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Wang B, Liu S, Li H, Dong W, Liu H, Zhang J, Tian C, Dong S. Facile Preparation of Carbohydrate-Containing Adjuvants Based on Self-Assembling Glycopeptide Conjugates. Angew Chem Int Ed Engl 2024; 63:e202309140. [PMID: 37950683 DOI: 10.1002/anie.202309140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2023] [Revised: 11/06/2023] [Accepted: 11/10/2023] [Indexed: 11/13/2023]
Abstract
Carbohydrates are intriguing biomolecules possessing diverse biological activities, including immune stimulating capability. However, their biomedical applications have been limited by their complex and heterogeneous structures. In this study, we have utilized a self-assembling glycopeptide conjugate (GPC) system to produce uniform nanoribbons appending homogeneous oligosaccharides with multivalency. This system successfully translates the nontrivial structural differences of oligomannoses into varied binding affinities to C-type lectin receptors (CLRs). We have shown that GPCs could promote the CLR-mediated endocytosis of ovalbumin (OVA) antigen, and two mannotriose-modified peptides F3m2 and F3m5 exhibit potent activity in inducing antigen-presenting cell maturation, as indicated by increased CD86 and MHCII expression. In vivo studies demonstrated that GPCs, combined with OVA antigen, significantly enhanced OVA-specific antibody production. Specifically, F3m2 and F3m5 exhibited the highest immunostimulatory effects, eliciting both Th1- and Th2-biased immune responses and promoting differentiation of CD4+ and CD8+ T cells. These findings highlight the potential of GPCs as vaccine adjuvants, and showcase their versatility in exploiting the biological functions of carbohydrates.
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Affiliation(s)
- Biao Wang
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Sijin Liu
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Haoting Li
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Weidong Dong
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Haiyun Liu
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Jun Zhang
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Chao Tian
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
| | - Suwei Dong
- State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, and School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
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Radine UK, Bumiller-Bini Hoch V, Boldt ABW, Zillikens D, Ludwig RJ, Hammers CM, Klinger M, Hundt JE. Electron microscopy of desmosomal structures in the pemphigus human skin organ culture model. Front Med (Lausanne) 2022; 9:997387. [DOI: 10.3389/fmed.2022.997387] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Accepted: 10/18/2022] [Indexed: 11/15/2022] Open
Abstract
Pemphigus is a chronic autoimmune skin blistering disease, characterized by acantholysis and by the production of autoantibodies directed against the structural desmosomal proteins desmoglein 1 (DSG1) and/or DSG3. Model systems allow the identification and testing of new therapeutic targets. Here, we evaluated ultrastructural desmosomal morphology in the human skin organ culture (HSOC) model injected with either anti-desmoglein (DSG) 1/3 single-chain variable fragment (scFv, termed Px4-3), Staphylococcus aureus exfoliative toxin (ETA) as a reference and positive control, and normal human IgG as a negative control. Each experimental condition was evaluated in abdominal skin biopsies from five different donors. After 24 h of incubation, we processed the samples for histological and ultrastructural electron microscopy analyses. We found that Px4-3 or ETA induced a loss of desmosomes and increased interdesmosomal widening, similar to patient skin biopsies and other pemphigus models. Thus, we propose the HSOC pemphigus model as an attractive tool to unravel novel therapeutic targets.
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Enterocytes in Food Hypersensitivity Reactions. Animals (Basel) 2021; 11:ani11092713. [PMID: 34573679 PMCID: PMC8466009 DOI: 10.3390/ani11092713] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2021] [Revised: 09/05/2021] [Accepted: 09/10/2021] [Indexed: 11/18/2022] Open
Abstract
Simple Summary Hypersensitivity to food, affecting both animals and humans, is increasing. Until a decade ago, it was thought that enterocytes, the most abundant constituent of the intestinal surface mucosa layer, served only to absorb digested food and prevent foreign and non-digested substances from passing below the intestinal layer. Growing evidence supports the involvement of enterocytes in immunological responses. Here, we present a comprehensive review of the new roles of enterocytes in food hypersensitivity conducted in animal models in order to better understand complicated immune pathological conditions. In addition, resources for further work in this area are suggested, along with a literature overview of the specific roles of enterocytes in maintaining oral tolerance. Lastly, it will be beneficial to investigate the various animal models involved in food hypersensitivity to reach the needed momentum necessary for the complete and profound understanding of the mechanisms of the ever-growing number of food allergies in animal and human populations. Abstract Food hypersensitivity reactions are adverse reactions to harmless dietary substances, whose causes are hidden within derangements of the complex immune machinery of humans and mammals. Until recently, enterocytes were considered as solely absorptive cells providing a physical barrier for unwanted lumen constituents. This review focuses on the enterocytes, which are the hub for innate and adaptive immune reactions. Furthermore, the ambiguous nature of enterocytes is also reflected in the fact that enterocytes can be considered as antigen-presenting cells since they constitutively express major histocompatibility complex (MHC) class II molecules. Taken together, it becomes clear that enterocytes have an immense role in maintaining oral tolerance to foreign antigens. In general, the immune system and its mechanisms underlying food hypersensitivity are still unknown and the involvement of components belonging to other anatomical systems, such as enterocytes, in these mechanisms make their elucidation even more difficult. The findings from studies with animal models provide us with valuable information about allergic mechanisms in the animal world, while on the other hand, these models are used to extrapolate results to the pathological conditions occurring in humans. There is a constant need for studies that deal with this topic and can overcome the glitches related to ethics in working with animals.
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Mowat AM. To respond or not to respond - a personal perspective of intestinal tolerance. Nat Rev Immunol 2019; 18:405-415. [PMID: 29491358 DOI: 10.1038/s41577-018-0002-x] [Citation(s) in RCA: 124] [Impact Index Per Article: 20.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
For many years, the intestine was one of the poor relations of the immunology world, being a realm inhabited mostly by specialists and those interested in unusual phenomena. However, this has changed dramatically in recent years with the realization of how important the microbiota is in shaping immune function throughout the body, and almost every major immunology institution now includes the intestine as an area of interest. One of the most important aspects of the intestinal immune system is how it discriminates carefully between harmless and harmful antigens, in particular, its ability to generate active tolerance to materials such as commensal bacteria and food proteins. This phenomenon has been recognized for more than 100 years, and it is essential for preventing inflammatory disease in the intestine, but its basis remains enigmatic. Here, I discuss the progress that has been made in understanding oral tolerance during my 40 years in the field and highlight the topics that will be the focus of future research.
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Affiliation(s)
- Allan McI Mowat
- Centre for Immunobiology, Institute of Infection, Immunity and Inflammation, College of Medicine, Veterinary Medicine and Life Sciences, University of Glasgow, Glasgow, UK.
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Intracellular Localization of Microbial Transglutaminase and Its Influence on the Transport of Gliadin in Enterocytes. J Pediatr Gastroenterol Nutr 2019; 68:e43-e50. [PMID: 30320664 DOI: 10.1097/mpg.0000000000002171] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
OBJECTIVE Celiac disease (CD) is a systemic inflammatory disorder, characterized by the destruction of duodenal epithelium. The CD8 T cells involved are associated with cross-presentation. In addition to other factors, the rising prevalence of CD might be induced by microbial transglutaminase (mTG) an enzyme frequently used in food production that shares enzymatic and antigenic properties of tissue transglutaminase (TG2), the autoantigen in CD. We hypothesized that mTG and gliadin are transported into the endoplasmic reticulum (ER), indicating cross-presentation of both antigens. METHODS Apical incubation of duodenal biopsies from CD and control patients was performed with mTG alone or with mTG and simultaneously with Frazer's fraction. Evaluation was carried out by immunofluorescence and electron microscopy. RESULTS Approximately 6% to 9% of the intracellular mTG and gliadin were transported to the ER of enterocytes. RACE cells (Rapid uptake of Antigen into the Cytosol of Enterocytes) displayed an enhanced antigen uptake into a dilated ER. mTG strongly localized at the basolateral membrane and the lamina propria. CONCLUSIONS mTG and gliadin are transported to the ER of enterocytes and to a greater extent to the ER of RACE cells, suggesting cross-presentation of exogenous antigens. The strong localization of mTG at the basolateral membrane and the lamina propria may also indicate a potential antigenic interaction with cells of the immune system. Since mTG may not only been taken up with food stuffs but could also be released by bacteria within the intestinal microbiota, further investigations are needed regarding the role of mTG in CD pathogenesis.
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Endocytosis in enterocytes. Wien Med Wochenschr 2016; 166:205-10. [DOI: 10.1007/s10354-016-0448-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Accepted: 02/18/2016] [Indexed: 10/22/2022]
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Gastrointestinal Digestion and Absorption of Pen j 1, a Major Allergen from Kuruma Prawn,Penaeus japonicus. Biosci Biotechnol Biochem 2014; 75:1249-58. [DOI: 10.1271/bbb.110021] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
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Bär F, Sina C, Hundorfean G, Pagel R, Lehnert H, Fellermann K, Büning J. Inflammatory bowel diseases influence major histocompatibility complex class I (MHC I) and II compartments in intestinal epithelial cells. Clin Exp Immunol 2013; 172:280-9. [PMID: 23574324 DOI: 10.1111/cei.12047] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/04/2012] [Indexed: 12/19/2022] Open
Abstract
Antigen presentation by intestinal epithelial cells (IEC) is crucial for intestinal homeostasis. Disturbances of major histocompatibility complex class I (MHC I)- and II-related presentation pathways in IEC appear to be involved in an altered activation of CD4(+) and CD8(+) T cells in inflammatory bowel disease. However, a comprehensive analysis of MHC I- and II-enriched compartments in IEC of the small and large bowel in the healthy state as opposed to inflammatory bowel diseases is lacking. The aim of this study was to characterize the subcellular expression of MHC I and II in the endocytic pathway of IEC throughout all parts of the intestinal tract, and to identify differences between the healthy state and inflammatory bowel diseases. Biopsies were taken by endoscopy from the duodenum, jejunum, ileum and colon in healthy individuals (n = 20). In Crohn's disease (CD), biopsies were obtained from the ileum and colon and within the colon from ulcerative colitis (UC) patients (n = 15). Analysis of IEC was performed by immunoelectron microscopy. MHC I and II were identified in early endosomes and multi-vesicular, multi-lamellar, electrondense and vacuolar late endosomes. Both molecules were enriched in multi-vesicular bodies. No differences were found between the distinct parts of the gut axis. In CD and UC the expression of MHC I and II showed a shift from multi-vesicular bodies towards the basolateral membranes. Within the multi-vesicular bodies, MHC I and II moved from internal vesicles to the limiting membranes upon inflammation in CD and UC. MHC I- and II-enriched compartments in IEC were identical in all parts of the small and large bowel. CD and UC appear to modulate the MHC I- and II-related presentation pathways of exogenous antigens in IEC.
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Affiliation(s)
- F Bär
- Department of Internal Medicine I, University Hospital of Schleswig-Holstein, Institute of Anatomy, University of Lübeck, Lübeck, Germany
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Li J, Geng S, Liu X, Liu H, Jin H, Liu CG, Wang B. DNA and protein co-administration induces tolerogenic dendritic cells through DC-SIGN mediated negative signals. Hum Vaccin Immunother 2013; 9:2237-45. [PMID: 24051433 PMCID: PMC3906410 DOI: 10.4161/hv.25011] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
We previously demonstrated that DNA and protein co-administration induced differentiation of immature dendritic cells (iDCs) into CD11c+CD40lowIL-10+ regulatory DCs (DCregs) via the caveolin-1 (Cav-1) -mediated signal pathway. Here, we demonstrate that production of IL-10 and the low expression of CD40 play a critical role in the subsequent induction of regulatory T cells (Tregs) by the DCregs. We observed that DNA and protein were co-localized with DC-SIGN in caveolae and early lysosomes in the treated DCs, as indicated by co-localization with Cav-1 and EEA-1 compartment markers. DNA and protein also co-localized with LAMP-2. Gene-array analysis of gene expression showed that more than a thousand genes were significantly changed by the DC co-treatment with DNA + protein compared with controls. Notably, the level of DC-SIGN expression was dramatically upregulated in pOVA + OVA co-treated DCs. The expression levels of Rho and Rho GNEF, the down-stream molecules of DC-SIGN mediated signal pathway, were also greatly upregulated. Further, the level of TLR9, the traditional DNA receptor, was significantly downregulated. These results suggest that DC-SIGN as the potential receptor for DNA and protein might trigger the negative pathway to contribute the induction of DCreg combining with Cav-1 mediated negative signal pathway.
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Affiliation(s)
- Jinyao Li
- Key laboratory of Medical Molecular Virology of MOE and MOH; Fudan University Shanghai Medical College; Shanghai, P.R. China
| | - Shuang Geng
- Key laboratory of Medical Molecular Virology of MOE and MOH; Fudan University Shanghai Medical College; Shanghai, P.R. China
| | - Xiuping Liu
- Department of Experimental Therapeutics; Division of Cancer Medicine; The University of Texas M.D. Anderson Cancer Center; Houston, TX USA
| | - Hu Liu
- State Key Laboratory for Agro-Biotechnology; Department of Microbiology and Immunology; College of Biological Science; China Agricultural University; Beijing, P.R. China
| | - Huali Jin
- State Key Laboratory for Agro-Biotechnology; Department of Microbiology and Immunology; College of Biological Science; China Agricultural University; Beijing, P.R. China
| | - Chang-Gong Liu
- Department of Experimental Therapeutics; Division of Cancer Medicine; The University of Texas M.D. Anderson Cancer Center; Houston, TX USA
| | - Bin Wang
- Key laboratory of Medical Molecular Virology of MOE and MOH; Fudan University Shanghai Medical College; Shanghai, P.R. China; State Key Laboratory for Agro-Biotechnology; Department of Microbiology and Immunology; College of Biological Science; China Agricultural University; Beijing, P.R. China
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Lübbing N, Barone MV, Rudloff S, Troncone R, Auricchio S, Zimmer KP. Correction of gliadin transport within enterocytes through celiac disease serum. Pediatr Res 2011; 70:357-62. [PMID: 21705964 DOI: 10.1203/pdr.0b013e31822a31e7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Celiac disease (CD) is caused by loss of tolerance toward gluten and related cereal products. The delivery of gliadin peptides (GP) to HLA-DR-positive late endosomes (LE) of enterocytes is required for antigen presentation and tolerance generation. We hypothesized that anti-gliadin antibodies in CD serum modify gliadin transport into LE within enterocytes. CD and control duodenal biopsies were incubated with digests of gluten as well as with serum of CD patients. Lissamin-labeled GP AA31-43 and AA56-68 were endocytozed by Caco-2 cells with serum of CD- or control patients. Colocalization of gliadin with the LE marker LAMP-2 and cathepsin D was determined and quantified on immunofluorescence and immunoelectron microscopical level. Up to 13% of internalized gliadin was located in LE of CD biopsies incubated with CD serum compared with less than 4% in CD biopsies without CD serum as well as in control biopsies. In Caco-2 cells, the colocalization coefficient of GP AA31-43 and LE was 0.82 with CD serum, 0.42 with control serum, and 0.48 with culture medium. Incubation with CD serum can direct GP AA31-43 into LE of enterocytes which is required for antigen presentation.
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Affiliation(s)
- Nico Lübbing
- Department of Pediatrics, Justus-Liebig-University Giessen, 35392 Giessen, Germany
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11
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Im JP, Ye BD, Kim JM, Jung HC, Song IS, Kim JS. Rectal administration of lipopolysaccharide and ovalbumin ameliorates acute murine colitis. Dig Dis Sci 2011; 56:2292-8. [PMID: 21359540 DOI: 10.1007/s10620-011-1630-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/15/2010] [Accepted: 02/14/2011] [Indexed: 12/09/2022]
Abstract
BACKGROUND Repeated challenges of lipopolysaccharide (LPS) could reduce the expression of proinflammatory cytokines in vitro, and oral administration of ovalbumin (OVA) induces mucosal tolerance in vivo. However, the effect of local administration of LPS and OVA on experimental colitis in vivo remains unknown. AIMS This study was performed to elucidate the effect of rectal administration of LPS and OVA on an acute murine colitis induced by dextran sulfate sodium (DSS). METHODS BALB/c mice were rectally administered LPS with or without OVA followed by 3% DSS. Colitis was assessed by disease activity index (DAI) including weight loss, stool consistency and rectal bleeding, and histopathology. Primary colon epithelial cells were isolated and the expression of Toll-like receptor 4 (TLR4) was examined using the Western blot analysis. IL-6, IFN-γ and IL-10 mRNA levels in colonic tissue were assessed using real-time RT-PCR. RESULTS LPS administration significantly attenuated the severity of acute DSS-induced colitis as assessed by DAI and histopathologic scoring compared with the control group. Combined treatment of LPS and OVA restored body weight loss and further ameliorated the severity of acute DSS colitis. LPS pretreatment regardless of OVA administration decreased TLR4 expression. LPS and OVA pretreatment reduced IL-6 and IFN-γ mRNA expression and increased IL-10 mRNA expression compared with controls. CONCLUSION Rectal administration of LPS attenuated acute murine colitis, possibly through TLR4 down-regulation, and combined treatment of OVA additionally ameliorated colonic inflammation associated with up-regulation of IL-10.
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Affiliation(s)
- Jong Pil Im
- Department of Internal Medicine Liver Research Institute, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul, 110-744, Korea
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Rescigno M. Functional specialization of antigen presenting cells in the gastrointestinal tract. Curr Opin Immunol 2010; 22:131-6. [DOI: 10.1016/j.coi.2009.12.007] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2009] [Revised: 12/22/2009] [Accepted: 12/23/2009] [Indexed: 01/21/2023]
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Büning J, von Smolinski D, Tafazzoli K, Zimmer KP, Strobel S, Apostolaki M, Kollias G, Heath JK, Ludwig D, Gebert A. Multivesicular bodies in intestinal epithelial cells: responsible for MHC class II-restricted antigen processing and origin of exosomes. Immunology 2009; 125:510-21. [PMID: 18710406 DOI: 10.1111/j.1365-2567.2008.02864.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
In normal conditions intestinal epithelial cells (IECs) constitutively stimulate regulatory CD4(+) T cells. However, in Crohn's disease (CD), this major histocompatibility complex (MHC) class II-restricted antigen presentation results in stimulation of proinflammatory CD4(+) T cells. We hypothesized that these alternative functions might be mediated by differential sorting and processing of antigens into distinct MHC II-enriched compartments (MIICs). Accordingly, we analysed the endocytic pathways of lumenally applied ovalbumin (OVA) in IECs of the jejunum and ileum of wild-type (WT) and TNFDeltaARE/WT mice that develop a CD-resembling ileitis. Using quantitative reverse transcription polymerase chain reaction, we found that messenger RNA levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-17 and interleukin-10 were significantly up-regulated in the inflamed ileum of TNFDeltaARE/WT mice, confirming CD-like inflammation. Fluorescence and immunoelectron microscopy revealed the presence of MHC II and invariant chain throughout the late endocytic compartments, with most molecules concentrated in the multivesicular bodies (MVB). OVA was targeted into MVB and, in contrast to other MIICs, accumulated in these structures within 120 min of exposure. The IEC-specific A33 antigen localized to internal vesicles of MVB and A33/class II-bearing exosomes were identified in intercellular spaces. Remarkably, the expression pattern of MHC II/invariant chain molecules and the trafficking of OVA were independent of mucosal inflammation and the specific region in the small intestine. MVB seem to be principally responsible for class II-associated antigen processing in IECs and to constitute the origin of MHC II-loaded exosomes. The distinctive functions of IECs in antigen presentation to CD4(+) T cells might arise as a result of differential processing within the MVB identified here.
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Affiliation(s)
- Jürgen Büning
- Department of Internal Medicine I, University Hospital of Schleswig-Holstein, Lübeck, Germany.
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Hundorfean G, Zimmer KP, Strobel S, Gebert A, Ludwig D, Büning J. Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis. Am J Physiol Gastrointest Liver Physiol 2007; 293:G798-808. [PMID: 17673546 DOI: 10.1152/ajpgi.00135.2007] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.
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Affiliation(s)
- Gheorghe Hundorfean
- Dept. of Internal Medicine I, Univ. Hospital of Schleswig-Holstein, Ratzeburger Allee 160, D-23538 Lübeck, Germany
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Abstract
The gastrointestinal tract represents the largest mucosal membrane surface in the human body. The immune system in the gut is the first line of host defense against mucosal microbial pathogens and it plays a crucial role in maintaining mucosal homeostasis. Membranous or microfold cells, commonly referred to as microfold cells, are specialized epithelial cells of the gut-associated lymphoid tissues (GALT) and they play a sentinel role for the intestinal immune system by delivering luminal antigens through the follicle-associated epithelium to the underlying immune cells. M cells sample and uptake antigens at their apical membrane, encase them in vesicles to transport them to the basolateral membrane of M cells, and from there deliver antigens to the nearby lymphocytes. On the flip side, some intestinal pathogens exploit M cells as their portal of entry to invade the host and cause infections. In this article, we briefly review our current knowledge on the morphology, development, and function of M cells, with an emphasis on their dual role in the pathogenesis of gut infection and in the development of host mucosal immunity.
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Mine Y, Yang M. Epitope characterization of ovalbumin in BALB/c mice using different entry routes. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2006; 1774:200-12. [PMID: 17236828 DOI: 10.1016/j.bbapap.2006.12.003] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Subscribe] [Scholar Register] [Received: 05/12/2006] [Revised: 12/04/2006] [Accepted: 12/05/2006] [Indexed: 02/04/2023]
Abstract
Ovalbumin (OVA) is known as a major allergen in egg white. A number of studies have reported the partial T and B cell epitope mapping of OVA using murine models and allergic patients' sera. Recently, we have reported the IgE-binding regions of the entire OVA molecule using egg allergic patients' sera. However, the entire epitope mapping of OVA in a murine model has not been completed yet. In the present study, BALB/c mice were administered a solution of OVA using three different entry routes (oral, intraperitoneal and subcutaneous) with their respective adjuvant (cholera toxin, aluminum hydroxide and Freund's adjuvant). Two nitrocellulose membranes containing 188 overlapping synthetic peptides (with a length of 12 amino acids and an offset of two amino acids) covering the primary sequence of OVA, were probed with the three different BALB/c mice antisera. Antisera obtained from orally challenged mice identified eight IgE epitope regions, i.e. I53D60; V77R84; S103E108; G127T136; E275V280; G301F306; I323A332 and A375S384, while sera raised by intraperitoneal and subcutaneous injections exhibited two (K55D60 and K277L282) and five (K55R58; G127T136; K279L282; T303S308 and I323A332) IgE binding sequences, respectively. The residues critical for the epitope-paratope interactions were finely characterized using the oral immunization serum. Analysis of IgE binding epitopes in mice provides us with potential strategies for design of specific immunotherapy.
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Affiliation(s)
- Yoshinori Mine
- Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
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Abstract
PURPOSE OF REVIEW The default response to protein antigens in the intestine is the induction of systemic and local hyporesponsiveness, ensuring the prevention of coeliac disease and food allergies. Interest is increasing in the role of dietary manipulation and probiotics in treating allergic and other diseases, but less is known about how these regimens might influence systemic and local immune responses. This paper addresses the mechanisms at the interface of innate and adaptive immunity that determine how the body responds to orally administered proteins and how local bacteria modify these. RECENT FINDINGS This paper discusses evidence that dendritic cells in the intestinal mucosa are the critical cells that take up dietary proteins and migrate to the draining mesenteric lymph node, where they induce regulatory CD4 T-cell differentiation. The properties of tolerized T cells are discussed and it is proposed that the gut microenvironment maintains homeostasis by conditioning dendritic cells to remain in a quiescent state. Inhibitory signalling by commensal bacteria possibly contributes to this process. SUMMARY A regulatory network controls how dietary antigens are taken up and presented to T lymphocytes by specialized antigen-presenting cells. Elucidating their nature and how they are influenced by external factors such as probiotics may help develop novel therapies for allergy and help understand diseases such as coeliac disease.
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Affiliation(s)
- Stephan Strobel
- Peninsula Postgraduate Health Institute, Peninsula Medical School, Plymouth, UK.
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Lin XP, Almqvist N, Telemo E. Human small intestinal epithelial cells constitutively express the key elements for antigen processing and the production of exosomes. Blood Cells Mol Dis 2006; 35:122-8. [PMID: 16027013 DOI: 10.1016/j.bcmd.2005.05.011] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2005] [Accepted: 05/20/2005] [Indexed: 12/11/2022]
Abstract
In humans, the small intestinal epithelial cells (IEC) have a high constitutive expression of MHC class II (MHC II), and contains lysosomes. The IEC also contains MHC II rich multivesicular compartments and has been shown to produce exosomes. This suggests a role for the IEC in antigen processing and presentation either directly or indirectly by the production of exosomes. However, the presence and localisation in the IEC of other key molecules involved in this process has not been studied previously. In the present work, we have investigated small intestinal biopsies from healthy adults and the HT29 IEC cell line with monoclonal antibodies against molecules involved in the antigen processing/presenting systems and molecules typically found on exosomes derived from professional APCs and IECs. Immunohistology was performed to study the expression and localisation of MHC II (HLA-DR), HLA-DM, MHC I (HLA-ABC), CD1d, Invariant chain, Lamp-1, CD68, CD63, B7.1, B7.2, ICAM-1, Cathepsin D/S/L and the IEC specific marker A33 in the IECs. We found that the IECs from the biopsies constitutively express MHC II, HLA-DM, MHC I, Invariant chain, Lamp-1, CD 68, CD63 and A33, and these markers were also found in the IFN-g treated HT-29 cells. All these molecules were found apically in the IECs of the biopsies, localised mainly in vesicular structures. Interestingly, in the baso-latereral area of the IEC, only MHC II, MHC I, Lamp 1, CD68, CD63 and A33 were found and also here with vesicular staining pattern which matches the molecules previously found on exosomes derived professional APCs and human IEC lines. CD1d, B7, ICAM-1, CD9 and cathepsin D and L were absent in the IEC compartment, but cathepsin S showed a relatively weak staining in the apical part of the IEC. The staining pattern and the morphological localisation of these markers suggest a prominent antigen processing/loading and trafficking compartment, and a possible baso-lateral release of exosomes in the normal human IEC.
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Affiliation(s)
- X P Lin
- Department of Rheumatology and Inflammation Research, Sahlgrenska Academy, University of Göteborg, Guldhedsg. 10, S-41346 Göteborg, Sweden
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19
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Abstract
Oral administration of a protein antigen generates a serum factor that induces tolerance when transferred into naïve recipients. This serum factor has been described in rats as consisting of exosome-like structures or tolerosomes, which express major histocompatibility complex class II molecules (MHCII) and mediate antigen-specific tolerance. In this study, we investigated the functions of serum-derived tolerosomes both in vivo and in vitro. Tolerosomes were purified from the 100,000 g pellet fraction of serum from ovalbumin (OVA)-fed mice. When transferred into naïve recipient mice, the tolerosomes mediated OVA-specific tolerance. We also found that tolerosomes from OVA-fed mice induced the activation of OVA-specific T cells both in vivo and in vitro. The inoculation of severe combined immunodeficiency (SCID) mice with an interferon-gamma-producing cell line normalized the expression of MHCII in the intestinal epithelial cells and restored their ability to generate tolerosomes. Syngeneic but not allogeneic transfer of tolerosomes from OVA-fed donors induced tolerance in the recipients. Our results show that tolerosomes can be isolated from mouse serum, that tolerosome-induced oral tolerance requires MHCII expression in intestinal epithelial cells, and that tolerosomes are functional only in syngeneic recipients.
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Affiliation(s)
- Sofia Ostman
- Department of Rheumatology and Inflammation Research, Göteborg University, Sweden
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20
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Abstract
Visceral sensitivity has been recognized over the last decade as a frequent pathophysiological component of functional bowel disorders. Studies in animals and humans have identified numerous neurotransmitters involved in the processing of sensations from the gut to the brain. However, up to now none of them has actually been proven to have a marked clinical efficacy and the benefit comes rather from their action of bowel disturbances. Reproducible tests are lacking to detect visceral hypersensitivity in humans and distension tests are difficult to undertake in a clinical setting. Therefore, abnormal visceral sensitivity may not be regarded as a tool to select IBS patients as candidates for a given treatment.
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Affiliation(s)
- Michel M Delvaux
- Department of Internal Medicine and Digestive Pathology, CHU de Nancy, Nancy, France.
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21
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Büning J, Hundorfean G, Schmitz M, Zimmer KP, Strobel S, Gebert A, Ludwig D. Antigen targeting to MHC class II-enriched late endosomes in colonic epithelial cells: trafficking of luminal antigens studied in vivo in Crohn's colitis patients. FASEB J 2005; 20:359-61. [PMID: 16373401 DOI: 10.1096/fj.05-4807fje] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
In Crohn's disease (CD), colonic epithelial cells (CECs) are suggested to stimulate pro-inflammatory CD4+ T cells. However, the endocytic pathways of luminal antigens involved in underlying MHC class II presentation by CECs remain unknown. Our aim was to elucidate antigen trafficking and associated MHC class II expression in CECs of CD patients in vivo. In CD patients (Crohn's colitis and remission) and healthy controls undergoing colonoscopy, ovalbumin (OVA) was sprayed onto inflamed or healthy mucosa. The subcellular localization of OVA and MHC class II was visualized in biopsies taken from OVA-incubated mucosa using fluorescence and cryoelectron microscopy. Targeting of OVA into late endosomes of CECs was found in healthy (controls and CD in remission) and inflamed mucosa (Crohn's colitis). MHC class II expression in CECs was not detected in healthy mucosa but strongly up-regulated during CD inflammation. Induced MHC class II in CECs was predominantly seen at basolateral membranes and in late endosomes, which were efficiently accessed by internalized OVA. Our data provide in vivo evidence that the endocytic pathway of luminal antigens in CECs of Crohn's colitis patients intersects MHC class II-enriched late endosomes and support the postulated role of CECs in MHC class II-associated antigen presentation during CD.
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Affiliation(s)
- Jürgen Büning
- Medizinische Klinik I and Institut für Anatomie, Universitätsklinikum Schleswig-Holstein, Lübeck, Germany.
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22
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Safadi R, Alvarez CE, Ohta M, Brimnes J, Kraus T, Mehal W, Bromberg J, Mayer L, Friedman SL. Enhanced oral tolerance in transgenic mice with hepatocyte secretion of IL-10. THE JOURNAL OF IMMUNOLOGY 2005; 175:3577-83. [PMID: 16148101 DOI: 10.4049/jimmunol.175.6.3577] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 +/- 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 +/- 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by approximately 33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-gamma secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.
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Affiliation(s)
- Rifaat Safadi
- Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY 10029, USA
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23
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Bagshaw RD, Mahuran DJ, Callahan JW. Lysosomal membrane proteomics and biogenesis of lysosomes. Mol Neurobiol 2005; 32:27-41. [PMID: 16077181 DOI: 10.1385/mn:32:1:027] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2004] [Accepted: 10/14/2004] [Indexed: 12/30/2022]
Abstract
This review focuses on events involved in the biogenesis of the lysosome. This organelle contains a diverse array of soluble, luminal proteins capable of digesting all the macromolecules in the cell. Altered function of lysosomes or its constituent enzymes has been implicated in a host of human pathologies, including storage diseases, cancer, and infectious and neurodegenerative diseases. Luminal enzymes are well-characterized, and aspects of how they are incorporated into lysosomes are known. However, little is known about the composition of the membrane surrounding the organelle or how the membrane is assembled. Our starting point to study lysosome biogenesis is to define the composition of the membrane by the use of proven methods for purification of lysosomes to near homogeneity and then to characterize membrane-associated and integral lysosomal membrane proteins. This has been achieved using advanced proteomics (electrophoretic or chromatographic separations of proteins followed by time-of-flight mass spectrometric identification of peptide sequences). To date, we have identified 55 proteins in the membrane-associated fraction and 215 proteins in the integral membrane. By applying these methods to mouse models of lysosome dysgenesis (such as BEIGE, Pale Ear, PEARL) that are related to human diseases such as Chediak-Higashi and Hermansky-Pudlak syndromes, it may be possible to define the membrane protein composition of lysosomes in each of these mutants and to determine how they differ from normal. Identifying proteins affected in the respective mutants may provide hints about how they are targeted to the lysosomal membrane and how failure to target them leads to disease; these features are pivotal to understanding lysosome biogenesis and have the potential to implicate lysosomes in a broad range of human pathologies.
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Affiliation(s)
- Richard D Bagshaw
- Research Institute, The Hospital for Sick Children, University of Toronto, Toronto, Canada
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24
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Kraus TA, Brimnes J, Muong C, Liu JH, Moran TM, Tappenden KA, Boros P, Mayer L. Induction of mucosal tolerance in Peyer's patch-deficient, ligated small bowel loops. J Clin Invest 2005; 115:2234-43. [PMID: 16041410 PMCID: PMC1177996 DOI: 10.1172/jci19102] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2003] [Accepted: 05/24/2005] [Indexed: 11/17/2022] Open
Abstract
To explore the requirement for M cells and the Peyer's patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyer's patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.
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Affiliation(s)
- Thomas A Kraus
- Immunobiology Center, Mount Sinai School of Medicine, New York, New York 10029, USA
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25
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Chirdo FG, Millington OR, Beacock-Sharp H, Mowat AM. Immunomodulatory dendritic cells in intestinal lamina propria. Eur J Immunol 2005; 35:1831-40. [PMID: 16010704 DOI: 10.1002/eji.200425882] [Citation(s) in RCA: 179] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
The lamina propria (LP) of the small intestine contains many dendritic cells (DC), which are likely to be in close contact with luminal antigens, but their role in intestinal immune responses has been overlooked. Here we show that after feeding mice ovalbumin (OVA), the majority of antigen uptake is associated with DC in the small intestinal LP, and we describe the isolation, purification and initial characterization of theses DC. We obtained >90% CD11c(+) DC using magnetic cell sorting, of which the majority were CD11b(+)CD8alpha(-), with smaller numbers of CD11b(-)CD8alpha(+) and CD11b(-)CD8alpha(-) DC as well as a distinct population of CD11c(int)class II MHC(lo) B220(+) DC. Freshly isolated LP DC expressed variable but generally low levels of CD40, CD80 and CD86, which were up-regulated by activation with LPS. LP DC were endocytic in vivo and in vitro and could present antigen to OVA-specific CD4(+) T cells in vitro. Antigen-loaded LP DC from OVA-fed mice also primed specific CD4(+) T cells in vivo and in vitro, but adoptive transfer of these DC into naive recipients induced hyporesponsiveness to subsequent challenge. LP DC also expressed significant levels of mRNA for IL-10 and type I IFN, but not IL-12, suggesting they may play a central and unique role in immune homeostasis in the gut.
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Affiliation(s)
- Fernando G Chirdo
- Division of Immunology, Infection and Inflammation, Western Infirmary, Glasgow, Scotland
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26
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Cheroutre H, Kronenberg M. Mucosal T lymphocytes--peacekeepers and warriors. ACTA ACUST UNITED AC 2005; 27:147-65. [PMID: 15931528 DOI: 10.1007/s00281-005-0205-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2005] [Accepted: 04/18/2005] [Indexed: 12/30/2022]
Abstract
Normal immune homeostasis of the intestine requires peaceful coexistence with commensal flora, combined with host defense against pathogens. Perhaps as a result of this unique dilemma, distinct populations of regulatory and effector T lymphocytes are found in the lamina propria and epithelium of the intestine. Here we summarize the properties and functions of these unusual T cells, and describe the molecular and cellular interactions that lead to their development and function. Some mucosal T cells, sometimes called type a, are conventional activated/memory T cells that have received instructions to migrate to the intestine during priming by dendritic cells in the mesenteric lymph node and elsewhere. Others, however, particularly subsets residing permanently in the epithelium, are intestine-specific T cell subpopulations generated by an atypical differentiation pathway.
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Affiliation(s)
- Hilde Cheroutre
- The La Jolla Institute for Allergy and Immunology, San Diego, CA, USA.
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27
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Rask C, Evertsson S, Telemo E, Wold AE. A Full Flora, but not Monocolonization by Escherichia coli or Lactobacilli, Supports Tolerogenic Processing of a Fed Antigen. Scand J Immunol 2005; 61:529-35. [PMID: 15963047 DOI: 10.1111/j.1365-3083.2005.01598.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Fed protein undergoes processing and coupling to major histocompatibility complex (MHC) II molecules during passage through the intestinal epithelium, generating a tolerogenic form of the antigen in serum. Transfer of this factor to naïve animals induces tolerance in the recipient. In this study, we investigate what impact colonization with Gram-positive (Lactobacillus plantarum) or Gram-negative (Escherichia coli) bacteria has on tolerogenic processing in the gut. Germ-free (GF), monocolonized or conventional mice were fed ovalbumin (OVA), and their serum was collected and transferred to naïve conventional recipients that were tested for delayed-type hypersensitivity against OVA after parenteral immunization. A transferable tolerogenic factor was produced by conventional mice, but not by mice that were germ free or monocolonized with either E. coli or L. plantarum. Conventional, but neither GF nor monocolonized mice showed upregulation of MHCII expression in the epithelium of small intestine. The results suggest that a complex intestinal microflora is needed to support oral tolerance development.
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Affiliation(s)
- C Rask
- Department of Clinical Bacteriology, Göteborg University, Göteborg, Sweden.
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28
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Büning J, Schmitz M, Repenning B, Ludwig D, Schmidt MA, Strobel S, Zimmer KP. Interferon-gamma mediates antigen trafficking to MHC class II-positive late endosomes of enterocytes. Eur J Immunol 2005; 35:831-42. [PMID: 15688349 DOI: 10.1002/eji.200425286] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
MHC class II-positive late endosomes of enterocytes are thought to be involved in antigen presentation to CD4(+) T cells. In contrast to enterocytes of BALB/c mice, severe combined immunodeficiency (SCID) enterocytes lack MHC class II expression and fail to transport internalized ovalbumin (OVA) into late endosomes. IFN-gamma is known to induce MHC class II in enterocytes and antigen targeting to late endosomes in macrophages. In this study, we investigated the influence of IFN-gamma and MHC class II on the processes of antigen traffic in enterocytes. Subcellular targeting of OVA and MHC class II expression within enterocytes were examined in SCID, IFN-gamma-treated SCID, BALB/c and C57BL/6 MHC class II knockout (KO) mice after a single feed with OVA. Sorting of OVA into late endosomes was found in enterocytes from BALB/c, C57BL/6 KO and IFN-gamma-stimulated SCID mice, but not from untreated SCID mice. MHC class II expression was restricted to enterocytes of IFN-gamma-treated SCID and BALB/c mice, present at basolateral membranes and within endosomal compartments. These enterocytes further revealed colocalization of class II antigens and OVA in endosomes. We suggest that antigen trafficking into late endosomes of enterocytes is mediated by IFN-gamma and occurs in the absence of MHC class II.
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Affiliation(s)
- Jürgen Büning
- Institut für Anatomie, Universität zu Lübeck, Lübeck, Germany
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29
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30
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Neutra MR, Kraehenbuhl JP. Cellular and Molecular Basis for Antigen Transport Across Epithelial Barriers. Mucosal Immunol 2005. [DOI: 10.1016/b978-012491543-5/50011-5] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
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31
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Chambers SJ, Wickham MSJ, Regoli M, Bertelli E, Gunning PA, Nicoletti C. Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut. Biochem Biophys Res Commun 2004; 325:1258-63. [PMID: 15555562 DOI: 10.1016/j.bbrc.2004.10.161] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2004] [Indexed: 10/26/2022]
Abstract
Although the route of sensitization to food allergens is still the subject of debate, it is generally accepted the gut immune system plays a pivotal role. However, hitherto the transport of allergens across the normal, pre-sensitized gut epithelium remained largely unknown. Our aim was to identify the route through which protein bodies and soluble proteins from digested peanuts penetrated the pre-sensitized gut epithelium in vivo and the specific cell types involved in the transport. Digestion of peanuts released a large number of protein bodies that are exclusively transported across the epithelium by specialized antigen-sampling M cells and delivered to the lymphoid tissue of Peyer's patch. Intracellular transport of soluble protein also occurred almost exclusively via M cells and it was negligible across absorptive enterocytes. We hypothesize that these conditions which are known to favour strongly the induction of immune responses rather than oral tolerance may play a significant role in the genesis of allergic reactions.
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Affiliation(s)
- Stephen J Chambers
- Programmes of Gastrointestinal Health and Function, Institute of Food Research, Norwich, UK
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32
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Abstract
The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.
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Affiliation(s)
- Ke Zen
- Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA.
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33
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Kersting S, Bruewer M, Schuermann G, Klotz A, Utech M, Hansmerten M, Krieglstein CF, Senninger N, Schulzke JD, Naim HY, Zimmer KP. Antigen transport and cytoskeletal characteristics of a distinct enterocyte population in inflammatory bowel diseases. THE AMERICAN JOURNAL OF PATHOLOGY 2004; 165:425-37. [PMID: 15277217 PMCID: PMC1618561 DOI: 10.1016/s0002-9440(10)63308-1] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Intestinal antigen uptake is enhanced in inflammatory bowel disease. We analyzed transcellular transport routes of antigens in different compartments of normal enterocytes and atypical intestinal epithelial cells called "rapid antigen uptake into the cytosol enterocytes" (RACE cells). These cells constitute a recently described population of enterocyte-derived cells, which are increased in inflammatory bowel disease. Mucosa of freshly resected specimens were incubated with the antigens ovalbumin or horseradish peroxidase. Ultrastructural labeling patterns of differentiation-dependent proteins, the brush-border enzyme sucrase-isomaltase and the cytoskeleton proteins villin and actin, were determined in enterocytes. Apoptosis was investigated biochemically and ultrastructurally by cleavage of caspase-3. Both antigens were transported to late endosomes and to trans-Golgi vesicles of enterocytes in inflammatory bowel disease and control specimens. Quantitative evaluation revealed a significantly increased transepithelial antigen transport in both compartments of RACE relative to normal enterocytes. Labeling densities for sucrase-isomaltase, villin, and actin were decreased in RACE relative to normal enterocytes. Caspase-3 was not increased in RACE cells relative to controls. RACE cells are characterized by increased antigen transport to late endosomes and the trans-Golgi network, a disassembled cytoskeleton and lower concentrations of proteins that are markers of cell differentiation.
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Affiliation(s)
- Sabine Kersting
- Department of Pediatrics, University of Muenster, Albert-Schweitzer-Str. 33, 48149 Muenster, Germany.
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34
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Schellscheidt J, Baas S, Schmid KW, Vormoor J, Zimmer KP. Identification of two distinct subpopulations in a ganglioneuroblastoma: lack of co-localization of vasoactive intestinal polypeptide and calcitonin in ganglionic cells at the ultrastructural level. ACTA ACUST UNITED AC 2003; 41:571-3. [PMID: 14595721 DOI: 10.1002/mpo.10427] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Affiliation(s)
- Jörn Schellscheidt
- Klinik und Poliklinik für Kinderheilkunde-Allgemeine Kinderheilkunde, Universitätsklinikum Münster, Münster, Germany
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35
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Abstract
The intestinal immune system has to discriminate between harmful and beneficial antigens. Although strong protective immunity is essential to prevent invasion by pathogens, equivalent responses against dietary proteins or commensal bacteria can lead to chronic disease. These responses are normally prevented by a complex interplay of regulatory mechanisms. This article reviews the unique aspects of the local microenvironment of the intestinal immune system and discuss how these promote the development of regulatory responses that ensure the maintenance of homeostasis in the gut.
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Affiliation(s)
- Allan McI Mowat
- Department of Immunology and Bacteriology, Western Infirmary, Glasgow G11 6NT, UK.
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36
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van Niel G, Heyman M. The epithelial cell cytoskeleton and intracellular trafficking. II. Intestinal epithelial cell exosomes: perspectives on their structure and function. Am J Physiol Gastrointest Liver Physiol 2002; 283:G251-5. [PMID: 12121870 DOI: 10.1152/ajpgi.00102.2002] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Intestinal epithelial cells (IEC) are located at the strategic interface between the external environment and the most extensive lymphoid compartment in the body. Besides their central role in the absorption of nutrients, they also provide sample information to the immune system on soluble or particulate antigens present in the intestinal lumen. Like professional antigen-presenting cells, IEC have recently been shown to secrete 30- to 90-nm diameter vesicles named exosomes from their apical and basolateral surfaces. These vesicles carry molecules that are implicated in adhesion and antigen presentation, such as major histocompatibility complex (MHC) class I molecules, MHC class II molecules, CD63, CD26/dipeptidyl-peptidase IV, tetraspan proteins, and A33 antigen. IEC exosomes therefore, constitute a link by which IEC may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.
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37
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Karlsson MR, Kahu H, Hanson LA, Telemo E, Dahlgren UI. An established immune response against ovalbumin is suppressed by a transferable serum factor produced after ovalbumin feeding: a role of CD25+ regulatory cells. Scand J Immunol 2002; 55:470-7. [PMID: 11975758 DOI: 10.1046/j.1365-3083.2002.t01-1-01079.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
We have previously demonstrated that rats fed ovalbumin (OVA) develop a tolerogenic activity in serum, which upon transfer induces tolerance to OVA and suppression of the immune response to a bystander antigen. Here, we have extended these studies and analysed if the tolerogenic activity in serum could suppress an established immune response in the recipients. Rats were immunized with OVA, 4 and 1 week prior to the transfer of serum from either OVA-fed or control animals. Rats that received serum from OVA-fed donors had significantly lower delayed-type hypersensitivity (DTH) reaction against OVA 1 week after the serum transfer compared with the controls, and the levels of immunoglobulin (IgG) anti-OVA antibodies were significantly lower 2 and 4 weeks after serum transfer. Monomeric OVA in amounts corresponding to the OVA transferred with serum did not induce the reduction of DTH response or IgG anti-OVA antibody levels. In vitro, the proliferation of OVA-stimulated spleen cells, taken from recipients of tolerogenic serum, was significantly lower compared with spleen cells from the controls. The in vitro suppression seemed to be mediated by a population of CD25+ cells, because the removal of such cells from OVA-stimulated spleen cell suspensions resulted in increased proliferation in cultures from rats receiving tolerogenic serum. Our results showed that the tolerogenic serum factor can suppress an established immune response in recipient animals, possibly through induction of regulatory CD25+ cells. Whether this capacity might be used to influence chronic inflammatory conditions needs to be investigated.
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Affiliation(s)
- M R Karlsson
- Department of Clinical Immunology, University of Göteborg, Göteborg, Sweden.
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Ständer S, Böhm M, Brzoska T, Zimmer KP, Luger T, Metze D. Expression of melanocortin-1 receptor in normal, malformed and neoplastic skin glands and hair follicles. Exp Dermatol 2002; 11:42-51. [PMID: 11952827 DOI: 10.1034/j.1600-0625.2002.110105.x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Melanocortin receptors (MC-Rs) are G-protein coupled receptors that mediate pleiotropic actions of melanocyte-stimulating hormones and adrenocorticotropin. There is increasing evidence that one of the five so far identified melanocortin receptors, i.e. melanocortin-1 receptor (MC-1R), has a more ubiquitous distribution in the skin than originally expected. In the present study, the expression of MC-1R in normal skin glands and hair follicles, various malformations and neoplasms with adnexal differentiation is described. Using an anti-MC-1R antibody directed against the amino acids 2-18 of the human MC-1R, specimens of normal healthy skin (n = 10) as well as hamartomas, cysts, hyperplasias, and benign or malignant neoplasms with eccrine, apocrine, sebaceous gland, and hair follicle differentiation (n = 98) were immunostained. MC-1R expression was widely preserved in various adnexal malformations and neoplasms as compared with normal skin and did not show major differences with regard to maturation of the neoplasms. The majority of adnexal epithelia showed an intracytoplasmically granular staining and, to a lesser extent, an intercellular staining pattern. Immunoelectron microscopical investigations revealed expression of MC-1R both along the cell surface and intracytoplasmically within tubular endosomes, the latter suggesting internalisation of the receptor. In conclusion, preserved MC-1R expression in adnexal epithelia suggests a functional role of proopiomelanocortin (POMC) in various malformations and neoplasms of the skin.
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Affiliation(s)
- Sonja Ständer
- Department of Dermatology, Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Münster, Germany.
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Abstract
The development of immunological tolerance to orally fed antigens depends on the sampling, processing and transportation events followed in the intestinal epithelium. We present here a description of a "tolerosome": a supra-molecular, exosome-like structure assembled in and released from the small intestinal epithelial cell. The tolerosome is a approximately 40 nm large vesicular structure that carries MHC class II (MHC II) with bound antigenic peptides sampled from the gut lumen. Tolerosomes isolated from serum shortly after antigen feeding or from an in vitro pulsed intestinal epithelial cell line are fully capable of inducing antigen specific tolerance in naive recipient animals. Purified tolerosomes represent a structure by which fed antigens can be efficiently presented to the immune system. Removal of the tolerosomes from serum by ultracentrifugation or absorption of MHC II results in abrogated tolerance development.
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Affiliation(s)
- M Karlsson
- Department of Clinical Immunology, University of Göteborg, Göteborg, Sweden
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van Niel G, Raposo G, Candalh C, Boussac M, Hershberg R, Cerf-Bensussan N, Heyman M. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 2001; 121:337-49. [PMID: 11487543 DOI: 10.1053/gast.2001.26263] [Citation(s) in RCA: 552] [Impact Index Per Article: 23.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/02/2022]
Abstract
BACKGROUND & AIMS Given the observations that intestinal epithelial cells (IECs) can present antigens to CD4(+) T lymphocytes and that professional antigen-presenting cells secrete exosomes (antigen-presenting vesicles), we hypothesized that IECs may secrete exosomes carrying molecules implicated in antigen presentation, which may be able to cross the basement membrane and convey immune information to noncontiguous immune cells. METHODS Human IEC lines HT29-19A and T84-DRB1*0401/CIITA were grown on microporous filters. Release of exosomes under basal or inflammatory conditions was evaluated in conditioned apical and basolateral media after differential ultracentrifugations. Morphologic and biochemical characterization of exosomes was performed using immunoelectron microscopy, Western blotting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS The intestinal cell lines released 30-90-nm-diameter vesicles from the apical and basolateral sides, and this release was significantly increased in the presence of interferon gamma. MHC class I, MHC class II, CD63, CD26/dipeptidyl-peptidase IV, and A33 antigen were present in epithelial-derived exosomes. CONCLUSIONS; Human IEC lines secrete exosomes bearing accessory molecules that may be involved in antigen presentation. These data are consistent with a model in which IECs may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.
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Affiliation(s)
- G van Niel
- INSERM E9925, Faculté Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
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