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Pham TT, Kim JY, Tuomivaara ST, Lee YI, Kim S, Wells L, Lim JM. Triplex glycan quantification by metabolic labeling with isotopically labeled glucose in yeast. Anal Chim Acta 2024; 1288:342114. [PMID: 38220268 DOI: 10.1016/j.aca.2023.342114] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2023] [Accepted: 12/04/2023] [Indexed: 01/16/2024]
Abstract
Mass spectrometry-based approaches encompass a powerful collection of tools for the analysis biological molecules, including glycans and glycoconjugates. Unlike most traditional bioanalytical methods focusing on these molecules, mass spectrometry is especially suited for multiplexing, by utilizing stable-isotope labeling. Indeed, stable isotope-based multiplexing can be regarded as the gold-standard approach in reducing noise and uncertainty in quantitative mass spectrometry and quantitative analyses generally. The increasing sophistication and depth of biological questions being asked continue to challenge the practitioners of mass spectrometry method development. To understand the biological relevance of glycans, many stable isotope labeling-based mass spectrometry methods have been developed. Based on the duplex MILPIG (metabolic isotope labeling of polysaccharides with isotopic glucose), we establish here a novel triplex isotope labeling method using baker's yeast as the model system. Two differentially isotope-labeled glucoses (medium: 1-13C1 and heavy: 1,2-13C2), in addition to natural abundance glucose (light), were successfully used to label each monosaccharide ring in N-linked glycans in three different cell culture conditions, that, after sample mixing, resulted in a predictable triplet spectrum amenable for relative quantitation. We demonstrate excellent accuracy and precision of relative quantitation for a 1:1:1 mixture of glycans labeled in such a fashion. In addition, we applied triplex MILPIG to interrogate differential N-glycan profiles in tunicamycin-treated and control yeast cells and show that different N-glycans respond differently to tunicamycin.
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Affiliation(s)
- Thao Thi Pham
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea
| | - Ji-Yeon Kim
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea
| | - Sami T Tuomivaara
- Department of Obstetrics, Gynecology & Reproductive Sciences, University of California San Francisco, San Francisco, CA, 94143, USA
| | - Yong-Ill Lee
- Department of Pharmaceutical Sciences, Pharmaceutical Technical University, Tashkent 100084, The Republic of Uzbekistan
| | - Seonghun Kim
- Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea
| | - Lance Wells
- Complex Carbohydrate Research Center, Department of Biochemistry and Molecular Biology, and Department of Chemistry, University of Georgia, Athens, Georgia, 30602, USA
| | - Jae-Min Lim
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea.
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2
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Ravindranath MH, Ravindranath NM, Selvan SR, Filippone EJ, Amato-Menker CJ, El Hilali F. Four Faces of Cell-Surface HLA Class-I: Their Antigenic and Immunogenic Divergence Generating Novel Targets for Vaccines. Vaccines (Basel) 2022; 10:vaccines10020339. [PMID: 35214796 PMCID: PMC8878457 DOI: 10.3390/vaccines10020339] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 02/07/2022] [Accepted: 02/17/2022] [Indexed: 12/19/2022] Open
Abstract
Leukocyte cell-surface HLA-I molecules, involved in antigen presentation of peptides to CD8+ T-cells, consist of a heavy chain (HC) non-covalently linked to β2-microglobulin (β2m) (Face-1). The HC amino acid composition varies across all six isoforms of HLA-I, while that of β2m remains the same. Each HLA-allele differs in one or more amino acid sequences on the HC α1 and α2 helices, while several sequences among the three helices are conserved. HCs without β2m (Face-2) are also observed on human cells activated by malignancy, viral transformation, and cytokine or chemokine-mediated inflammation. In the absence of β2m, the monomeric Face-2 exposes immunogenic cryptic sequences on these cells as confirmed by HLA-I monoclonal antibodies (LA45, L31, TFL-006, and TFL-007). Furthermore, such exposure enables dimerization between two Face-2 molecules by SH-linkage, salt linkage, H-bonding, and van der Waal forces. In HLA-B27, the linkage between two heavy chains with cysteines at position of 67 of the amino acid residues was documented. Similarly, several alleles of HLA-A, B, C, E, F and G express cysteine at 67, 101, and 164, and additionally, HLA-G expresses cysteine at position 42. Thus, the monomeric HC (Face-2) can dimerize with another HC of its own allele, as homodimers (Face-3), or with a different HC-allele, as heterodimers (Face-4). The presence of Face-4 is well documented in HLA-F. The post-translational HLA-variants devoid of β2m may expose several cryptic linear and non-linear conformationally altered sequences to generate novel epitopes. The objective of this review, while unequivocally confirming the post-translational variants of HLA-I, is to highlight the scientific and clinical importance of the four faces of HLA and to prompt further research to elucidate their functions and their interaction with non-HLA molecules during inflammation, infection, malignancy and transplantation. Indeed, these HLA faces may constitute novel targets for passive and active specific immunotherapy and vaccines.
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Affiliation(s)
- Mepur H. Ravindranath
- Department of Hematology and Oncology, Children’s Hospital, Los Angeles, CA 90027, USA
- Emeritus Research Scientist at Terasaki Foundation Laboratory, Santa Monica, CA 90064, USA
- Correspondence:
| | - Narendranath M. Ravindranath
- Norris Dental Science Center, Herman Ostrow School of Dentistry, University of Southern California, Los Angeles, CA 90089, USA;
| | | | - Edward J. Filippone
- Division of Nephrology, Department of Medicine, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA 19145, USA;
| | - Carly J. Amato-Menker
- Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, WV 26506, USA;
| | - Fatiha El Hilali
- The Faculty of Medicine and Pharmacy of Laayoune, Ibn Zohr University, Agadir 70000, Morocco;
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Brazil JC, Parkos CA. Finding the sweet spot: glycosylation mediated regulation of intestinal inflammation. Mucosal Immunol 2022; 15:211-222. [PMID: 34782709 PMCID: PMC8591159 DOI: 10.1038/s41385-021-00466-8] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Revised: 10/11/2021] [Accepted: 10/14/2021] [Indexed: 02/04/2023]
Abstract
Glycans are essential cellular components that facilitate a range of critical functions important for tissue development and mucosal homeostasis. Furthermore, specific alterations in glycosylation represent important diagnostic hallmarks of cancer that contribute to tumor cell dissociation, invasion, and metastasis. However, much less is known about how glycosylation contributes to the pathobiology of inflammatory mucosal diseases. Here we will review how epithelial and immune cell glycosylation regulates gut homeostasis and how inflammation-driven changes in glycosylation contribute to intestinal pathobiology.
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Affiliation(s)
- Jennifer C. Brazil
- grid.214458.e0000000086837370Department of Pathology, University of Michigan, Ann Arbor, MI 48109 USA
| | - Charles A. Parkos
- grid.214458.e0000000086837370Department of Pathology, University of Michigan, Ann Arbor, MI 48109 USA
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4
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Kim JY, Joo WH, Shin DS, Lee YI, Teo CF, Lim JM. Metabolic labeling of glycans with isotopic glucose for quantitative glycomics in yeast. Anal Biochem 2021; 621:114152. [PMID: 33726981 DOI: 10.1016/j.ab.2021.114152] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2020] [Revised: 02/23/2021] [Accepted: 03/02/2021] [Indexed: 12/29/2022]
Abstract
Changes in glycan levels could directly affect the biochemical properties of glycoproteins and thus influence their physiological functions. In order to decode the correlation of glycan prevalence with their physiological contribution, many mass spectrometry (MS) and stable isotope labeling-based methods have been developed for the relative quantification of glycans. In this study, we expand the quantitative glycomic toolbox with the addition of optimized Metabolic Isotope Labeling of Polysaccharides with Isotopic Glucose (MILPIG) approach in baker's yeast (Saccharomyces cerevisiae). We demonstrate that culturing baker's yeast in the presence of carbon-13 labeled glucose (1-13C1) leads to effective incorporation of carbon-13 to both N-linked and O-linked glycans. We established that metabolic incorporation of isotope-labeled glucose at a concentration of 5 mg/mL for three days is required for an accurate quantitative analysis with optimal isotopic cluster distribution of glycans. To validate the robustness of the method, we performed the analysis by 1:1 mixing of normal and isotope-labeled glycans, and obtained excellent linear calibration curves from various analytes. Finally, we quantitated the inhibitory effect of tunicamycin, a N-linked glycosylation inhibitor, to glycan expression profile in yeast.
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Affiliation(s)
- Ji-Yeon Kim
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea
| | - Woo Hong Joo
- Department of Biology, Changwon National University, Changwon, 51140, Republic of Korea
| | - Dong-Soo Shin
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea
| | - Yong-Ill Lee
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea
| | - Chin Fen Teo
- Departments of Physiology, Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, 94158, USA
| | - Jae-Min Lim
- Department of Chemistry, Changwon National University, Changwon, 51140, Republic of Korea.
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Jokkel Z, Piroska M, Szalontai L, Hernyes A, Tarnoki DL, Tarnoki AD. Twin and family studies on epigenetics of autoimmune diseases. TWIN AND FAMILY STUDIES OF EPIGENETICS 2021:169-191. [DOI: 10.1016/b978-0-12-820951-6.00009-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2025]
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Jeong S, Lim J. Isotope Labeling of
N
‐linked Glycans by Transglycosylation of
Endo‐M
and Isotopic Glucose for Quantitative Glycomics. B KOREAN CHEM SOC 2020. [DOI: 10.1002/bkcs.12120] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Affiliation(s)
- Su‐Hee Jeong
- Department of Chemistry Changwon National University Gyeongnam 51140 South Korea
| | - Jae‐Min Lim
- Department of Chemistry Changwon National University Gyeongnam 51140 South Korea
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Kudelka MR, Stowell SR, Cummings RD, Neish AS. Intestinal epithelial glycosylation in homeostasis and gut microbiota interactions in IBD. Nat Rev Gastroenterol Hepatol 2020; 17:597-617. [PMID: 32710014 PMCID: PMC8211394 DOI: 10.1038/s41575-020-0331-7] [Citation(s) in RCA: 185] [Impact Index Per Article: 37.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 06/05/2020] [Indexed: 02/08/2023]
Abstract
Inflammatory bowel disease (IBD) affects 6.8 million people globally. A variety of factors have been implicated in IBD pathogenesis, including host genetics, immune dysregulation and gut microbiota alterations. Emerging evidence implicates intestinal epithelial glycosylation as an underappreciated process that interfaces with these three factors. IBD is associated with increased expression of truncated O-glycans as well as altered expression of terminal glycan structures. IBD genes, glycosyltransferase mislocalization, altered glycosyltransferase and glycosidase expression and dysbiosis drive changes in the glycome. These glycan changes disrupt the mucus layer, glycan-lectin interactions, host-microorganism interactions and mucosal immunity, and ultimately contribute to IBD pathogenesis. Epithelial glycans are especially critical in regulating the gut microbiota through providing bacterial ligands and nutrients and ultimately determining the spatial organization of the gut microbiota. In this Review, we discuss the regulation of intestinal epithelial glycosylation, altered epithelial glycosylation in IBD and mechanisms for how these alterations contribute to disease pathobiology. We hope that this Review provides a foundation for future studies on IBD glycosylation and the emergence of glycan-inspired therapies for IBD.
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Affiliation(s)
- Matthew R Kudelka
- Medical Scientist Training Program, Emory University School of Medicine, Atlanta, GA, USA
- Department of Internal Medicine, Weill Cornell Medicine, New York, NY, USA
| | - Sean R Stowell
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
| | - Richard D Cummings
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Andrew S Neish
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA.
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Groux-Degroote S, Cavdarli S, Uchimura K, Allain F, Delannoy P. Glycosylation changes in inflammatory diseases. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2019; 119:111-156. [PMID: 31997767 DOI: 10.1016/bs.apcsb.2019.08.008] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in a number of inflammatory diseases. Pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of glycan chains, inducing the expression of specific carbohydrate antigens at the cell surface that can be recognized by different types of lectins or by bacterial adhesins, contributing to the development of diseases. Glycosylation can also regulate biological functions of immune cells by recruiting leukocytes to inflammation sites with pro- or anti-inflammatory effects. Cell surface proteoglycans provide a large panel of binding sites for many mediators of inflammation, and regulate their bio-availability and functions. In this review, we summarize the current knowledge of the glycosylation changes occurring in mucin type O-linked glycans, glycosaminoglycans, as well as in glycosphingolipids, with a particular focus on cystic fibrosis and neurodegenerative diseases, and their consequences on cell interactions and disease progression.
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Affiliation(s)
- Sophie Groux-Degroote
- University Lille, CNRS, UMR 8576 - UGSF - Unite de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Sumeyye Cavdarli
- University Lille, CNRS, UMR 8576 - UGSF - Unite de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Kenji Uchimura
- University Lille, CNRS, UMR 8576 - UGSF - Unite de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Fabrice Allain
- University Lille, CNRS, UMR 8576 - UGSF - Unite de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
| | - Philippe Delannoy
- University Lille, CNRS, UMR 8576 - UGSF - Unite de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France
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Blakeley-Ruiz JA, Erickson AR, Cantarel BL, Xiong W, Adams R, Jansson JK, Fraser CM, Hettich RL. Metaproteomics reveals persistent and phylum-redundant metabolic functional stability in adult human gut microbiomes of Crohn's remission patients despite temporal variations in microbial taxa, genomes, and proteomes. MICROBIOME 2019; 7:18. [PMID: 30744677 PMCID: PMC6371617 DOI: 10.1186/s40168-019-0631-8] [Citation(s) in RCA: 41] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/21/2018] [Accepted: 01/17/2019] [Indexed: 05/11/2023]
Abstract
BACKGROUND The gut microbiome plays a fundamental role in the human host's overall health by contributing key biological functions such as expanded metabolism and pathogen defense/immune control. In a healthy individual, the gut microbiome co-exists within the human host in a symbiotic, non-inflammatory relationship that enables mutual benefits, such as microbial degradation of indigestible food products into small molecules that the host can utilize, and enhanced pathogen defense. In abnormal conditions, such as Crohn's disease, this favorable metabolic relationship breaks down and a variety of undesirable activities result, including chronic inflammation and other health-related issues. It has been difficult, however, to elucidate the overall functional characteristics of this relationship because the microbiota can vary substantially in composition for healthy humans and possibly even more in individuals with gut disease conditions such as Crohn's disease. Overall, this suggests that microbial membership composition may not be the best way to characterize a phenotype. Alternatively, it seems to be more informative to examine and characterize the functional composition of a gut microbiome. Towards that end, this study examines 25 metaproteomes measured in several Crohn's disease patients' post-resection surgery across the course of 1 year, in order to examine persistence of microbial taxa, genes, proteins, and metabolic functional distributions across time in individuals whose microbiome might be more variable due to the gut disease condition. RESULTS The measured metaproteomes were highly personalized, with all the temporally-related metaproteomes clustering most closely by individual. In general, the metaproteomes were remarkably distinct between individuals and to a lesser extent within individuals. This prompted a need to characterize the metaproteome at a higher functional level, which was achieved by annotating identified protein groups with KEGG orthologous groups to infer metabolic modules. At this level, similar and redundant metabolic functions across multiple phyla were observed across time and between individuals. Tracking through these various metabolic modules revealed a clear path from carbohydrate, lipid, and amino acid degradation to central metabolism and finally the production of fermentation products. CONCLUSIONS The human gut metaproteome can vary quite substantially across time and individuals. However, despite substantial intra-individual variation in the metaproteomes, there is a clear persistence of conserved metabolic functions across time and individuals. Additionally, the persistence of these core functions is redundant across multiple phyla but is not always observable in the same sample. Finally, the gut microbiome's metabolism is not driven by a set of discrete linear pathways but a web of interconnected reactions facilitated by a network of enzymes that connect multiple molecules across multiple pathways.
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Affiliation(s)
- J Alfredo Blakeley-Ruiz
- Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA
- Graduate School of Genome Science & Technology, University of Tennessee, Knoxville, TN, 37996, USA
| | - Alison R Erickson
- Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA
- Current address: Harvard Medical School, Cell Biology, 240 Longwood Avenue, Boston, MA, 02115, USA
| | - Brandi L Cantarel
- Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
- Current address: Lyda Hill Department of Bioinformatics, UT Southwestern Medical Center, Dallas, TX, 75390, USA
| | - Weili Xiong
- Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA
- Current address: U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, MD, 20740, USA
| | - Rachel Adams
- Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA
| | - Janet K Jansson
- Biological Sciences Division, Pacific Northwest National Lab, Richland, WA, 99352, USA
| | - Claire M Fraser
- Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
- Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, 21201, USA
| | - Robert L Hettich
- Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA.
- Graduate School of Genome Science & Technology, University of Tennessee, Knoxville, TN, 37996, USA.
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10
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Markovic BS, Milosavljevic N, Arsenijevic A, Gazdic M, Lukic ML, Volarevic V. Bacterial Flora Play Important Roles in Acute Dextran Sulphate Sodium-Induced Colitis But Are Not Involved in Gal-3 Dependent Modulation of Colon Inflammation. SERBIAN JOURNAL OF EXPERIMENTAL AND CLINICAL RESEARCH 2017. [DOI: 10.1515/sjecr-2017-0022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Abstract
An altered immune response to normal gut microflora is important for the pathogenesis of ulcerative colitis (UC). Galectin- 3 (Gal-3) is an endogenous lectin that plays an important pro-inflammatory role in the induction phase of acute colitis by promoting activation of the NLRP3 infl ammasome and production of IL-1β in macrophages. By using dextran sulphate sodium (DSS) induced colitis, a well-established animal model of UC, we determined whether Gal-3 affects the function of colon infiltrating macrophages by interfering with intestinal microfl ora. Our results showed that genetic deletion of Gal-3 significantly attenuates DSS-induced colitis by down-regulating infiltration of phagocytic cells (neutrophils, macrophages and dendritic cells) in colon tissue of DSS-treated mice, and this correlated with differences in bacterial flora of the gut. Antibiotic treatment attenuates DSS-induced colitis in WT and Gal-3-/- mice without affecting differences between the groups. In conclusion, Gram negative bacterial flora play an important role in DSS-induced acute colitis of mice but are not involved in Gal-3 dependent modulation of colon inflammation.
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Affiliation(s)
- Bojana Simovic Markovic
- Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac , Serbia
| | - Neda Milosavljevic
- Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac , Serbia
| | - Aleksandar Arsenijevic
- Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac , Serbia
| | - Marina Gazdic
- Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac , Serbia
| | - Miodrag L. Lukic
- Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac , Serbia
| | - Vladislav Volarevic
- Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Kragujevac , Serbia
- 69 Svetozara Markovica Street, 34000 Kragujevac , Serbia
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Sicard JF, Le Bihan G, Vogeleer P, Jacques M, Harel J. Interactions of Intestinal Bacteria with Components of the Intestinal Mucus. Front Cell Infect Microbiol 2017; 7:387. [PMID: 28929087 PMCID: PMC5591952 DOI: 10.3389/fcimb.2017.00387] [Citation(s) in RCA: 299] [Impact Index Per Article: 37.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2017] [Accepted: 08/18/2017] [Indexed: 12/19/2022] Open
Abstract
The human gut is colonized by a variety of large amounts of microbes that are collectively called intestinal microbiota. Most of these microbial residents will grow within the mucus layer that overlies the gut epithelium and will act as the first line of defense against both commensal and invading microbes. This mucus is essentially formed by mucins, a family of highly glycosylated protein that are secreted by specialize cells in the gut. In this Review, we examine how commensal members of the microbiota and pathogenic bacteria use mucus to their advantage to promote their growth, develop biofilms and colonize the intestine. We also discuss how mucus-derived components act as nutrient and chemical cues for adaptation and pathogenesis of bacteria and how bacteria can influence the composition of the mucus layer.
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Affiliation(s)
- Jean-Félix Sicard
- Centre de Recherche en Infectiologie Porcine et Aviaire, Faculté de Médecine Vétérinaire, Université de MontréalSaint-Hyacinthe, QC, Canada
| | - Guillaume Le Bihan
- Centre de Recherche en Infectiologie Porcine et Aviaire, Faculté de Médecine Vétérinaire, Université de MontréalSaint-Hyacinthe, QC, Canada
| | - Philippe Vogeleer
- Centre de Recherche en Infectiologie Porcine et Aviaire, Faculté de Médecine Vétérinaire, Université de MontréalSaint-Hyacinthe, QC, Canada
| | - Mario Jacques
- Regroupement de Recherche Pour un Lait de Qualité Optimale (Op+Lait), Faculté de Médecine Vétérinaire, Université de MontréalSaint-Hyacinthe, QC, Canada
| | - Josée Harel
- Centre de Recherche en Infectiologie Porcine et Aviaire, Faculté de Médecine Vétérinaire, Université de MontréalSaint-Hyacinthe, QC, Canada
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12
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Generali E, Ceribelli A, Stazi MA, Selmi C. Lessons learned from twins in autoimmune and chronic inflammatory diseases. J Autoimmun 2017; 83:51-61. [PMID: 28431796 DOI: 10.1016/j.jaut.2017.04.005] [Citation(s) in RCA: 78] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2017] [Accepted: 04/10/2017] [Indexed: 12/16/2022]
Abstract
Autoimmunity and chronic inflammation recognize numerous shared factors and, as a result, the resulting diseases frequently coexist in the same patients or respond to the same treatments. Among the convenient truths of autoimmune and chronic inflammatory diseases, there is now agreement that these are complex conditions in which the individual genetic predisposition provides a rate of heritability. The concordance rates in monozygotic and dizygotic twins allows to estimate the weight of the environment in determining disease susceptibility, despite recent data supporting that only a minority of immune markers depend on hereditary factors. Concordance rates in monozygotic and dizygotic twins should be evaluated over an observation period to minimize the risk of false negatives and this is well represented by type I diabetes mellitus. Further, concordance rates in monozygotic twins should be compared to those in dizygotic twins, which share 50% of their genes, as in regular siblings, but also young-age environmental factors. Twin studies have been extensively performed in several autoimmune conditions and cumulatively suggest that some diseases, i.e. celiac disease and psoriasis, are highly genetically determined, while rheumatoid arthritis or systemic sclerosis have a limited role for genetics. These observations are necessary to interpret data gathered by genome-wide association studies of polymorphisms and DNA methylation in MZ twins. New high-throughput technological platforms are awaited to provide new insights into the mechanisms of disease discordance in twins beyond strong associations such as those with HLA alleles.
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Affiliation(s)
- Elena Generali
- Division of Rheumatology and Clinical Immunology, Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Angela Ceribelli
- Division of Rheumatology and Clinical Immunology, Humanitas Research Hospital, Rozzano, Milan, Italy
| | - Maria Antonietta Stazi
- Italian Twin Registry, Centre for Behavioural Sciences and Mental Health, Istituto Superiore di Sanità, Rome, Italy
| | - Carlo Selmi
- Division of Rheumatology and Clinical Immunology, Humanitas Research Hospital, Rozzano, Milan, Italy; BIOMETRA Department, University of Milan, Milan, Italy.
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Role of Cytokine-Induced Glycosylation Changes in Regulating Cell Interactions and Cell Signaling in Inflammatory Diseases and Cancer. Cells 2016; 5:cells5040043. [PMID: 27916834 PMCID: PMC5187527 DOI: 10.3390/cells5040043] [Citation(s) in RCA: 63] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Revised: 11/23/2016] [Accepted: 11/24/2016] [Indexed: 12/17/2022] Open
Abstract
Glycosylation is one of the most important modifications of proteins and lipids, and cell surface glycoconjugates are thought to play important roles in a variety of biological functions including cell-cell and cell-substrate interactions, bacterial adhesion, cell immunogenicity and cell signaling. Alterations of glycosylation are observed in number of diseases such as cancer and chronic inflammation. In that context, pro-inflammatory cytokines have been shown to modulate cell surface glycosylation by regulating the expression of glycosyltransferases involved in the biosynthesis of carbohydrate chains. These changes in cell surface glycosylation are also known to regulate cell signaling and could contribute to disease pathogenesis. This review summarizes our current knowledge of the glycosylation changes induced by pro-inflammatory cytokines, with a particular focus on cancer and cystic fibrosis, and their consequences on cell interactions and signaling.
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14
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Gibold L, Garenaux E, Dalmasso G, Gallucci C, Cia D, Mottet-Auselo B, Faïs T, Darfeuille-Michaud A, Nguyen HTT, Barnich N, Bonnet R, Delmas J. The Vat-AIEC protease promotes crossing of the intestinal mucus layer by Crohn's disease-associated Escherichia coli. Cell Microbiol 2015; 18:617-31. [PMID: 26499863 DOI: 10.1111/cmi.12539] [Citation(s) in RCA: 65] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2015] [Revised: 10/07/2015] [Accepted: 10/20/2015] [Indexed: 12/21/2022]
Abstract
The aetiology of Crohn's disease (CD) involves disorders in host genetic factors and intestinal microbiota. Adherent-invasive Escherichia coli (AIEC) are receiving increased attention because in studies of mucosa-associated microbiota, they are more prevalent in CD patients than in healthy subjects. AIEC are associated both with ileal and colonic disease phenotypes. In this study, we reported a protease called Vat-AIEC from AIEC that favours the mucosa colonization. The deletion of the Vat-AIEC-encoding gene resulted in an adhesion-impaired phenotype in vitro and affected the colonization of bacteria in contact with intestinal epithelial cells in a murine intestinal loop model, and also their gut colonization in vivo. Furthermore, unlike LF82Δvat-AIEC, wild-type AIEC reference strain LF82 was able to penetrate a mucus column extensively and promoted the degradation of mucins and a decrease in mucus viscosity. Vat-AIEC transcription was stimulated by several chemical conditions found in the ileum environment. Finally, the screening of E. coli strains isolated from CD patients revealed a preferential vat-AIEC association with AIEC strains belonging to the B2 phylogroup. Overall, this study revealed a new component of AIEC virulence that might favour their implantation in the gut of CD patients.
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Affiliation(s)
- Lucie Gibold
- Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France.,Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Estelle Garenaux
- Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Guillaume Dalmasso
- Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Camille Gallucci
- Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France
| | - David Cia
- Equipe Biophysique Neurosensorielle, Faculté de Pharmacie, Université d'Auvergne, UMR INSERM 1107, Clermont-Ferrand, France
| | - Benoit Mottet-Auselo
- Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France.,Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Tiphanie Faïs
- Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France.,Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Arlette Darfeuille-Michaud
- Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Hang Thi Thu Nguyen
- Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Nicolas Barnich
- Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Richard Bonnet
- Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France.,Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
| | - Julien Delmas
- Laboratoire de Bactériologie, Centre Hospitalo-Universitaire Clermont-Ferrand, Clermont-Ferrand, France.,Microbes, Intestins, Inflammation et Susceptibilité de l'Hôte, Université d'Auvergne, INSERM U1071, INRA USC2018, Clermont-Ferrand, France
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15
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Abstract
Nanoscale systems are currently under investigation for multiple different diagnostic and therapeutic applications. These systems can be used to identify pathologically changed tissues or to selectively deliver drugs to these sites; both applications have an extremely high potential to ameliorate therapeutic outcomes for patients. Tissues as well as single cells can be targeted because of the small size of these systems, which enables enhanced diagnosis and increased specificity of therapy. Drug loads can be delivered directly to the site of action, which can result in a reduction in incidence and severity of adverse systemic effects. Several nano-based platform technologies are currently under investigation for use in therapeutic approaches, mainly for anti-inflammatory and anti-cancer therapies. Although many nanoscale systems show promising therapeutic outcomes in preclinical studies, only a limited number are ready for clinical use. This Review will discuss the diverse nanomaterials currently available and the first specific uses for select gastroenterological and hepatological pathologies. The discussion of diagnostic and therapeutic applications will consider realities of market introduction of these sometimes very complex systems in light of remaining regulatory challenges and hurdles for industrial production.
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16
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Aleandri M, Conte MP, Simonetti G, Panella S, Celestino I, Checconi P, Marazzato M, Longhi C, Goldoni P, Nicoletti M, Barnich N, Palamara AT, Schippa S, Nencioni L. Influenza A virus infection of intestinal epithelial cells enhances the adhesion ability of Crohn's disease associated Escherichia coli strains. PLoS One 2015; 10:e0117005. [PMID: 25706391 PMCID: PMC4338238 DOI: 10.1371/journal.pone.0117005] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2014] [Accepted: 12/17/2014] [Indexed: 12/19/2022] Open
Abstract
Modifications of intestinal glycoreceptors expression, in particular CEACAM6, typically found in ileal Crohn's disease (CD), favor, among the commensal species of microbiota, the enrichment in Escherichia coli. Removal of protein glycosidic residues by neuraminidase, a sialidase typical of influenza virus, increases adhesion ability of Escherichia coli to Caco-2 intestinal cells. In this study we investigated whether influenza virus infection of human intestinal epithelial cells could influence the adhesiveness of different Escherichia coli strains isolated from CD patients by altering surface glycoreceptors. Influenza virus infection of intestinal cells increased exposure of galactose and mannose residues on the cell surface. In particular, glycoreceptors Thomsen-Friedenreich and CEACAM6 were over-expressed in influenza virus infected cells. In the same experimental conditions, a significant increase in bacterial adhesiveness was observed, independently of their own adhesive ability. The increase was reverted by treatment with anti-TF and anti-CEACAM6 antibodies. Interestingly, influenza virus was able to efficiently replicate in human primary intestinal cells leading to TF exposure. Finally, intestinal infected cells produced high levels of pro-inflammatory cytokines compared to control. Overall these data suggest that influenza virus infection, could constitute an additional risk factor in CD patients.
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Affiliation(s)
- Marta Aleandri
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
| | - Maria Pia Conte
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
| | - Giovanna Simonetti
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
| | - Simona Panella
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
- IRCCS San Raffaele Pisana, Telematic University, Rome, Italy, Rome, Italy
| | - Ignacio Celestino
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
- CEINGE-Biotecnologie Avanzate, Naples, Italy
| | - Paola Checconi
- Department of Public Health and Infectious Diseases, Pasteur Institute Cenci Bolognetti Foundation, “Sapienza” University, Rome, Italy
| | | | - Catia Longhi
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
| | - Paola Goldoni
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
| | - Mauro Nicoletti
- Department of Experimental Sciences and Clinics “G. D’Annunzio” University, Chieti, Italy
| | - Nicolas Barnich
- Clermont University, M2iSH, UMR 1071 INSERM/University of Auvergne, Clermont-Ferrand, France
| | - Anna Teresa Palamara
- IRCCS San Raffaele Pisana, Telematic University, Rome, Italy, Rome, Italy
- Department of Public Health and Infectious Diseases, Pasteur Institute Cenci Bolognetti Foundation, “Sapienza” University, Rome, Italy
| | - Serena Schippa
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
| | - Lucia Nencioni
- Department of Public Health and Infectious Diseases, “Sapienza” University, Rome, Italy
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17
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Theodoratou E, Campbell H, Ventham NT, Kolarich D, Pučić-Baković M, Zoldoš V, Fernandes D, Pemberton IK, Rudan I, Kennedy NA, Wuhrer M, Nimmo E, Annese V, McGovern DPB, Satsangi J, Lauc G. The role of glycosylation in IBD. Nat Rev Gastroenterol Hepatol 2014; 11:588-600. [PMID: 24912389 DOI: 10.1038/nrgastro.2014.78] [Citation(s) in RCA: 121] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
A number of genetic and immunological studies give impetus for investigating the role of glycosylation in IBD. Experimental mouse models have helped to delineate the role of glycosylation in intestinal mucins and to explore the putative pathogenic role of glycosylation in colitis. These experiments have been extended to human studies investigating the glycosylation patterns of intestinal mucins as well as levels of glycans of serum glycoproteins and expression of glycan receptors. These early human studies have generated interesting hypotheses regarding the pathogenic role of glycans in IBD, but have generally been restricted to fairly small underpowered studies. Decreased glycosylation has been observed in the intestinal mucus of patients with IBD, suggesting that a defective inner mucus layer might lead to increased bacterial contact with the epithelium, potentially triggering inflammation. In sera, decreased galactosylation of IgG has been suggested as a diagnostic marker for IBD. Advances in glycoprofiling technology make it technically feasible and affordable to perform high-throughput glycan pattern analyses and to build on previous work investigating a much wider range of glycan parameters in large numbers of patients.
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Affiliation(s)
- Evropi Theodoratou
- Centre for Population Health Sciences, University of Edinburgh, Teviot Place, EH8 9AG, Edinburgh, UK
| | - Harry Campbell
- Centre for Population Health Sciences, University of Edinburgh, Teviot Place, EH8 9AG, Edinburgh, UK
| | - Nicholas T Ventham
- Centre for Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Edinburgh, UK
| | - Daniel Kolarich
- Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1 OT Golm, 14476, Potsdam, Germany
| | | | - Vlatka Zoldoš
- University of Zagreb, Faculty of Science, Horvatovac 102a, 10000 Zagreb, Croatia
| | | | - Iain K Pemberton
- IP Research Consulting SAS, 34 Rue Carnot, 93160 Noisy-le-Grand, Paris, France
| | - Igor Rudan
- Centre for Population Health Sciences, University of Edinburgh, Teviot Place, EH8 9AG, Edinburgh, UK
| | - Nicholas A Kennedy
- Centre for Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Edinburgh, UK
| | - Manfred Wuhrer
- Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, Netherlands
| | - Elaine Nimmo
- Centre for Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Edinburgh, UK
| | - Vito Annese
- Department of Medical and Surgical Specialities, Division of Gastroenterology, AOU Careggi University Hospital, Largo Brambilla 13, 50139 Florence, Italy
| | - Dermot P B McGovern
- F.Widjaja Family Foundation Inflammatory Bowel and Immunobiology Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Suite D4063, Los Angeles, CA 90048, USA
| | - Jack Satsangi
- Centre for Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Edinburgh, UK
| | - Gordan Lauc
- Department of Biochemistry and Molecular Biology, University of Zagreb Faculty of Pharmacy and Biochemistry, Trg maršala Tita 14, 10000 Zagreb, Croatia
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18
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Merga Y, Campbell BJ, Rhodes JM. Mucosal barrier, bacteria and inflammatory bowel disease: possibilities for therapy. Dig Dis 2014; 32:475-83. [PMID: 24969297 DOI: 10.1159/000358156] [Citation(s) in RCA: 134] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The mucosal barrier has three major components, the mucus layer, the epithelial glycocalyx and the surface epithelium itself, whose integrity largely depends on tight junction function. In health, there is relatively little direct interaction between the luminal microbiota and the epithelium - the continuous mucus layer in the colon keeps the surface epithelium out of contact with bacteria and the ileo-caecal valve ensures that the distal small intestine is relatively microbe free. Most interaction takes place at the Peyer's patches in the distal ileum and their smaller colonic equivalents, the lymphoid follicles. Peyer's patches are overlain by a 'dome' epithelium, 5% of whose cells are specialised M (microfold) epithelial cells, which act as the major portal of entry for bacteria. There are no goblet cells in the dome epithelium and M cells have a very sparse glycocalyx allowing easy microbial interaction. It is intriguing that the typical age range for the onset of Crohn's disease (CD) is similar to the age at which the number of Peyer's patches is greatest. Peyer's patches are commonly the sites of the initial lesions in CD and the 'anti-pancreatic' antibody associated with CD has been shown to have as its epitope the glycoprotein 2 that is the receptor for type-1 bacterial fimbrial protein (fimH) on M cells. There are many reasons to believe that the mucosal barrier is critically important in the pathogenesis of inflammatory bowel disease (IBD). These include (i) associations between both CD and ulcerative colitis (UC) with genes that are relevant to the mucosal barrier; (ii) increased intestinal permeability in unaffected relatives of CD patients; (iii) increased immune reactivity against bacterial antigens, and (iv) animal models in which altered mucosal barrier, e.g. denudation of the mucus layer associated with oral dextran sulphate in rodents, induces colitis. Whilst some IBD patients may have genetic factors leading to weakening of the mucosal barrier, it is likely that environmental factors may be even more important. Some may be subtle and indirect, e.g. the effects of stress on the mucosa barrier, whilst others may be more obvious, e.g. the effect of pathogen-related gastroenteritis, known often to act as trigger for IBD relapse. We have also been very interested in the potentially harmful effects of ingested detergents - either by contamination of cutlery by inadequate rinsing or via ingestion of processed foods containing permitted emulsifiers. In vitro and ex vivo studies show that even very small trace amounts of these surfactants can greatly increase bacterial translocation. Implications for therapy are not yet so obvious. We advise our IBD patients to avoid processed foods containing emulsifiers and to rinse their dishes well - whilst accepting that there is no direct evidence yet to support this. Therapies that aim to enhance the mucosal barrier have yet to come to market, but trials of enteric-delivered phosphatidylcholine in UC are promising. The faecal concentration of mucus-degrading bacterial enzymes (particularly proteases, sulphatases and sialidases) correlates with disease activity in UC, and these represent good targets for therapy.
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Affiliation(s)
- Yvette Merga
- Department of Gastroenterology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK
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19
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Jiang K, Rankin CR, Nava P, Sumagin R, Kamekura R, Stowell SR, Feng M, Parkos CA, Nusrat A. Galectin-3 regulates desmoglein-2 and intestinal epithelial intercellular adhesion. J Biol Chem 2014; 289:10510-10517. [PMID: 24567334 DOI: 10.1074/jbc.m113.538538] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The desmosomal cadherins, desmogleins, and desmocollins mediate strong intercellular adhesion. Human intestinal epithelial cells express the desmoglein-2 isoform. A proteomic screen for Dsg2-associated proteins in intestinal epithelial cells identified a lectin referred to as galectin-3 (Gal3). Gal3 bound to N-linked β-galactosides in Dsg2 extracellular domain and co-sedimented with caveolin-1 in lipid rafts. Down-regulation of Gal3 protein or incubation with lactose, a galactose-containing disaccharide that competitively inhibits galectin binding to Dsg2, decreased intercellular adhesion in intestinal epithelial cells. In the absence of functional Gal3, Dsg2 protein was internalized from the plasma membrane and degraded in the proteasome. These results report a novel role of Gal3 in stabilizing a desmosomal cadherin and intercellular adhesion in intestinal epithelial cells.
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Affiliation(s)
- Kun Jiang
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Carl R Rankin
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Porfirio Nava
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322; Department of Physiology, Biophysics and Neuroscience, CINVESTAV IPN., Av. IPN 2508, Col. San Pedro Zacatenco, 07360 Ciudad de México, Distrito Federal, México
| | - Ronen Sumagin
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Ryuta Kamekura
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Sean R Stowell
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Mingli Feng
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Charles A Parkos
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322
| | - Asma Nusrat
- Department of Pathology and Laboratory Medicine, Epithelial Pathobiology and Mucosal Inflammation Research Unit, Emory University, Atlanta, Georgia 30322.
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20
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Antoni L, Nuding S, Wehkamp J, Stange EF. Intestinal barrier in inflammatory bowel disease. World J Gastroenterol 2014; 20:1165-1179. [PMID: 24574793 PMCID: PMC3921501 DOI: 10.3748/wjg.v20.i5.1165] [Citation(s) in RCA: 293] [Impact Index Per Article: 26.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Revised: 11/08/2013] [Accepted: 12/13/2013] [Indexed: 02/06/2023] Open
Abstract
A complex mucosal barrier protects as the first line of defense the surface of the healthy intestinal tract from adhesion and invasion by luminal microorganisms. In this review, we provide an overview about the major components of this protective system as for example an intact epithelium, the synthesis of various antimicrobial peptides (AMPs) and the formation of the mucus layer. We highlight the crucial importance of their correct functioning for the maintenance of a proper intestinal function and the prevention of dysbiosis and disease. Barrier disturbances including a defective production of AMPs, alterations in thickness or composition of the intestinal mucus layer, alterations of pattern-recognition receptors, defects in the process of autophagy as well as unresolved endoplasmic reticulum stress result in an inadequate host protection and are thought to play a crucial role in the pathogenesis of the inflammatory bowel diseases Crohn’s disease and ulcerative colitis.
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21
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Wang C, Wu Z, Yuan J, Wang B, Zhang P, Zhang Y, Wang Z, Huang L. Simplified Quantitative Glycomics Using the Stable Isotope Label Girard’s Reagent P by Electrospray Ionization Mass Spectrometry. J Proteome Res 2013; 13:372-84. [DOI: 10.1021/pr4010647] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Affiliation(s)
- Chengjian Wang
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
| | - Zhiyu Wu
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
| | - Jiangbei Yuan
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
| | - Bo Wang
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
| | - Ping Zhang
- Chemistry
and Chemical Engineering School, Xianyang Normal University, Xianyang 712000, China
| | - Ying Zhang
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
| | - Zhongfu Wang
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
| | - Linjuan Huang
- Educational
Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, College of Life Science, Northwest University, Xi’an 710069, China
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22
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Caputo I, Secondo A, Lepretti M, Paolella G, Auricchio S, Barone MV, Esposito C. Gliadin peptides induce tissue transglutaminase activation and ER-stress through Ca2+ mobilization in Caco-2 cells. PLoS One 2012; 7:e45209. [PMID: 23049776 PMCID: PMC3458012 DOI: 10.1371/journal.pone.0045209] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2012] [Accepted: 08/16/2012] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Celiac disease (CD) is an intestinal inflammatory condition that develops in genetically susceptible individuals after exposure to dietary wheat gliadin. The role of post-translational modifications of gliadin catalyzed by tissue transglutaminase (tTG) seems to play a crucial role in CD. However, it remains to be established how and where tTG is activated in vivo. We have investigated whether gliadin peptides modulate intracellular Ca(2+) homeostasis and tTG activity. METHODS/PRINCIPAL FINDINGS We studied Ca(2+) homeostasis in Caco-2 cells by single cell microfluorimetry. Under our conditions, A-gliadin peptides 31-43 and 57-68 rapidly mobilized Ca(2+) from intracellular stores. Specifically, peptide 31-43 mobilized Ca(2+) from the endoplasmic reticulum (ER) and mitochondria, whereas peptide 57-68 mobilized Ca(2+) only from mitochondria. We also found that gliadin peptide-induced Ca(2+) mobilization activates the enzymatic function of intracellular tTG as revealed by in situ tTG activity using the tTG substrate pentylamine-biotin. Moreover, we demonstrate that peptide 31-43, but not peptide 57-68, induces an increase of tTG expression. Finally, we monitored the expression of glucose-regulated protein-78 and of CCAAT/enhancer binding protein-homologous protein, which are two biochemical markers of ER-stress, by real-time RT-PCR and western blot. We found that chronic administration of peptide 31-43, but not of peptide 57-68, induces the expression of both genes. CONCLUSIONS By inducing Ca(2+) mobilization from the ER, peptide 31-43 could promote an ER-stress pathway that may be relevant in CD pathogenesis. Furthermore, peptides 31-43 and 57-68, by activating intracellular tTG, could alter inflammatory key regulators, and induce deamidation of immunogenic peptides and gliadin-tTG crosslinking in enterocytes and specialized antigen-presenting cells.
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Affiliation(s)
- Ivana Caputo
- Department of Chemistry and Biology, University of Salerno, Fisciano, Salerno, Italy.
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23
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Larsson JMH, Karlsson H, Crespo JG, Johansson MEV, Eklund L, Sjövall H, Hansson GC. Altered O-glycosylation profile of MUC2 mucin occurs in active ulcerative colitis and is associated with increased inflammation. Inflamm Bowel Dis 2011; 17:2299-307. [PMID: 21290483 DOI: 10.1002/ibd.21625] [Citation(s) in RCA: 247] [Impact Index Per Article: 17.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/11/2010] [Accepted: 12/06/2010] [Indexed: 12/12/2022]
Abstract
BACKGROUND The MUC2 mucin organizes the two mucus layers in the colon. This mucin carries a large number of O-glycans that are assumed to be attachment sites for the commensal flora found in the outer mucus layer. METHODS Single biopsies from the sigmoid colon of controls (25) and patients with inactive (13) or active (15) ulcerative colitis (UC) were collected during routine colonoscopy. The insoluble MUC2 mucin was prepared and separated by gel electrophoresis, its relative amount estimated, its O-glycans released, and glycans analyzed by novel sensitive glycomics chromatography / mass spectrometry providing information on glycan structures and relative abundances. The glycosylation pattern was related to the degree of mucosal inflammation and clinical severity of the disease. RESULTS The relative abundance of MUC2 showed high individual variability. Two major glycan profiles were found; a normal pattern in the control and inactive UC patients and an aberrant profile in patients with active colitis with an increase in a subset of the smaller glycans and a decrease of several complex glycans. The magnitude of this phenomenon was significantly related to both the degree of inflammation in the biopsies and also to some extent the severity of disease course. The aberrant profile was further shown to be reversible upon remission. CONCLUSIONS In the majority of the active UC patients MUC2 mucin has an altered glycan profile as compared to inactive UC and control patients. Patients with strong alterations in the glycan pattern tended to have a more severe disease course.
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Affiliation(s)
- Jessica M Holmn Larsson
- Department of Medical Biochemistry, University of Gothenburg, Gothenburg, Sweden, Sahlgrens' University Hospital, Gothenburg, Sweden
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24
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Desantis S, Zizza S, Accogli G, Tufarelli V, Laudadio V. Morphometric features and glycoconjugate pattern of rabbit intestine are affected by particle size of pelleted diets. Anat Rec (Hoboken) 2011; 294:1875-89. [PMID: 21965045 DOI: 10.1002/ar.21477] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2011] [Revised: 07/20/2011] [Accepted: 07/26/2011] [Indexed: 11/09/2022]
Abstract
Feed particle size effects on morphology and glycoconjugate pattern was investigated in the rabbit intestine. Rabbits fed with fine particles (2 mm) displayed more irregularly shaped, higher duodenal villi and deeper crypts in distal colon as well as higher number of goblet cells than coarse (8 mm) fed ones. Brush border expressed: (i) in duodenum, neutral/sulfated glycoconjugates and glycans binding MAL II, SNA, Con A than KOH-sialidase-PNA and DBA reactivity in fine and coarse fed rabbits, respectively, (ii) in cecum, mainly sulfoglycans in coarse fed rabbits, MAL II and PNA staining in all samples, and (iii) in distal colon few sulfoglycans and MAL II reactivity. Enterocytes bound MAL II in duodenum, Con A in cecum, DBA, and Con A in distal colon of all rabbits, SNA in distal colon of coarse fed ones. Brunner's glands displayed high presence of acidic/sulfated mucins in fine fed rabbits, neutral glycoconjugates and reactivity with MAL II, SNA, PNA, KOH-sialidase-PNA, and Con A in all rabbits. Goblet cells exhibited: (i) in duodenum neutral and sulfomucins as well as MAL II and KOH-sialidase-PNA staining, than SNA and DBA in fine and coarse fed rabbits, respectively, (ii) in cecum sulfated glycans, MAL II, SNA, KOH-sialidase-PNA, DBA reactivity, and (iii) in distal colon acidic/sulfomucins, MAL II and SNA staining, and DBA reactivity in fine fed specimens. Crypt cells exhibited neutral and PNA reactive glycoconjugates in the cecum. In the distal colon also acidic/sulfated glycans, and MAL II, KOH-sialidase-PNA, DBA; SNA staining showed weaker reactivity in fine fed rabbits, which bound Con A.
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Lepage P, Häsler R, Spehlmann ME, Rehman A, Zvirbliene A, Begun A, Ott S, Kupcinskas L, Doré J, Raedler A, Schreiber S. Twin study indicates loss of interaction between microbiota and mucosa of patients with ulcerative colitis. Gastroenterology 2011; 141:227-36. [PMID: 21621540 DOI: 10.1053/j.gastro.2011.04.011] [Citation(s) in RCA: 450] [Impact Index Per Article: 32.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2010] [Revised: 04/02/2011] [Accepted: 04/08/2011] [Indexed: 12/11/2022]
Abstract
BACKGROUND & AIMS Interactions between genetic and environmental factors are believed to be involved in onset and initiation of inflammatory bowel disease. We analyzed the interaction between gastrointestinal mucosal microbiota and host genes in twin pairs discordant for ulcerative colitis (UC) to study the functional interaction between microbiota and mucosal epithelium. METHODS Biopsy were collected from sigmoid colon of UC patients and their healthy twins (discordant twin pairs) and from twins without UC. Microbiota profiles were determined from analysis of 16S ribosomal DNA libraries; messenger RNA profiles were determined by microarray analysis. RESULTS Patients with UC had dysbiotic microbiota, characterized by less bacterial diversity and more Actinobacteria and Proteobacteria than that of their healthy siblings; healthy siblings from discordant twins had more bacteria from the Lachnospiraceae and Ruminococcaceae families than twins who were both healthy. In twins who were both healthy, 34 mucosal transcripts correlated with bacterial genera, whereas only 25 and 11 correlated with bacteria genera in healthy individuals and their twins with UC, respectively. Transcripts related to oxidative and immune responses were differentially expressed between patients with UC and their healthy twins. CONCLUSIONS The transcriptional profile of the mucosa appears to interact with the colonic microbiota; this interaction appears to be lost in colon of patients with UC. Bacterial functions, such as butyrate production, might affect mucosal gene expression. Patients with UC had different gene expression profiles and lower levels of biodiversity than their healthy twins, as well as unusual aerobic bacteria. Patients with UC had lower percentages of potentially protective bacterial species than their healthy twins.
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Affiliation(s)
- Patricia Lepage
- Institute of Clinical Molecular Biology, Christian-Albrechts University-Kiel, Kiel, Germany
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Ryan SO, Bonomo JA, Zhao F, Cobb BA. MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation. ACTA ACUST UNITED AC 2011; 208:1041-53. [PMID: 21502329 PMCID: PMC3092352 DOI: 10.1084/jem.20100508] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
N-linked glycans on class II MHC molecules are required for the presentation of glycoantigens, but not peptide antigens. N-linked glycans are thought to protect class II major histocompatibility complex (MHC) molecules (MHCII) from proteolytic cleavage and assist in arranging proteins within the immune synapse, but were not thought to directly participate in antigen presentation. Here, we report that antigen-presenting cells (APCs) lacking native complex N-glycans showed reduced MHCII binding and presentation of the T cell activating glycoantigen (GlyAg) polysaccharide A from Bacteroides fragilis but not conventional peptides. APCs lacking native N-glycans also failed to mediate GlyAg-driven T cell activation but activated T cells normally with protein antigen. Mice treated with the mannosidase inhibitor kifunensine to prevent the formation of complex N-glycans were unable to expand GlyAg-specific T cells in vivo upon immunization, yet adoptive transfer of normally glycosylated APCs into these animals overcame this defect. Our findings reveal that MHCII N-glycosylation directly impacts binding and presentation of at least one class of T cell–dependent antigen.
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Affiliation(s)
- Sean O Ryan
- Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA
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Greer JB, O'Keefe SJ. Microbial induction of immunity, inflammation, and cancer. Front Physiol 2011; 1:168. [PMID: 21423403 PMCID: PMC3059938 DOI: 10.3389/fphys.2010.00168] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2010] [Accepted: 12/23/2010] [Indexed: 12/13/2022] Open
Abstract
The human microbiota presents a highly active metabolic that influences the state of health of our gastrointestinal tracts as well as our susceptibility to disease. Although much of our initial microbiota is adopted from our mothers, its final composition and diversity is determined by environmental factors. Westernization has significantly altered our microbial function. Extensive experimental and clinical evidence indicates that the westernized diet, rich in animal products and low in complex carbohydrates, plus the overuse of antibiotics and underuse of breastfeeding, leads to a heightened inflammatory potential of the microbiota. Chronic inflammation leads to the expression of certain diseases in genetically predisposed individuals. Antibiotics and a “clean” environment, termed the “hygiene hypothesis,” has been linked to the rise in allergy and inflammatory bowel disease, due to impaired beneficial bacterial exposure and education of the gut immune system, which comprises the largest immune organ within the body. The elevated risk of colon cancer is associated with the suppression of microbial fermentation and butyrate production, as butyrate provides fuel for the mucosa and is anti-inflammatory and anti-proliferative. This article will summarize the work to date highlighting the complicated and dynamic relationship between the gut microbiota and immunity, inflammation and carcinogenesis.
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Affiliation(s)
- Julia B Greer
- Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Pittsburgh School of Medicine Pittsburgh, PA, USA
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Friswell M, Campbell B, Rhodes J. The role of bacteria in the pathogenesis of inflammatory bowel disease. Gut Liver 2010; 4:295-306. [PMID: 20981205 DOI: 10.5009/gnl.2010.4.3.295] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/10/2010] [Accepted: 04/06/2010] [Indexed: 12/19/2022] Open
Abstract
Crohn's disease (CD) and ulcerative colitis (UC) have features that suggest bacterial involvement, and all genetic models of inflammatory bowel disease (IBD) require the presence of commensal bacteria. CD is associated with innate immune response genes such as NOD2/CARD15 and the autophagy genes ATG16L1 and IRGM. However, IBD responds to immunosuppression, suggesting that any bacteria involved are not acting as conventional pathogens. Molecular techniques are rapidly advancing our knowledge of the gut microbiota. In CD there is reduced diversity, and notably a reduction in the probiotic Faecalibacterium prausnitzii, the presence of which in the terminal ileum is associated with a reduced risk of recurrence following surgery. There is also a consistent increase in mucosa-associated Escherichia coli with an "adherent and invasive" phenotype, which allows them to replicate inside macrophages and induce granulomas. Speculation that CD could be caused by the Mycobacterium avium subspecies paratuberculosis (MAP) continues. The response to antitumor necrosis factor treatments suggests that, if relevant at all, MAP is not acting as a conventional pathogen. However, there is increased colonization by MAP in CD, and there is evidence that it could have an indirect effect mediated by the suppression of macrophage function. UC relapse is frequently associated with infection by pathogens, but there is less evidence for involvement of a specific bacterial species. Poor barrier integrity followed by an inflammatory reaction to bacterial components, with chronicity maintained by an autoimmune process, seems a plausible pathogenic model. Bacterial theories of pathogenesis are now becoming testable by targeted therapeutic interventions.
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Affiliation(s)
- Melissa Friswell
- Gastroenterology Research Unit, University of Liverpool School of Clinical Sciences, Liverpool, UK
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Smoking and inflammatory bowel diseases: what in smoking alters the course? Int J Colorectal Dis 2010; 25:671-80. [PMID: 20333390 DOI: 10.1007/s00384-010-0925-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 02/25/2010] [Indexed: 02/08/2023]
Abstract
Epidemiological studies provide strong evidence to confirm the correlation between cigarette smoking and inflammatory bowel diseases. This relationship is proved to be positive in Crohn's disease and negative in ulcerative colitis. What in smoking alters the course of inflammatory bowel diseases is still a mystery. Different smoking parts have different and may be opponent actions. Smoking has dual effects. Some of its activities are, sometimes, constructive as they are working in an antagonistic manner to the mechanism of the disease, such as reducing rectal blood flow and accordingly less recruitments of inflammatory mediators to the area of inflammation, enhancement of mucosal production, and consequently, strengthening the membranes, and inhibition of pro-inflammatory mediators' liberation and activity in subjects with ulcerative colitis. Yet the outcome of smoking actions may be affected by the existence of other cofactors. Odd factors, such as shortage of zinc in subjects with Crohn's disease, may facilitate liberation of pro-inflammatory mediators and their activities and accordingly exacerbates symptoms.
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McGuckin MA, Eri RD, Das I, Lourie R, Florin TH. ER stress and the unfolded protein response in intestinal inflammation. Am J Physiol Gastrointest Liver Physiol 2010; 298:G820-32. [PMID: 20338921 DOI: 10.1152/ajpgi.00063.2010] [Citation(s) in RCA: 148] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Endoplasmic reticulum (ER) stress is a phenomenon that occurs when excessive protein misfolding occurs during biosynthesis. ER stress triggers a series of signaling and transcriptional events known as the unfolded protein response (UPR). The UPR attempts to restore homeostasis in the ER but if unsuccessful can trigger apoptosis in the stressed cells and local inflammation. Intestinal secretory cells are susceptible to ER stress because they produce large amounts of complex proteins for secretion, most of which are involved in mucosal defense. This review focuses on ER stress in intestinal secretory cells and describes how increased protein misfolding could occur in these cells, the process of degradation of misfolded proteins, the major molecular elements of the UPR pathway, and links between the UPR and inflammation. Evidence is reviewed from mouse models and human inflammatory bowel diseases that ties ER stress and activation of the UPR with intestinal inflammation, and possible therapeutic approaches to ameliorate ER stress are discussed.
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Affiliation(s)
- Michael A McGuckin
- Mucosal Diseases Program, Mater Medical Research Institute, Mater Health Services, South Brisbane, Qld 4029, Australia.
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Braun A, Treede I, Gotthardt D, Tietje A, Zahn A, Ruhwald R, Schoenfeld U, Welsch T, Kienle P, Erben G, Lehmann WD, Fuellekrug J, Stremmel W, Ehehalt R. Alterations of phospholipid concentration and species composition of the intestinal mucus barrier in ulcerative colitis: a clue to pathogenesis. Inflamm Bowel Dis 2009; 15:1705-20. [PMID: 19504612 DOI: 10.1002/ibd.20993] [Citation(s) in RCA: 148] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
BACKGROUND Phospholipids are essential for the normal function of the intestinal mucus barrier. The objective of this study was to systematically investigate phospholipids in the intestinal mucus of humans suffering from inflammatory bowel diseases, where a barrier defect is strongly supposed to be pathogenetic. METHODS Optimal mucus recovery was first validated in healthy mice and the method was then transferred to the endoscopic acquisition of ileal and colonic mucus from 21 patients with ulcerative colitis (UC), 10 patients with Crohn's disease (CD), and 29 healthy controls. Nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to determine phosphatidylcholine (PC), lysophosphatidylcholine (LPC), and sphingomyelin (SM) in lipid extracts of mucus specimens. RESULTS Human and rodent mucus contained very similar phospholipid species. In the ileal and colonic mucus from patients suffering from UC, the concentration of PC was highly significantly lower (607 +/- 147 pmol/100 microg protein and 745 +/- 148 pmol/100 microg protein) compared to that of patients with CD (3223 +/- 1519 pmol/100 microg protein and 2450 +/- 431 pmol/100 microg protein) and to controls (3870 +/- 760 pmol/100 microg protein and 2790 +/- 354 pmol/100 microg protein); overall, P = 0.0002 for ileal specimens and P < 0.0001 for colonic specimens. Independent of disease activity, patients suffering from UC showed an increased saturation grade of PC fatty acid residues and a higher LPC-to-PC ratio. CONCLUSIONS The intestinal mucus barrier of patients with UC is significantly altered concerning its phospholipid concentration and species composition. These alterations may be very important for the pathogenesis of this disease and underline new therapeutic strategies.
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Affiliation(s)
- Annika Braun
- Department of Gastroenterology, University Hospital Heidelberg, Heidelberg, Germany
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Guzman-Aranguez A, Mantelli F, Argüeso P. Mucin-type O-glycans in tears of normal subjects and patients with non-Sjögren's dry eye. Invest Ophthalmol Vis Sci 2009; 50:4581-7. [PMID: 19407012 PMCID: PMC2751810 DOI: 10.1167/iovs.09-3563] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
PURPOSE O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study was conducted to identify the repertoire of O-glycans in the tear film and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögren's dry eye. METHODS Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium by using a glycogene microarray database. Lectin-binding analyses were performed with agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. RESULTS The O-glycan profile of human tears consisted primarily of core 1 (Gal beta 1-3GalNAc alpha 1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, with alpha2-6-sialyl core 1 being the predominant O-glycan structure in human tears (48%). Four families of glycosyltransferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included 13 polypeptide-GalNAc-transferases (GALNT), the core 1 beta-3-galactosyltransferase (T-synthase), three alpha2-6-sialyltransferases (ST6GalNAc), and two alpha2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and patients with dry eye, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. CONCLUSIONS This study identified the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögren's dry eye showed no alterations in composition and amount of O-glycans in the tear fluid.
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Affiliation(s)
- Ana Guzman-Aranguez
- Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA
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Xia B, Feasley CL, Sachdev GP, Smith DF, Cummings RD. Glycan reductive isotope labeling for quantitative glycomics. Anal Biochem 2009; 387:162-70. [PMID: 19454239 DOI: 10.1016/j.ab.2009.01.028] [Citation(s) in RCA: 106] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2008] [Revised: 01/16/2009] [Accepted: 01/19/2009] [Indexed: 12/12/2022]
Abstract
Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [(12)C(6)]aniline and [(13)C(6)]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.
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Affiliation(s)
- Baoyun Xia
- Department of Biochemistry and Glycomics Center at Emory University, Emory University School of Medicine, Atlanta, GA 30322, USA
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Abstract
Similar changes in glycosylation occur in the colonic epithelium in inflammatory conditions such as ulcerative colitis and Crohn's disease and also in colon cancer and precancerous adenomatous polyps. They include reduced length of O-glycans, reduced sulfation, increased sialylation and increased expression of oncofetal carbohydrate antigens, such as sialyl-Tn (sialylalpha2-6GalNAc), and the TF antigen (Thomsen-Friedenreich antigen) Galbeta1-3GalNAcalpha-Ser/Thr. The changes affect cell surface as well as secreted glycoproteins and mediate altered interactions between the epithelium and lectins of dietary, microbial or human origin. Different TF-binding lectins cause diverse effects on epithelial cells, reflecting subtle differences in binding specificities e.g. for sialylated TF; some of these interactions, such as with the TF-binding peanut lectin that resists digestion, may be biologically significant. Increased TF expression by cancer cells also allows interaction with the human galactose-binding lectin, galectin-3. This lectin has increased concentration in the sera of patients with metastatic cancer and binds TF on cancer cell surface MUC1 (mucin 1), causing clustering of MUC1 and revealing underlying adhesion molecules which promote adhesion to endothelium. This is likely to be an important mechanism in cancer metastasis and represents a valid therapeutic target. Tools are now available to allow fast and accurate elucidation of glycosylation changes in epithelial disease, characterization of their potential lectin ligands, whether dietary, microbial or human, and determination of the functional significance of their interactions. This should prove a very fruitful area for future research with relevance to infectious, inflammatory and cancerous diseases of the epithelia.
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Gersemann M, Becker S, Kübler I, Koslowski M, Wang G, Herrlinger KR, Griger J, Fritz P, Fellermann K, Schwab M, Wehkamp J, Stange EF. Differences in goblet cell differentiation between Crohn's disease and ulcerative colitis. Differentiation 2008; 77:84-94. [PMID: 19281767 DOI: 10.1016/j.diff.2008.09.008] [Citation(s) in RCA: 219] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2008] [Revised: 06/23/2008] [Accepted: 07/02/2008] [Indexed: 02/06/2023]
Abstract
Goblet cells are mucin-secreting intestinal cells forming the mucus layer that protects the mucosal surface. Ulcerative colitis (UC) has been associated with a defective colonic mucus layer and a reduced number of goblet cells. In experimental animals, colonic goblet cell differentiation is regulated by interacting transcription factors Hath1, KLF4 and the Notch, as well as Wnt pathways, whereas data in humans are limited. We investigated goblet cell differentiation factors and mucins in controls and in inflammatory bowel diseases (IBDs). We performed real-time PCR for Hath1, KLF4, several ligands, receptors and target genes of the Notch and Wnt pathways, as well as several mucins in biopsies from the sigmoid colon of controls (n=21), Crohn's disease (CD, n=48) and UC (n=40). In addition, Hath1 protein was quantitated with Western blot and localized with immunohistochemistry. Notably, the degree of inflammation as measured by IL-8 and histology was similar in both disease entities. The proportion of goblet cells was lowered in both IBDs, but specifically diminished in the upper third of the crypt in UC. Comparable levels of inflammation induced both Hath1 (2.0-fold, p<0.001) and KLF4 (1.8-fold for KLF4, p=0.031) mRNA expression in CD but not in UC (0.8-0.9-fold, ns). The differential induction was confirmed for Hath1 protein using Western blot. Hath1 immunostaining was found mostly in the lower half of the colonic crypts. Hath1, KLF4 and the Notch target gene Hes1 were significantly (p<0.001) and positively correlated. Moreover, both Hath1 and KLF4 were correlated (p<0.001) with MUC1, MUC2 as well as MUC4 in all control and IBD cohorts. The results indicate that both transcription factors are key regulators of goblet cell differentiation and mucin formation in the human colon. Conspicuously, inflammation is associated with an enhanced goblet cell differentiation in CD but not in UC, a defect possibly of pathogenic importance.
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Affiliation(s)
- Michael Gersemann
- Department of Internal Medicine I, Robert Bosch Hospital, Stuttgart, Germany
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Pathogenic Escherichia coli in inflammatory bowel diseases Proceedings of the 1st International Meeting on E. coli and IBD, June 2007, Lille, France. J Crohns Colitis 2008; 2:255-62. [PMID: 21172221 DOI: 10.1016/j.crohns.2008.02.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2008] [Accepted: 02/28/2008] [Indexed: 02/08/2023]
Abstract
Several different groups have recently reported the presence of pathogenic E. coli associated with ileal and/or colonic mucosa of patients with inflammatory bowel diseases. Given the important role of the gut microflora and enteropathogens in the initiation and perpetuation of intestinal inflammation, important issues now arise. Are these IBD associated E. coli pathogenic? Have they evolved from commensal bacteria? What is their reservoir? Are these bacteria sufficient to drive IBD pathogenesis? Which immunological defects may predispose to colonization by such bacteria? In June 2007, clinicians and basic scientists met in Lille, France with the goal of exchanging ideas and materials on this emerging topic. State-of-the-art lectures given by widely recognized international experts were associated with debates, case discussions and expert opinions. This paper summarizes most data that were exchanged during this day and represents an update on the potential role of pathogenic E. coli in inflammatory bowel diseases.
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Ardesjö B, Portela-Gomes GM, Rorsman F, Gerdin E, Lööf L, Grimelius L, Kämpe O, Ekwall O. Immunoreactivity against Goblet cells in patients with inflammatory bowel disease. Inflamm Bowel Dis 2008; 14:652-61. [PMID: 18213698 DOI: 10.1002/ibd.20370] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
BACKGROUND A number of autoantibodies have been reported in inflammatory bowel disease (IBD). The aim of this study was to investigate to what extent sera from patients with IBD contain autoantibodies directed against normal human gastrointestinal mucosa. METHODS Samples of sera from 50 patients with IBD and 50 healthy subjects were used for immunostaining of normal and affected human gastrointestinal tissues. RESULTS Eighty-four percent of the sera from IBD patients showed immunoreactivity against goblet cells in the appendix compared with 8% of the sera from healthy subjects. Goblet cell reactivity of IBD patient sera varied between regions in the gastrointestinal tract. Sera from healthy subjects only reacted with goblet cells in the appendix. In the colon and the appendix, goblet cell reactivity of IBD sera was generally weak at the base of the crypts and gradually increased toward the lumen. Three IBD sera samples reacted with gastrin cells in the antrum. In colon biopsies from patients with ulcerative colitis, immunoreactivity against the remaining goblet cells showed an inverse correlation with inflammatory activity. CONCLUSIONS These findings suggest that immunoreactivity against goblet cells may be of central importance in the pathogenesis of IBD. Identification of goblet cell antigens could lead to a better understanding of IBD and provide a new diagnostic tool.
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Affiliation(s)
- Brita Ardesjö
- Department of Medical Sciences, Uppsala University, Uppsala, Sweden.
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Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, Thornton DJ, Png CW, Crockford TL, Cornall RJ, Adams R, Kato M, Nelms KA, Hong NA, Florin THJ, Goodnow CC, McGuckin MA. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med 2008; 5:e54. [PMID: 18318598 PMCID: PMC2270292 DOI: 10.1371/journal.pmed.0050054] [Citation(s) in RCA: 565] [Impact Index Per Article: 33.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/14/2007] [Accepted: 01/17/2008] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. METHODS AND FINDINGS By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis. CONCLUSIONS Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
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Affiliation(s)
- Chad K Heazlewood
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Matthew C Cook
- Immunology and Inflammation Group, Phenomix Australia, Acton, Australia
| | - Rajaraman Eri
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Gareth R Price
- Molecular Genetics Team, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Sharyn B Tauro
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Douglas Taupin
- Gastroenterology Unit, Canberra Hospital, Woden, Australia
| | - David J Thornton
- Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
| | - Chin Wen Png
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Tanya L Crockford
- Nuffield Dept of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
| | - Richard J Cornall
- Nuffield Dept of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
| | - Rachel Adams
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Masato Kato
- Dendritic Cell Program, Mater Medical Research Institute, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Keats A Nelms
- Immunology and Inflammation Group, Phenomix Australia, Acton, Australia
| | - Nancy A Hong
- Phenomix Corporation, San Diego, California, United States of America
| | - Timothy H. J Florin
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
| | - Christopher C Goodnow
- Division of Immunology and Genetics and Australian Phenomics Facility, John Curtin School of Medical Research, The Australian National University, Canberra, Australia
| | - Michael A McGuckin
- Mucin and IBD Research Teams, Mucosal Diseases Program, Mater Medical Research Institute, and the University of Queensland, Aubigny Place, Mater Health Services, South Brisbane, Queensland, Australia
- * To whom correspondence should be addressed. E-mail:
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39
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Abstract
INTRODUCTION/BACKGROUND Inflammatory bowel disease (IBD) results from complex interactions between: host genome, immune system, mucosa, bacteria, and environment. SOURCES OF DATA Review of PubMed database using search terms 'bacteria and inflammatory bowel disease' and 'genetics and inflammatory bowel disease'. PubMed 'related reference' feature and references from retrieved articles were examined. AREAS OF AGREEMENT IBD results from interaction between the microbiota of the gut and the immune system. Key gene defects associated with IBD are involved in bacterial recognition and processing. The environment at least modifies and may determine pathogenesis. AREAS OF CONTROVERSY It has been disputed whether the primary defect in IBD is immunological or bacterial, and which bacteria are key. GROWING POINTS/AREAS FOR RESEARCH: 'M cells', the specialized epithelial cells that overlie Peyer's patches, are a major interface between gut bacteria and the immune system. Improved understanding is needed of the bacteria involved in IBD pathogenesis, their genotypes and phenotypes, their portal of entry and their mechanism for escaping attack from the immune system. Bacterial ligands involved in bacteria-epithelial adhesion are emerging, and molecular techniques are rapidly increasing our knowledge of the human intestinal microbiota.
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Affiliation(s)
- Paul Knight
- School of Clinical Sciences, University of Liverpool, Nuffield Building, Crown Street, Liverpool L693BX, UK
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40
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Abstract
PURPOSE OF REVIEW Recently published studies presenting novel and relevant information on defensins and other antimicrobial peptides in Crohn's disease and ulcerative colitis are reviewed. RECENT FINDINGS Different clinical localizations of Crohn's disease are associated with different deficiencies in epithelial and leukocyte antimicrobial peptide expression. As compared with ulcerative colitis, Crohn's disease of the colon is characterized by an impaired induction of beta defensins, and antimicrobial antiproteases elafin and SLPI, as well as the cathelicidin LL37. The attenuated induction of beta defensins is linked to fewer gene copy numbers in this locus, which is associated with colonic but not ileal Crohn's disease. In contrast, ileal Crohn's disease patients are characterized by a reduced antibacterial activity and a specific reduction of ileal Paneth cell defensins. This decrease is independent of the grade of histological inflammation and cannot be found in inflammation controls. Thus, some of these defects can be explained either by direct or indirect genetic mechanisms and appear to be primary. SUMMARY Unlike ulcerative colitis, ileal and colonic Crohn's disease are characterized by localized deficiencies of antibacterial peptides. Understanding the precise molecular mechanisms of the defective antibacterial barrier function might provide new therapeutic directions.
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Affiliation(s)
- Jan Wehkamp
- Robert Bosch Hospital and Dr Margarete Fischer Bosch Institute of Clinical Pharmacology and University of Tübingen, Stuttgart, Germany
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41
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Abstract
Mucosa‐associated E Coli are reported to be increased in Crohn's disease
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Affiliation(s)
- Jonathan M Rhodes
- School of Clinical Sciences, University of Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA.
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42
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Van Limbergen J, Russell RK, Nimmo ER, Ho GT, Arnott ID, Wilson DC, Satsangi J. Genetics of the innate immune response in inflammatory bowel disease. Inflamm Bowel Dis 2007; 13:338-55. [PMID: 17206667 DOI: 10.1002/ibd.20096] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The discovery of nucleotide-binding oligomerization domain 2/caspase recruitment domain-containing protein 15 (NOD2/CARD15) as the first susceptibility gene in Crohn's disease (CD) has shifted the focus of research into the pathogenesis of inflammatory bowel disease (IBD) firmly to the innate immune response and the integrity of the epithelial barrier. The subsequent implication in IBD of variant alleles of OCTN, DLG5, MDR1, and TLRs has provided further support for a new, more complex model of innate immunity function in the gastrointestinal tract. In this review, we examine the recent advances in our understanding of the influence of genetics of the innate immune response on IBD. We will focus on germline variation of genes encoding pathogen-recognition receptors, proteins involved in epithelial homeostasis and secreted antimicrobial proteins.
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Affiliation(s)
- Johan Van Limbergen
- Gastrointestinal Unit, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK.
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