOI: 10.1111/apt.13780] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/10/2015] [Revised: 12/03/2015] [Accepted: 08/04/2016] [Indexed: 12/11/2022]
Abstract
BACKGROUND A total proctocolectomy followed by ileal pouch-anal anastomosis is a potentially curative surgery for ulcerative colitis or familial adenomatous polyposis. About 5-35% of patients with ulcerative colitis and 0-11% of patients with familial adenomatous polyposis develop subsequent inflammation of the ileal pouch termed pouchitis. AIM To provide a comprehensive analysis of the research studying the possible pathogenesis of pouchitis. The goals were to identify promising areas of investigation, to help focus clinicians, researchers and patients on how to better understand and then potentially manage ileal pouchitis, and to provide avenues for future research investigations. METHODS This review examined manuscripts from 1981 to 2015 that discussed and/or proposed hypotheses with supportive evidence for the potential underlying pathogenic mechanism for pouchitis. RESULTS The pathogenesis of pouchitis is not definitively understood, but various hypotheses have been proposed, including (i) recurrence of ulcerative colitis, (ii) dysbiosis of the ileal pouch microbiota, (iii) deprivation of nutritional short-chain fatty acids, (iv) mucosal ischaemia and oxygen-free radical injury, (v) host genetic susceptibility and (vi) immune dysregulation. However, none of these alone are able to fully explain pouchitis pathogenesis. CONCLUSIONS Pouchitis, similar to inflammatory bowel disease, is a complex disorder that is not caused by any one single factor. More likely, pouchitis occurs through a combination of both dysregulated host inflammatory mechanisms and interaction with luminal microbiota.
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Affiliation(s)
- Kathleen M. Schieffer
- Department of Surgery, Division of Colon and Rectal Surgery, The Pennsylvania State University, College of Medicine, Hershey, PA, USA 17033
| | - Emmanuelle D. Williams
- Department of Medicine, Division of Gastroenterology, The Pennsylvania State University, College of Medicine, Hershey, PA, USA 17033
| | - Gregory S. Yochum
- Department of Surgery, Division of Colon and Rectal Surgery, The Pennsylvania State University, College of Medicine, Hershey, PA, USA 17033,Department of Biochemistry & Molecular Biology, The Pennsylvania State University, College of Medicine, Hershey, PA, USA 17033
| | - Walter A. Koltun
- Department of Surgery, Division of Colon and Rectal Surgery, The Pennsylvania State University, College of Medicine, Hershey, PA, USA 17033
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Landy J, Ronde E, English N, Clark SK, Hart AL, Knight SC, Ciclitira PJ, Al-Hassi HO. Tight junctions in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer. World J Gastroenterol 2016; 22:3117-3126. [PMID: 27003989 PMCID: PMC4789987 DOI: 10.3748/wjg.v22.i11.3117] [Citation(s) in RCA: 354] [Impact Index Per Article: 39.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Revised: 09/19/2015] [Accepted: 12/21/2015] [Indexed: 02/06/2023] Open
Abstract
Inflammatory bowel diseases are characterised by inflammation that compromises the integrity of the epithelial barrier. The intestinal epithelium is not only a static barrier but has evolved complex mechanisms to control and regulate bacterial interactions with the mucosal surface. Apical tight junction proteins are critical in the maintenance of epithelial barrier function and control of paracellular permeability. The characterisation of alterations in tight junction proteins as key players in epithelial barrier function in inflammatory bowel diseases is rapidly enhancing our understanding of critical mechanisms in disease pathogenesis as well as novel therapeutic opportunities. Here we give an overview of recent literature focusing on the role of tight junction proteins, in particular claudins, in inflammatory bowel diseases and inflammatory bowel disease associated colorectal cancer.
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Markov AG, Falchuk EL, Kruglova NM, Radloff J, Amasheh S. Claudin expression in follicle-associated epithelium of rat Peyer's patches defines a major restriction of the paracellular pathway. Acta Physiol (Oxf) 2016; 216:112-9. [PMID: 26228735 DOI: 10.1111/apha.12559] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Revised: 05/19/2015] [Accepted: 07/24/2015] [Indexed: 12/17/2022]
Abstract
AIM Members of the tight junction protein family of claudins have been demonstrated to specifically determine paracellular permeability of the intestinal epithelium. In small intestinal mucosa, which is generally considered to be a leaky epithelium, Peyer's patches are a primary part of the immune system. The aim of this study was to analyse the tight junctional barrier of follicle-associated epithelium covering Peyer's patches (lymphoid follicles). METHODS Employing small intestinal tissue specimens of male Wistar rats, electrophysiological analyses including the Ussing chamber technique, marker flux measurements and one-path impedance spectroscopy were performed. Morphometry of HE-stained tissue sections was taken into account. Claudin expression and localization was analysed by immunoblotting and confocal laser scanning immunofluorescence microscopy. RESULTS Almost twofold higher parameters of epithelial and transepithelial tissue resistance and a markedly lower permeability for the paracellular permeability markers 4 and 20 kDa FITC-dextran were detected in follicle-associated epithelium compared to neighbouring villous epithelium. Analysis of claudin expression and localization revealed a stronger expression of major sealing proteins in follicle-associated epithelium, including claudin-1, claudin-4, claudin-5 and claudin-8. Therefore, the specific expression and localization of claudins is in accordance with barrier properties of follicle-associated epithelium vs. neighbouring villous epithelium. CONCLUSION We demonstrate that follicle-associated epithelium is specialized to ensure maximum restriction of the epithelial paracellular pathway in Peyer's patches by selective sealing of tight junctions. This results in an exclusive transcellular pathway of epithelial cells as the limiting and mandatory route for a controlled presentation of antigens to the underlying lymphocytes under physiological conditions.
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Affiliation(s)
- A. G. Markov
- Institute of General Physiology; Biological Faculty; St. Petersburg State University; St. Petersburg Russia
| | - E. L. Falchuk
- Institute of General Physiology; Biological Faculty; St. Petersburg State University; St. Petersburg Russia
| | - N. M. Kruglova
- Institute of General Physiology; Biological Faculty; St. Petersburg State University; St. Petersburg Russia
| | - J. Radloff
- Institute of Veterinary Physiology; Freie Universität Berlin; Berlin Germany
| | - S. Amasheh
- Institute of Veterinary Physiology; Freie Universität Berlin; Berlin Germany
- Institute of Clinical Physiology; Charité - Universitätsmedizin Berlin; Berlin Germany
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Zezos P, Saibil F. Inflammatory pouch disease: The spectrum of pouchitis. World J Gastroenterol 2015; 21:8739-8752. [PMID: 26269664 PMCID: PMC4528017 DOI: 10.3748/wjg.v21.i29.8739] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Revised: 04/29/2015] [Accepted: 06/26/2015] [Indexed: 02/06/2023] Open
Abstract
Restorative proctocolectomy with ileal-pouch anal anastomosis (IPAA) is the operation of choice for medically refractory ulcerative colitis (UC), for UC with dysplasia, and for familial adenomatous polyposis (FAP). IPAA can be a treatment option for selected patients with Crohn's colitis without perianal and/or small bowel disease. The term "pouchitis" refers to nonspecific inflammation of the pouch and is a common complication in patients with IPAA; it occurs more often in UC patients than in FAP patients. This suggests that the pathogenetic background of UC may contribute significantly to the development of pouchitis. The symptoms of pouchitis are many, and can include increased bowel frequency, urgency, tenesmus, incontinence, nocturnal seepage, rectal bleeding, abdominal cramps, and pelvic discomfort. The diagnosis of pouchitis is based on the presence of symptoms together with endoscopic and histological evidence of inflammation of the pouch. However, "pouchitis" is a general term representing a wide spectrum of diseases and conditions, which can emerge in the pouch. Based on the etiology we can sub-divide pouchitis into 2 groups: idiopathic and secondary. In idiopathic pouchitis the etiology and pathogenesis are still unclear, while in secondary pouchitis there is an association with a specific causative or pathogenetic factor. Secondary pouchitis can occur in up to 30% of cases and can be classified as infectious, ischemic, non-steroidal anti-inflammatory drugs-induced, collagenous, autoimmune-associated, or Crohn's disease. Sometimes, cuffitis or irritable pouch syndrome can be misdiagnosed as pouchitis. Furthermore, idiopathic pouchitis itself can be sub-classified into types based on the clinical pattern, presentation, and responsiveness to antibiotic treatment. Treatment differs among the various forms of pouchitis. Therefore, it is important to establish the correct diagnosis in order to select the appropriate treatment and further management. In this editorial, we present the spectrum of pouchitis and the specific features related to the diagnosis and treatment of the various forms.
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Claudin-related intestinal diseases. Semin Cell Dev Biol 2015; 42:30-8. [PMID: 25999319 DOI: 10.1016/j.semcdb.2015.05.006] [Citation(s) in RCA: 79] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2015] [Revised: 05/09/2015] [Accepted: 05/12/2015] [Indexed: 02/07/2023]
Abstract
With up to 200 m(2) the human intestine is the organ with the largest absorptive surface of the body. It is lined by a single layer of epithelial cells that separates the host from the environment. The intestinal epithelium provides both, selective absorption of nutrients, ions, and water but also a highly effective barrier function which includes the first line of defense against environmental antigens. The paracellular part of this barrier function is provided by tight junction (TJ) proteins, especially the large family of claudins. Changes in abundance or molecular structure of claudins can generally result in three typical effects, (i) decreased absorptive passage, (ii) increased secretory passage of small solutes and water causing leak flux diarrhea and (iii) increased absorptive passage of macromolecules which may induce inflammatory processes. Several intestinal diseases are associated with such changes that can result in intestinal inflammation and symptoms like weight loss, abdominal pain or diarrhea. This review summarizes our current knowledge on barrier dysfunction and claudin dysregulation in several intestinal diseases gastroenterologists are often faced with, like inflammatory bowel disease, microscopic colitis, celiac disease, irritable bowel syndrome, gallstones and infectious diseases like HIV enteropathy, Campylobacter jejuni and Clostridium perfringens infection.
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Luettig J, Rosenthal R, Barmeyer C, Schulzke JD. Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation. Tissue Barriers 2015; 3:e977176. [PMID: 25838982 DOI: 10.4161/21688370.2014.977176] [Citation(s) in RCA: 197] [Impact Index Per Article: 19.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2014] [Accepted: 10/10/2014] [Indexed: 02/07/2023] Open
Abstract
The epithelial tight junction determines the paracellular water and ion movement in the intestine and also prevents uptake of larger molecules, including antigens, in an uncontrolled manner. Claudin-2, one of the 27 mammalian claudins regulating that barrier function, forms a paracellular channel for small cations and water. It is typically expressed in leaky epithelia like proximal nephron and small intestine and provides a major pathway for the paracellular transport of sodium, potassium, and fluid. In intestinal inflammation (Crohn's disease, ulcerative colitis), immune-mediated diseases (celiac disease), and infections (HIV enteropathy), claudin-2 is upregulated in small and large intestine and contributes to diarrhea via a leak flux mechanism. In parallel to that upregulation, other epithelial and tight junctional features are altered and the luminal uptake of antigenic macromolecules is enhanced, for which claudin-2 may be partially responsible through induction of tight junction strand discontinuities.
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Key Words
- AP, activator protein
- CARD15, caspase recruitment domain-containing protein 15
- Crohn's disease
- DSS, dextran sodium sulfate
- ECL, extracellular loop
- ERK, extracellular-regulated kinase
- HIV
- HIV, human immunodeficiency virus
- HNF, hepatocyte nuclear factor
- IBD, inflammatory bowel disease
- IFN, interferon
- IFNγ
- IL, interleukin
- JAM, junctional adhesion molecule
- JNK, c-jun N-terminal kinase
- LPS, lipopolysaccharides
- MAPK, mitogen-activated protein kinase
- MDCK, Madine Darby canine kidney
- MLC, myosin light chain
- NFκB, nuclear factor kappa B
- NOD2, nucleotide-binding oligomerization domain-containing protein 2
- PI3K, phosphatidyl-inositol-3-kinase
- ROCK, Rho kinase
- Rho, ras homolog
- STAT, signal transducers and activators of transcription
- TEER, transepithelial electrical resistance
- TJ, tight junction
- TNBS, 2,4,6-trinitrobenzene sulfonic acid
- TNF, tumor necrosis factor
- TNFα
- Tat, trans-activator of transcription
- Vpr, viral protein r; ZO, zonula occludens
- celiac disease
- claudin-2
- gp, glycoprotein
- inflammatory bowel disease
- tight junction
- ulcerative colitis
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Affiliation(s)
- J Luettig
- Institute of Clinical Physiology; Department of Gastroenterology; Charité ; Berlin, Germany
| | - R Rosenthal
- Institute of Clinical Physiology; Department of Gastroenterology; Charité ; Berlin, Germany
| | - C Barmeyer
- Institute of Clinical Physiology; Department of Gastroenterology; Charité ; Berlin, Germany
| | - J D Schulzke
- Institute of Clinical Physiology; Department of Gastroenterology; Charité ; Berlin, Germany
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Markov AG, Aschenbach JR, Amasheh S. Claudin clusters as determinants of epithelial barrier function. IUBMB Life 2015; 67:29-35. [PMID: 25788154 DOI: 10.1002/iub.1347] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2014] [Accepted: 01/05/2015] [Indexed: 12/18/2022]
Abstract
Claudins are tetraspan tight junction proteins which have been attributed to primarily determine epithelial barrier function in a wide variety of different organs and tissues. Among this protein family with currently 27 members, single claudins contribute in an organ- and tissue-specific manner to defined properties such as cation-, anion- or water-selective pore functions, sealing functions or ambiguous functions. As the size of tight junction strand particles visualized by freeze-fracture electron microscopy have a diameter of approximately 10 nm, multimeric assembly of tight junction proteins appears to be a basic principle for barrier formation. Moreover, expression patterns of different tissues showed that single claudins appear to specifically co-localize with other claudins, which indicates a cluster formation within tight junction strand particles with a fixed stoichiometry. This review provides a critical view on the current understanding of tight junction protein co-localization within strands. We analyze how tissue specific differences of claudin functions could be dependent on their specific partners for barrier formation. Furthermore, a model of claudin clusters as structural and functional units within tight junction strands is provided.
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Affiliation(s)
- Alexander G Markov
- Department of General Physiology, St. Petersburg State University, St. Petersburg, Russia
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Abstract
BACKGROUND Tight junction proteins (TJPs) and dendritic cells (DC) are critical in the pathogenesis of inflammatory bowel diseases. The ileal pouch formed by restorative proctocolectomy provides a unique human model for studying the pathogenesis of inflammatory bowel diseases. Data implicate the microbiota in the pathogenesis of pouchitis, while the role of innate immune factors remains unclear. We performed longitudinal and cross-sectional studies of patients after restorative proctocolectomy and assessed TJP and DC characteristics in the ileal pouch. METHODS Mucosal biopsies were taken from the ileal pouch of patients with ulcerative colitis (UC) and familial adenomatous polyposis (n = 8). Of patients with UC, one group (n = 5) was followed longitudinally over the first year after ileostomy closure, another group had pouchitis (n = 15), and another group no inflammation (n = 18). Dendritic cell phenotype and epithelial cell TJP expression were assessed using flow cytometric analysis. RESULTS Increased epithelial expression of the "pore-forming" TJP claudin 2, and DC expression of gut-homing markers CCR 9 and integrin β7, occurred early after ileostomy closure. In patients with UC with pouchitis, epithelial expression of ZO-1 and claudin 1 were reduced, DC were activated with increased CD40, and Toll-like receptor 4 expression increased. In pouchitis, DC expressing CCR 9 were decreased, whereas DC expressing β7 increased. CONCLUSIONS Abnormalities were found in TJP expression in the pouch of patients with UC, in particular, increased expression of the pore-forming claudin 2 as an early event in the development of pouch inflammation and an aberrant DC phenotype was characterized in the ileal pouch of patients with UC.
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Markov AG, Amasheh S. Tight junction physiology of pleural mesothelium. Front Physiol 2014; 5:221. [PMID: 25009499 PMCID: PMC4067758 DOI: 10.3389/fphys.2014.00221] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Accepted: 05/27/2014] [Indexed: 01/12/2023] Open
Abstract
Pleura consists of visceral and parietal cell layers, producing a fluid, which is necessary for lubrication of the pleural space. Function of both mesothelial cell layers is necessary for the regulation of a constant pleural fluid volume and composition to facilitate lung movement during breathing. Recent studies have demonstrated that pleural mesothelial cells show a distinct expression pattern of tight junction proteins which are known to ubiquitously determine paracellular permeability. Most tight junction proteins provide a sealing function to epithelia, but some have been shown to have a paracellular channel function or ambiguous properties. Here we provide an in-depth review of the current knowledge concerning specific functional contribution of these proteins determining transport and barrier function of pleural mesothelium.
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Affiliation(s)
- Alexander G Markov
- Department of General Physiology, St. Petersburg State University St. Petersburg, Russia
| | - Salah Amasheh
- Department of Veterinary Medicine, Institute of Veterinary Physiology, Freie Universität Berlin Berlin, Germany
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Markov AG, Falchuk EL, Kruglova NM, Rybalchenko OV, Fromm M, Amasheh S. Comparative analysis of theophylline and cholera toxin in rat colon reveals an induction of sealing tight junction proteins. Pflugers Arch 2014; 466:2059-65. [DOI: 10.1007/s00424-014-1460-z] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2013] [Accepted: 01/22/2014] [Indexed: 10/25/2022]
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Abstract
Epithelial transport relies on the proper function and regulation of the tight junction (TJ), other-wise uncontrolled paracellular leakage of solutes and water would occur. They also act as a fence against mixing of membrane proteins of the apical and basolateral side. The proteins determining paracellular transport consist of four transmembrane regions, intracellular N and C terminals, one intracellular and two extracellular loops (ECLs). The ECLs interact laterally and with counterparts of the neighboring cell and by this achieve a general sealing function. Two TJ protein families can be distinguished, claudins, comprising 27 members in mammals, and TJ-associated MARVEL proteins (TAMP), comprising occludin, tricellulin, and MarvelD3. They are linked to a multitude of TJ-associated regulatory and scaffolding proteins. The major TJ proteins are classified according to the physiological role they play in enabling or preventing paracellular transport. Many TJ proteins have sealing functions (claudins 1, 3, 5, 11, 14, 19, and tricellulin). In contrast, a significant number of claudins form channels across TJs which feature selectivity for cations (claudins 2, 10b, and 15), anions (claudin-10a and -17), or are permeable to water (claudin-2). For several TJ proteins, function is yet unclear as their effects on epithelial barriers are inconsistent (claudins 4, 7, 8, 16, and occludin). TJs undergo physiological and pathophysiological regulation by altering protein composition or abundance. Major pathophysiological conditions which involve changes in TJ protein composition are (1) effects of pathogens binding to TJ proteins, (2) altered TJ protein composition during inflammation and infection, and (3) altered TJ protein expression in cancers.
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Affiliation(s)
- Dorothee Günzel
- Institute of Clinical Physiology, Charité, Universtätsmedizin Berlin, Campus Benjamin Franklin, Freie Universität/Humboldt-Universität, Berlin, Germany
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Wasserman M, Hyman N, Iyer A, Wilcox R, Osler T. The natural history of anal transition zone inflammation and possible relationship to pouchitis: a long-term longitudinal study. Colorectal Dis 2013; 15:1493-8. [PMID: 23777389 DOI: 10.1111/codi.12322] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2013] [Accepted: 03/21/2013] [Indexed: 12/21/2022]
Abstract
AIM Reservoir ileitis (pouchitis) is the most common complication after pelvic pouch surgery for ulcerative colitis and the aetiology remains largely unknown. The anal transition zone (ATZ) contains the only remaining colonic epithelium after ileal pouch anal anastomosis (IPAA) and may provide important clues as to whether ulcerative colitis and pouchitis share a common pathogenesis. The aim of this study was to evaluate longitudinally the long-term histological changes in the ATZ and their relationship to the incidence of pouchitis. METHOD Patients with a double-stapled IPAA for ulcerative colitis at an academic medical centre with at least 10 years of clinical and histological follow-up were identified from a prospective database. Annual ATZ and pouch biopsies were taken and interpreted by two expert gastrointestinal pathologists. ATZ histological variability score, the incidence of pouchitis, and function were correlated over time. ATZ biopsies were scored from one to three based on the extent of inflammation. RESULTS Sixteen of the 114 patients having IPAA fulfilled the criteria for admission to the study. There were 179 biopsies of the ATZ. All exhibited variability in ATZ histology over time and 81% had a 2-unit change in their inflammatory score. There was no correlation between pouchitis and histological severity score of the ATZ. Similarly, function over time did not vary with the intensity of ATZ inflammation. CONCLUSION ATZ inflammation varies substantially over time in most patients. But these changes from year to year did not correlate with function or the occurrence of pouchitis.
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Affiliation(s)
- M Wasserman
- Department of Surgery, University of Vermont College of Medicine, Burlington, VT, USA
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Xue LY, Ouyang Q, Zhou XG, Huang ZH, Chen W, Chen M, Yu LM. Bacterial immune interaction in experimental colitis. J Dig Dis 2013; 14:526-35. [PMID: 23734583 DOI: 10.1111/1751-2980.12079] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
OBJECTIVES This study aimed to analyze the effects of 5-aminosalicylic acid (5-ASA) on intestinal microbiota and immune regulation in inflammatory bowel disease (IBD) and to investigate the correlation between intestinal microbiota and immune factors. METHODS Colitis in mice was induced by oxazolone. The community composition of luminal and mucosal microbiota was analyzed by a terminal restriction fragment length polymorphism. The expression of occludin, toll-like receptor (TLR)-2, TLR-4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 proteins were measured by immunohistochemistry and Western blot. Linear correlation between intestinal microbial community and the severity of the colitis or intestinal microbial community and expressions of immune factors were determined. RESULTS Protective bacteria decreased while aggressive bacteria increased in the colitis group. The richness and diversity of both luminal and mucosal microbiota decreased in the colitis group the decrease was enhanced in the 5-ASA-treated group. The diversity of mucosal microbiota significantly correlated with the extent of the colitis. Expressions of occludin, TLR-2, TLR-4, tumor necrosis factor-α and NF-κB p65 were significantly correlated with the diversity of mucosal microbiota. CONCLUSIONS Mucosal microbiota are important in the pathogenesis of IBD. 5-ASA increases protective bacteria but decreases aggressive bacteria, thus inducing the new intestinal microbial homeostasis.
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Affiliation(s)
- Lin Yun Xue
- Department of Gastroenterology, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China
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Lu Z, Ding L, Lu Q, Chen YH. Claudins in intestines: Distribution and functional significance in health and diseases. Tissue Barriers 2013; 1:e24978. [PMID: 24478939 PMCID: PMC3879173 DOI: 10.4161/tisb.24978] [Citation(s) in RCA: 189] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2013] [Revised: 05/06/2013] [Accepted: 05/08/2013] [Indexed: 02/08/2023] Open
Abstract
Intestines are organs that not only digest food and absorb nutrients, but also provide a defense barrier against pathogens and noxious agents ingested. Tight junctions (TJs) are the most apical component of the junctional complex, providing one form of cell-cell adhesion in enterocytes and playing a critical role in regulating paracellular barrier permeability. Alteration of TJs leads to a number of pathophysiological diseases causing malabsorption of nutrition and intestinal structure disruption, which may even contribute to systemic organ failure. Claudins are the major structural and functional components of TJs with at least 24 members in mammals. Claudins have distinct charge-selectivity, either by tightening the paracellular pathway or functioning as paracellular channels, regulating ions and small molecules passing through the paracellular pathway. In this review, we have discussed the functions of claudin family members, their distribution and localization in the intestinal tract of mammals, their alterations in intestine-related diseases and chemicals/agents that regulate the expression and localization of claudins as well as the intestinal permeability, which provide a therapeutic view for treating intestinal diseases.
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Affiliation(s)
- Zhe Lu
- Department of Basic Medicine; Hangzhou Normal University, Hangzhou, PR China ; Department of Anatomy and Cell Biology; Brody School of Medicine; East Carolina University; Greenville, NC USA
| | - Lei Ding
- Department of Anatomy and Cell Biology; Brody School of Medicine; East Carolina University; Greenville, NC USA ; Department of Oncology; Beijing Shijitan Hospital; Capital Medical University; Beijing, PR China
| | - Qun Lu
- Department of Anatomy and Cell Biology; Brody School of Medicine; East Carolina University; Greenville, NC USA
| | - Yan-Hua Chen
- Department of Anatomy and Cell Biology; Brody School of Medicine; East Carolina University; Greenville, NC USA
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Wang X, Valenzano MC, Mercado JM, Zurbach EP, Mullin JM. Zinc supplementation modifies tight junctions and alters barrier function of CACO-2 human intestinal epithelial layers. Dig Dis Sci 2013; 58:77-87. [PMID: 22903217 DOI: 10.1007/s10620-012-2328-8] [Citation(s) in RCA: 81] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/16/2011] [Accepted: 07/17/2012] [Indexed: 12/12/2022]
Abstract
BACKGROUND Zinc deficiency is known to result in epithelial barrier leak in the GI tract. Precise effects of zinc on epithelial tight junctions (TJs) are only beginning to be described and understood. Along with nutritional regimens like methionine-restriction and compounds such as berberine, quercetin, indole, glutamine and rapamycin, zinc has the potential to function as a TJ modifier and selective enhancer of epithelial barrier function. AIMS The purpose of this study was to determine the effects of zinc-supplementation on the TJs of a well-studied in vitro GI model, CACO-2 cells. METHODS Barrier function was assessed electrophysiologically by measuring transepithelial electrical resistance (Rt), and radiochemically, by measuring transepithelial (paracellular) diffusion of 14C-D-mannitol and 14C-polyethyleneglycol. TJ composition was studied by Western immunoblot analyses of occludin, tricellulin and claudins-1 to -5 and -7. RESULTS Fifty- and 100-μM zinc concentrations (control medium is 2 μM) significantly increase Rt but simultaneously increase paracellular leak to D-mannitol. Claudins 2 and 7 are downregulated in total cell lysates, while occludin, tricellulin and claudins-1, -3, -4 and -5 are unchanged. Claudins-2 and -7 as well as tricellulin exhibit decreased cytosolic content as a result of zinc supplementation. CONCLUSIONS Zinc alters CACO-2 TJ composition and modifies TJ barrier function selectively. Zinc is one of a growing number of "nutraceutical" substances capable of enhancing epithelial barrier function, and may find use in countering TJ leakiness induced in various disease states.
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Affiliation(s)
- Xuexuan Wang
- Lankenau Institute for Medical Research, 100 W Lancaster Ave, Wynnewood, PA 19096, USA
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Lameris AL, Huybers S, Kaukinen K, Mäkelä TH, Bindels RJ, Hoenderop JG, Nevalainen PI. Expression profiling of claudins in the human gastrointestinal tract in health and during inflammatory bowel disease. Scand J Gastroenterol 2013. [PMID: 23205909 DOI: 10.3109/00365521.2012.741616] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
BACKGROUND Claudins, being part of the tight junction protein family, partially determine the integrity and paracellular permeability of the intestinal epithelium. The aim of this study was twofold. First, the authors set out to create an overview of claudin mRNA expression along the proximal-distal axis of the healthy human intestine. Second, the authors aimed to analyze expression levels of claudins in patients with active and inactive inflammatory bowel diseases (IBD) such as Crohn's disease or ulcerative colitis (UC). METHODS mRNA expression levels of claudins were determined in gastrointestinal biopsies from healthy patients as well as patients diagnosed with IBD using SybrGreen real-time PCR. RESULTS Claudins show distinct expression patterns throughout the gastrointestinal tract. Some claudins show a proximal expression pattern, such as CLDN18 which is solely expressed in the stomach, and CLDN2 and -15 that are predominantly expressed in the proximal parts of the gastrointestinal tract. Other claudins, such as CLDN3, -4, -7 and -8, are predominantly expressed in the distal parts of the gastrointestinal tract or show a ubiquitous expression pattern throughout the entire gastrointestinal tract, which is the case for CLDN12. In addition, we show that changes in claudin expression in IBD are dependent on gastrointestinal location and inflammatory activity. CONCLUSIONS This study provides detailed mRNA expression patterns of various claudins throughout the human gastrointestinal tract. Analysis of expression levels of claudins in patients with CD, active and inactive UC shows that changes in expression are confined to specific intestinal segments and strongly depend on inflammatory activity.
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Affiliation(s)
- Anke L Lameris
- Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre , The Netherlands
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Schulzke JD, Günzel D, John LJ, Fromm M. Perspectives on tight junction research. Ann N Y Acad Sci 2012; 1257:1-19. [PMID: 22671584 DOI: 10.1111/j.1749-6632.2012.06485.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The tight junction connects neighboring epithelial or endothelial cells. As a general function, it seals the paracellular pathway and thus prevents back-leakage of just transported solutes and water. However, not all tight junctions are merely tight: some tight junction proteins build their own transport pathways by forming channels selective for small cations, anions, or water. Two families of tight junction proteins have been identified, claudins (27 members in mammals) and tight junction-associated MARVEL proteins ((TAMPs) occludin, tricellulin, and MarvelD3); an additional, structurally different, junction protein is junction adhesion molecule (JAM). Besides classification by genetic or molecular kinship, classification of tight junction proteins has been suggested according to permeability attributes. Recent studies describe specific cis and trans interactions and manifold physiologic regulations of claudins and TAMPs. In many inflammatory and infectious diseases they are found to be altered, for example, causing adversely increased permeability. Currently, attempts are being made to alter the paracellular barrier for therapeutic interventions or for transiently facilitating drug uptake. This overview concludes with a list of open questions and future topics in tight junction research.
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Affiliation(s)
- Jörg-Dieter Schulzke
- Department of Gastroenterology, Infectious Diseases, and Rheumatology, Division of Nutritional Medicine, Charité, Universitätsmedizin Berlin, Germany.
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Alhamoruni A, Wright KL, Larvin M, O'Sullivan SE. Cannabinoids mediate opposing effects on inflammation-induced intestinal permeability. Br J Pharmacol 2012; 165:2598-610. [PMID: 21745190 DOI: 10.1111/j.1476-5381.2011.01589.x] [Citation(s) in RCA: 105] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND AND PURPOSE Activation of cannabinoid receptors decreases emesis, inflammation, gastric acid secretion and intestinal motility. The ability to modulate intestinal permeability in inflammation may be important in therapy aimed at maintaining epithelial barrier integrity. The aim of the present study was to determine whether cannabinoids modulate the increased permeability associated with inflammation in vitro. EXPERIMENTAL APPROACH Confluent Caco-2 cell monolayers were treated for 24 h with IFNγ and TNFα (10 ng·mL(-1) ). Monolayer permeability was measured using transepithelial electrical resistance and flux measurements. Cannabinoids were applied either apically or basolaterally after inflammation was established. Potential mechanisms of action were investigated using antagonists for CB(1) , CB(2) , TRPV1, PPARγ and PPARα. A role for the endocannabinoid system was established using inhibitors of the synthesis and degradation of endocannabinoids. KEY RESULTS Δ(9) -Tetrahydrocannabinol (THC) and cannabidiol accelerated the recovery from cytokine-induced increased permeability; an effect sensitive to CB(1) receptor antagonism. Anandamide and 2-arachidonylglycerol further increased permeability in the presence of cytokines; this effect was also sensitive to CB(1) antagonism. No role for the CB(2) receptor was identified in these studies. Co-application of THC, cannabidiol or a CB(1) antagonist with the cytokines ameliorated their effect on permeability. Inhibiting the breakdown of endocannabinoids worsened, whereas inhibiting the synthesis of endocannabinoids attenuated, the increased permeability associated with inflammation. CONCLUSIONS AND IMPLICATIONS These findings suggest that locally produced endocannabinoids, acting via CB(1) receptors play a role in mediating changes in permeability with inflammation, and that phytocannabinoids have therapeutic potential for reversing the disordered intestinal permeability associated with inflammation. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
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Affiliation(s)
- A Alhamoruni
- School of Graduate Entry Medicine & Health, Derby City General Hospital, University of Nottingham, Derby, UK
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Rosenthal R, Heydt MS, Amasheh M, Stein C, Fromm M, Amasheh S. Analysis of absorption enhancers in epithelial cell models. Ann N Y Acad Sci 2012; 1258:86-92. [DOI: 10.1111/j.1749-6632.2012.06562.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
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Markov AG, Kruglova NM, Fomina YA, Fromm M, Amasheh S. Altered expression of tight junction proteins in mammary epithelium after discontinued suckling in mice. Pflugers Arch 2012; 463:391-8. [PMID: 21975594 DOI: 10.1007/s00424-011-1034-2] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2011] [Revised: 09/09/2011] [Accepted: 09/19/2011] [Indexed: 02/04/2023]
Abstract
Milk production is modulated by the paracellular barrier function of tight junction (TJ) proteins located in the mammary epithelium. The aim of our study was the molecular analysis of TJs in native lactating murine mammary gland epithelium as this process may strongly challenge epithelial barrier properties and regulation. Mammary gland tissue specimens from lactating control mice and animals after a 20-h interruption of suckling were prepared; histological analyses were performed by light and electron microscopy; and expression of TJ proteins was detected by PCR, Western blotting, immunofluorescent staining, and confocal laser scanning microscopy. Discontinuation of suckling resulted in a substantial accumulation of milk in mammary glands, an increase of alveolar size, and a flattening of epithelial cells without effects on inflammatory indicators. In control tissues, PCR and Western blots showed signals for occludin, and claudin-1, -2, -3, -4, -5, -7, -8, -15, and -16. After a 20-h accumulation of milk, expression of two sealing TJ proteins, claudin-1 and -3, was markedly increased, whereas two TJ proteins involved in cation transport, claudin-2 and -16, were reduced. Real-time PCR validated increased transcripts of claudin-1 and claudin-3. During extension of mammary glands in the process of lactation, claudin-1 and -3 are markedly induced and claudin-2 and -16 are decreased. Volume and composition of milk might be strongly dependent on this counter-regulation of sealing claudins with permeability-mediating claudins, indicating a physiological process of a tightening of TJs against a back-leak of solutes and ions from the alveolar lumen.
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Affiliation(s)
- Alexander G Markov
- Biological and Soil Faculty, St. Petersburg University, Universitetskaya nab. 7/9, 199034 St. Petersburg, Russia
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Landy J, Al-Hassi HO, McLaughlin SD, Knight SC, Ciclitira PJ, Nicholls RJ, Clark SK, Hart AL. Etiology of pouchitis. Inflamm Bowel Dis 2012; 18:1146-55. [PMID: 22021180 DOI: 10.1002/ibd.21911] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/18/2011] [Accepted: 09/06/2011] [Indexed: 12/16/2022]
Abstract
Restorative proctocolectomy with ileal-pouch anal anastomosis (RPC) is the operation of choice for ulcerative colitis (UC) patients requiring surgery. It is also used for patients with familial adenomatous polyposis (FAP). Pouchitis accounts for 10% of pouch failures. It is an idiopathic inflammatory condition that may occur in up to 50% of patients after RPC for UC. It is rarely seen in FAP patients after RPC. The etiology of pouchitis remains unclear. An overlap with UC is suggested by the frequency with which pouchitis affects patients with UC compared with FAP patients. There is significant clinical evidence implicating bacteria in the pathogenesis of pouchitis. Studies using culture and molecular methods demonstrate a dysbiosis of the pouch microbiota in pouchitis. Risk factors, genetic associations, and serological markers of pouchitis suggest that the interactions between the host immune responses and the pouch microbiota underlie the etiology of this idiopathic inflammatory condition. Here we present a detailed review of the data focusing on the pouch microbiota and the immune responses that support this hypothesis. We also discuss the contribution of luminal metabolic factors and the epithelial membrane in the etiology of this inflammatory process. The ileoanal pouch offers a unique opportunity to study the inter-relationships between the gut microbiota and host immune responses from before the onset of disease. For this reason the study of pouchitis could serve as a human model that significantly enhances our understanding of inflammatory bowel diseases in general.
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Affiliation(s)
- J Landy
- Department of Gastroenterology St Mark's Hospital, Harrow, London, UK
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Amasheh S, Fromm M, Günzel D. Claudins of intestine and nephron - a correlation of molecular tight junction structure and barrier function. Acta Physiol (Oxf) 2011; 201:133-40. [PMID: 20518752 DOI: 10.1111/j.1748-1716.2010.02148.x] [Citation(s) in RCA: 110] [Impact Index Per Article: 7.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
A prerequisite of epithelial transport is a paracellular barrier function, which seals the tissue against an uncontrolled leak flux. Moreover, selective paracellular permeability has been shown to be crucial for physiological epithelial transport function. Claudins are tetraspan tight junction proteins which play a major role in paracellular ion permeability across epithelia. The multigene family consists of 24 members and several splice variants which show distinct tissue-specific expression profiles. Moreover, in diseases associated with a loss of barrier function such as forms of inflammatory bowel disease, the expression of claudins is altered. Functional characterization of single claudins revealed specific contribution to barrier properties in epithelia. This review gives an overview on the exploration of molecular structure and barrier function along the intestine and nephron, which not only share mechanisms of selective restriction of the paracellular pathway but also exhibit distinct organ-specific characteristics.
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Affiliation(s)
- S Amasheh
- Institute of Clinical Physiology, Charité, Berlin, Germany.
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Markov AG, Voronkova MA, Volgin GN, Yablonsky PK, Fromm M, Amasheh S. Tight junction proteins contribute to barrier properties in human pleura. Respir Physiol Neurobiol 2010; 175:331-5. [PMID: 21187167 DOI: 10.1016/j.resp.2010.12.012] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2010] [Revised: 12/20/2010] [Accepted: 12/20/2010] [Indexed: 10/18/2022]
Abstract
The permeability of pleural mesothelium helps to control the volume and composition of the liquid lubricating pleural surfaces. Information on pleural barrier function in health and disease, however, is scarce. Tissue specimens of human pleura were mounted in Ussing chambers for measurement of transmesothelial resistance. Expression of tight junction (TJ) proteins was studied by Western blots and immune fluorescence confocal microscopy. Both visceral and parietal pleura showed barrier properties represented by transmesothelial resistance. Occludin, claudin-1, -3, -5, and -7, were detected in visceral pleura. In parietal pleura, the same TJ proteins were detected, except claudin-7. In tissues from patients with pleural inflammation these tightening claudins were decreased and in visceral pleura claudin-2, a paracellular channel former, became apparent. We report that barrier function in human pleura coincides with expression of claudins known to be key determinants of epithelial barrier properties. In inflamed tissue, claudin expression indicates a reduced barrier function.
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Affiliation(s)
- Alexander G Markov
- Biological and Soil Faculty, St. Petersburg State University, St. Petersburg, Russia
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Segmental expression of claudin proteins correlates with tight junction barrier properties in rat intestine. J Comp Physiol B 2010; 180:591-8. [DOI: 10.1007/s00360-009-0440-7] [Citation(s) in RCA: 100] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2009] [Revised: 12/06/2009] [Accepted: 12/14/2009] [Indexed: 10/20/2022]
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Bücker R, Schumann M, Amasheh S, Schulzke JD. Claudins in Intestinal Function and Disease. CURRENT TOPICS IN MEMBRANES 2010. [DOI: 10.1016/s1063-5823(10)65009-0] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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