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Find video protocols related to scientific articles indexed in Pubmed.
A click-and-release approach to CO prodrugs.
Chem. Commun. (Camb.)
PUBLISHED: 11-07-2014
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Carbon monoxide belongs to the family of signaling molecules and has been shown to possess therapeutic effects. Similar to NO, safe delivery of CO is a key issue in developing CO-based therapeutics. Herein we report a "click and release" CO-prodrug approach, which allows the release of CO under physiological conditions without the need for light irradiation. The system releases CO in a triggered and controllable manner and possesses the potential of tunable release rates.
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A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-14-2014
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Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
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Glycoprotein CD98 as a receptor for colitis-targeted delivery of nanoparticle.
J Mater Chem B Mater Biol Med
PUBLISHED: 04-15-2014
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Treatment strategies for inflammatory bowel disease have been constrained by limited therapeutic efficacy and serious adverse effects owing to a lack of receptor for targeted drug delivery to the inflamed colon. Upon inflammation, CD98 expression is highly elevated in colonic epithelial cells and infiltrating immune cells. To investigate whether CD98 can be used as a colitis-targeted delivery receptor, we constructed CD98 Fab'-bearing quantum dots (QDs)-loaded nanoparticles (Fab'-NPs). The resultant Fab'-NPs had desired particle size (~458 nm) with a narrow size distribution and zeta-potential (approximately +19 mV), low cytotoxicity, and excellent fluorescence properties. Electron microscopy images provided direct evidence for the well-dispersed distribution of QDs within spherical Fab'-NPs. Cellular uptake experiments demonstrated that Fab'-NPs were efficiently internalized into Colon-26 and RAW 264.7 cells through the CD98-mediated endocytosis pathway, and showed that the targeting effect of CD98 Fab' markedly increased their cellular uptake efficiency compared with control pegylated QDs-loaded NPs (PEG-NPs). Furthermore, ex vivo studies showed much more effective accumulation of Fab'-NPs in colitis tissue than that of PEG-NPs. These findings suggest that because of inflammation-dependent over-expression of CD98, active colitis-targeted delivery can be accomplished using NPs decorated with CD98 antibody.
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Genetic deletion of Klf4 in the mouse intestinal epithelium ameliorates dextran sodium sulfate-induced colitis by modulating the NF-?B pathway inflammatory response.
Inflamm. Bowel Dis.
PUBLISHED: 04-01-2014
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Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in the differentiated epithelial cells lining of the intestine. Under physiological conditions, KLF4 inhibits cell proliferation. Conversely, KLF4 mediates proinflammatory signaling in macrophages and its overexpression in the esophageal epithelium activates cytokines, leading to inflammation-mediated esophageal squamous cell cancer formation in mice. Here, we tested whether KLF4 has a proinflammatory activity in experimental colitis in mice.
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Longitudinal study of circulating protein biomarkers in inflammatory bowel disease.
J Proteomics
PUBLISHED: 03-15-2014
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Inflammatory bowel diseases (IBDs) are chronic and progressive inflammatory disorders of the gastrointestinal tract. In IBD, protein serological biomarkers could be relevant tools for assessing disease activity, performing early-stage diagnosis and managing the treatment. Using the interleukin-10 knockout (IL-10(-/-)) mouse, a model that develops a time-dependent IBD-like disorder that predominates in the colon; we performed longitudinal studies of circulating protein biomarkers in IBD. Circulating protein profiles in serum samples collected from 30-, 93-, to 135-day-old IL-10(-/-) mice were investigated using two-dimensional differential gel electrophoresis and MALDI-TOF/TOF tandem mass spectrometry. A total of 15 different proteins were identified and confirmed by ELISA and Western blot to be differentially accumulated in serum samples from mid- to late-stage IL-10(-/-) mice compared to early non-inflamed IL-10(-/-) mice. The use of another model of colitis and an extra-intestinal inflammation model validated this biomarker panel and demonstrated that comprised some global inflammatory markers, some intestinal inflammation-specific markers and some chronic intestinal inflammation markers. Statistical analyses using misclassification error rate charts validated the use of these identified proteins as powerful biomarkers of colitis. Unlike standard biomarker screening studies, our analyses identified a panel of proteins that allowed the definition of protein signatures that reflect colitis status.
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Fab'-bearing siRNA TNF?-loaded nanoparticles targeted to colonic macrophages offer an effective therapy for experimental colitis.
J Control Release
PUBLISHED: 03-07-2014
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Patients suffering from inflammatory bowel disease (IBD) are currently treated by systemic drugs that can have significant side effects. Thus, it would be highly desirable to target TNF? siRNA (a therapeutic molecule) to the inflamed tissue. Here, we demonstrate that TNF? siRNA can be efficiently loaded into nanoparticles (NPs) made of poly (lactic acid) poly (ethylene glycol) block copolymer (PLA-PEG), and that grafting of the Fab' portion of the F4/80 Ab (Fab'-bearing) onto the NP surface via maleimide/thiol group-mediated covalent bonding improves the macrophage (MP)-targeting kinetics of the NPs to RAW264.7 cells in vitro. Direct binding was shown between MPs and the Fab'-bearing NPs. Next, we orally administered hydrogel (chitosan/alginate)-encapsulated Fab'-bearing TNF?-siRNA-loaded NPs to 3% dextran sodium sulfate (DSS)-treated mice and investigated the therapeutic effect on colitis. In vivo, the release of TNF?-siRNA-loaded NPs into the mouse colon attenuated colitis more efficiently when the NPs were covered with Fab'-bearing, compared to uncovered NPs. All DSS-induced parameters of colonic inflammation (e.g., weight loss, myeloperoxidase activity, and I?b? accumulation) were more attenuated Fab'-bearing NPs loaded with TNF? siRNA than without the Fab'-bearing. Grafting the Fab'-bearing onto the NPs improved the kinetics of endocytosis as well as the MP-targeting ability, as indicated by flow cytometry. Collectively, our results show that Fab'-bearing PLA-PEG NPs are powerful and efficient nanosized tools for delivering siRNAs into colonic macrophages.
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Micheliolide, a new sesquiterpene lactone that inhibits intestinal inflammation and colitis-associated cancer.
Lab. Invest.
PUBLISHED: 02-13-2014
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Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal (GI) tract associated with an increased risk of colorectal cancer (CRC). Current treatments for both IBD and colitis-associated CRC suffer from numerous side effects. Parthenolide (PTL) is a sesquiterpene lactone with anti-inflammatory activity, and previous studies have demonstrated that PTL is a potent inhibitor of the NF-?B pathway. Micheliolide (MCL), substantially more stable than PTL in vivo, was recently developed, and this study aimed to decipher its suitability as therapeutic tool for IBD and IBD-associated diseases. Similar to PTL, MCL inhibited NF-?B activation and subsequent pro-inflammatory pathways activation in vitro. Pro-drug forms of both compounds inhibited the DSS-induced colitis when administrated intraperitoneally or encapsulated in a polysaccharide gel designed to release drugs in the colon. Interestingly, MCL was found to attenuate carcinogenesis in AOM/DSS-induced CRC, thus providing new candidate for the treatment of inflammatory bowel disease and CRC.
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Nanoparticles with surface antibody against CD98 and carrying CD98 small interfering RNA reduce colitis in mice.
Gastroenterology
PUBLISHED: 01-28-2014
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Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis.
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Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, plays an important role in cell-to-cell communication during colitis.
PLoS ONE
PUBLISHED: 01-01-2014
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PepT1 is a member of the proton-oligopeptide cotransporter family SLC15, which mediates the transport of di/tripeptides from intestinal lumen into epithelial cells. MicroRNAs (miRNAs), a small noncoding RNAs (21-23 nucleotides), post-transcriptionally regulate gene expression by binding to the 3'-untranslated regions (UTRs) of their target mRNAs. Although the role of most miRNAs remains elusive, they have been implicated in vital cellular functions such as intestinal epithelial cells differentiation, proliferation, and apoptosis. In the present study, we investigated the effect of intestinal epithelial PepT1 expression on microRNA (miRNA) expression/secretion in the colons of control mice and in mice with experimentally induced colonic inflammation (colitis). The colonic miRNA expression was deregulated in both colitis and control mice but the deregulation of miRNA expression/secretion was specific to colonic tissue and did not affect other tissues such as spleen and liver. Intestinal epithelial PepT1-dependent deregulation of colonic miRNA expression not only affects epithelial cells but also other cell types, such as intestinal macrophages. Importantly, we found the miRNA 23b which was known to be involved in inflammatory bowel disease was secreted and transported between cells to impose a gene-silencing effect on recipient intestinal macrophages. Based on our data, we may conclude that the expression of a specific protein, PepT1, in the intestine affects local miRNA expression/secretion in the colon on a tissue specific manner and may play an important role during the induction and progression of colitis. Colonic miRNA expression/secretion, regulated by intestinal epithelial PepT1, could play a crucial role in cell-to-cell communication during colitis.
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Tumor necrosis factor-neuropeptide Y cross talk regulates inflammation, epithelial barrier functions, and colonic motility.
Inflamm. Bowel Dis.
PUBLISHED: 10-11-2013
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Neuro-immune interactions play a significant role in regulating the severity of inflammation. Our previous work demonstrated that neuropeptide Y (NPY) is upregulated in the enteric nervous system during murine colitis and that NPY knockout mice exhibit reduced inflammation. Here, we investigated if NPY expression during inflammation is induced by tumor necrosis factor (TNF), the main proinflammatory cytokine.
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Mannosylated bioreducible nanoparticle-mediated macrophage-specific TNF-? RNA interference for IBD therapy.
Biomaterials
PUBLISHED: 05-13-2013
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The application of RNA interference (RNAi) for in?ammatory bowel disease (IBD) therapy has been limited by the lack of non-cytotoxic, efficient and targetable small interfering RNA (siRNA) carriers. TNF-? is the major pro-inflammatory cytokine mainly secreted by macrophages during IBD. Here, a mannosylated bioreducible cationic polymer (PPM) was synthesized and further spontaneously assembled nanoparticles (NPs) assisted by sodium triphosphate (TPP). The TPP-PPM/siRNA NPs exhibited high uniformity (polydispersity index = 0.004), a small particle size (211-275 nm), excellent bioreducibility, and enhanced cellular uptake. Additionally, the generated NPs had negative cytotoxicity compared to control NPs fabricated by branched polyethylenimine (bPEI, 25 kDa) or Oligofectamine (OF) and siRNA. In vitro gene silencing experiments revealed that TPP-PPM/TNF-? siRNA NPs with a weight ratio of 40:1 showed the most efficient inhibition of the expression and secretion of TNF-? (approximately 69.9%, which was comparable to the 71.4% obtained using OF/siRNA NPs), and its RNAi efficiency was highly inhibited in the presence of mannose (20 mm). Finally, TPP-PPM/siRNA NPs showed potential therapeutic effects on colitis tissues, remarkably reducing TNF-? level. Collectively, these results suggest that non-toxic TPP-PPM/siRNA NPs can be exploited as efficient, macrophage-targeted carriers for IBD therapy.
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NF-?B pathway in colitis-associated cancers.
Transl Gastrointest Cancer
PUBLISHED: 04-30-2013
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Colitis-associated cancer is the subtype of colorectal cancer that is associated with inflammatory bowel diseases including ulcerative colitis and Crohns disease. Colorectal cancer is one of the most commonly diagnosed cancers, and is the third leading cause of cancer death in developed countries. Of the signaling pathways involved in colonic inflammation, that triggered by NF-?B plays a key role. A relationship between inflammation and cancer is now well documented. Moreover, the association between NF-?B activity and cancer development has been intensively investigated. The present review focuses on the activity of the NF-?B signaling pathway in colitis-associated carcinogenesis. The pivotal roles played by this pathway in apoptosis, tumor promotion, and tumor maintenance strongly suggest that inhibitors of the pathway would be powerful anti-cancer agents.
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Targeting Intestinal Inflammation With CD98 siRNA/PEI-loaded Nanoparticles.
Mol. Ther.
PUBLISHED: 04-17-2013
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Intestinal CD98 expression plays a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in intestinal cells therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as inflammatory bowel disease. Here, the advantages of nanoparticles (NPs) are used, including their ability to easily pass through physiological barriers and evade phagocytosis, high loading concentration, rapid kinetics of mixing and resistance to degradation. Using physical chemistry characterizations techniques, CD98 siRNA/polyethyleneimine (PEI)-loaded NPs was characterized (diameter of ~480?nm and a zeta potential of -5.26 mV). Interestingly, CD98 siRNA can be electrostatically complexed by PEI and thus protected from RNase. In addition, CD98 siRNA/PEI-loaded NPs are nontoxic and biocompatible with intestinal cells. Oral administration of CD98/PEI-loaded NPs encapsulated in a hydrogel reduced CD98 expression in mouse colonic tissues and decreased dextran sodium sulfate-induced colitis in a mouse model. Finally, flow cytometry showed that CD98 was effectively downregulated in the intestinal epithelial cells and intestinal macrophages of treated mice. Finally, the results collectively demonstrated the therapeutic effect of "hierarchical nano-micro particles" with colon-homing capabilities and the ability to directly release "molecularly specific" CD98 siRNA in colonic cells, thereby decreasing colitis.
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Nanotechnology in diagnostics and therapeutics for gastrointestinal disorders.
Dig Liver Dis
PUBLISHED: 02-27-2013
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This review describes the state of the art in nanoparticle and nanodevice applications for medical diagnosis and disease treatment. Nanodevices, such as cantilevers, have been integrated into high-sensitivity disease marker diagnostic detectors and devices, are stable over long periods of time, and display reliable performance properties. Nanotechnology strategies have been applied to therapeutic purposes as well. For example, nanoparticle-based delivery systems have been developed to protect drugs from degradation, thereby reducing the required dose and dose frequency, improving patient comfort and convenience during treatment, and reducing treatment expenses. The main objectives for integrating nanotechnologies into diagnostic and therapeutic applications in the context of intestinal diseases are reviewed.
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PepT1 expressed in immune cells has an important role in promoting the immune response during experimentally induced colitis.
Lab. Invest.
PUBLISHED: 02-12-2013
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We and others have shown that the dipeptide cotransporter PepT1 is expressed in immune cells, including macrophages that are in close contact with the lamina propria of the small and large intestines. In the present study, we used PepT1-knockout (KO) mice to explore the role played by PepT1 in immune cells during dextran sodium sulfate (DSS)-induced colitis. DSS treatment caused less severe body weight loss, diminished rectal bleeding, and less diarrhea in PepT1-KO mice than in wild-type (WT) animals. A histological examination of colonic sections revealed that the colonic architecture was less disrupted and the extent of immune cell infiltration into the mucosa and submucosa following DSS treatment was reduced in PepT1-KO mice compared with WT animals. Consistent with these results, the DSS-induced colitis increase in colonic myeloperoxidase activity was significantly less in PepT1-KO mice than in WT littermates. The colonic levels of mRNAs encoding the inflammatory cytokines CXCL1, interleukin (IL)-6, monocyte chemotactic protein-1, IL-12, and interferon-? were significantly lower in DSS-treated PepT1-KO mice than in DSS-treated WT animals. Colonic immune cells from WT had significantly higher level of proinflammatory cytokines then PepT1 KO. In addition, we observed that knocking down the PepT1 expression decreases chemotaxis of immune cells recruited during intestinal inflammation. Antibiotic treatment before DSS-induced colitis eliminated the differential expression of inflammatory cytokines between WT and PepT1-KO mice. In conclusion, PepT1 in immune cells regulates the secretion of proinflammatory cytokines triggered by bacteria and/or bacterial products, and thus has an important role in the induction of colitis. PepT1 may transport small bacterial products, such as muramyl dipeptide and the tripeptide L-Ala-gamma-D-Glu-meso-DAP, into macrophages. These materials may be sensed by members of the nucleotide-binding site-leucine-rich repeat family of intracellular receptors, ultimately resulting in altered homeostasis of the intestinal microbiota.
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Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA.
BMC Res Notes
PUBLISHED: 01-31-2013
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Dextran sodium sulfate (DSS) is commonly used in mouse studies to induce a very reproducible colitis that effectively mimics the clinical and histological features of human inflammatory bowel disease (IBD) patients, especially ulcerative colitis. However, the mechanisms of action of DSS remain poorly understood, and observations by our laboratory and other groups indicate that DSS contamination of colonic tissues from DSS-treated mice potently inhibits the quantitative reverse-transcription polymerase chain reaction (qRT-PCR) amplification of mRNA.
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Intestinal epithelial CD98 directly modulates the innate host response to enteric bacterial pathogens.
Infect. Immun.
PUBLISHED: 01-07-2013
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CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. Enteropathogenic Escherichia coli (EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactions in vitro and ex vivo and examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected with Citrobacter rodentium as an in vivo model. In vivo studies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment of C. rodentium in mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue of C. rodentium-infected hCD98 Tg mice compared to that of WT mice. Ex vivo studies correlate with the in vivo data. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down. In vitro surface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentium proteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.
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The role and pathophysiological relevance of membrane transporter PepT1 in intestinal inflammation and inflammatory bowel disease.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 12-22-2011
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Intestinal inflammation is characterized by epithelial disruption, leading to loss of barrier function and the recruitment of immune cells, including neutrophils. Although the mechanisms are not yet completely understood, interactions between environmental and immunological factors are thought to be critical in the initiation and progression of intestinal inflammation. In recent years, it has become apparent that the di/tripeptide transporter PepT1 may play an important role in the pathogenesis of such inflammation. In healthy individuals, PepT1 is primarily expressed in the small intestine and transports di/tripeptides for metabolic purposes. However, during chronic inflammation such as that associated with inflammatory bowel disease, PepT1 expression is upregulated in the colon, wherein the protein is normally expressed either minimally or not at all. Several recent studies have shown that PepT1 binds to and transports various bacterial di/tripeptides into colon cells, leading to activation of downstream proinflammatory responses via peptide interactions with innate immune receptors. In the present review, we examine the relationship between colonic PepT1-mediated peptide transport in the colon and activation of innate immune responses during disease. It is important to understand the mechanisms of PepT1 action during chronic intestinal inflammation to develop future therapies addressing inappropriate immune activation in the colon.
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ADAM15 to ?5?1 integrin switch in colon carcinoma cells: a late event in cancer progression associated with tumor dedifferentiation and poor prognosis.
Int. J. Cancer
PUBLISHED: 11-09-2011
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ADAM15, a member of the A Disintegrin And Metalloproteinase (ADAM) family, is a membrane protein containing an adhesion domain that binds to ?5?1 integrin through a unique RGD domain. ADAM15, expressed by human normal colonocytes, is involved in epithelial wound healing and tissue remodeling in inflammatory bowel disease. The aims of our study were (i) to analyze ADAM15 expression in a series of colon carcinomas and paired normal mucosa and (ii) to integrate the spatial relationship of ADAM15 with its binding partners ?5?1 integrin, a mesenchymal marker, as well as with other adhesion molecules, ?3?1 integrin and E-cadherin. A series of 94 colon carcinomas of the non other specified category were graded according to the World Health Organization classification. Immunohistochemistry was performed on frozen tissue sections using antibodies directed to ADAM15, ?5?1 and ?3?1 integrins, and E-cadherin. ADAM15 was quantified at the mRNA level. Finally, promoter methylation of ADAM15 was examined as well as the microsatellite instability status (MSS/MSI). Thirty-six percent of colorectal carcinomas displayed a reduced expression of ADAM15 in cancer cells, confirmed at the mRNA level in most cases, without promoter methylation. ADAM15 down-regulation was associated with histologically poorly differentiated carcinomas. In addition, it was associated with the acquisition of ?5?1 by cancer cells and down-regulation of ?3?1 integrin and E-cadherin. Finally this profile that includes characteristic of epithelial to mesenchymal transition is a late progression event of colon cancer with a poor prognosis.
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L-Ala-?-D-Glu-meso-diaminopimelic acid (DAP) interacts directly with leucine-rich region domain of nucleotide-binding oligomerization domain 1, increasing phosphorylation activity of receptor-interacting serine/threonine-protein kinase 2 and its interacti
J. Biol. Chem.
PUBLISHED: 07-12-2011
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The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-?-D-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-?B activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a K(d) value of 34.5 ?M. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a K(d) value of 4.13 ?M. However, NOD1/RICK binding was of higher affinity (K(d) of 3.26 ?M) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity.
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Overexpression of Ste20-related proline/alanine-rich kinase exacerbates experimental colitis in mice.
J. Immunol.
PUBLISHED: 06-24-2011
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Inflammatory bowel disease, mainly Crohns disease and ulcerative colitis, are characterized by epithelial barrier disruption and altered immune regulation. Colonic Ste20-like proline/alanine-rich kinase (SPAK) plays a role in intestinal inflammation, but its underlying mechanisms need to be defined. Both SPAK-transfected Caco2-BBE cells and villin-SPAK transgenic (TG) FVB/6 mice exhibited loss of intestinal barrier function. Further studies demonstrated that SPAK significantly increased paracellular intestinal permeability to FITC-dextran. In vivo studies using the mouse models of colitis induced by dextran sulfate sodium (DSS) and trinitrobenzene sulfonic acid showed that TG FVB/6 mice were more susceptible to DSS and trinitrobenzene sulfonic acid treatment than wild-type FVB/6 mice, as demonstrated by clinical and histological characteristics and enzymatic activities. Consistent with this notion, we found that SPAK increased intestinal epithelial permeability, which likely facilitated the production of inflammatory cytokines in vitro and in vivo, aggravated bacterial translocation in TG mice under DSS treatment, and consequently established a context favorable for the triggering of intestinal inflammation cascades. In conclusion, overexpression of SPAK inhibits maintenance of intestinal mucosal innate immune homeostasis, which makes regulation of SPAK important to attenuate pathological responses in inflammatory bowel disease.
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The PepT1-NOD2 signaling pathway aggravates induced colitis in mice.
Gastroenterology
PUBLISHED: 04-06-2011
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The human di/tripeptide transporter human intestinal H-coupled oligonucleotide transporter (hPepT1) is abnormally expressed in colons of patients with inflammatory bowel disease, although its exact role in pathogenesis is unclear. We investigated the contribution of PepT1 to intestinal inflammation in mouse models of colitis and the involvement of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway in the pathogenic activity of colonic epithelial hPepT1.
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Microbiota modulate host gene expression via microRNAs.
PLoS ONE
PUBLISHED: 03-28-2011
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Microbiota are known to modulate host gene expression, yet the underlying molecular mechanisms remain elusive. MicroRNAs (miRNAs) are importantly implicated in many cellular functions by post-transcriptionally regulating gene expression via binding to the 3-untranslated regions (3-UTRs) of the target mRNAs. However, a role for miRNAs in microbiota-host interactions remains unknown. Here we investigated if miRNAs are involved in microbiota-mediated regulation of host gene expression. Germ-free mice were colonized with the microbiota from pathogen-free mice. Comparative profiling of miRNA expression using miRNA arrays revealed one and eight miRNAs that were differently expressed in the ileum and the colon, respectively, of colonized mice relative to germ-free mice. A computational approach was then employed to predict genes that were potentially targeted by the dysregulated miRNAs during colonization. Overlapping the miRNA potential targets with the microbiota-induced dysregulated genes detected by a DNA microarray performed in parallel revealed several host genes that were regulated by miRNAs in response to colonization. Among them, Abcc3 was identified as a highly potential miRNA target during colonization. Using the murine macrophage RAW 264.7 cell line, we demonstrated that mmu-miR-665, which was dysregulated during colonization, down-regulated Abcc3 expression by directly targeting the Abcc3 3-UTR. In conclusion, our study demonstrates that microbiota modulate host microRNA expression, which could in turn regulate host gene expression.
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Notch1 regulates the effects of matrix metalloproteinase-9 on colitis-associated cancer in mice.
Gastroenterology
PUBLISHED: 03-08-2011
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Inflammatory bowel disease increases the risks of colon cancer and colitis-associated cancer (CAC). Epithelial cell-derived matrix metalloproteinase (MMP)-9 mediates inflammation during acute colitis and the cleavage and activation of the transcription factor Notch1, which prevents differentiation of progenitor cells into goblet cells. However, MMP-9 also protects against the development of CAC and acts as a tumor suppressor. We investigated the mechanisms by which MMP-9 protects against CAC in mice.
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Functional TNF? gene silencing mediated by polyethyleneimine/TNF? siRNA nanocomplexes in inflamed colon.
Biomaterials
PUBLISHED: 03-05-2011
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During inflammatory bowel disease, TNF? is the major pro-inflammatory cytokine mainly secreted from macrophages and dendritic cells. Here, we have demonstrated that TNF? siRNA/polyethyleneimine loaded into polylactide at an optimal concentration of 20 g/L nanoparticles covered with polyvinyl alcohol are efficiently taken up by inflamed macrophages and inhibit TNF? secretion by the macrophages. Those nanoparticles have a diameter of ?380 nm and zeta potential of -8 mV at pH 7.2, and are non-cytotoxic. Complexation, interactions and protection from RNAse between TNF? siRNA and polyethyleneimine were higher than those using chitosan. Importantly, complexation between TNF? siRNA and polyethyleneimine facilitated higher rates of siRNA loading into nanoparticles, compared to Chi or free siRNA mixed with Lipofectamine. Oral administration of encapsulated TNF? siRNA-loaded nanoparticles specifically reduced the TNF? expression/secretion in colonic tissue in LPS-treated mice. In conclusion, we have shown: (1) that proposed TNF? siRNA-loaded NPs are prepared via a non-denaturing synthetic process; (2) a high encapsulation rate of TNF? siRNA complexed to polyethyleneimine into NPs; (3) effective enzymatic protection of TNF? siRNA by polyethyleneimine; (4) non-cytotoxicity and biodegradability of nanoparticles loaded with polyethyleneimine/TNF? siRNA; and (5) in vitro and in vivo significant anti-inflammatory effects at low TNF? siRNA dose that is specific and restricted to the colonic cells. Our results collectively indicate that polyethyleneimine/TNF? siRNA nanocomplexes represent an efficient therapeutic option for diseases such as IBD.
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CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice.
J. Clin. Invest.
PUBLISHED: 01-26-2011
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Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.
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Nanomedicine in GI.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 12-09-2010
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Recent advances in nanotechnology offer new hope for disease detection, prevention, and treatment. Nanomedicine is a rapidly evolving field wherein targeted therapeutic approaches using nanotechnology based on the pathophysiology of gastrointestinal diseases are being developed. Nanoparticle vectors capable of delivering drugs specifically and exclusively to regions of the gastrointestinal tract affected by disease for a prolonged period of time are likely to significantly reduce the side effects of existing otherwise effective treatments. This review aims at integrating various applications of the most recently developed nanomaterials that have tremendous potential for the detection and treatment of gastrointestinal diseases.
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MicroRNA-92b regulates expression of the oligopeptide transporter PepT1 in intestinal epithelial cells.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 10-28-2010
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MicroRNAs (miRNAs), which are noncoding RNAs that posttranscriptionally inhibit expression of target genes, have recently emerged as important regulators of many cellular functions such as cell differentiation. The epithelial di/tripeptide membrane transporter PepT1 is expressed in highly differentiated cells (the villous tip) but not in undifferentiated cells (the crypt) of the small intestine. Here, we investigated the regulation of PepT1 expression by miRNAs and its functional consequences. We observed a reverse correlation between the expression levels of PepT1 and mature miRNA-92b (miR-92b) during the differentiation of intestinal epithelial Caco2-BBE cells, suggesting a miR-92b-mediated regulation of PepT1 expression. We demonstrate that miR-92b suppressed PepT1 expression at both mRNA and protein levels, with subsequent reduced PepT1 transport activity, in Caco2-BBE cells by directly targeting the PepT1 3-untranslated region. In addition, miR-92b suppresses bacterial peptide-induced proinflammatory responses in intestinal epithelial cells by inhibiting PepT1 expression. Altogether, our study provides for the first time evidence for the regulation of PepT1 expression at a posttranscriptional level by miRNAs in intestinal epithelial cells during pathophysiological states.
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Orally delivered thioketal nanoparticles loaded with TNF-?-siRNA target inflammation and inhibit gene expression in the intestines.
Nat Mater
PUBLISHED: 08-19-2010
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Small interfering RNAs (siRNAs) directed against proinflammatory cytokines have the potential to treat numerous diseases associated with intestinal inflammation; however, the side-effects caused by the systemic depletion of cytokines demands that the delivery of cytokine-targeted siRNAs be localized to diseased intestinal tissues. Although various delivery vehicles have been developed to orally deliver therapeutics to intestinal tissue, none of these strategies has demonstrated the ability to protect siRNA from the harsh environment of the gastrointestinal tract and target its delivery to inflamed intestinal tissue. Here, we present a delivery vehicle for siRNA, termed thioketal nanoparticles (TKNs), that can localize orally delivered siRNA to sites of intestinal inflammation, and thus inhibit gene expression in inflamed intestinal tissue. TKNs are formulated from a polymer, poly-(1,4-phenyleneacetone dimethylene thioketal), that degrades selectively in response to reactive oxygen species (ROS). Therefore, when delivered orally, TKNs release siRNA in response to the abnormally high levels of ROS specific to sites of intestinal inflammation. Using a murine model of ulcerative colitis, we demonstrate that orally administered TKNs loaded with siRNA against the proinflammatory cytokine tumour necrosis factor-alpha (TNF-?) diminish TNF-? messenger RNA levels in the colon and protect mice from ulcerative colitis.
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Nanoparticle-based therapeutic delivery of prohibitin to the colonic epithelial cells ameliorates acute murine colitis.
Inflamm. Bowel Dis.
PUBLISHED: 07-26-2010
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Intestinal epithelial expression of antioxidants and nuclear factor kappa B (NF-?B) contribute to mucosal barrier integrity and epithelial homeostasis, two key events in the pathogenesis of inflammatory bowel disease (IBD). Genetic restoration of intestinal epithelial prohibitin 1 (PHB) levels during experimental colitis reduces the severity of disease through sustained epithelial antioxidant expression and reduced NF-?B activation. To determine the therapeutic potential of restoring epithelial PHB during experimental colitis in mice, we assessed two methods of PHB colonic mucosal delivery: adenovirus-directed administration by enema and poly(lactic acid) nanoparticle (NPs) delivery by gavage.
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PepT1 mediates transport of the proinflammatory bacterial tripeptide L-Ala-{gamma}-D-Glu-meso-DAP in intestinal epithelial cells.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 06-17-2010
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PepT1 is a di/tripeptide transporter highly expressed in the small intestine, but poorly or not expressed in the colon. However, during chronic inflammation, such as inflammatory bowel disease, PepT1 expression is induced in the colon. Commensal bacteria that colonize the human colon produce a large amount of di/tripeptides. To date, two bacterial peptides (N-formylmethionyl-leucyl-phenylalanine and muramyl dipeptide) have been identified as substrates of PepT1. We hypothesized that the proinflammatory tripeptide l-Ala-gamma-d-Glu-meso-DAP (Tri-DAP), a breakdown product of bacterial peptidoglycan, is transported into intestinal epithelial cells via PepT1. We found that uptake of glycine-sarcosine, a specific substrate of PepT1, in intestinal epithelial Caco2-BBE cells was inhibited by Tri-DAP in a dose-dependent manner. Tri-DAP induced activation of NF-kappaB and MAP kinases, consequently leading to production of the proinflammatory cytokine interleukin-8. Tri-DAP-induced inflammatory response in Caco2-BBE cells was significantly suppressed by silencing of PepT1 expression by using PepT1-shRNAs in a tetracycline-regulated expression (Tet-off) system. Colonic epithelial HT29-Cl.19A cells, which do not express PepT1 under basal condition, were mostly insensitive to Tri-DAP-induced inflammation. However, HT29-Cl.19A cells exhibited proinflammatory response to Tri-DAP upon stable transfection with a plasmid encoding PepT1. Accordingly, Tri-DAP significantly increased keratinocyte-derived chemokine production in colonic tissues from transgenic mice expressing PepT1 in intestinal epithelial cells. Finally, Tri-DAP induced a significant drop in intracellular pH in intestinal epithelial cells expressing PepT1, but not in cells that did not express PepT1. Our data collectively support the classification of Tri-DAP as a novel substrate of PepT1. Given that PepT1 is highly expressed in the colon during inflammation, PepT1-mediated Tri-DAP transport may occur more effectively during such conditions, further contributing to intestinal inflammation.
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MicroRNAs determine human intestinal epithelial cell fate.
Differentiation
PUBLISHED: 04-13-2010
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MicroRNAs (miRNAs) are small, non-coding RNA molecules that post-transcriptionally regulate gene expression. Evidence has shown that miRNAs play important roles in various cellular processes, including proliferation, differentiation and survival. The intestinal epithelium is regenerated throughout life, and enterocytes undergo differentiation during migration along the crypt/villus axis. Our study aimed at establishing the expression profiles of miRNAs during intestinal epithelial cell (IEC) differentiation and determining a miRNA "signature" that distinguishes between small and large IECs. MiRNA arrays were employed to profile miRNA expression in two IEC models: the enterocyte-like Caco2-BBE and the colonocyte-like HT29-Cl.19A cell lines. Microarray data showed that in both cell lineages, the differentiated stage exhibited a different miRNA expression profile from undifferentiated stage. Interestingly, Caco2-BBE cells were distinguished from HT29-Cl.19A cells by their unique miRNA expression profile. Notably, HT29-Cl.19A cells exhibited down-regulation of miR-1269 and up-regulation of miR-99b and miR-125a-5p compared with Caco2-BBE cells. Most importantly, transfection of Caco2-BBE cells with mature miR-99b, mature miR-125a-5p and antisense of mature miR-1269 decreased growth rate and trans-epithelial resistance of the cells, indicating their shift toward HT29-Cl.19A cell phenotype. In conclusion, our study shows that miRNAs might play a role in determining the unique physiological characteristics of IECs.
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Adenosine 2B receptor expression is post-transcriptionally regulated by microRNA.
J. Biol. Chem.
PUBLISHED: 04-13-2010
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We have reported that epithelial adenosine 2B receptor (A(2B)AR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor alpha (TNF-alpha). Here, we examined the mechanism that governs A(2B)AR expression during colitis. A 1.4-kb sequence of the A(2B)AR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3-untranslated region (UTR) of A(2B)AR mRNA was examined by cloning this 3-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-alpha induced a 35-fold increase in A(2B)AR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A(2B)AR mRNA following TNF-alpha treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3-UTR of the A(2B)AR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-alpha reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A(2B)AR levels by >60%. Blockade of miR27b increased A(2B)AR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A(2B)AR mRNA. Collectively, these data demonstrate that TNF-alpha-induced A(2B)AR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A(2B)AR expression in murine colitis.
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Matrix metalloproteinase-9 functions as a tumor suppressor in colitis-associated cancer.
Cancer Res.
PUBLISHED: 01-12-2010
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There is a well-documented association of matrix metalloproteinase-9 (MMP-9) and receptor Notch-1 overexpression in colon cancer. We recently showed that MMP-9 is also upregulated in colitis, where it modulates tissue damage and goblet cell differentiation via proteolytic cleavage of Notch-1. In this study, we investigated whether MMP-9 is critical for colitis-associated colon cancer (CAC). Mice that are wild type (WT) or MMP-9 nullizygous (MMP-9(-/-)) were used for in vivo studies and the human enterocyte cell line Caco2-BBE was used for in vitro studies. CAC was induced in mice using an established carcinogenesis protocol that involves exposure to azoxymethane followed by treatment with dextran sodium sulfate. MMP-9(-/-) mice exhibited increased susceptibility to CAC relative to WT mice. Elevations in tumor multiplicity, size, and mortality were associated with increased proliferation and decreased apoptosis. Tumors formed in MMP-9(-/-) mice exhibited expression of p21(WAF1/Cip1) and increased expression of beta-catenin relative to WT mice. In vitro studies of MMP-9 overexpression showed increased Notch-1 activation with a reciprocal decrease in beta-catenin. Notch and beta-catenin/Wnt signaling have crucial roles in determining differentiation and carcinogenesis in gut epithelia. Despite being a mediator of proinflammatory responses in colitis, MMP-9 plays a protective role and acts as a tumor suppressor in CAC by modulating Notch-1 activation, thereby resulting in activation of p21(WAF1/Cip1) and suppression of beta-catenin.
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MicroRNA-7 modulates CD98 expression during intestinal epithelial cell differentiation.
J. Biol. Chem.
PUBLISHED: 11-04-2009
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The transmembrane glycoprotein CD98 regulates multiple cellular functions, including extracellular signaling, epithelial cell adhesion/polarity, amino acid transport, and cell-cell interactions. MicroRNAs post-transcriptionally regulate gene expression, thereby functioning as modulators of numerous cellular processes, such as cell differentiation, proliferation, and apoptosis. Here, we investigated if microRNAs regulate CD98 expression during intestinal epithelial cell differentiation and inflammation. We found that microRNA-7 repressed CD98 expression in Caco2-BBE cells by directly targeting the 3-untranslated region of human CD98 mRNA. Expression of CD98 was decreased, whereas that of microRNA-7 was increased in well-differentiated Caco2-BBE cells compared with undifferentiated cells. Undifferentiated crypt cells isolated from mouse jejunum showed higher CD98 levels and lower levels of mmu-microRNA-706, a murine original microRNA candidate for CD98, than well-differentiated villus cells. Importantly, microRNA-7 decreased Caco2-BBE cell attachment on laminin-1, and CD98 overexpression recovered this inhibition, suggesting that microRNA-7 modulates epithelial cell adhesion to extracellular matrix, which in turn could affect proliferation and differentiation during the migration of enterocytes across the crypt-villus axis, by regulating CD98 expression. In a pathological context, the pro-inflammatory cytokine interleukin 1-beta increased CD98 expression in Caco2-BBE cells by decreasing microRNA-7 levels. Consistent with the in vitro findings, microRNA-7 levels were decreased in actively inflamed Crohn disease colonic tissues, where CD98 expression was up-regulated, compared with normal tissues. Together, these results reveal a novel mechanism underlying regulation of CD98 expression during patho-physiological states. This study raises microRNAs as a promising target for therapeutic modulations of CD98 expression in intestinal inflammatory disorders.
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Prohibitin inhibits tumor necrosis factor alpha-induced nuclear factor-kappa B nuclear translocation via the novel mechanism of decreasing importin alpha3 expression.
Mol. Biol. Cell
PUBLISHED: 08-26-2009
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Expression of prohibitin 1 (PHB), a multifunctional protein in the cell, is decreased during inflammatory bowel disease (IBD). Little is known regarding the regulation and role of PHB during intestinal inflammation. We examined the effect of tumor necrosis factor alpha (TNF-alpha), a cytokine that plays a central role in the pathogenesis of IBD, on PHB expression and the effect of sustained PHB expression on TNF-alpha activation of nuclear factor-kappa B (NF-kappaB) and epithelial barrier dysfunction, two hallmarks of intestinal inflammation. We show that TNF-alpha decreased PHB protein and mRNA abundance in intestinal epithelial cells in vitro and in colon mucosa in vivo. Sustained expression of prohibitin in intestinal epithelial cells in vitro and in vivo (prohibitin transgenic mice, PHB TG) resulted in a marked decrease in TNF-alpha-induced nuclear translocation of the NF-kappaB protein p65, NF-kappaB/DNA binding, and NF-kappaB-mediated transcriptional activation despite robust IkappaB-alpha phosphorylation and degradation and increased cytosolic p65. Cells overexpressing PHB were protected from TNF-alpha-induced increased epithelial permeability. Expression of importin alpha3, a protein involved in p50/p65 nuclear import, was decreased in cells overexpressing PHB and in colon mucosa of PHB TG mice. Restoration of importin alpha3 levels sustained NF-kappaB activation by TNF-alpha during PHB transfection. These results suggest that PHB inhibits NF-kappaB nuclear translocation via a novel mechanism involving alteration of importin alpha3 levels. TNF-alpha decreases PHB expression in intestinal epithelial cells and restoration of PHB expression in these cells can protect against the deleterious effects of TNF-alpha and NF-kappaB on barrier function.
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The RNA binding protein CsrA is a pleiotropic regulator of the locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli.
Infect. Immun.
PUBLISHED: 07-06-2009
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The attaching and effacing (A/E) pathogen enteropathogenic Escherichia coli (EPEC) forms characteristic actin-filled membranous protrusions upon infection of host cells termed pedestals. Here we examine the role of the RNA binding protein CsrA in the expression of virulence genes and proteins that are necessary for pedestal formation. The csrA mutant was defective in forming actin pedestals on epithelial cells and in disrupting transepithelial resistance across polarized epithelial cells. Consistent with reduced pedestal formation, secretion of the translocators EspA, EspB, and EspD and the effector Tir was substantially reduced in the csrA mutant. Purified CsrA specifically bound to the sepL espADB mRNA leader, and the corresponding transcript levels were reduced in the csrA mutant. In contrast, Tir synthesis was unaffected in the csrA mutant. Reduced secretion of Tir appeared to be in part due to decreased synthesis of EscD, an inner membrane architectural protein of the type III secretion system (TTSS) and EscF, a protein that forms the protruding needle complex of the TTSS. These effects were not mediated through the locus of enterocyte effacement (LEE) transcriptional regulator GrlA or Ler. In contrast to the csrA mutant, multicopy expression of csrA repressed transcription from LEE1, grlRA, LEE2, LEE5, escD, and LEE4, an effect mediated by GrlA and Ler. Consistent with its role in other organisms, CsrA also regulated flagellar motility and glycogen levels. Our findings suggest that CsrA governs virulence factor expression in an A/E pathogen by regulating mRNAs encoding translocators, effectors, or transcription factors.
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Drug-loaded nanoparticles targeted to the colon with polysaccharide hydrogel reduce colitis in a mouse model.
Gastroenterology
PUBLISHED: 06-26-2009
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One of the challenges to treating inflammatory bowel disease (IBD) is to target the site of inflammation. We engineered nanoparticles (NPs) to deliver an anti-inflammatory tripeptide Lys-Pro-Val (KPV) to the colon and assessed its therapeutic efficacy in a mouse model of colitis.
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Temporal and spatial analysis of clinical and molecular parameters in dextran sodium sulfate induced colitis.
PLoS ONE
PUBLISHED: 05-27-2009
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Inflammatory bowel diseases (IBD), including mainly ulcerative colitis (UC) and Crohns disease (CD), are inflammatory disorders of the gastrointestinal tract caused by an interplay of genetic and environmental factors. Murine colitis model induced by Dextran Sulfate Sodium (DSS) is an animal model of IBD that is commonly used to address the pathogenesis of IBD as well as to test efficacy of therapies. In this study we systematically analyzed clinical parameters, histological changes, intestinal barrier properties and cytokine profile during the colitic and recovery phase.
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Adenosine 2B receptors (A(2B)AR) on enteric neurons regulate murine distal colonic motility.
FASEB J.
PUBLISHED: 04-08-2009
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Delayed colonic emptying leading to constipation is a significant health concern. We investigated the role of adenosine 2B receptor (A(2B)AR) in modulating distal colonic motility using wild-type and A(2B)AR-knockout (A(2B)AR(-/-)) mice. Colon motility was assessed using stool characteristics and colonic transit. Distal colonic ganglia, isolated by laser capture microdissection, were tested for A(2B)AR expression by RT-PCR. The distal colon contraction and relaxation responses were assessed by electrical field stimulation (EFS) in presence of A(2B)AR agonists, antagonists or inhibitors of nitric oxide (NO) and guanylate cyclase. Nitrite levels were measured in enteric neuronal cultures exposed to A(2B)AR agonists/antagonists. A(2B)AR(-/-) mice display increased stool retention, decreased stool frequency, delayed colonic emptying, and decreased circular muscle relaxation. RT-PCR identified A(2B)AR expression in distal colonic ganglia. EFS studies revealed that enteric neuronal A(2B)AR is essential for distal colonic relaxation, and A(2B)AR antagonists can inhibit relaxation. Enteric neurons stimulated with A(2B)AR agonists produced more nitrite than cultures treated with antagonists. We demonstrate an essential role of A(2B)AR in regulating distal colon relaxation, as A(2B)AR activation is linked to NO signaling. Hence targeting the colonic A(2B)AR could represent a novel therapeutic strategy to treat constipation.
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Interleukin-1 receptor signaling protects mice from lethal intestinal damage caused by the attaching and effacing pathogen Citrobacter rodentium.
Infect. Immun.
PUBLISHED: 02-21-2009
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Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to the intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes a breach of the intestinal epithelial barrier, leading to colitis via a vigorous inflammatory response resulting in diarrhea and a protective antibody response that clears the pathogen. Here we show that interleukin-1 receptor (IL-1R) signaling protects mice following infection with C. rodentium. Upon infection, mice lacking the type I IL-1R exhibit increased mortality together with severe colitis characterized by intramural colonic bleeding and intestinal damage including gangrenous mucosal necrosis, phenotypes also evident in MyD88-deficient mice. However, unlike MyD88(-/-) mice, IL-1R(-/-) mice do not exhibit increased pathogen loads in the colon, delays in the recruitment of innate immune cells such as neutrophils, or defects in the capacity to replace damaged enterocytes. Further, we demonstrate that IL-1R(-/-) mice have an increased predisposition to intestinal damage caused by C. rodentium but not to that caused by chemical irritants, such as dextran sodium sulfate. Together, these data suggest that IL-1R signaling regulates the susceptibility of the intestinal epithelia to damage caused by C. rodentium.
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Prohibitin is a novel regulator of antioxidant response that attenuates colonic inflammation in mice.
Gastroenterology
PUBLISHED: 02-18-2009
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Increased free radicals and/or impaired antioxidant defenses have been shown to play a pathogenetic role in human and animal models of inflammatory bowel disease. Our previous studies showed that prohibitin (PHB) levels are decreased during colitis and that cultured intestinal epithelial cells overexpressing PHB are protected from oxidative stress. This study investigated the effect of intestinal epithelial cell-specific PHB overexpression on oxidative stress associated with experimental colitis and the potential mechanism by which PHB functions as an antioxidant using PHB transgenic mice.
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Ste20-related proline/alanine-rich kinase (SPAK) regulated transcriptionally by hyperosmolarity is involved in intestinal barrier function.
PLoS ONE
PUBLISHED: 02-02-2009
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The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohns disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-kappaB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-kappaB and Sp1 to bind to their binding sites. Knock-down of either NF-kappaB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-kappaB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function.
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Matrix metalloproteinase-9-mediated tissue injury overrides the protective effect of matrix metalloproteinase-2 during colitis.
Am. J. Physiol. Gastrointest. Liver Physiol.
PUBLISHED: 01-28-2009
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Matrix metalloproteinases (MMP) play an important role in pathogenesis of inflammatory bowel disease (IBD). Two known gelatinases, MMP-2 and MMP-9, are upregulated during IBD. Epithelial-derived MMP-9 is an important mediator of tissue injury in colitis, whereas MMP-2 protects against tissue damage and maintains gut barrier function. It has been suggested that developing strategies to block MMP-9 activity in the gut might be of benefit to IBD. However, given that MMP-2 and MMP-9 are structurally similar, such approaches would also likely inhibit MMP-2. Thus, to gain insight into outcome of inhibiting both MMP-2 and MMP-9, MMP-2(-/-)/MMP-9(-/-) double knockout mice (dKO) lacking both MMP-2 and MMP-9 were used in this study. Three models of murine colitis were used: dextran sodium sulfate (DSS), Salmonella typhimurium (S.T.), and trinitrobenzene sulfonic acid (TNBS). Our data demonstrate that MMP-2 and MMP-9 activities were highly upregulated in wild-type (WT) mice treated with DSS, S.T., or TNBS whereas dKO mice were resistant to the development of colitis. WT mice had extensive inflammation and tissue damage compared with dKO mice as suggested by histological assessment and myeloperoxidase activity. In conclusion, these results suggest an overriding role of MMP-9 in mediating tissue injury compared with the protective role of MMP-2 in development of colitis. Thus inhibition of MMP-9 may be beneficial in treatment of colitis even if resulting in inhibition of MMP-2.
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Oral colon-specific therapeutic approaches toward treatment of inflammatory bowel disease.
Expert Opin Drug Deliv
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Inflammatory bowel disease (IBD) is a chronic relapsing idiopathic disease. In clinical terms, most patients require lifelong medication associated with possible unpleasant adverse effects. Oral colon-specific drug delivery systems are designed to deliver therapeutic drugs to the inflamed colon to target pathophysiological manifestations of IBD. The aim is to maintain the drug with proper concentration in the inflamed colon, to enhance drug residence time and to minimize drug absorption by healthy tissues.
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NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis.
Mol. Microbiol.
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Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F.?tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.
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A(?B)AR expression in non-immune cells plays an important role in the development of murine colitis.
Dig Liver Dis
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Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis.
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Intestinal epithelial cell-specific CD98 expression regulates tumorigenesis in Apc(Min/+) mice.
Lab. Invest.
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The transmembrane glycoprotein CD98 regulates integrin signaling that in turn controls cell proliferation and survival. CD98 expression is upregulated in various carcinomas, including colorectal cancer. Recently, by generating gain- and loss-of-function mouse models featuring genetic manipulation of CD98 expression specifically in intestinal epithelial cells (IECs), we have explored the crucial role of CD98 in the regulation of intestinal homeostasis and inflammation-associated tumorigenesis. In the present study, we investigated the contribution of CD98 to intestinal tumorigenesis in Apc(Min/+) mice and the underlying mechanism of action. Mice featuring IEC-specific CD98 overexpression (Tg animals) were crossed with Apc(Min/+) mice, and the characteristics of intestinal adenoma formation were assessed. Compared with Apc(Min/+) mice, Tg/Apc(Min/+) animals exhibited increases in both intestinal tumor incidence and tumor size; these parameters correlated with enhanced proliferation and decreased apoptosis of IECs. IEC-specific CD98 overexpression resulted in increased synthesis of the oncogenic proteins c-myc and cyclin-D1 in Apc(Min/+) mice, independently of the Wnt-APC-?-catenin pathway, suggesting the implication of CD98 overexpression-mediated Erk activation. IEC-specific CD98 overexpression enhanced the production of proinflammatory cytokines and chemokines that are crucial for tumorigenesis. We validated our results in mice exhibiting IEC-specific CD98 downregulation (CD98(flox/+)VillinCre animals). IEC-specific CD98 downregulation efficiently attenuated tumor incidence and growth in Apc(Min/+) mice. The reduction of intestinal tumorigenesis upon IEC-specific CD98 downregulation was caused by the attenuation of IEC proliferation and cytokine/chemokine production. In conclusion, we show that CD98 exerts an oncogenic activity in terms of intestinal tumorigenesis, via an ability to regulate tumor growth and survival.
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Gastrointestinal delivery of anti-inflammatory nanoparticles.
Meth. Enzymol.
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The concept of nanomedicine has risen to be the future of medicine. Advantages of using nanoobjects as vectors for drug delivery systems are numerous, such as fewer side effects due to a low drug dose, and high specificity between drug and target. Unlike systemic therapy, targeting a specific target is more efficient and less costly. In inflammatory bowel disease, including ulcerative colitis and Crohn disease, the colon represents the targeted organ. A large number of drugs are candidates for loading into nanoparticles (NPs). Small molecules, such as tripeptides and siRNA, or larger molecules, such as proteins (hormones, antibodies (Ab), etc.), can be encapsulated alone or in a complex form inside the NPs. In our studies, once NPs are synthesized and loaded with anti-inflammatory compounds, they are delivered to the colon. An efficient technique has been developed for specific NP targeting to digestive tract regions, including the colon, using a hydrogel based on electrostatic interactions between positive ions and negative polysaccharides. An in situ double cross-linking process, mediated by Ca²? and SO?²?, of chitosan and alginate administered to the mouse gastrointestinal (GI) tract by double gavage, is used for gel formation. When the drug is given in NPs, NPs are targeted to the colon, and NP degradation by aggressive environmental conditions in the GI tract is significantly reduced. Using a biomaterial (hydrogel) associated with nanotechnology, lower doses of drug can be loaded efficiently and delivered to the colon to reduce colonic inflammation.
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Intestinal epithelial CD98 synthesis specifically modulates expression of colonic microRNAs during colitis.
Am. J. Physiol. Gastrointest. Liver Physiol.
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The transmembrane glycoprotein CD98 is known to be involved in intestinal inflammation. In the present study, we found that CD98 overexpression in intestinal epithelial cells does not normally affect the expression of colonic (epithelial and immune cell) microRNAs (miRNAs), small noncoding RNAs that posttranscriptionally regulate a wide variety of biological processes. However, upon dextran sulfate sodium (DSS) treatment, the expression of several colonic miRNAs, but not miRNAs from other tissues such as liver and spleen, were differentially regulated in mice overexpressing CD98 in epithelial cells compared with wild-type (WT) animals. For example, the level of colonic miRNA 132 was not affected by DSS treatment in WT animals but was upregulated in mice overexpressing CD98 in intestinal epithelial cells. Other colonic miRNAs, including colonic miRNA 23a and 23b, were downregulated in WT animals after DSS treatment but not in colonic epithelial cell CD98-overexpressing mice. Interestingly, the expression of potential miRNA target genes affected intestinal epithelial cells that overexpress CD98 and cell types that did not overexpress CD98 but were in close proximity to CD98-overexpressing intestinal epithelial cells. Taken together, these observations show that the combination of an inflammatory context and intestinal epithelial cell expression of CD98 affects the regulation of miRNA expression in colonic epithelial and immune cells. This is new evidence that protein expression modulates miRNA expression and suggests the existence of regulatory crosstalk between proteins and miRNAs in diseases such as colitis.
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Homeostatic and innate immune responses: role of the transmembrane glycoprotein CD98.
Cell. Mol. Life Sci.
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The transmembrane glycoprotein CD98 is a potential regulator of multiple functions, including integrin signaling and amino acid transport. Abnormal expression or function of CD98 and disruption of the interactions between CD98 and its binding partners result in defects in cell homeostasis and immune responses. Indeed, expression of CD98 has been correlated with diseases such as inflammation and tumor metastasis. Modulation of CD98 expression and/or function therefore represents a promising therapeutic strategy for the treatment and prevention of such pathologies. Herein, we review the role of CD98 with focus on its functional importance in homeostasis and immune responses, which could help to better understand the pathogenesis of CD98-associated diseases.
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Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon.
PLoS ONE
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Inflammatory bowel diseases (IBDs), primarily ulcerative colitis and Crohns disease, are inflammatory disorders caused by multiple factors. Research on IBD has often used the dextran sodium sulfate (DSS)-induced colitis mouse model. DSS induces in vivo but not in vitro intestinal inflammation. In addition, no DSS-associated molecule (free glucose, sodium sulfate solution, free dextran) induces in vitro or in vivo intestinal inflammation. We find that DSS but not dextran associated molecules established linkages with medium-chain-length fatty acids (MCFAs), such as dodecanoate, that are present in the colonic lumen. DSS complexed to MCFAs forms nanometer-sized vesicles ~200 nm in diameter that can fuse with colonocyte membranes. The arrival of nanometer-sized DSS/MCFA vesicles in the cytoplasm may activate intestinal inflammatory signaling pathways. We also show that the inflammatory activity of DSS is mediated by the dextran moieties. The deleterious effect of DSS is localized principally in the distal colon, therefore it will be important to chemically modify DSS to develop materials beneficial to the colon without affecting colon-targeting specificity.
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