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Sample records for intestinal epithelial cells

  1. Type 3 innate lymphoid cells maintain intestinal epithelial stem cells after tissue damage

    NARCIS (Netherlands)

    P. Aparicio-Domingo (Patricia); M. Romera-Hernandez (Monica); J.J. Karrich (Julien J.); F.H.J. Cornelissen (Ferry); N. Papazian (Natalie); D.J. Lindenbergh-Kortleve (Dicky); J.A. Butler (James A.); L. Boon (Louis); M. Coles (Mark); J.N. Samsom (Janneke); T. Cupedo (Tom)

    2015-01-01

    textabstractDisruption of the intestinal epithelial barrier allows bacterial translocation and predisposes to destructive inflammation. To ensure proper barrier composition, crypt-residing stem cells continuously proliferate and replenish all intestinal epithelial cells within days. As a consequence

  2. Intestinal Epithelial Cells Synthesize Glucocorticoids and Regulate T Cell Activation

    Science.gov (United States)

    Cima, Igor; Corazza, Nadia; Dick, Bernhard; Fuhrer, Andrea; Herren, Simon; Jakob, Sabine; Ayuni, Erick; Mueller, Christoph; Brunner, Thomas

    2004-01-01

    Glucocorticoids (GCs) are important steroid hormones with widespread activities in metabolism, development, and immune regulation. The adrenal glands are the major source of GCs and release these hormones in response to psychological and immunological stress. However, there is increasing evidence that GCs may also be synthesized by nonadrenal tissues. Here, we report that the intestinal mucosa expresses steroidogenic enzymes and releases the GC corticosterone in response to T cell activation. T cell activation causes an increase in the intestinal expression of the steroidogenic enzymes required for GC synthesis. In situ hybridization analysis revealed that these enzymes are confined to the crypt region of the intestinal epithelial layer. Surprisingly, in situ–produced GCs exhibit both an inhibitory and a costimulatory role on intestinal T cell activation. In the absence of intestinal GCs in vivo, activation by anti-CD3 injection resulted in reduced CD69 expression and interferon-γ production by intestinal T cells, whereas activation by viral infection led to increased T cell activation. We conclude that the intestinal mucosa is a potent source of immunoregulatory GCs. PMID:15596520

  3. Attachment of Giardia lamblia to rat intestinal epithelial cells.

    OpenAIRE

    Inge, P M; Edson, C M; Farthing, M J

    1988-01-01

    The human enteric protozoan, Giardia lamblia, has surface membrane lectin activity which mediates parasite adherence to erythrocytes. To determine whether an intestinal binding site exists for this lectin we have studied the interaction in vitro between axenically cultured Giardia trophozoites and isolated rat intestinal epithelial cells. Scanning electron microscopy showed that Giardia attached to the apical microvillus membrane and basolateral membrane of rat enterocytes. Any location on th...

  4. Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Andrea C. Lei-Leston

    2017-09-01

    Full Text Available Pattern recognition receptors (PRR, such as NOD-like receptors (NLRs, sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host–lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

  5. Inhibition of EV71 by curcumin in intestinal epithelial cells

    Science.gov (United States)

    Chio, Chi-Chong; Lin, Jhao-Yin

    2018-01-01

    EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections. PMID:29370243

  6. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine

    NARCIS (Netherlands)

    Klunder, Leon J.; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C. D.

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we

  7. Serratia marcescens is injurious to intestinal epithelial cells.

    Science.gov (United States)

    Ochieng, John B; Boisen, Nadia; Lindsay, Brianna; Santiago, Araceli; Ouma, Collins; Ombok, Maurice; Fields, Barry; Stine, O Colin; Nataro, James P

    2014-01-01

    Diarrhea causes substantial morbidity and mortality in children in low-income countries. Although numerous pathogens cause diarrhea, the etiology of many episodes remains unknown. Serratia marcescens is incriminated in hospital-associated infections, and HIV/AIDS associated diarrhea. We have recently found that Serratia spp. may be found more commonly in the stools of patients with diarrhea than in asymptomatic control children. We therefore investigated the possible enteric pathogenicity of S. marcescens in vitro employing a polarized human colonic epithelial cell (T84) monolayer. Infected monolayers were assayed for bacterial invasion, transepithelial electrical resistance (TEER), cytotoxicity, interleukin-8 (IL-8) release and morphological changes by scanning electron microscopy. We observed significantly greater epithelial cell invasion by S. marcescens compared to Escherichia coli strain HS (p = 0.0038 respectively). Cell invasion was accompanied by reduction in TEER and secretion of IL-8. Lactate dehydrogenase (LDH) extracellular concentration rapidly increased within a few hours of exposure of the monolayer to S. marcescens. Scanning electron microscopy of S. marcescens-infected monolayers demonstrated destruction of microvilli and vacuolization. Our results suggest that S. marcescens interacts with intestinal epithelial cells in culture and induces dramatic alterations similar to those produced by known enteric pathogens.

  8. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    OpenAIRE

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; De Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing with the epithelial lineage. However, the functional relevance of these observations is unknown. In the present study we employ a model system in which we cannot only detect cell fusion but also exam...

  9. Fusion of intestinal epithelial cells with bone marrow derived cells is dispensable for tissue homeostasis

    NARCIS (Netherlands)

    de Jong, Joan H.; Rodermond, Hans M.; Zimberlin, Cheryl D.; Lascano, Valeria; de Sousa E Melo, Felipe; Richel, Dick J.; Medema, Jan Paul; Vermeulen, Louis

    2012-01-01

    The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing

  10. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    Directory of Open Access Journals (Sweden)

    Z. Liu

    2010-05-01

    Full Text Available Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines, allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39 and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV, may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

  11. Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

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    Colin R Lickwar

    2017-08-01

    Full Text Available The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development

  12. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with the cell membrane. This involves a large number (17 million per cell) of high affinity binding sites which belong to a single class. Binding of biologically active 125 I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected in the isolated cells. The response (elevation of cellular cAMP) of the enterocytes to cholera toxin is linear with time for 40-50 min and causes a six- to eight-fold increase over control levels at steady stae. cAMP and agents that increase cAMP production inhibit Cl - -independent Na + influx into the isolated enterocytes whereas chlorporomazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na + entry. Correlation between cellular cAMP levels and the magnitude of Na + influx into the enterocytes provides evidence for a cAMP-mediated control of intestinal Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT and Na + during induction of intestinal secretion. The effect of cAMP on Na + but no Cl - influx in our villus cell preparation can be partially explained in terms of a cAMP-regulated Na + /H + neutral exchange system

  13. Action of cholera toxin in the intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with the cell membrane. This involves a large number (17 million per cell) of high affinity binding sites which belong to a single class. Binding of biologically active /sup 125/I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected in the isolated cells. The response (elevation of cellular cAMP) of the enterocytes to cholera toxin is linear with time for 40-50 min and causes a six- to eight-fold increase over control levels at steady stae. cAMP and agents that increase cAMP production inhibit Cl/sup -/-independent Na/sup +/ influx into the isolated enterocytes whereas chlorporomazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na/sup +/ entry. Correlation between cellular cAMP levels and the magnitude of Na/sup +/ influx into the enterocytes provides evidence for a cAMP-mediated control of intestinal Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT and Na/sup +/ during induction of intestinal secretion. The effect of cAMP on Na/sup +/ but no Cl/sup -/ influx in our villus cell preparation can be partially explained in terms of a cAMP-regulated Na/sup +//H/sup +/ neutral exchange system.

  14. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

    OpenAIRE

    Liu, Z.; Zhang, P.; Zhou, Y.; Qin, H.; Shen, T.

    2010-01-01

    Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithel...

  15. Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system.

    Science.gov (United States)

    Jijon, H B; Suarez-Lopez, L; Diaz, O E; Das, S; De Calisto, J; Yaffe, M B; Pittet, M J; Mora, J R; Belkaid, Y; Xavier, R J; Villablanca, E J

    2017-11-15

    Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4 + goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.Mucosal Immunology advance online publication, 15 November 2017; doi:10.1038/mi.2017.91.

  16. Mechanisms of Cell Polarity-Controlled Epithelial Homeostasis and Immunity in the Intestine.

    Science.gov (United States)

    Klunder, Leon J; Faber, Klaas Nico; Dijkstra, Gerard; van IJzendoorn, Sven C D

    2017-07-05

    Intestinal epithelial cell polarity is instrumental to maintain epithelial homeostasis and balance communications between the gut lumen and bodily tissue, thereby controlling the defense against gastrointestinal pathogens and maintenance of immune tolerance to commensal bacteria. In this review, we highlight recent advances with regard to the molecular mechanisms of cell polarity-controlled epithelial homeostasis and immunity in the human intestine. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

  17. Enteric glial cells and their role in the intestinal epithelial barrier

    OpenAIRE

    Yu, Yan-Bo; Li, Yan-Qing

    2014-01-01

    The intestinal epithelium constitutes a physical and functional barrier between the external environment and the host organism. It is formed by a continuous monolayer of intestinal epithelial cells maintained together by intercellular junctional complex, limiting access of pathogens, toxins and xenobiotics to host tissues. Once this barrier integrity is disrupted, inflammatory disorders and tissue injury are initiated and perpetuated. Beneath the intestinal epithelial cells lies a population ...

  18. Krüppel-like factor 4 regulates intestinal epithelial cell morphology and polarity.

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    Tianxin Yu

    Full Text Available Krüppel-like factor 4 (KLF4 is a zinc finger transcription factor that plays a vital role in regulating cell lineage differentiation during development and maintaining epithelial homeostasis in the intestine. In normal intestine, KLF4 is predominantly expressed in the differentiated epithelial cells. It has been identified as a tumor suppressor in colorectal cancer. KLF4 knockout mice demonstrated a decrease in number of goblet cells in the colon, and conditional ablation of KLF4 from the intestinal epithelium led to altered epithelial homeostasis. However, the role of KLF4 in differentiated intestinal cells and colon cancer cells, as well as the mechanism by which it regulates homeostasis and represses tumorigenesis in the intestine is not well understood. In our study, KLF4 was partially depleted in the differentiated intestinal epithelial cells by a tamoxifen-inducible Cre recombinase. We found a significant increase in the number of goblet cells in the KLF4-deleted small intestine, suggesting that KLF4 is not only required for goblet cell differentiation, but also required for maintaining goblet cell numbers through its function in inhibiting cell proliferation. The number and position of Paneth cells also changed. This is consistent with the KLF4 knockout study using villin-Cre [1]. Through immunohistochemistry (IHC staining and statistical analysis, we found that a stem cell and/or tuft cell marker, DCAMKL1, and a proliferation marker, Ki67, are affected by KLF4 depletion, while an enteroendocrine cell marker, neurotensin (NT, was not affected. In addition, we found KLF4 depletion altered the morphology and polarity of the intestinal epithelial cells. Using a three-dimensional (3D intestinal epithelial cyst formation assay, we found that KLF4 is essential for cell polarity and crypt-cyst formation in human colon cancer cells. These findings suggest that, as a tumor suppressor in colorectal cancer, KLF4 affects intestinal epithelial cell

  19. Drug permeation across intestinal epithelial cells using porous silicon nanoparticles.

    Science.gov (United States)

    Bimbo, Luis M; Mäkilä, Ermei; Laaksonen, Timo; Lehto, Vesa-Pekka; Salonen, Jarno; Hirvonen, Jouni; Santos, Hélder A

    2011-04-01

    Mesoporous silicon particles hold great potential in improving the solubility of otherwise poorly soluble drugs. To effectively translate this feature into the clinic, especially via oral or parenteral administration, a thorough understanding of the interactions of the micro- and nanosized material with the physiological environment during the delivery process is required. In the present study, the behaviour of thermally oxidized porous silicon particles of different sizes interacting with Caco-2 cells (both non-differentiated and polarized monolayers) was investigated in order to establish their fate in a model of intestinal epithelial cell barrier. Particle interactions and TNF-α were measured in RAW 264.7 macrophages, while cell viabilities, reactive oxygen species and nitric oxide levels, together with transmission electron microscope images of the polarized monolayers, were assessed with both the Caco-2 cells and RAW 264.7 macrophages. The results showed a concentration and size dependent influence on cell viability and ROS-, NO- and TNF-α levels. There was no evidence of the porous nanoparticles crossing the Caco-2 cell monolayers, yet increased permeation of the loaded poorly soluble drug, griseofulvin, was shown. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Action of cholera toxin in the intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with a large number of high affinity binding sites in the cell membrane. Binding of 125 I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected. The response (elevation of cellular cAMP) is linear with time for 40 to 50 min and causes a six- to eight-fold increase over control levels (10 to 15 picomole cAMP/mg cellular protein) at steady state. cAMP and agents that increase cAMP production inhibit Cl - -independent Na + influx into the isolated enterocytes whereas chlorpromazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na + entry. Correlation between cellular cAMP levels and the magnitude of Na + influx provides evidence for a cAMP-mediated control of intestinal Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretion. The effect of cAMP on Na + but not Cl - influx preparations can be partially explained in terms of a cAMP-regulated Na + /H + neutral exchange system. Data on the coupling relationship between Na + transport and the intra- and extracellular pH in the enterocytes show that an amiloride-sensitive electroneutral Na + /H + exchange process occurs. This coupling between Na + and H + is partially inhibited by CT and dbcAMP, suggesting that the Na + /H + exchange may be a cAMP-regulated process. 31 references, 32 figures, 5 tables

  1. Arginine consumption by the intestinal parasite Giardia intestinalis reduces proliferation of intestinal epithelial cells.

    Science.gov (United States)

    Stadelmann, Britta; Merino, María C; Persson, Lo; Svärd, Staffan G

    2012-01-01

    In the field of infectious diseases the multifaceted amino acid arginine has reached special attention as substrate for the hosts production of the antimicrobial agent nitric oxide (NO). A variety of infectious organisms interfere with this part of the host immune response by reducing the availability of arginine. This prompted us to further investigate additional roles of arginine during pathogen infections. As a model we used the intestinal parasite Giardia intestinalis that actively consumes arginine as main energy source and secretes an arginine-consuming enzyme, arginine deiminase (ADI). Reduced intestinal epithelial cell (IEC) proliferation is a common theme during bacterial and viral intestinal infections, but it has never been connected to arginine-consumption. Our specific question was thereby, whether the arginine-consumption by Giardia leads to reduced IEC proliferation, in addition to NO reduction. In vitro cultivation of human IEC lines in arginine-free or arginine/citrulline-complemented medium, as well as in interaction with different G. intestinalis isolates, were used to study effects on host cell replication by MTT assay. IEC proliferation was further analyzed by DNA content analysis, polyamine measurements and expressional analysis of cell cycle regulatory genes. IEC proliferation was reduced upon arginine-withdrawal and also in an arginine-dependent manner upon interaction with G. intestinalis or addition of Giardia ADI. We show that arginine-withdrawal by intestinal pathogens leads to a halt in the cell cycle in IECs through reduced polyamine levels and upregulated cell cycle inhibitory genes. This is of importance with regards to intestinal tissue homeostasis that is affected through reduced cell proliferation. Thus, the slower epithelial cell turnover helps the pathogen to maintain a more stable niche for colonization. This study also shows why supplementation therapy of diarrhea patients with arginine/citrulline is helpful and that

  2. Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells

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    Bhattacharya, Sujoy; Ray, Ramesh M.; Johnson, Leonard R.

    2014-01-01

    Homeostasis of the gastrointestinal epithelium is dependent upon a balance between cell proliferation and apoptosis. Cyclin-dependent kinases (Cdks) are well known for their role in cell proliferation. Previous studies from our group have shown that polyamine-depletion of intestinal epithelial cells (IEC-6) decreases cyclin-dependent kinase 2 (Cdk2) activity, increases p53 and p21Cip1 protein levels, induces G1 arrest, and protects cells from camptothecin (CPT)-induced apoptosis. Although emerging evidence suggests that members of the Cdk family are involved in the regulation of apoptosis, their roles directing apoptosis of IEC-6 cells are not known. In this study, we report that inhibition of Cdk1, 2, and 9 (with the broad range Cdk inhibitor, AZD5438) in proliferating IEC-6 cells triggered DNA damage, activated p53 signaling, inhibited proliferation, and induced apoptosis. By contrast, inhibition of Cdk2 (with NU6140) increased p53 protein and activity, inhibited proliferation, but had no effect on apoptosis. Notably, AZD5438 sensitized, whereas, NU6140 rescued proliferating IEC-6 cells from CPT-induced apoptosis. However, in colon carcinoma (Caco2) cells with mutant p53, treatment with either AZD5438 or NU6140 blocked proliferation, albeit more robustly with AZD5438. Both Cdk inhibitors induced apoptosis in Caco2 cells in a p53-independent manner. In serum starved quiescent IEC-6 cells, both AZD5438 and NU6140 decreased TNF- /CPT-induced activation of p53 and, consequently, rescued cells from apoptosis, indicating that sustained Cdk activity is required for apoptosis of quiescent cells. Furthermore, AZD5438 partially reversed the protective effect of polyamine depletion whereas NU6140 had no effect. Together, these results demonstrate that Cdks possess opposing roles in the control of apoptosis in quiescent and proliferating cells. In addition, Cdk inhibitors uncouple proliferation from apoptosis in a p53-dependent manner. PMID:24242917

  3. Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.

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    Nan Ye Lei

    Full Text Available Intestinal epithelial stem cells (ISCs are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

  4. Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.

    Science.gov (United States)

    Lei, Nan Ye; Jabaji, Ziyad; Wang, Jiafang; Joshi, Vaidehi S; Brinkley, Garrett J; Khalil, Hassan; Wang, Fengchao; Jaroszewicz, Artur; Pellegrini, Matteo; Li, Linheng; Lewis, Michael; Stelzner, Matthias; Dunn, James C Y; Martín, Martín G

    2014-01-01

    Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

  5. HDAC1 and HDAC2 restrain the intestinal inflammatory response by regulating intestinal epithelial cell differentiation.

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    Naomie Turgeon

    Full Text Available Acetylation and deacetylation of histones and other proteins depends on histone acetyltransferases and histone deacetylases (HDACs activities, leading to either positive or negative gene expression. HDAC inhibitors have uncovered a role for HDACs in proliferation, apoptosis and inflammation. However, little is known of the roles of specific HDACs in intestinal epithelial cells (IEC. We investigated the consequences of ablating both HDAC1 and HDAC2 in murine IECs. Floxed Hdac1 and Hdac2 homozygous mice were crossed with villin-Cre mice. Mice deficient in both IEC HDAC1 and HDAC2 weighed less and survived more than a year. Colon and small intestinal sections were stained with hematoxylin and eosin, or with Alcian blue and Periodic Acid Schiff for goblet cell identification. Tissue sections from mice injected with BrdU for 2 h, 14 h and 48 h were stained with anti-BrdU. To determine intestinal permeability, 4-kDa FITC-labeled dextran was given by gavage for 3 h. Microarray analysis was performed on total colon RNAs. Inflammatory and IEC-specific gene expression was assessed by Western blot or semi-quantitative RT-PCR and qPCR with respectively total colon protein and total colon RNAs. HDAC1 and HDAC2-deficient mice displayed: 1 increased migration and proliferation, with elevated cyclin D1 expression and phosphorylated S6 ribosomal protein, a downstream mTOR target; 2 tissue architecture defects with cell differentiation alterations, correlating with reduction of secretory Paneth and goblet cells in jejunum and goblet cells in colon, increased expression of enterocytic markers such as sucrase-isomaltase in the colon, increased expression of cleaved Notch1 and augmented intestinal permeability; 3 loss of tissue homeostasis, as evidenced by modifications of claudin 3 expression, caspase-3 cleavage and Stat3 phosphorylation; 4 chronic inflammation, as determined by inflammatory molecular expression signatures and altered inflammatory gene expression

  6. Binding of cholera toxin B subunit to intestinal epithelial cells.

    Science.gov (United States)

    Navolotskaya, Elena V; Sadovnikov, Vladimir B; Lipkin, Valery M; Zav'yalov, Vladimir P

    2018-03-01

    We have prepared 125 I-labeled cholera toxin B subunit ( 125 I-labeled CT-B, a specific activity of 98Ci/mmol) and found that it binds to rat IEC-6 and human Caco-2 intestinal epithelial cells with high affinity (K d 3.6 and 3.7nM, respectively). The binding of labeled protein was completely inhibited by unlabeled thymosin-α 1 (TM-α 1 ), interferon-α 2 (IFN-α 2 ), and the synthetic peptide LKEKK that corresponds to residues 16-20 in TM-α 1 and 131-135 in IFN-α 2 , but was not inhibited by the synthetic peptide KKEKL with inverted amino acid sequence (K i >10μM). Thus, TM-α 1 , IFN-α 2 , and the peptide: LKEKK bind with high affinity and specificity to the cholera toxin receptor on IEC-6 and Caco-2 cells. It was found that CT-B and the peptide: LKEKK at concentrations of 10-1000nM increased in a dose-dependent manner the nitric oxide production and the soluble guanylate cyclase activity in IEC-6 and Caco-2 cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Enteric glial cells and their role in the intestinal epithelial barrier.

    Science.gov (United States)

    Yu, Yan-Bo; Li, Yan-Qing

    2014-08-28

    The intestinal epithelium constitutes a physical and functional barrier between the external environment and the host organism. It is formed by a continuous monolayer of intestinal epithelial cells maintained together by intercellular junctional complex, limiting access of pathogens, toxins and xenobiotics to host tissues. Once this barrier integrity is disrupted, inflammatory disorders and tissue injury are initiated and perpetuated. Beneath the intestinal epithelial cells lies a population of astrocyte-like cells that are known as enteric glia. The morphological characteristics and expression markers of these enteric glia cells were identical to the astrocytes of the central nervous system. In the past few years, enteric glia have been demonstrated to have a trophic and supporting relationship with intestinal epithelial cells. Enteric glia lesions and/or functional defects can be involved in the barrier dysfunction. Besides, factors secreted by enteric glia are important for the regulation of gut barrier function. Moreover, enteric glia have an important impact on epithelial cell transcriptome and induce a shift in epithelial cell phenotype towards increased cell adhesion and cell differentiation. Enteric glia can also preserve epithelial barrier against intestinal bacteria insult. In this review, we will describe the current body of evidence supporting functional roles of enteric glia on intestinal barrier.

  8. Action of cholera toxin in the intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hyun, C.S.

    1982-01-01

    The primary event in the action of cholera toxin on the isolated chick intestinal epithelial cell is its interaction with a large number of high affinity binding sites in the cell membrane. Binding of /sup 125/I-labeled toxin is rapid, temperature-dependent, reversible, and saturable over a wide range of concentrations and includes only a small contribution from nonspecific sites. A characteristic lag phase of 10 min occurs following the complete binding of toxin before any increase in cellular cAMP levels can be detected. The response (elevation of cellular cAMP) is linear with time for 40 to 50 min and causes a six- to eight-fold increase over control levels (10 to 15 picomole cAMP/mg cellular protein) at steady state. cAMP and agents that increase cAMP production inhibit Cl/sup -/-independent Na/sup +/ influx into the isolated enterocytes whereas chlorpromazine (CPZ) which completely abolishes toxin-induced elevation of cAMP both reverses and prevents the cAMP-mediated inhibition of Na/sup +/ entry. Correlation between cellular cAMP levels and the magnitude of Na/sup +/ influx provides evidence for a cAMP-mediated control of intestinal Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na/sup +/ during induction of intestinal secretion. The effect of cAMP on Na/sup +/ but not Cl/sup -/ influx preparations can be partially explained in terms of a cAMP-regulated Na/sup +//H/sup +/ neutral exchange system. Data on the coupling relationship between Na/sup +/ transport and the intra- and extracellular pH in the enterocytes show that an amiloride-sensitive electroneutral Na/sup +//H/sup +/ exchange process occurs. This coupling between Na/sup +/ and H/sup +/ is partially inhibited by CT and dbcAMP, suggesting that the Na/sup +//H/sup +/ exchange may be a cAMP-regulated process. 31 references, 32 figures, 5 tables.

  9. Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

    NARCIS (Netherlands)

    Roubos-van den Hil, P.J.; Nout, M.J.R.; Beumer, R.R.; Meulen, van der J.; Zwietering, M.H.

    2009-01-01

    Aims: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and

  10. Plasticity of intestinal epithelial cells in regeneration and cancer

    NARCIS (Netherlands)

    Tetteh, Paul W.

    2015-01-01

    Cellular plasticity refers to the ability of a cell to change its fate or identity in response to external or intrinsic factors. Regeneration of the intestinal epithelium after injury is driven mainly by plasticity of crypt stem cells that can rapidly divide to replace all the lost cells. Stem cell

  11. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform

    Directory of Open Access Journals (Sweden)

    Kenji Kozuka

    2017-12-01

    Full Text Available Summary: We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound. : Siegel and colleagues describe their development of a human and mouse intestinal epithelial cell monolayer platform that maintains the cellular, molecular, and functional characteristics of tissue for each intestinal segment. They demonstrate the platform's application to drug discovery by screening a library of over 2,000 compounds to identify an inhibitor of potassium ion transport in the mouse distal colon. Keywords: intestinal epithelium, organoids, monolayer, colon, small intestine, phenotype screening assays, enteroid, colonoid

  12. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    International Nuclear Information System (INIS)

    Horita, Nobukatsu; Tsuchiya, Kiichiro; Hayashi, Ryohei; Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro; Okamoto, Ryuichi; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-01-01

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus

  13. Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

    Energy Technology Data Exchange (ETDEWEB)

    Horita, Nobukatsu [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Tsuchiya, Kiichiro, E-mail: kii.gast@tmd.ac.jp [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Hayashi, Ryohei [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Department of Gastroenterology and Metabolism, Hiroshima University (Japan); Fukushima, Keita; Hibiya, Shuji; Fukuda, Masayoshi; Kano, Yoshihito; Mizutani, Tomohiro; Nemoto, Yasuhiro; Yui, Shiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan); Okamoto, Ryuichi; Nakamura, Tetsuya [Department of Advanced Therapeutics for Gastrointestinal Diseases, Graduate School, Tokyo Medical and Dental University (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University (Japan)

    2014-11-28

    Highlights: • Lentivirus mixed with Matrigel enables direct infection of intestinal organoids. • Our original approach allows the marking of a single stem cell in a crypt. • Time-lapse imaging shows the dynamics of a single stem cell. • Our lentivirus transgene system demonstrates plural long-lived stem cells in a crypt. - Abstract: Background and aims: The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. Methods: Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. Results: We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1 year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. Conclusions: The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

  14. Effect of neuronal PC12 cells on the functional properties of intestinal epithelial Caco-2 cells.

    Science.gov (United States)

    Satsu, Hideo; Yokoyama, Tatsuya; Ogawa, Nobumasa; Fujiwara-Hatano, Yoko; Shimizu, Makoto

    2003-06-01

    The effect of neuronal cells on the functional properties of intestinal epithelial cells was examined by using an in vitro coculture system. Two cell lines, Caco-2 and PC12, were respectively used as intestinal epithelial and enteric neuronal cell models. Coculture of differentiated Caco-2 cells with PC12 caused a significant decrease in the transepithelial electrical resistance (TER) value of the Caco-2 monolayer. The permeability to lucifer yellow (LY) was also significantly increased, suggesting that the tight junction (TJ) of the Caco-2 monolayers was modulated by coculturing with PC12. To identify the TJ-modulating factor presumably secreted from PC12, the effects of the major neurotransmitters on the TER value and LY transport were examined, but no influence was apparent. The TJ-modulating effect of PC12 was prevented by exposing PC12 to cycloheximide, suggesting that new protein synthesis in PC12 was necessary for this regulation.

  15. Mechanism of leukotriene D4 inhibition of Na-alanine cotransport in intestinal epithelial cells

    OpenAIRE

    Talukder, Jamilur R.; Kekuda, Ramesh; Saha, Prosenjit; Sundaram, Uma

    2008-01-01

    In a rabbit model of chronic intestinal inflammation, we previously demonstrated inhibition of neutral Na-amino acid cotransport. The mechanism of the inhibition was secondary to a decrease in the affinity for amino acid rather than the number of cotransporters. Since leukotriene (LT)D4 is known to be elevated in enterocytes during chronic intestinal inflammation, we used rat intestinal epithelial cell (IEC-18) monolayers to determine the mechanism of regulation of Na-alanine cotransport (ala...

  16. Lactobacillus rhamnosus GG supernatant upregulates serotonin transporter expression in intestinal epithelial cells and mice intestinal tissues.

    Science.gov (United States)

    Wang, Y M; Ge, X Z; Wang, W Q; Wang, T; Cao, H L; Wang, B L; Wang, B M

    2015-09-01

    The role that probiotics play in relieving irritable bowel syndrome (IBS) has been demonstrated; however, the mechanism by which IBS is affected remains unclear. In this study, serotonin transporter (SERT) mRNA and serotonin transporter protein (SERT-P) levels in HT-29, Caco-2 cells, and mice intestinal tissues were examined after treatment with Lactobacillus rhamnosus GG supernatant (LGG-s). HT-29 and Caco-2 cells were treated with different concentrations of LGG-s for 12 and 24 h and C57BL/6 mice received supplements of different concentrations for 4 weeks. SERT mRNA and SERT-P levels were detected by real-time PCR and Western blotting. SERT mRNA and SERT-P levels in HT-29 and Caco-2 cells were higher than those in the control 24 h after treatment. Undiluted LGG-s upregulated SERT mRNA levels by 9.4-fold in the first week, which dropped in the second week. The double-diluted LGG-s upregulated SERT mRNA by 2.07-fold in the first week; levels dropped to 1.75-fold within the second week and under base expression levels by the third week, while they again climbed to 1.56-fold in the fourth week. The triple-diluted LGG-s could not upregulate SERT mRNA expression until the end of the fourth week. The SERT-P levels in the double-diluted LGG-s group were higher than that in the control but fluctuated with time. SERT-P levels in the triple-diluted LGG-s were higher than that in the control in the last 2 weeks and increased with time. LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues. © 2015 John Wiley & Sons Ltd.

  17. Cdx2 modulates proliferation in normal human intestinal epithelial crypt cells

    International Nuclear Information System (INIS)

    Escaffit, Fabrice; Pare, Frederic; Gauthier, Remy; Rivard, Nathalie; Boudreau, Francois; Beaulieu, Jean-Francois

    2006-01-01

    The homeobox gene Cdx2 is involved in the regulation of the expression of intestine specific markers such as sucrase-isomaltase and lactase-phlorizin hydrolase. Previous studies performed with immortalized or transformed intestinal cell lines have provided evidence that Cdx2 can promote morphological and functional differentiation in these experimental models. However, no data exist concerning the implication of this factor in normal human intestinal cell physiology. In the present work, we have investigated the role of Cdx2 in normal human intestinal epithelial crypt (HIEC) cells that lack this transcription factor. The establishment of HIEC cells expressing Cdx2 in an inducible manner shows that forced expression of Cdx2 significantly alters the proliferation of intestinal crypt cells and stimulates dipeptidylpeptidase IV expression but is not sufficient to trigger intestinal terminal differentiation. These observations suggest that Cdx2 requires additional factors to activate the enterocyte differentiation program in normal undifferentiated cells

  18. Reversible effect of dextran sodium sulfate on mucus secreting intestinal epithelial cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, V

    2016-01-01

    investigated effects of increasing doses of DSS on viability and integrity of these intestinal epithelial cells. For cell viability studies, cells were treated with DSS solutions for 24 or 48 h and viability was measured fluorometrically by PicoGreen double-stranded DNA quantitation. HT29-MTX-E12 cells were...... provide valuable insight into a possible mechanism for dextran sodium sulfate (DSS)–induced colitis of importance for the design of subsequent in vivo studies. To develop a new in vitro IBD model with DSS-induced inflammation in human mucus-secreting intestinal epithelial cells (HT29-MTX-E12), we first...... affects the viability and disrupts the intestinal barrier function of HT29-MTX-E12 monolayers, a main feature observed in IBD. Furthermore, the harmful effect of DSS is reversible, suggesting that recovery of intestinal integrity after DSS treatment by potential therapeutic drugs can be studied in vitro....

  19. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    Energy Technology Data Exchange (ETDEWEB)

    Tang, J.; Jiang, Y. [Southern Medical University, Nanfang Hospital, Department of Anesthesia, Guangzhou, China, Department of Anesthesia, Nanfang Hospital, Southern Medical University, Guangzhou (China); Tang, Y.; Chen, B. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China); Sun, X. [Laboratory of Traditional Chinese Medicine Syndrome, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou (China); Su, L.; Liu, Z. [Guangzhou General Hospital of Guangzhou Military Command, Department of Intensive Care Unit, Guangzhou, China, Department of Intensive Care Unit, Guangzhou General Hospital of Guangzhou Military Command, Guangzhou (China)

    2013-06-25

    Gut-derived endotoxin and pathogenic bacteria have been proposed as important causative factors of morbidity and death during heat stroke. However, it is still unclear what kind of damage is induced by heat stress. In this study, the rat intestinal epithelial cell line (IEC-6) was treated with heat stress or a combination of heat stress and lipopolysaccharide (LPS). In addition, propofol, which plays an important role in anti-inflammation and organ protection, was applied to study its effects on cellular viability and apoptosis. Heat stress, LPS, or heat stress combined with LPS stimulation can all cause intestinal epithelial cell damage, including early apoptosis and subsequent necrosis. However, propofol can alleviate injuries caused by heat stress, LPS, or the combination of heat stress and LPS. Interestingly, propofol can only mitigate LPS-induced intestinal epithelial cell apoptosis, and has no protective role in heat-stress-induced apoptosis. This study developed a model that can mimic the intestinal heat stress environment. It demonstrates the effects on intestinal epithelial cell damage, and indicated that propofol could be used as a therapeutic drug for the treatment of heat-stress-induced intestinal injuries.

  20. Effects of propofol on damage of rat intestinal epithelial cells induced by heat stress and lipopolysaccharides

    International Nuclear Information System (INIS)

    Tang, J.; Jiang, Y.; Tang, Y.; Chen, B.; Sun, X.; Su, L.; Liu, Z.

    2013-01-01

    Gut-derived endotoxin and pathogenic bacteria have been proposed as important causative factors of morbidity and death during heat stroke. However, it is still unclear what kind of damage is induced by heat stress. In this study, the rat intestinal epithelial cell line (IEC-6) was treated with heat stress or a combination of heat stress and lipopolysaccharide (LPS). In addition, propofol, which plays an important role in anti-inflammation and organ protection, was applied to study its effects on cellular viability and apoptosis. Heat stress, LPS, or heat stress combined with LPS stimulation can all cause intestinal epithelial cell damage, including early apoptosis and subsequent necrosis. However, propofol can alleviate injuries caused by heat stress, LPS, or the combination of heat stress and LPS. Interestingly, propofol can only mitigate LPS-induced intestinal epithelial cell apoptosis, and has no protective role in heat-stress-induced apoptosis. This study developed a model that can mimic the intestinal heat stress environment. It demonstrates the effects on intestinal epithelial cell damage, and indicated that propofol could be used as a therapeutic drug for the treatment of heat-stress-induced intestinal injuries

  1. Mapping of HNF4alpha target genes in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Boyd, Mette; Bressendorff, Simon; Moller, Jette

    2009-01-01

    not previously been described as being regulated by HNF4alpha. The 1,541 genes contributed significantly to gene ontology (GO) pathways categorized by lipid and amino acid transport and metabolism. An analysis of the homeodomain transcription factor Cdx-2 (CDX2), the disaccharidase trehalase (TREH......ABSTRACT: BACKGROUND: The role of HNF4alpha has been extensively studied in hepatocytes and pancreatic beta-cells, and HNF4alpha is also regarded as key regulator of intestinal epithelial cell differentiation as well. The aim of the present work is to identify novel HNF4alpha target genes...... in the human intestinal epithelial cells in order to elucidate the role of HNF4alpha in the intestinal differentiation progress. METHODS: We have performed a ChIP-chip analysis of the human intestinal cell line Caco-2 in order to make a genome-wide identification of HNF4alpha binding to promoter regions...

  2. Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells.

    Science.gov (United States)

    Roubos-van den Hil, P J; Nout, M J R; Beumer, R R; van der Meulen, J; Zwietering, M H

    2009-03-01

    This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases.

  3. Does dietary fibre stimulate intestinal epithelial cell proliferation in germ free rats?

    OpenAIRE

    Goodlad, R A; Ratcliffe, B; Fordham, J P; Wright, N A

    1989-01-01

    The aim of the present experiment was to investigate the role of hind gut fermentation in the proliferative response of the intestinal epithelium to dietary fibre. We have previously shown that refeeding starved rats with an elemental diet supplemented with fermentable dietary fibre (but not inert bulk) is capable of stimulating intestinal epithelial cell proliferation throughout the gastrointestinal tract. Three groups of 10 germ free (GF) rats and three groups of 10 conventional (CV) rats, ...

  4. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy

    NARCIS (Netherlands)

    Grabinger, T.; Luks, L.; Kostadinova, F.; Zimberlin, C.; Medema, J. P.; Leist, M.; Brunner, T.

    2014-01-01

    Intestinal epithelial cells (IECs) not only have a critical function in the absorption of nutrients, but also act as a physical barrier between our body and the outside world. Damage and death of the epithelial cells lead to the breakdown of this barrier function and inflammation due to access of

  5. Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

    Science.gov (United States)

    Zhu, Shu; Ding, Siyuan; Wang, Penghua; Wei, Zheng; Pan, Wen; Palm, Noah W; Yang, Yi; Yu, Hua; Li, Hua-Bing; Wang, Geng; Lei, Xuqiu; de Zoete, Marcel R; Zhao, Jun; Zheng, Yunjiang; Chen, Haiwei; Zhao, Yujiao; Jurado, Kellie A; Feng, Ningguo; Shan, Liang; Kluger, Yuval; Lu, Jun; Abraham, Clara; Fikrig, Erol; Greenberg, Harry B; Flavell, Richard A

    2017-06-29

    Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

  6. Bioavailability of genistein, daidzein, and their glycosides in intestinal epithelial Caco-2 cells

    NARCIS (Netherlands)

    Steensma, A.; Noteborn, H.P.J.M.; Jagt, van der R.C.M.; Polman, Th.H.G.; Mengelers, M.J.B.; Kuiper, H.A.

    1999-01-01

    In this study information was obtained on bioavailability of genistein, daidzein and their glycosides in human intestinal epithelial Caco-2 cells grown on semi-permeable filters. The integrity of Caco-2 monolayers was confirmed by transepithelial electrical resistance measurements and by

  7. Campylobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures

    NARCIS (Netherlands)

    R.P.L. Louwen (Rogier); E.E.S. Nieuwenhuis (Edward); L. van Marrewijk (Leonie); D. Horst-Kreft (Deborah); L.F. de Ruiter (Lilian); A.P. Heikema (Astrid); W.J.B. van Wamel (Willem); J.A. Wagenaar (Jaap); H.P. Endtz (Hubert); J.N. Samsom (Janneke); P. van Baarlen (Peter); A.S. Akhmanova (Anna); A.F. van Belkum (Alex)

    2012-01-01

    textabstractTranslocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether

  8. Characterizing microbiota-independent effects of oligosaccharides on intestinal epithelial cells

    NARCIS (Netherlands)

    Akbari, Peyman; Fink-Gremmels, Johanna; Willems, Rianne H.A.M.; Difilippo, Elisabetta; Schols, Henk A.; Schoterman, Margriet H.C.; Garssen, Johan; Braber, Saskia

    2017-01-01

    Purpose: The direct effects of galacto-oligosaccharides (GOS), including Vivinal® GOS syrup (VGOS) and purified Vivinal® GOS (PGOS), on the epithelial integrity and corresponding interleukin-8 (IL-8/CXCL8) release were examined in a Caco-2 cell model for intestinal barrier dysfunction. To

  9. Molecular and cellular studies on the absorption, function, and safety of food components in intestinal epithelial cells.

    Science.gov (United States)

    Satsu, Hideo

    2017-03-01

    The intestinal tract comes into direct contact with the external environment despite being inside the body. Intestinal epithelial cells, which line the inner face of the intestinal tract, have various important functions, including absorption of food substances, immune functions such as cytokine secretion, and barrier function against xenobiotics by means of detoxification enzymes. It is likely that the functions of intestinal epithelial cells are regulated or modulated by these components because they are frequently exposed to food components at high concentrations. This review summarizes our research on the interaction between intestinal epithelial cells and food components at cellular and molecular levels. The influence of xenobiotic contamination in foods on the cellular function of intestinal epithelial cells is also described in this review.

  10. Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells.

    Science.gov (United States)

    Lukyanenko, Valeriy; Malyukova, Irina; Hubbard, Ann; Delannoy, Michael; Boedeker, Edgar; Zhu, Chengru; Cebotaru, Liudmila; Kovbasnjuk, Olga

    2011-11-01

    Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.

  11. A20 restricts wnt signaling in intestinal epithelial cells and suppresses colon carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Ling Shao

    Full Text Available Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Here, we have studied the role of A20 (also known as TNFAIP3, a ubiquitin-editing enzyme that restricts NFκB and cell death signaling, in intestinal homeostasis and tumorigenesis. We have found that A20 expression is consistently reduced in human colonic adenomas than in normal colonic tissues. To further investigate A20's potential roles in regulating colon carcinogenesis, we have generated mice lacking A20 specifically in intestinal epithelial cells and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APC(min. While A20(FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20(FL/FL villin-Cre APC(min/+ mice contain far greater numbers and larger colonic polyps than control APC(min mice. We find that A20 binds to the β-catenin destruction complex and restricts canonical wnt signaling by supporting ubiquitination and degradation of β-catenin in intestinal epithelial cells. Moreover, acute deletion of A20 from intestinal epithelial cells in vivo leads to enhanced expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer. Taken together, these findings demonstrate new roles for A20 in restricting β-catenin signaling and preventing colon tumorigenesis.

  12. Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells.

    Science.gov (United States)

    Atarashi, Koji; Tanoue, Takeshi; Ando, Minoru; Kamada, Nobuhiko; Nagano, Yuji; Narushima, Seiko; Suda, Wataru; Imaoka, Akemi; Setoyama, Hiromi; Nagamori, Takashi; Ishikawa, Eiji; Shima, Tatsuichiro; Hara, Taeko; Kado, Shoichi; Jinnohara, Toshi; Ohno, Hiroshi; Kondo, Takashi; Toyooka, Kiminori; Watanabe, Eiichiro; Yokoyama, Shin-Ichiro; Tokoro, Shunji; Mori, Hiroshi; Noguchi, Yurika; Morita, Hidetoshi; Ivanov, Ivaylo I; Sugiyama, Tsuyoshi; Nuñez, Gabriel; Camp, J Gray; Hattori, Masahira; Umesaki, Yoshinori; Honda, Kenya

    2015-10-08

    Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Long-term Renewable Human Intestinal Epithelial Stem Cells as Monolayers: A Potential for Clinical Use

    Science.gov (United States)

    Scott, Andrew; Rouch, Joshua D; Jabaji, Ziyad; Khalil, Hassan A; Solorzano, Sergio; Lewis, Michael; Martín, Martín G.; Stelzner, Matthias G.; Dunn, James C.Y.

    2016-01-01

    Purpose Current culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel. Methods Cadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4–5 days. Results Intestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids. Conclusion This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation. PMID:26995514

  14. Mammalian intestinal epithelial cells in primary culture: a mini-review.

    Science.gov (United States)

    Kaeffer, Bertrand

    2002-03-01

    Epithelial cells lining the digestive tract represent a highly organized system built up by multipotent stem cells. A process of asymmetric mitosis produces a population of proliferative cells that are rapidly renewed and migrate along the crypt-villus axis, differentiating into functional mature cells before dying and exfoliating into the intestinal lumen. Isolated crypts or epithelial cells retaining high viability can be prepared within a few h after tissue sampling. After cells are cultured in serum-free media, short-term studies (16-48 h) can be conducted for endocrinology, energy metabolism, or programmed cell death. However, long-term primary culture of intestinal cells (up to 10 d) is still difficult despite progress in isolation methodologies and manipulation of the cell microenvironment. The main problem in developing primary culture is the lack of structural markers specific to the stem cell compartment. The design of a microscopic multidimensional analytic system to record the expression profiles of biomarkers all along the living intestinal crypt should improve basic knowledge of the survival and growth of adult crypt stem cells, and the selection of totipotent embryonic stem cells capable of differentiating into intestinal tissues should facilitate studies of the genomic basis of endodermal tissue differentiation.

  15. Establishment of primary bovine intestinal epithelial cell culture and clone method.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, Ming-Mei; Sui, Yang-Nan; Zhao, Guo-Qi

    2017-01-01

    The aim of this study was to establish bovine intestinal epithelial cell (BIEC) line and provide a novel clone cell method. Although various strategies of bovine cell culture and clone techniques have been reported, these methods remain not established. Here, we culture successfully primary BIECs and establish a novel clone cell method. Our result showed that BIECs could be successfully cultured and passaged about generation 5. These cellular aggregates and clusters were adherent loosely at day 2 of culture. Cell aggregates and clusters start to proliferate after approximately 4 d. The BIECs showed positive reaction against cytokeratin 18, E-cadherin, and characteristics of epithelial-like morphology. In addition, the fatty acid-binding proteins (FABPs), villin, and intestinal peptidase (IP) band were positive in BIECs. Our results suggest that the establishment of culturing and clone BIEC methods will apply to isolate and clone other primary cells. These BIECs could therefore contribute to the study of bovine intestinal nutrient absorption and regulation, immune regulation, and the pathogenesis of the bovine intestinal disease, which will provide intestinal cell model in vitro.

  16. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.

    Science.gov (United States)

    Uekawa, Atsushi; Yamanaka, Hitoki; Lieben, Liesbet; Kimira, Yoshifumi; Uehara, Mariko; Yamamoto, Yoko; Kato, Shigeaki; Ito, Kosei; Carmeliet, Geert; Masuyama, Ritsuko

    2018-01-05

    Extracellular low phosphate strongly enhances intestinal calcium absorption independently of active vitamin D [1,25(OH) 2 D 3 ] signaling, but the underlying mechanisms remain poorly characterized. To elucidate the phosphate-dependent regulation of calcium transport, we investigated part of the enteral environment that is involved in 1,25(OH) 2 D 3 -independent calcium absorption, which responds to dietary phosphate levels in mice that lack intestinal vitamin D receptor ( Vdr) activity. Impaired calcium absorption in intestinal Vdr-null mice was improved by dietary phosphate restriction. Accordingly, calcium transport in cultured intestinal epithelial cells was increased when the apical side was exposed to low phosphate levels (0.5 mM) compared with normal or high phosphate levels (1.0 or 5.0 mM, respectively). Mechanistically, low phosphate increased ATP in the apical side medium and allowed calcium entry into epithelial cells via the P2X7 purinoreceptor, which results in increased calcium transport. We found that luminal ATP was regulated by the release and degradation of ATP at the epithelium, and phosphate restriction increased ATP release from epithelial cells via connexin-43 hemichannels. Furthermore, ATP degradation by ectonucleotide pyrophosphatase-1 was reduced, which was caused by the reduction of the MAPK cascade. These findings indicate that luminal ATP metabolism regulates transcellular calcium transport in the intestine by an 1,25(OH) 2 D 3 -independent mechanism in response to dietary phosphate levels.-Uekawa, A., Yamanaka, H., Lieben, L., Kimira, Y., Uehara, M., Yamamoto, Y., Kato, S., Ito, K., Carmeliet, G., Masuyama, R. Phosphate-dependent luminal ATP metabolism regulates transcellular calcium transport in intestinal epithelial cells.

  17. Intestinal Epithelial Cell Endoplasmic Reticulum Stress and Inflammatory Bowel Disease Pathogenesis: An Update Review

    Directory of Open Access Journals (Sweden)

    Xiaoshi Ma

    2017-10-01

    Full Text Available The intestinal epithelial cells serve essential roles in maintaining intestinal homeostasis, which relies on appropriate endoplasmic reticulum (ER function for proper protein folding, modification, and secretion. Exogenous or endogenous risk factors with an ability to disturb the ER function can impair the intestinal barrier function and activate inflammatory responses in the host. The last decade has witnessed considerable progress in the understanding of the functional role of ER stress and unfolded protein response (UPR in the gut homeostasis and its significant contribution to the pathogenesis of inflammatory bowel disease (IBD. Herein, we review recent evidence supporting the viewpoint that deregulation of ER stress and UPR signaling in the intestinal epithelium, including the absorptive cells, Paneth cells, goblet cells, and enteroendocrine cells, mediates the action of genetic or environmental factors driving colitis in experimental animals and IBD patients. In addition, we highlight pharmacologic application of chaperones or small molecules that enhance protein folding and modification capacity or improve the function of the ER. These molecules represent potential therapeutic strategies in the prevention or treatment of IBD through restoring ER homeostasis in intestinal epithelial cells.

  18. Epithelial Cell Damage Activates Bactericidal/Permeability Increasing-Protein (BPI Expression in Intestinal Epithelium

    Directory of Open Access Journals (Sweden)

    Arjun Balakrishnan

    2017-08-01

    Full Text Available As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn’s Disease, Ulcerative colitis, and Infectious enteritis’s. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin. Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium and Shigella flexneri whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.

  19. Epithelial WNT Ligands Are Essential Drivers of Intestinal Stem Cell Activation

    Directory of Open Access Journals (Sweden)

    Winnie Y. Zou

    2018-01-01

    Full Text Available Intestinal stem cells (ISCs maintain and repair the intestinal epithelium. While regeneration after ISC-targeted damage is increasingly understood, injury-repair mechanisms that direct regeneration following injuries to differentiated cells remain uncharacterized. The enteric pathogen, rotavirus, infects and damages differentiated cells while sparing all ISC populations, thus allowing the unique examination of the response of intact ISC compartments during injury-repair. Upon rotavirus infection in mice, ISC compartments robustly expand and proliferating cells rapidly migrate. Infection results specifically in stimulation of the active crypt-based columnar ISCs, but not alternative reserve ISC populations, as is observed after ISC-targeted damage. Conditional ablation of epithelial WNT secretion diminishes crypt expansion and ISC activation, demonstrating a previously unknown function of epithelial-secreted WNT during injury-repair. These findings indicate a hierarchical preference of crypt-based columnar cells (CBCs over other potential ISC populations during epithelial restitution and the importance of epithelial-derived signals in regulating ISC behavior.

  20. Antigen presentation by small intestinal epithelial cells uniquely enhances IFN-γ secretion from CD4{sup +} intestinal intraepithelial lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hatano, Ryo; Yamada, Kiyoshi; Iwamoto, Taku; Maeda, Nana; Emoto, Tetsuro; Shimizu, Makoto; Totsuka, Mamoru, E-mail: atotuka@mail.ecc.u-tokyo.ac.jp

    2013-06-14

    Highlights: •Small intestinal epithelial cells (sIECs). •sIECs are able to induce antigen specific proliferation of CD4{sup +} IELs. •sIECs induce markedly enhanced IFN-γ secretion by CD4{sup +} IELs. •Induction of enhanced IFN-γ secretion by sIECs is uniquely observed in CD4{sup +} IELs. -- Abstract: Small intestinal epithelial cells (sIECs) express major histocompatibility complex class II molecules even in a normal condition, and are known to function as antigen presenting cells (APCs) at least in vitro. These findings raised the possibility that sIECs play an important role in inducing immune responses against luminal antigens, especially those of intestinal intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs). We herein showed that antigenic stimulation with sIECs induced markedly greater secretion of interferon-gamma (IFN-γ) by CD4{sup +} IELs, but not interleukin (IL)-4, IL-10 and IL-17 although the proliferative response was prominently lower than that with T cell-depleted splenic APCs. In contrast, no enhanced IFN-γ secretion by CD4{sup +} LPLs and primed splenic CD4{sup +} T cells was observed when stimulated with sIECs. Taken together, these results suggest that sIECs uniquely activate CD4{sup +} IELs and induce remarkable IFN-γ secretion upon antigenic stimulation in vivo.

  1. Modulation of Intestinal Epithelial Cell Proliferation, Migration, and Differentiation In Vitro by Astragalus Polysaccharides

    Science.gov (United States)

    Zhang, Chun Li; Ren, Hui Jun; Liu, Meng Meng; Li, Xiao Gai; Sun, De Li; Li, Nan; Ming, Liang

    2014-01-01

    Previous studies have shown that Astragalus polysaccharides (APS) can be used to treat general gastrointestinal disturbances including intestinal mucosal injury. However, the mechanism by which APS mediate this effect is unclear. In the present study, the effects of APS on proliferation, migration, and differentiation of intestinal epithelial cells (IEC-6) were assessed using an in vitro wounding model and colorimetric thiazolyl blue (MTT) assays. The effect of APS on IEC-6 cell differentiation was observed using a light microscope and scanning electron microscope, and the expression of differentiation-specific markers of IEC-6 cells, such as cytokeratin 18 (CK18), alkaline phosphatase (ALP), tight junction protein ZO-2, and sucrase-isomaltase (SI), was determined by immunofluorescence assay (IFA) and real-time PCR. In addition, APS-induced signaling pathways in IEC-6 cells were characterized. Our results indicated that APS significantly enhance migration and proliferation of IEC-6 cells in vitro. APS-treated IEC-6 cells have numerous microvilli on their apical surface and also highly express CK18, ALP, ZO-2, and SI. Moreover, APS-treated IEC-6 cells, in which the activity and expression level of ornithine decarboxylase (ODC) were significantly elevated, also exhibited an increase in cellular putrescine, whereas no significant increase in TGF-β levels was observed. These findings suggest that APS may enhance intestinal epithelial cell proliferation, migration, and differentiation in vitro by stimulating ODC gene expression and activity and putrescine production, independent of TGF-β. Exogenous administration of APS may provide a new approach for modulating intestinal epithelial wound restitution in vivo. PMID:25157577

  2. Bile acid receptor TGR5 overexpression is associated with decreased intestinal mucosal injury and epithelial cell proliferation in obstructive jaundice.

    Science.gov (United States)

    Ji, Chen-Guang; Xie, Xiao-Li; Yin, Jie; Qi, Wei; Chen, Lei; Bai, Yun; Wang, Na; Zhao, Dong-Qiang; Jiang, Xiao-Yu; Jiang, Hui-Qing

    2017-04-01

    Bile acids stimulate intestinal epithelial proliferation in vitro. We sought to investigate the role of the bile acid receptor TGR5 in the protection of intestinal epithelial proliferation in obstructive jaundice. Intestinal tissues and serum samples were obtained from patients with malignant obstructive jaundice and from bile duct ligation (BDL) rats. Intestinal permeability and morphological changes in the intestinal mucosa were observed. The functions of TGR5 in cell proliferation in intestinal epithelial injury were determined by overexpression or knockdown studies in Caco-2 and FHs 74 Int cells pretreated with lipopolysaccharide (LPS). Internal biliary drainage was superior to external biliary drainage in recovering intestinal permeability and mucosal histology in patients with obstructive jaundice. In BDL rats, feeding of chenodeoxycholic acid (CDCA) decreased intestinal mucosa injury. The levels of PCNA, a marker of proliferation, increased in response to CDCA feeding and were paralleled by elevated TGR5 expression. CDCA upregulated TGR5 expression and promoted proliferation in Caco-2 and FHs 74 Int cells pretreated with LPS. Overexpression of TGR5 resulted in increased PCNA, cell viability, EdU incorporation, and the proportion of cells in S phase, whereas knockdown of TGR5 had the opposite effect. Our data indicate that bile acids promote intestinal epithelial cell proliferation and decrease mucosal injury by upregulating TGR5 expression in obstructive jaundice. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Intestinal epithelial cell caveolin 1 regulates fatty acid and lipoprotein cholesterol plasma levels.

    Science.gov (United States)

    Otis, Jessica P; Shen, Meng-Chieh; Quinlivan, Vanessa; Anderson, Jennifer L; Farber, Steven A

    2017-03-01

    Caveolae and their structural protein caveolin 1 (CAV1) have roles in cellular lipid processing and systemic lipid metabolism. Global deletion of CAV1 in mice results in insulin resistance and increases in atherogenic plasma lipids and cholesterol, but protects from diet-induced obesity and atherosclerosis. Despite the fundamental role of the intestinal epithelia in the regulation of dietary lipid processing and metabolism, the contributions of CAV1 to lipid metabolism in this tissue have never been directly investigated. In this study the cellular dynamics of intestinal Cav1 were visualized in zebrafish and the metabolic contributions of CAV1 were determined with mice lacking CAV1 in intestinal epithelial cells (CAV1 IEC-KO ). Live imaging of Cav1-GFP and fluorescently labeled caveolae cargos shows localization to the basolateral and lateral enterocyte plasma membrane (PM), suggesting Cav1 mediates transport between enterocytes and the submucosa. CAV1 IEC-KO mice are protected from the elevation in circulating fasted low-density lipoprotein (LDL) cholesterol associated with a high-fat diet (HFD), but have increased postprandial LDL cholesterol, total free fatty acids (FFAs), palmitoleic acid, and palmitic acid. The increase in circulating FAs in HFD CAV1 IEC-KO mice is mirrored by decreased hepatic FAs, suggesting a non-cell-autonomous role for intestinal epithelial cell CAV1 in promoting hepatic FA storage. In conclusion, CAV1 regulates circulating LDL cholesterol and several FA species via the basolateral PM of enterocytes. These results point to intestinal epithelial cell CAV1 as a potential therapeutic target to lower circulating FFAs and LDL cholesterol, as high levels are associated with development of type II diabetes and cardiovascular disease. © 2017. Published by The Company of Biologists Ltd.

  4. Autophagy protects intestinal epithelial cells against deoxynivalenol toxicity by alleviating oxidative stress via IKK signaling pathway.

    Science.gov (United States)

    Tang, Yulong; Li, Jianjun; Li, Fengna; Hu, Chien-An A; Liao, Peng; Tan, Kunrong; Tan, Bie; Xiong, Xia; Liu, Gang; Li, Tiejun; Yin, Yulong

    2015-12-01

    Autophagy is an intracellular process of homeostatic degradation that promotes cell survival under various stressors. Deoxynivalenol (DON), a fungal toxin, often causes diarrhea and disturbs the homeostasis of the intestinal system. To investigate the function of intestinal autophagy in response to DON and associated mechanisms, we firstly knocked out ATG5 (autophagy-related gene 5) in porcine intestinal epithelial cells (IPEC-J2) using CRISPR-Cas9 technology. When treated with DON, autophagy was induced in IPEC-J2 cells but not in IPEC-J2.Atg5ko cells. The deficiency in autophagy increased DON-induced apoptosis in IPEC-J2.atg5ko cells, in part, through the generation of reactive oxygen species (ROS). The cellular stress response can be restored in IPEC-J2.atg5ko cells by overexpressing proteins involved in protein folding. Interestingly, we found that autophagy deficiency downregulated the expression of endoplasmic reticulum folding proteins BiP and PDI when IPEC-J2.atg5ko cells were treated with DON. In addition, we investigated the molecular mechanism of autophagy involved in the IKK, AMPK, and mTOR signaling pathway and found that Bay-117082 and Compound C, specific inhibitors for IKK and AMPK, respectively, inhibited the induction of autophagy. Taken together, our results suggest that autophagy is pivotal for protection against DON in pig intestinal cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Distinct Roles for Intestinal Epithelial Cell-Specific Hdac1 and Hdac2 in the Regulation of Murine Intestinal Homeostasis.

    Science.gov (United States)

    Gonneaud, Alexis; Turgeon, Naomie; Boudreau, François; Perreault, Nathalie; Rivard, Nathalie; Asselin, Claude

    2016-02-01

    The intestinal epithelium responds to and transmits signals from the microbiota and the mucosal immune system to insure intestinal homeostasis. These interactions are in part conveyed by epigenetic modifications, which respond to environmental changes. Protein acetylation is an epigenetic signal regulated by histone deacetylases, including Hdac1 and Hdac2. We have previously shown that villin-Cre-inducible intestinal epithelial cell (IEC)-specific Hdac1 and Hdac2 deletions disturb intestinal homeostasis. To determine the role of Hdac1 and Hdac2 in the regulation of IEC function and the establishment of the dual knockout phenotype, we have generated villin-Cre murine models expressing one Hdac1 allele without Hdac2, or one Hdac2 allele without Hdac1. We have also investigated the effect of short-term deletion of both genes in naphtoflavone-inducible Ah-Cre and tamoxifen-inducible villin-Cre(ER) mice. Mice with one Hdac1 allele displayed normal tissue architecture, but increased sensitivity to DSS-induced colitis. In contrast, mice with one Hdac2 allele displayed intestinal architecture defects, increased proliferation, decreased goblet cell numbers as opposed to Paneth cells, increased immune cell infiltration associated with fibrosis, and increased sensitivity to DSS-induced colitis. In comparison to dual knockout mice, intermediary activation of Notch, mTOR, and Stat3 signaling pathways was observed. While villin-Cre(ER) Hdac1 and Hdac2 deletions led to an impaired epithelium and differentiation defects, Ah-Cre-mediated deletion resulted in blunted proliferation associated with the induction of a DNA damage response. Our results suggest that IEC determination and intestinal homeostasis are highly dependent on Hdac1 and Hdac2 activity levels, and that changes in the IEC acetylome may alter the mucosal environment. © 2015 Wiley Periodicals, Inc.

  6. Heat shock protein 70-dependent protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells.

    Science.gov (United States)

    Qin, Ying; Naito, Yuji; Handa, Osamu; Hayashi, Natsuko; Kuki, Aiko; Mizushima, Katsura; Omatsu, Tatsushi; Tanimura, Yuko; Morita, Mayuko; Adachi, Satoko; Fukui, Akifumi; Hirata, Ikuhiro; Kishimoto, Etsuko; Nishikawa, Taichiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Yagi, Nobuaki; Kokura, Satoshi; Yoshikawa, Toshikazu

    2011-11-01

    Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic

  7. A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC

    OpenAIRE

    Minghetti, Matteo; Drieschner, Carolin; Bramaz, Nadine; Schug, Hannah; Schirmer, Kristin

    2017-01-01

    The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow tr...

  8. Lactobacillus reuteri glyceraldehyde-3-phosphate dehydrogenase functions in adhesion to intestinal epithelial cells.

    Science.gov (United States)

    Zhang, Wen-Ming; Wang, Hai-Feng; Gao, Kan; Wang, Cong; Liu, Li; Liu, Jian-Xin

    2015-05-01

    This study was aimed to identify key surface proteins mediating the adhesion of lactobacilli to intestinal epithelial cells. By using Caco-2 and IPEC-J2 cells labeled with sulfo-NHS-biotin in the western blotting, a protein band of an approximately 37 kDa was detected on the surface layer of Lactobacillus reuteri strains ZJ616, ZJ617, ZJ621, and ZJ623 and Lactobacillus rhamnosus GG. Mass spectrometry analysis using the adhesion-related protein from L. reuteri ZJ617 showed that it was 100% homologous to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. reuteri JCM 1112 (GenBank: YP_001841377). The ability of L. reuteri ZJ617 to adhere to epithelial cells decreased significantly by treatment with LiCl or by blocking with an anti-GAPDH antibody, in comparison with the untreated strain (p reuteri ZJ617. The results indicated that the GAPDH protein of L. reuteri ZJ617 acts as an adhesion component that plays an important role in binding to the intestinal epithelial cells.

  9. Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Junqiang; Doi, Hiroshi [Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, California (United States); Saar, Matthias; Santos, Jennifer [Department of Urology, School of Medicine, Stanford University, Stanford, California (United States); Li, Xuejun; Peehl, Donna M. [Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, California (United States); Knox, Susan J., E-mail: sknox@stanford.edu [Department of Radiation Oncology, School of Medicine, Stanford University, Stanford, California (United States)

    2013-12-01

    Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome. The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy.

  10. Radioprotection and Cell Cycle Arrest of Intestinal Epithelial Cells by Darinaparsin, a Tumor Radiosensitizer

    International Nuclear Information System (INIS)

    Tian, Junqiang; Doi, Hiroshi; Saar, Matthias; Santos, Jennifer; Li, Xuejun; Peehl, Donna M.; Knox, Susan J.

    2013-01-01

    Purpose: It was recently reported that the organic arsenic compound darinaparsin (DPS) is a cytotoxin and radiosensitizer of tumor cells in vitro and in subcutaneous xenograft tumors. Surprisingly, it was also found that DPS protects normal intestinal crypt epithelial cells (CECs) from clonogenic death after ionizing radiation (IR). Here we tested the DPS radiosensitizing effect in a clinically relevant model of prostate cancer and explored the radioprotective effect and mechanism of DPS on CECs. Methods and Materials: The radiation modification effect of DPS was tested in a mouse model of orthotopic xenograft prostate cancer and of IR-induced acute gastrointestinal syndrome. The effect of DPS on CEC DNA damage and DNA damage responses was determined by immunohistochemistry. Results: In the mouse model of IR-induced gastrointestinal syndrome, DPS treatment before IR accelerated recovery from body weight loss and increased animal survival. DPS decreased post-IR DNA damage and cell death, suggesting that the radioprotective effect was mediated by enhanced DNA damage repair. Shortly after DPS injection, significant cell cycle arrest was observed in CECs at both G1/S and G2/M checkpoints, which was accompanied by the activation of cell cycle inhibitors p21 and growth arrest and DNA-damage-inducible protein 45 alpha (GADD45A). Further investigation revealed that DPS activated ataxia telangiectasia mutated (ATM), an important inducer of DNA damage repair and cell cycle arrest. Conclusions: DPS selectively radioprotected normal intestinal CECs and sensitized prostate cancer cells in a clinically relevant model. This effect may be, at least in part, mediated by DNA damage response activation and has the potential to significantly increase the therapeutic index of radiation therapy

  11. Mechanism of leukotriene D4 inhibition of Na-alanine cotransport in intestinal epithelial cells.

    Science.gov (United States)

    Talukder, Jamilur R; Kekuda, Ramesh; Saha, Prosenjit; Sundaram, Uma

    2008-07-01

    In a rabbit model of chronic intestinal inflammation, we previously demonstrated inhibition of neutral Na-amino acid cotransport. The mechanism of the inhibition was secondary to a decrease in the affinity for amino acid rather than the number of cotransporters. Since leukotriene (LT)D4 is known to be elevated in enterocytes during chronic intestinal inflammation, we used rat intestinal epithelial cell (IEC-18) monolayers to determine the mechanism of regulation of Na-alanine cotransport (alanine, serine, cysteine transporter 1: ASCT1) by LTD4. Na-alanine cotransport was inhibited by LTD4 in IEC-18 cells. The mechanism of inhibition of ASCT1 (solute carrier, SLC1A4) by LTD4 is secondary to a decrease in the affinity of the cotransporter for alanine without a significant change in cotransporter numbers and is not secondary to an alteration in the Na+ extruding capacity of the cells. Real-time quantitative PCR and Western blot analysis results indicate that ASCT1 message and protein levels are also unchanged in LTD4-treated IEC-18 cells. These results indicate that LTD4 inhibits Na-dependent neutral amino acid cotransport in IEC. The mechanism of inhibition is secondary to a decrease in the affinity for alanine, which is identical to that seen in villus cells from the chronically inflamed rabbit small intestine, where LTD4 levels are significantly increased.

  12. FOXA2 regulates a network of genes involved in critical functions of human intestinal epithelial cells.

    Science.gov (United States)

    Gosalia, Nehal; Yang, Rui; Kerschner, Jenny L; Harris, Ann

    2015-07-01

    The forkhead box A (FOXA) family of pioneer transcription factors is critical for the development of many endoderm-derived tissues. Their importance in regulating biological processes in the lung and liver is extensively characterized, though much less is known about their role in intestine. Here we investigate the contribution of FOXA2 to coordinating intestinal epithelial cell function using postconfluent Caco2 cells, differentiated into an enterocyte-like model. FOXA2 binding sites genome-wide were determined by ChIP-seq and direct targets of the factor were validated by ChIP-qPCR and siRNA-mediated depletion of FOXA1/2 followed by RT-qPCR. Peaks of FOXA2 occupancy were frequent at loci contributing to gene ontology pathways of regulation of cell migration, cell motion, and plasma membrane function. Depletion of both FOXA1 and FOXA2 led to a significant reduction in the expression of multiple transmembrane proteins including ion channels and transporters, which form a network that is essential for maintaining normal ion and solute transport. One of the targets was the adenosine A2B receptor, and reduced receptor mRNA levels were associated with a functional decrease in intracellular cyclic AMP. We also observed that 30% of FOXA2 binding sites contained a GATA motif and that FOXA1/A2 depletion reduced GATA-4, but not GATA-6 protein levels. These data show that FOXA2 plays a pivotal role in regulating intestinal epithelial cell function. Moreover, that the FOXA and GATA families of transcription factors may work cooperatively to regulate gene expression genome-wide in the intestinal epithelium. Copyright © 2015 the American Physiological Society.

  13. Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.

    Science.gov (United States)

    Powell, Anne E; Anderson, Eric C; Davies, Paige S; Silk, Alain D; Pelz, Carl; Impey, Soren; Wong, Melissa H

    2011-02-15

    The most deadly phase in cancer progression is attributed to the inappropriate acquisition of molecular machinery leading to metastatic transformation and spread of disease to distant organs. Although it is appreciated that metastasis involves epithelial-mesenchymal interplay, the underlying mechanism defining this process is poorly understood. Specifically, how cancer cells evade immune surveillance and gain the ability to navigate the circulatory system remains a focus. One possible mechanism underlying metastatic conversion is fusion between blood-derived immune cells and cancer cells. While this notion is a century old, in vivo evidence that cell fusion occurs within tumors and imparts genetic or physiologic changes remains controversial. We have previously demonstrated in vivo cell fusion between blood cells and intestinal epithelial cells in an injury setting. Here, we hypothesize that immune cells, such as macrophages, fuse with tumor cells imparting metastatic capabilities by transferring their cellular identity. We used parabiosis to introduce fluorescent-labeled bone marrow-derived cells to mice with intestinal tumors, finding that fusion between circulating blood-derived cells and tumor epithelium occurs during the natural course of tumorigenesis. Moreover, we identify the macrophage as a key cellular partner for this process. Interestingly, cell fusion hybrids retain a transcriptome identity characteristic of both parental derivatives, while also expressing a unique subset of transcripts. Our data supports the novel possibility that tumorigenic cell fusion may impart physical behavior attributed to migratory macrophages, including navigation of circulation and immune evasion. As such, cell fusion may represent a promising novel mechanism underlying the metastatic conversion of cancer cells. ©2011 AACR.

  14. Commensal Streptococcus salivarius Modulates PPARγ Transcriptional Activity in Human Intestinal Epithelial Cells.

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    Benoît Couvigny

    Full Text Available The impact of commensal bacteria in eukaryotic transcriptional regulation has increasingly been demonstrated over the last decades. A multitude of studies have shown direct effects of commensal bacteria from local transcriptional activity to systemic impact. The commensal bacterium Streptococcus salivarius is one of the early bacteria colonizing the oral and gut mucosal surfaces. It has been shown to down-regulate nuclear transcription factor (NF-кB in human intestinal cells, a central regulator of the host mucosal immune system response to the microbiota. In order to evaluate its impact on a further important transcription factor shown to link metabolism and inflammation in the intestine, namely PPARγ (peroxisome proliferator-activated receptor, we used human intestinal epithelial cell-lines engineered to monitor PPARγ transcriptional activity in response to a wide range of S. salivarius strains. We demonstrated that different strains from this bacterial group share the property to inhibit PPARγ activation independently of the ligand used. First attempts to identify the nature of the active compounds showed that it is a low-molecular-weight, DNase-, proteases- and heat-resistant metabolite secreted by S. salivarius strains. Among PPARγ-targeted metabolic genes, I-FABP and Angptl4 expression levels were dramatically reduced in intestinal epithelial cells exposed to S. salivarius supernatant. Both gene products modulate lipid accumulation in cells and down-regulating their expression might consequently affect host health. Our study shows that species belonging to the salivarius group of streptococci impact both host inflammatory and metabolic regulation suggesting a possible role in the host homeostasis and health.

  15. Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D.

    Science.gov (United States)

    Anabazhagan, Arivarasu N; Chatterjee, Ishita; Priyamvada, Shubha; Kumar, Anoop; Tyagi, Sangeeta; Saksena, Seema; Alrefai, Waddah A; Dudeja, Pradeep K; Gill, Ravinder K

    2017-03-16

    The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin

  16. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang [Department of General Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Yang, Hua, E-mail: hwbyang@126.com [Department of General Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing 400037 (China)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. Black-Right-Pointing-Pointer The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. Black-Right-Pointing-Pointer GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. Black-Right-Pointing-Pointer GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  17. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    International Nuclear Information System (INIS)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang; Yang, Hua

    2012-01-01

    Highlights: ► Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. ► The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. ► GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. ► GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  18. Uptake of Gold Nanoparticles by Intestinal Epithelial Cells: Impact of Particle Size on Their Absorption, Accumulation, and Toxicity.

    Science.gov (United States)

    Yao, Mingfei; He, Lili; McClements, David Julian; Xiao, Hang

    2015-09-16

    Inorganic nanomaterials have been increasingly utilized in many consumer products, which has led to concerns about their potential toxicity. At present, there is limited knowledge about the gastrointestinal fate and cytotoxicity of ingested inorganic nanoparticles. This study determined the influence of particle size and concentration of gold nanoparticles (AuNPs) on their absorption, accumulation, and cytotoxicity in model intestinal epithelial cells. As the mean particle diameter of the AuNPs decreased (from 100 to 50 to 15 nm), their rate of absorption by the intestinal epithelium cells increased, but their cellular accumulation in the epithelial cells decreased. Moreover, accumulation of AuNPs caused cytotoxicity in the intestinal epithelial cells, which was evidenced by depolarization of mitochondria membranes. These results provide important insights into the relationship between the dimensions of AuNPs and their gastrointestinal uptake and potential cytotoxicity.

  19. Effects of the ionising radiations on the structure and the function of the intestinal epithelial cell

    International Nuclear Information System (INIS)

    Haton, C.

    2005-06-01

    The intestinal mucosa is a particularly radio-sensitive tissue and damage may occur following either accidental or therapeutic exposure. the deleterious actions of ionizing radiation are linked to the formation of sometimes overwhelming quantities of reactive oxygen species (R.O.S.). Production of R.O.S. is both direct and indirect from the secondary effects of irradiation. A better comprehension of the underlying mechanisms of injury will lead to more adapted therapeutic approaches to limit the harmful effects of irradiation. The homeostasis of the intestinal epithelium is regulated by three factors: proliferation, apoptosis and differentiation. these three factors were studied using the cell model, HT29, in order to analyze modulations of this balance after irradiation. our results, in agreement with other data, showed the establishment of mitotic delay. This arrest of proliferation was followed by apoptosis to be the major mechanism leading to cell death in this model. thus, for the first time, we have shown that irradiated intestinal epithelial cells preserve their capacity to differentiate. This indicates, although indirectly, that intestinal cells have and preserve an intrinsic capacity restore a functional epithelium. R.O.S. are considered as intermediates between the physical nature of radiations and biological responses. It seems essential to understand anti-oxidant mechanisms used by the cell for defence against the deleterious effects of R.O.S post exposure. This study of several anti-oxidant defence mechanisms of intestinal mucosa, was carried out in vivo in the mouse at different times following abdominal irradiation. We observed an early mitochondrial response in the hours following irradiation revealing this organelle as a particular target. We demonstrated a strong alteration of anti-oxidant capacity as revealed by a decrease in S.O.D.s, catalase and an increase of the G.P.X.s and M.T.s. A part of these modifications appeared to depend on an

  20. Loss of Survivin in Intestinal Epithelial Progenitor Cells Leads to Mitotic Catastrophe and Breakdown of Gut Immune Homeostasis

    Directory of Open Access Journals (Sweden)

    Eva Martini

    2016-02-01

    Full Text Available A tightly regulated balance of proliferation and cell death of intestinal epithelial cells (IECs is essential for maintenance of gut homeostasis. Survivin is highly expressed during embryogenesis and in several cancer types, but little is known about its role in adult gut tissue. Here, we show that Survivin is specifically expressed in transit-amplifying cells and Lgr5+ stem cells. Genetic loss of Survivin in IECs resulted in destruction of intestinal integrity, mucosal inflammation, and death of the animals. Survivin deletion was associated with decreased epithelial proliferation due to defective chromosomal segregation. Moreover, Survivin-deficient animals showed induced phosphorylation of p53 and H2AX and increased levels of cell-intrinsic apoptosis in IECs. Consequently, induced deletion of Survivin in Lgr5+ stem cells led to cell death. In summary, Survivin is a key regulator of gut tissue integrity by regulating epithelial homeostasis in the stem cell niche.

  1. Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells.

    Science.gov (United States)

    Miller, Kelly A; Chaand, Mudit; Gregoire, Stacy; Yoshida, Takeshi; Beck, Lisa A; Ivanov, Andrei I; Dziejman, Michelle

    2016-12-01

    AM-19226 is a pathogenic, non-O1/non-O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co-culturing the Caco2-BBE human intestinal epithelial cell line with AM-19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2-BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell-cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM-19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner. © 2016 John Wiley & Sons Ltd.

  2. Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells

    Science.gov (United States)

    Miller, Kelly A.; Chaand, Mudit; Gregoire, Stacy; Yoshida, Takeshi; Beck, Lisa; Ivanov, Andrei I.; Dziejman, Michelle

    2016-01-01

    Summary AM-19226 is a pathogenic, non-O1/non-O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co-culturing the Caco2-BBE human intestinal epithelial cell line with AM-19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2-BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell-cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM-19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner. PMID:27302486

  3. Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

    Science.gov (United States)

    Milovic, Vladan; Turchanowa, Lyudmila; Stein, Jürgen; Caspary, Wolfgang F.

    2001-01-01

    AIM: To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption. METHODS: The transepithelial transport and the cellular accumulation of putrescine was measured using Caco-2 cell monolayers grown on permeable filters. RESULTS: Transepithelial transport of putrescine in physiological concentrations ( > 0.5 mM) from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical to basolateral direction.EGF enhanced putrescine accumulation in Caco-2 cells by four fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of S adenosylmethionine decarboxylase. However, EGF did not have any significant influence on putrescine flux across the Caco- 2 cell monolayers. Excretion of putrescine from Caco-2 cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber, contributed hundreds of micromoles polyamines to the basolateral chamber. CONCLUSION: Transepithelial transport of putrescine across Caco-2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF. PMID:11819759

  4. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

    DEFF Research Database (Denmark)

    Rosenstiel, Philip; Sina, Christian; End, Caroline

    2007-01-01

    Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes...... the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene...... for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF...

  5. Regulation of DMBT1 via NOD2 and TLR4 in intestinal epithelial cells modulates bacterial recognition and invasion

    DEFF Research Database (Denmark)

    Rosenstiel, Philip; Sina, Christian; End, Caroline

    2007-01-01

    Mucosal epithelial cell layers are constantly exposed to a complex resident microflora. Deleted in malignant brain tumors 1 (DMBT1) belongs to the group of secreted scavenger receptor cysteine-rich proteins and is considered to be involved in host defense by pathogen binding. This report describes...... for the intracellular pathogen receptor NOD2 via NF-kappaB activation. DMBT1 is strongly up-regulated in the inflamed intestinal mucosa of Crohn's disease patients with wild-type, but not with mutant NOD2. We show that DMBT1 inhibits cytoinvasion of Salmonella enterica and LPS- and muramyl dipeptide-induced NF...... the regulation and function of DMBT1 in intestinal epithelial cells, which form the primary immunological barrier for invading pathogens. We report that intestinal epithelial cells up-regulate DMBT1 upon proinflammatory stimuli (e.g., TNF-alpha, LPS). We demonstrate that DMBT1 is a target gene...

  6. Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Del Regno, Marisanta; Adesso, Simona; Popolo, Ada [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Quaroni, Andrea [Department of Biomedical Sciences, Cornell University, Veterinary Research Tower, Cornell University, Ithaca, NY 14853–6401 (United States); Autore, Giuseppina [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy); Severino, Lorella [Department of Pathology and Animal Health, Division of Toxicology, School of Veterinary Medicine, University of Naples “Federico II”, Via Delpino 1, 80137 Naples (Italy); Marzocco, Stefania, E-mail: smarzocco@unisa.it [Department of Pharmacy, School of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132–84084 Fisciano, Salerno (Italy)

    2015-06-01

    Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. - Highlights: • Nivalenol induces oxidative stress in intestinal epithelial cells (IECs). • Nivalenol increases deoxynivalenol pro-oxidant effects in IECs. • Nivalenol and deoxynivalenol trigger antioxidant response IECs. • These results indicate the importance of mycotoxins co-contamination.

  7. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia.

    Directory of Open Access Journals (Sweden)

    Simon A W G Dello

    Full Text Available OBJECTIVES: The intermittent Pringle maneuver (IPM is frequently applied to minimize blood loss during liver transection. Clamping the hepatoduodenal ligament blocks the hepatic inflow, which leads to a non circulating (hepatosplanchnic outflow. Also, IPM blocks the mesenteric venous drainage (as well as the splenic drainage with raising pressure in the microvascular network of the intestinal structures. It is unknown whether the IPM is harmful to the gut. The aim was to investigate intestinal epithelial cell damage reflected by circulating intestinal fatty acid binding protein levels (I-FABP in patients undergoing liver resection with IPM. METHODS: Patients who underwent liver surgery received total IPM (total-IPM or selective IPM (sel-IPM. A selective IPM was performed by selectively clamping the right portal pedicle. Patients without IPM served as controls (no-IPM. Arterial blood samples were taken immediately after incision, ischemia and reperfusion of the liver, transection, 8 hours after start of surgery and on the first post-operative day. RESULTS: 24 patients (13 males were included. 7 patients received cycles of 15 minutes and 5 patients received cycles of 30 minutes of hepatic inflow occlusion. 6 patients received cycles of 15 minutes selective hepatic occlusion and 6 patients underwent surgery without inflow occlusion. Application of total-IPM resulted in a significant increase in I-FABP 8 hours after start of surgery compared to baseline (p<0.005. In the no-IPM group and sel-IPM group no significant increase in I-FABP at any time point compared to baseline was observed. CONCLUSION: Total-IPM in patients undergoing liver resection is associated with a substantial increase in arterial I-FABP, pointing to intestinal epithelial injury during liver surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01099475.

  8. A gene expression programme induced by bovine colostrum whey promotes growth and wound-healing processes in intestinal epithelial cells.

    Science.gov (United States)

    Blais, M; Pouliot, Y; Gauthier, S; Boutin, Y; Lessard, M

    2014-01-01

    Bovine colostrum is well known for its beneficial properties on health and development. It contains a wide variety of bioactive ingredients that are known to promote a number of cellular processes. Therefore the use of colostrum whey as a feed additive to promote intestinal health has been proposed, yet little is known about mechanisms implicated in its beneficial properties on intestinal epithelial cells. In the present paper, casein were removed from bovine colostrum and the remaining liquid, rich in bioactive compounds, was evaluated for its capacity to modulate cellular processes in porcine intestinal epithelial cell line IPEC-J2 and human colon adenocarcinoma cell line Caco-2/15. First, we verified the effect of colostrum whey and cheese whey on processes involved in intestinal wound healing, including cell proliferation, attachment, morphology and migration. Our results showed that colostrum whey promoted proliferation and migration, and decreased specifically the attachment of Caco-2/15 cells on the culture dish. On the other hand, cheese whey induced proliferation and morphological changes in IPEC-J2 cells, but failed to induce migration. The gene expression profile of IPEC-J2 cells following colostrum whey treatment was evaluated by microarray analysis. Results revealed that the expression of a significant number of genes involved in cell migration, adhesion and proliferation was indeed affected in colostrum whey-treated cells. In conclusion, colostrum specific bioactive content could be beneficial for intestinal epithelial cell homoeostasis by controlling biological processes implicated in wound healing through a precise gene expression programme.

  9. Coating with luminal gut-constituents alters adherence of nanoparticles to intestinal epithelial cells

    Directory of Open Access Journals (Sweden)

    Heike Sinnecker

    2014-12-01

    Full Text Available Background: Anthropogenic nanoparticles (NPs have found their way into many goods of everyday life. Inhalation, ingestion and skin contact are potential routes for NPs to enter the body. In particular the digestive tract with its huge absorptive surface area provides a prime gateway for NP uptake. Considering that NPs are covered by luminal gut-constituents en route through the gastrointestinal tract, we wanted to know if such modifications have an influence on the interaction between NPs and enterocytes.Results: We investigated the consequences of a treatment with various luminal gut-constituents on the adherence of nanoparticles to intestinal epithelial cells. Carboxylated polystyrene particles 20, 100 and 200 nm in size represented our anthropogenic NPs, and differentiated Caco-2 cells served as model for mature enterocytes of the small intestine. Pretreatment with the proteins BSA and casein consistently reduced the adherence of all NPs to the cultured enterocytes, while incubation of NPs with meat extract had no obvious effect on particle adherence. In contrast, contact with intestinal fluid appeared to increase the particle-cell interaction of 20 and 100 nm NPs.Conclusion: Luminal gut-constituents may both attenuate and augment the adherence of NPs to cell surfaces. These effects appear to be dependent on the particle size as well as on the type of interacting protein. While some proteins will rather passivate particles towards cell attachment, possibly by increasing colloid stability or camouflaging attachment sites, certain components of intestinal fluid are capable to modify particle surfaces in such a way that interactions with cellular surface structures result in an increased binding.

  10. Primary culture of intestinal epithelial cells as a potential model for Toxoplasma gondii enteric cycle studies

    Directory of Open Access Journals (Sweden)

    Marcos de Assis Moura

    2009-09-01

    Full Text Available The primary culture of intestinal epithelial cells from domestic cats is an efficient cellular model to study the enteric cycle of Toxoplasma gondii in a definitive host. The parasite-host cell ratio can be pointed out as a decisive factor that determines the intracellular fate of bradyzoites forms. The development of the syncytial-like forms of T. gondii was observed using the 1:20 bradyzoite-host cell ratio, resulting in similar forms described in in vivo systems. This alternative study potentially opens up the field for investigation into the molecular aspects of this interaction. This can contribute to the development of new strategies for intervention of a main route by which toxoplasmosis spreads.

  11. Na+/H+ Exchanger Regulates Amino Acid-Mediated Autophagy in Intestinal Epithelial Cells

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    Huiying Shi

    2017-08-01

    Full Text Available Background/Aims: Dysfunctional autophagy has been reported to be associated with aberrant intestinal metabolism. Amino acids can regulate autophagic activity in intestinal epithelial cells (IECs. Na+/H+-exchanger 3 (NHE3 has been found to participate in the absorption of amino acids in the intestine, but whether NHE3 is involved in the regulation of autophagy in IECs is unclear. Methods: In the present study, an amino acid starvation-induced autophagic model was established. Then, the effects of alanine and proline with or without the NHE inhibitor 5-(N-ethyl-N-isopropyl amiloride (EIPA were evaluated. Autophagy was examined based on the microtubule-associated light chain 3 (LC3 levels, transmission electron microscopy (TEM, tandem GFP-mCherry-LC3 construct, sequestosome-1 (SQSTM1, P62 mRNA and protein levels, and autophagy-related gene (ATG 5, 7, and 12 expression levels. The autophagic flux was evaluated as the ratio of yellow (autophagosomes to red (autolysosomes LC3 puncta. Results: Following amino acid starvation, we found the LC3-II and ATG expression levels were enhanced in the IEC-18 cells. An increase in the number of autophagic vacuoles was concomitantly observed by TEM and confocal microscopy. Based on the results, supplementation with either alanine or proline depressed autophagy in the IEC-18 cells. Consistent with the elevated LC3-II levels, ATG expression increased upon NHE3 inhibition. Moreover, the mCherry-GFP-LC3 autophagic puncta representing both autophagosomes and autolysosomes per cell increased after EIPA treatment. Conclusions: These results demonstrate that NHE (most likely NHE3 may participate in the amino acid regulation of autophagy in IECs, which would aid in the design of better treatments for intestinal inflammation.

  12. Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Mandayam O. Nandan

    2015-01-01

    Full Text Available Krüppel-like factor 5 (KLF5 is a pro-proliferative transcription factor that is expressed in dividing epithelial cells of the intestinal crypt. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5 has been identified as a stem cell marker in both small intestinal and colonic epithelial cells. To determine whether KLF5 regulates proliferation of intestinal stem cells, we investigated the effects of Klf5 deletion specifically from the intestinal stem cells in adult mice. Mice with inducible intestinal stem cell-specific deletion of Klf5 (Lgr5-Klf5fl/fl were injected with tamoxifen for 5 consecutive days to induce Lgr5-driven Cre expression. Intestinal and colonic tissues were examined by immunohistochemistry at various time points up to 112 days following start of tamoxifen treatment. Klf5 is co-localized in the crypt-based columnar (CBC cells that express Lgr5. By 11 days following the start of tamoxifen treatment, Lgr5-positive crypts from which Klf5 was deleted exhibited a loss of proliferation that was accompanied by an increase in apoptosis. Beginning at 14 days following the start of tamoxifen treatment, both Klf5 expression and proliferation were re-established in the transit-amplifying epithelial cells but not in the Lgr5-positive CBC cells. By 112 days post-treatment, up to 90% of the Lgr5-positive cells from which Klf5 was deleted were lost from the intestinal crypts. These results indicate a critical role for KLF5 in the survival and maintenance of intestinal stem cells.

  13. Histidine deficiency attenuates cell viability in rat intestinal epithelial cells by apoptosis via mitochondrial dysfunction

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    Tatsunobu Matsui, M.S.

    2017-06-01

    Conclusions: This is the first report showing that histidine deficiency reduced cell viability and induced apoptosis in IEC-6 cells, and that a small amount of histidine supplementation prevented and improved the IEC-6 cell injury. This is a potential new clinical treatment against intestinal and/or gastric cell injury that would improve the patient's quality of life.

  14. Celiac anti-type 2 transglutaminase antibodies induce phosphoproteome modification in intestinal epithelial Caco-2 cells.

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    Gaetana Paolella

    Full Text Available BACKGROUND: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2 activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line. METHODS AND PRINCIPAL FINDINGS: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins, three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis. CONCLUSIONS: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here

  15. A gene expression programme induced by bovine colostrum whey promotes growth and wound-healing processes in intestinal epithelial cells

    OpenAIRE

    Blais, M.; Pouliot, Y.; Gauthier, S.; Boutin, Y.; Lessard, M.

    2014-01-01

    Bovine colostrum is well known for its beneficial properties on health and development. It contains a wide variety of bioactive ingredients that are known to promote a number of cellular processes. Therefore the use of colostrum whey as a feed additive to promote intestinal health has been proposed, yet little is known about mechanisms implicated in its beneficial properties on intestinal epithelial cells. In the present paper, casein were removed from bovine colostrum and the remaining liqui...

  16. E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells.

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    Peter Schierack

    Full Text Available BACKGROUND: The probiotic Escherichia coli strain Nissle 1917 (EcN has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. CONCLUSIONS: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion.

  17. Pseudomonas fluorescens alters epithelial permeability and translocates across Caco-2/TC7 intestinal cells

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    Madi Amar

    2010-11-01

    Full Text Available Abstract Background Pseudomonas fluorescens has long been considered as a psychrotrophic microorganism. Recently, we have shown that clinical strains of P. fluorescens (biovar 1 are able to adapt at a growth temperature of 37°C or above and induce a specific inflammatory response. Interestingly, a highly specific antigen of P. fluorescens, I2, is detected in the serum of patients with Crohn's disease but the possible role of this bacterium in the disease has not yet been explored. In the present study, we examined the ability of a psychrotrophic and a clinical strain of P. fluorescens to modulate the permeability of a Caco-2/TC7 intestinal epithelial model, reorganize the actin cytoskeleton, invade the target cells and translocate across the epithelium. The behaviour of these two strains was compared to that of the well known opportunistic pathogen P. aeruginosa PAO1. Results Both strains of P. fluorescens were found to decrease the transepithelial resistance (TER of Caco-2/TC7 differentiated monolayers. This was associated with an increase in paracellular permeability and F-actin microfilaments rearrangements. Moreover, the invasion and translocation tests demonstrated that the two strains used in this study can invade and translocate across the differentiated Caco-2/TC7 cell monolayers. Conclusions The present work shows for the first time, that P. fluorescens is able to alter the intestinal epithelial barrier function by disorganizing the F-actin microfilament network. Moreover, we reveal that independently of their origins, the two P. fluorescens strains can translocate across differentiated Caco-2/TC7 cell monolayers by using the transcellular pathway. These findings could, at least in part, explain the presence of the P. fluorescens specific I2 antigen in the serum of patients with Crohn's disease.

  18. Progressive Depletion of Rough Endoplasmic Reticulum in Epithelial Cells of the Small Intestine in Monosodium Glutamate Mice Model of Obesity

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    Kazuhiko Nakadate

    2016-01-01

    Full Text Available Chronic obesity is a known risk factor for metabolic syndrome. However, little is known about pathological changes in the small intestine associated with chronic obesity. This study investigated cellular and subcellular level changes in the small intestine of obese mice. In this study, a mouse model of obesity was established by early postnatal administration of monosodium glutamate. Changes in body weight were monitored, and pathological changes in the small intestine were evaluated using hematoxylin-eosin and Nissl staining and light and electron microscopy. Consequently, obese mice were significantly heavier compared with controls from 9 weeks of age. Villi in the small intestine of obese mice were elongated and thinned. There was reduced hematoxylin staining in the epithelium of the small intestine of obese mice. Electron microscopy revealed a significant decrease in and shortening of rough endoplasmic reticulum in epithelial cells of the small intestine of obese mice compared with normal mice. The decrease in rough endoplasmic reticulum in the small intestine epithelial cells of obese mice indicates that obesity starting in childhood influences various functions of the small intestine, such as protein synthesis, and could impair both the defense mechanism against invasion of pathogenic microbes and nutritional absorption.

  19. Induction of P-glycoprotein expression and function in human intestinal epithelial cells (T84).

    Science.gov (United States)

    Haslam, I S; Jones, K; Coleman, T; Simmons, N L

    2008-10-01

    Intestinal induction of Pgp is known to limit the oral availability of certain drug compounds and give rise to detrimental drug-drug interactions. We have investigated the induction of P-glycoprotein (Pgp; MDR1) activity in a human intestinal epithelial cell line (T84) following pre-exposure to a panel of drug compounds, reported to be Pgp substrates, inhibitors or inducers. Human MDR1-transfected MDCKII epithelial monolayers were used to assess Pgp substrate interactions and inhibition of digoxin secretion by the selected drug compounds. The T84 cell line was used to assess induction of Pgp-mediated digoxin secretion following pre-exposure to the same compounds. Changes in gene expression (MDR1, MRP2, PXR and CAR) were determined by quantitative RT-PCR. Net transepithelial digoxin secretion was increased (1.3 fold, n=6, Pfollowing pre-exposure to the PXR activator hyperforin (100nM, 72h), as was MDR1 mRNA expression (3.0 fold, n=4, P<0.05). A number of Pgp substrates (quinidine, amprenavir, irinotecan, topotecan, atorvastatin and erythromycin) induced net digoxin secretion, as did the non-Pgp substrate artemisinin. Various non-Pgp substrates demonstrated inhibition of digoxin secretion (verapamil, mifepristone, clotrimazole, mevastatin, diltiazem and isradipine) but did not induce Pgp-mediated digoxin secretion. Of the compounds that increased Pgp secretion, quinidine, topotecan, atorvastatin and amprenavir pre-exposure also elevated MDR1 mRNA levels, whereas erythromycin, irinotecan and artemisinin displayed no change in transcript levels. This indicates possible post-translational regulation of digoxin secretion. Finally, a strong correlation between drug modulation of MRP2 and PXR mRNA expression levels was evident.

  20. Toxicological interactions between the mycotoxins deoxynivalenol, nivalenol and their acetylated derivatives in intestinal epithelial cells.

    Science.gov (United States)

    Alassane-Kpembi, Imourana; Puel, Olivier; Oswald, Isabelle P

    2015-08-01

    In case of mycotoxin contaminations, food and feedstuff are usually contaminated by more than one toxin. However toxicological data concerning the effects of mycotoxin combinations are sparse. The intestinal epithelium is the first barrier against food contaminants and this constantly renewing organ is particularly sensitive to mycotoxins. The aim of this study was to investigate the effects of deoxynivalenol (DON) and four other type B trichothecenes (TCTB), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX) alone or in combination on intestinal epithelial cells. Proliferating, non-transformed IPEC-1 cells were exposed to increasing doses of TCTB, alone or in binary mixtures and mycotoxin-induced cytotoxicity was measured with MTT test. The toxicological interactions were assessed using the isobologram-Combination index method. The five tested mycotoxins and their mixtures had a dose-dependent effect on the proliferating enterocytes. DON-NIV, DON-15-ADON and 15-ADON-3-ADON combinations were synergistic, with magnitude of synergy for 10 % cytotoxicity ranging from 2 to 7. The association between DON and 3-ADON also demonstrated a synergy but only at high doses, at lower doses antagonism was noted. Additivity was observed between NIV and FX, and antagonism between DON and FX. These results indicate that the simultaneous presence of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. This synergy should be taken into account considering the frequent co-occurrence of TCTB in the diet.

  1. Three-Dimensional Organotypic Co-Culture Model of Intestinal Epithelial Cells and Macrophages to Study "Salmonella Enterica" Colonization Patterns

    Science.gov (United States)

    Ott, Mark; Yang, J; Barilla, J.; Crabbe, A.; Sarker, S. F.; Liu, Y.

    2017-01-01

    Three-dimensional/3-D organotypic models of human intestinal epithelium mimic the differentiated form and function of parental tissues often not exhibited by 2-D monolayers and respond to Salmonella in ways that reflect in vivo infections. To further enhance the physiological relevance of 3-D models to more closely approximate in vivo intestinal microenvironments during infection, we developed and validated a novel 3-D intestinal co-culture model containing multiple epithelial cell types and phagocytic macrophages, and applied to study enteric infection by different Salmonella pathovars.

  2. Transporter mRNA expression in a conditionally immortalized rat small intestine epithelial cell line (TR-SIE).

    Science.gov (United States)

    Hosoya, Ken-ichi; Tomi, Masatoshi; Takayama, Megumi; Komokata, Yuko; Nakai, Daisuke; Tokui, Taro; Nishimura, Kenji; Ueda, Masatsugu; Obinata, Masuo; Hori, Satoko; Ohtsuki, Sumio; Amidon, Gordon L; Terasaki, Tetsuya

    2004-08-01

    Small intestine epithelial cell lines (TR-SIE), which are established from the small intestine of transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), were used to characterize the mRNA expression of small intestine transporters. TR-SIE cells had a polygonal morphology and expressed cytokeratin protein and villin mRNA. Although the large T-antigen was strongly expressed at 33 degrees C, this was reduced at 37 and 39 degrees C. Concomitantly, the cell growth was arrested at 37 and 39 degrees C compared with that at 33 degrees C, suggesting that TR-SIE cells are conditionally immortalized cell lines. RT-PCR analysis revealed that TR-SIE cells expressed ABCB1 (mdr1a and mdr1b), ABCB4 (mdr2), ABCC2 (mrp2), ABCC6 (mrp6), ABCG1, ABCG2 (bcrp/mxr), Slc21a7 (Oatp3), Slc15a1 (PepT1), and Slc16a1 (Mct1). Conditionally immortalized rat small intestine epithelial cell lines were established from tsA58 Tg rats and expressed the mRNA of intestinal transporters.

  3. Effects of the Probiotic Enterococcus faecium and Pathogenic Escherichia coli Strains in a Pig and Human Epithelial Intestinal Cell Model

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    Ulrike Lodemann

    2015-01-01

    Full Text Available The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC and enteropathogenic Escherichia coli (EPEC. Porcine (IPEC-J2 and human (Caco-2 intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods.

  4. Processing of whey modulates proliferative and immune functions in intestinal epithelial cells.

    Science.gov (United States)

    Nguyen, Duc Ninh; Sangild, Per T; Li, Yanqi; Bering, Stine B; Chatterton, Dereck E W

    2016-02-01

    Whey protein concentrate (WPC) is often subjected to heat treatment during industrial processing, resulting in protein denaturation and loss of protein bioactivity. We hypothesized that WPC samples subjected to different degrees of thermal processing are associated with different levels of bioactive proteins and effects on proliferation and immune response in intestinal epithelial cells (IEC). The results showed that low-heat-treated WPC had elevated levels of lactoferrin and transforming growth factor-β2 compared with that of standard WPC. The level of aggregates depended on the source of whey, with the lowest level being found in WPC derived from acid whey. Following acid activation, WPC from acid whey enhanced IEC proliferation compared with WPC from sweet whey or nonactivated WPC. Low-heat-treated WPC from acid whey induced greater secretion of IL-8 in IEC than either standard WPC from acid whey or low-heat-treated WPC from sweet whey. Following acid activation (to activate growth factors), low-heat-treated WPC from sweet whey induced higher IL-8 levels in IEC compared with standard WPC from sweet whey. In conclusion, higher levels of bioactive proteins in low-heat-treated WPC, especially from acid whey, may enhance proliferation and cytokine responses of IEC. These considerations could be important to maintain optimal bioactivity of infant formulas, including their maturational and immunological effects on the developing intestine. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells.

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    Yuan, Q; Ray, R M; Viar, M J; Johnson, L R

    2001-01-01

    Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.

  6. Effects of biodegradable Mg–6Zn alloy extracts on apoptosis of intestinal epithelial cells

    International Nuclear Information System (INIS)

    Wang Zhanhui; Yan Jun; Li Jianan; Zheng Qi; Wang Zhigang; Zhang Xiaonong; Zhang Shaoxiang

    2012-01-01

    Highlights: ► We evaluated the effects of Mg–6Zn alloys on apoptosis of IEC-6 cells. ► The apoptosis was evaluated by investigating the expression of caspase-1 and Bcl-2. ► The IEC-6 cells displayed better cell functions in 60% or 20% extract. ► The conspicuous alkaline environment is disadvantageous to apoptosis of IEC cells. ► The excessive Mg concentration is disadvantageous to apoptosis of IEC-6 cells. - Abstract: In this study, intestinal epithelial cells (IEC)-6 were cultured in different concentration extracts of Mg–6Zn alloys for different time periods. To achieve a total of three concentrations (100%, 60% and 20% concentration), the extracts were serially diluted with Dulbecco's modified Eagle medium High Glucose to observe a dose–response relationship. We studied the indirect effects of Mg–6Zn alloys on IEC-6 cells apoptosis. The apoptosis of IEC-6 cells was measured using flow cytometry. And the apoptosis of IEC-6 cells was evaluated by investigating the expression of caspase-1and Bcl-2 using real-time polymerase chain reaction (PCR) and Western blotting tests. It was found that the levels of apoptosis in IEC-6 cells cultured in 100% Mg–6Zn alloy extracts were significantly higher than those in 60% and 20% extracts; the 100% extract can down-regulate expression of Bcl-2 after culture. The in vitro results indicated that the conspicuous alkaline environment and excessive Mg concentration, even Zn concentration caused by rapid corrosion of Mg–6Zn alloys promote IEC-6 cells apoptosis, although further experiments will be necessary to formally prove our conclusions. Therefore, the adjustment of the degradation rate is needed for using Mg–Zn alloy as a surgical suture material.

  7. Visualization of probiotic-mediated Ca2+ signaling in intestinal epithelial cells in vivo

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    Takahiro Adachi

    2016-12-01

    Full Text Available Probiotics, such as lactic acid bacteria (LAB and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs, because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60 transgenic mouse line and established 5D (x, y, z, time, and Ca2+ intravital imaging systems of lymphoid tissues including those in Peyer’s patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed Bacillus subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions, and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

  8. Morphogenesis and proliferative rule of porcine transmissible gastroenteritis virus in porcine intestinal epithelial cells.

    Science.gov (United States)

    Song, Zhenhui; Dai, Xianjin; Ye, Cuifang; Li, Yuntian; Wang, Li; Hu, Yang

    2016-12-01

    To gain a better understanding of the replication, proliferation and infection characteristics of porcine transmissible gastroenteritis virus (TGEV) in porcine intestinal epithelial cells (IECs), this study established a cell model of IECs infected with the Chongqing (CQ) strain of TGEV. The morphogenesis and proliferative rule of TGEV in porcine IECs were investigated using transmission electron microscopy, indirect immunofluorescence assays and real-time fluorescence quantitative PCR. Observations under the TEM indicated that the enveloped viral particles were roughly spherical, with diameters of between 80 and 120nm. The virions entered porcine IECs by membrane fusion and the mature viruses in the vacuoles were transported to the cell membrane before release. The results also showed that from 0 to 12h after TGEV infection of porcine IECs, the intracellular viral RNA content did not change significantly. Logarithmic growth occurred from 12 to 36h, after which it gradually decreased. Moreover, the extracellular RNA content began to rise at 24h after inoculation and then reduced gradually at approximately 48h. This study provided a theoretical foundation for further study on the infection characteristics of TGEV in target cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis.

    Science.gov (United States)

    Zou, Tongtong; Rao, Jaladanki N; Liu, Lan; Xiao, Lan; Cui, Yu-Hong; Jiang, Zhengran; Ouyang, Miao; Donahue, James M; Wang, Jian-Ying

    2012-07-01

    Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

  10. Acetonic Extract from the Feijoa sellowiana Berg. Fruit Exerts Antioxidant Properties and Modulates Disaccharidases Activities in Human Intestinal Epithelial Cells.

    Science.gov (United States)

    Turco, Fabio; Palumbo, Ilaria; Andreozzi, Paolo; Sarnelli, Giovanni; De Ruberto, Francesca; Esposito, Giuseppe; Basile, Adriana; Cuomo, Rosario

    2016-08-01

    Feijoa sellowiana fruit has been shown to possess various biological activities, such as anti-bacterial and anti-cancer properties, in a variety of cellular models, but its activity on human intestinal epithelial cells has never been tested. The purpose of this study was to investigate the effects of the acetonic extract of F. sellowiana fruits on the viability, membrane peroxidation, disaccharidases activities and proliferation of in vitro models of human intestinal epithelial cells. To obtain this goal, Caco-2 and HT-29 cells were exposed to the acetonic extract for 24 h. Cell proliferation, viability, lactase and sucrase-isomaltase activity and H2 O2 -induced membrane lipid peroxidation were tested. We found that, compared to control conditions, the acetonic extract significantly increased lactase and sucrase-isomaltase activity in Caco-2, but not HT-29, cells, decreased proliferation, had no effects on viability and restored lipid peroxidation in both cell models. This study suggests that the acetonic extract improves lactase and sucrase-isomaltase activity, inhibits cell proliferation, have no cytotoxic effects and prevent lipid peroxidation of intestinal epithelial cells. These effects may be exploited in case of disaccharidases deficit and also as an adjuvant treatment of diseases related to oxidative stress. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Interferon-γ induces expression of MHC class II on intestinal epithelial cells and protects mice from colitis.

    Directory of Open Access Journals (Sweden)

    Christoph Thelemann

    Full Text Available Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD and involve CD4(+ T cells, which are activated by major histocompatibility complex class II (MHCII molecules on antigen-presenting cells (APCs. However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC affects CD4(+ T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL-10 receptor-blocking antibodies (anti-IL10R mAb. To assess the role of interferon (IFN-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+ T-helper type (Th1 cells - but not group 3 innate lymphoid cells (ILCs or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+ T cells and forkhead box P3 (FoxP3(+ regulatory T (Treg cells. IFN-γ produced mainly by CD4(+ T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

  12. A fish intestinal epithelial barrier model established from the rainbow trout (Oncorhynchus mykiss) cell line, RTgutGC.

    Science.gov (United States)

    Minghetti, Matteo; Drieschner, Carolin; Bramaz, Nadine; Schug, Hannah; Schirmer, Kristin

    2017-12-01

    The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na + /K + -ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion.

  13. The Nucleotide Synthesis Enzyme CAD Inhibits NOD2 Antibacterial Function in Human Intestinal Epithelial Cells

    Science.gov (United States)

    Richmond, Amy L.; Kabi, Amrita; Homer, Craig R.; García, Noemí Marina; Nickerson, Kourtney P.; NesvizhskiI, Alexey I.; Sreekumar, Arun; Chinnaiyan, Arul M.; Nuñez, Gabriel; McDonald, Christine

    2013-01-01

    BACKGROUND & AIMS Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn’s disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS Carbamoyl phosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD. PMID:22387394

  14. Excreted/secreted Trichuris suis products reduce barrier function and suppress inflammatory cytokine production of intestinal epithelial cells

    DEFF Research Database (Denmark)

    Hiemstra, I. H.; Klaver, E. J.; Vrijland, K.

    2014-01-01

    . The intestinal epithelium forms an efficient barrier between the intestinal lumen containing the microbial flora and helminths, and dendritic cells (DCs) present in the lamina propria that determine the TH response. Here, we investigated how excreted/secreted (E/S) products of T. suis affect the barrier function...... of intestinal epithelial cells (IECs) in order to reach the DCs and modulate the immune response. We show that T. suis E/S products reduce the barrier function and the expression of the tight junction proteins EMP-1 and claudin-4 in IEC CMT93/69 monolayers in a glycan-dependent manner. This resulted...... in an increased passage of soluble compounds to the basolateral side that affected DC function. In addition, T. suis E/S suppressed LPS-induced pro-inflammatory cytokine production by CMT93/69 cells, whereas the production of the TH2 response-inducing cytokine thymic stromal lymphopoietin (TSLP) was induced. Our...

  15. Nivalenol induces oxidative stress and increases deoxynivalenol pro-oxidant effect in intestinal epithelial cells.

    Science.gov (United States)

    Del Regno, Marisanta; Adesso, Simona; Popolo, Ada; Quaroni, Andrea; Autore, Giuseppina; Severino, Lorella; Marzocco, Stefania

    2015-06-01

    Mycotoxins are secondary fungal metabolites often found as contaminants in almost all agricultural commodities worldwide, and the consumption of food or feed contaminated by mycotoxins represents a major risk for human and animal health. Reactive oxygen species are normal products of cellular metabolism. However, disproportionate generation of reactive oxygen species poses a serious problem to bodily homeostasis and causes oxidative tissue damage. In this study we analyzed the effect of two trichothecenes mycotoxins: nivalenol and deoxynivalenol, alone and in combination, on oxidative stress in the non-tumorigenic intestinal epithelial cell line IEC-6. Our results indicate the pro-oxidant nivalenol effect in IEC-6, the stronger pro-oxidant effect of nivalenol when compared to deoxynivalenol and, interestingly, that nivalenol increases deoxynivalenol pro-oxidative effects. Mechanistic studies indicate that the observed effects were mediated by NADPH oxidase, calcium homeostasis alteration, NF-kB and Nrf2 pathways activation and by iNOS and nitrotyrosine formation. The toxicological interaction by nivalenol and deoxynivalenol reported in this study in IEC-6, points out the importance of the toxic effect of these mycotoxins, mostly in combination, further highlighting the risk assessment process of these toxins that are of growing concern. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Persistent Transmissible Gastroenteritis Virus Infection Enhances Enterotoxigenic Escherichia coli K88 Adhesion by Promoting Epithelial-Mesenchymal Transition in Intestinal Epithelial Cells.

    Science.gov (United States)

    Xia, Lu; Dai, Lei; Yu, Qinghua; Yang, Qian

    2017-11-01

    Transmissible gastroenteritis virus (TGEV) is a coronavirus characterized by diarrhea and high morbidity rates, and the mortality rate is 100% in piglets less than 2 weeks old. Pigs infected with TGEV often suffer secondary infection by other pathogens, which aggravates the severity of diarrhea, but the mechanisms remain unknown. Here, we hypothesized that persistent TGEV infection stimulates the epithelial-mesenchymal transition (EMT), and thus enterotoxigenic Escherichia coli (ETEC) can more easily adhere to generating cells. Intestinal epithelial cells are the primary targets of TGEV and ETEC infections. We found that TGEV can persistently infect porcine intestinal columnar epithelial cells (IPEC-J2) and cause EMT, consistent with multiple changes in key cell characteristics. Infected cells display fibroblast-like shapes; exhibit increases in levels of mesenchymal markers with a corresponding loss of epithelial markers; have enhanced expression levels of interleukin-1β (IL-1β), IL-6, IL-8, transforming growth factor β (TGF-β), and tumor necrosis factor alpha (TNF-α) mRNAs; and demonstrate increases in migratory and invasive behaviors. Additional experiments showed that the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways via TGF-β is critical for the TGEV-mediated EMT process. Cellular uptake is also modified in cells that have undergone EMT. TGEV-infected cells have higher levels of integrin α5 and fibronectin and exhibit enhanced ETEC K88 adhesion. Reversal of EMT reduces ETEC K88 adhesion and inhibits the expression of integrin α5 and fibronectin. Overall, these results suggest that TGEV infection induces EMT in IPEC-J2 cells, increasing the adhesion of ETEC K88 in the intestine and facilitating dual infection. IMPORTANCE Transmissible gastroenteritis virus (TGEV) causes pig diarrhea and is often followed by secondary infection by other pathogens. In this study, we showed

  17. Characterization of human, mouse, and rat cultures of enteric glial cells and their effect on intestinal epithelial cells.

    Science.gov (United States)

    Soret, R; Coquenlorge, S; Cossais, F; Meurette, G; Rolli-Derkinderen, M; Neunlist, M

    2013-11-01

    Enteric glial cells (EGC) are major regulators of neuronal and intestinal epithelial cell (IEC) functions. Simple isolation methods of EGC, especially human tissues, remain scarce and limit their study. We present herein a method to isolate EGC and we characterize EGC phenotype and their functional impact on IEC. Longitudinal muscle and myenteric plexus preparations of rat, mouse, or human intestine were obtained by microdissection. After mechanical and enzymatic dissociation, individual ganglionic or interganglionic structures were seeded into plates, maintained in culture several weeks and passaged up to 4 times. Purity of cultures was assessed by immunocytochemistry using antibodies against glial fibrillary acidic protein (GFAP), S100β and Sox10 or smooth muscle actin. Effects of adenosine triphosphate (ATP) on intracellular Ca²⁺ signaling in EGC were studied. Co-cultures of EGC with IEC line, Caco-2, were performed for 2-6 days to analyze their impact on monolayer resistance, cell proliferation, and cell spreading. More than 80% of DAPI-positive cells were GFAP, S100β, and Sox10-immunoreactive. EGC expressed these glial markers over 4 consecutive passages, and the majority of them responded to ATP by an increase in intracellular Ca²⁺ concentration. In addition, rat, mouse, and human EGC increased intestinal barrier resistance, IEC size, and reduced IEC number. We have developed a simple method to isolate and culture human, rat, or mouse EGC. EGC exhibit similar functional properties on the intestinal barrier independently of the species. This study sets the basis for exploring glial biology and functions in human health and diseases. © 2013 John Wiley & Sons Ltd.

  18. Determination of Autophagy in the Caco-2 Spontaneously Differentiating Model of Intestinal Epithelial Cells.

    Science.gov (United States)

    Tunçer, Sinem; Banerjee, Sreeparna

    2017-08-27

    The Caco-2 colorectal cancer cell line is widely used as a model for intestinal differentiation and barrier function. These cells, upon reaching confluency, spontaneously differentiate into enterocyte-like cells, synthesize intestinal enzymes, and form domes. Caco-2 cells also undergo autophagy in the course of differentiation. The criteria to establish the induction of autophagy in cells are already well established. Here, we describe the protocol for the spontaneous differentiation of Caco-2 cells and the detection of autophagy using Western blot, flow cytometry, and immunofluorescence.

  19. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  20. A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

    Science.gov (United States)

    Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

  1. Cyanidin-3-O-Glucoside Modulates the In Vitro Inflammatory Crosstalk between Intestinal Epithelial and Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Daniela Ferrari

    2017-01-01

    Full Text Available Intestinal epithelium represents a protective physical barrier and actively contributes to the mucosal immune system. Polarized basolateral intestinal secretion of inflammatory mediators, followed by activation of NF-κB signaling and inflammatory pathways in endothelial cells, efficiently triggers extravasation of neutrophils from the vasculature, therefore contributing to the development and maintenance of intestinal inflammation. Proper regulation of NF-κB activation at the epithelial interface is crucial for the maintenance of physiological tissue homeostasis. Many papers reported that anthocyanins, a group of compounds belonging to flavonoids, possess anti-inflammatory effects and modulate NF-κB activity. In this study, by using a coculture in vitro system, we aimed to evaluate the effects of TNF-α-stimulated intestinal cells on endothelial cells activation, as well as the protective effects of cyanidin-3-glucoside (C3G. In this model, TNF-α induced nuclear translocation of NF-κB and TNF-α and IL-8 gene expression in Caco-2 cells, whereas C3G pretreatment dose-dependently reduced these effects. Furthermore, TNF-α-stimulated Caco-2 cells induced endothelial cells activation with increased E-selectin and VCAM-1 mRNA, leukocyte adhesion, and NF-κB levels in HUVECs, which were inhibited by C3G. We demonstrated that selective inhibition of the NF-κB pathway in epithelial cells represents the main mechanism by which C3G exerts these protective effects. Thus, anthocyanins could contribute to the management of chronic gut inflammatory diseases.

  2. Suppressive effect of nobiletin and epicatechin gallate on fructose uptake in human intestinal epithelial Caco-2 cells.

    Science.gov (United States)

    Satsu, Hideo; Awara, Sohei; Unno, Tomonori; Shimizu, Makoto

    2018-04-01

    Inhibition of excessive fructose intake in the small intestine could alleviate fructose-induced diseases such as hypertension and non-alcoholic fatty liver disease. We examined the effect of phytochemicals on fructose uptake using human intestinal epithelial-like Caco-2 cells which express the fructose transporter, GLUT5. Among 35 phytochemicals tested, five, including nobiletin and epicatechin gallate (ECg), markedly inhibited fructose uptake. Nobiletin and ECg also inhibited the uptake of glucose but not of L-leucine or Gly-Sar, suggesting an inhibitory effect specific to monosaccharide transporters. Kinetic analysis further suggested that this reduction in fructose uptake was associated with a decrease in the apparent number of cell-surface GLUT5 molecules, and not with a change in the affinity of GLUT5 for fructose. Lastly, nobiletin and ECg suppressed the permeation of fructose across Caco-2 cell monolayers. These findings suggest that nobiletin and ECg are good candidates for preventing diseases caused by excessive fructose intake.

  3. Transcriptional response of HT-29 intestinal epithelial cells to human and bovine milk oligosaccharides.

    Science.gov (United States)

    Lane, Jonathan A; O'Callaghan, John; Carrington, Stephen D; Hickey, Rita M

    2013-12-01

    Human milk oligosaccharides (HMO) have been shown to interact directly with immune cells. However, large quantities of HMO are required for intervention or clinical studies, but these are unavailable in most cases. In this respect, bovine milk is potentially an excellent source of commercially viable analogues of these unique molecules. In the present study, we compared the transcriptional response of colonic epithelial cells (HT-29) to the entire pool of HMO and bovine colostrum oligosaccharides (BCO) to determine whether the oligosaccharides from bovine milk had effects on gene expression that were similar to those of their human counterparts. Gene set enrichment analysis of the transcriptional data revealed that there were a number of similar biological processes that may be influenced by both treatments including a response to stimulus, signalling, locomotion, and multicellular, developmental and immune system processes. For a more detailed insight into the effects of milk oligosaccharides, the effect on the expression of immune system-associated glycogenes was chosen as a case study when performing validation studies. Glycogenes in the current context are genes that are directly or indirectly regulated in the presence of glycans and/or glycoconjugates. RT-PCR analysis revealed that HMO and BCO influenced the expression of cytokines (IL-1β, IL-8, colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF2), IL-17C and platelet factor 4 (PF4)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 6 (CXCL6), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X3-C motif) ligand 1 (CX3CL1) and CXCL2) and cell surface receptors (interferon γ receptor 1 (IFNGR1), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and IL-10 receptor α (IL10RA)). The present study suggests

  4. Mesenchymal stem cells enhance the viability and proliferation of human fetal intestinal epithelial cells following hypoxic injury via paracrine mechanisms.

    Science.gov (United States)

    Weil, Brent R; Markel, Troy A; Herrmann, Jeremy L; Abarbanell, Aaron M; Meldrum, Daniel R

    2009-08-01

    Mesenchymal stem cells (MSCs) may be used to treat injured tissues. The ability of MSCs to treat injured fetal intestinal epithelial cells (FIEs), similar to those in infants with necrotizing enterocolitis, has not been elucidated. We hypothesized that MSCs would enhance FIE viability and proliferation after hypoxic injury via paracrine mechanisms. LLC-PK1 cells (differentiated control [DC]) and human MSCs were exposed to 1 hour of hypoxia. Cells were reoxygenated for 24 hours and cell-free conditioned media were collected. Human FIEs were exposed to 1 hour of hypoxia and plated for experiments. FIEs were reoxygenated in nonconditioned media, DC-conditioned media, or MSC-conditioned media. Supernatants were analyzed for interleukin-6 (IL-6), hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) via enzyme-linked immunosorbent assay. Cell viability was assessed by trypan blue exclusion and cell counting. Proliferation was determined via 5-bromo-2'-deoxyuridine (BrdU). Expression of caspases-3 and -8 was determined via Western blot. FIEs reoxygenated in MSC-conditioned media demonstrated enhanced viability and increased proliferation after hypoxic injury. Enhanced FIE viability and proliferation were associated with increased IL-6, HGF, and VEGF, as well as decreased expression of caspase-3. MSCs may increase the viability and proliferative capacity of FIEs after hypoxic injury via the paracrine release of IL-6, HGF, and VEGF, as well as downregulation of apoptotic signaling.

  5. Effect of fluoride on the intestinal epithelial cell brush border membrane

    Energy Technology Data Exchange (ETDEWEB)

    Rastogi, R.; Upreti, R.K.; Kidwai, A.M.

    1987-07-01

    Fluoride consumed by man and animals is chiefly absorbed in the intestine. Chronic fluoride exposure causes mottled teeth and osteosclerosis. Over-fluoridation (126 mM) of drinking water have been reported to cause nausea, vomiting and diarrhea. Furthermore, the effect of acute and low concentrations of fluoride on gastric secretion, ion transport and other disorders have also been studied. Fluoride also causes alterations in the permeability of membranes and membrane bound enzymes. The intestinal cell lining plays an important role in digestion and absorption. It automatically becomes the most exposed site of contact to fluoride following ingestion. Earlier study have shown significant alterations in the formation of lipid peroxides in rat intestine following oral administration of fluoride. The present study was undertaken to investigate the damage of rat intestinal epithelium in situ caused by relatively high and low fluoride concentrations.

  6. Kefir-isolated bacteria and yeasts inhibit Shigella flexneri invasion and modulate pro-inflammatory response on intestinal epithelial cells.

    Science.gov (United States)

    Bolla, P A; Abraham, A G; Pérez, P F; de Los Angeles Serradell, M

    2016-02-01

    The aim of this work was to evaluate the ability of a kefir-isolated microbial mixture containing three bacterial and two yeast strains (MM) to protect intestinal epithelial cells against Shigella flexneri invasion, as well as to analyse the effect on pro-inflammatory response elicited by this pathogen. A significant decrease in S. flexneri strain 72 invasion was observed on both HT-29 and Caco-2 cells pre-incubated with MM. Pre-incubation with the individual strains Saccharomyces cerevisiae CIDCA 8112 or Lactococcus lactis subsp. lactis CIDCA 8221 also reduced the internalisation of S. flexneri into HT-29 cells although in a lesser extent than MM. Interestingly, Lactobacillus plantarum CIDCA 83114 exerted a protective effect on the invasion of Caco-2 and HT-29 cells by S. flexneri. Regarding the pro-inflammatory response on HT-29 cells, S. flexneri infection induced a significant activation of the expression of interleukin 8 (IL-8), chemokine (C-C motif) ligand 20 (CCL20) and tumour necrosis factor alpha (TNF-α) encoding genes (Pkefir, resulted in inhibition of S. flexneri internalisation into human intestinal epithelial cells, along with the inhibition of the signalling via NF-κB that in turn led to the attenuation of the inflammatory response.

  7. Influence of Physicochemical Properties and PEG Modification of Magnetic Liposomes on Their Interaction with Intestinal Epithelial Caco-2 Cells.

    Science.gov (United States)

    Kono, Yusuke; Jinzai, Hitomi; Kotera, Yota; Fujita, Takuya

    2017-12-01

    The present study aimed to investigate the effect of particle size (100, 500 nm), surface charge (cationic, neutral and anionic) and polyethylene glycol (PEG) modification of magnetic liposomes on their interaction with the human intestinal epithelial cell line, Caco-2. The cellular associated amount of all the magnetic liposomes was significantly increased by the presence of a magnetic field. The highest association and internalization into Caco-2 cells was observed with magnetic cationic liposomes. Moreover, small magnetic liposomes were more efficiently associated and taken up into the cells, than large ones. In contrast, PEG modification significantly attenuated the enhancing effect of the magnetic field on the cellular association of magnetic liposomes. We also found that magnetic cationic liposomes had the highest retention properties to Caco-2 cells. Moreover, the retention of large magnetic liposomes to the cells was much longer than that of small ones. In addition, magnetic cationic and neutral liposomes had relatively high stability in Caco-2 cells, whereas magnetic anionic liposomes rapidly degraded. These results indicate that the physicochemical properties and PEG modification of magnetic liposomes greatly influences their intestinal epithelial transport.

  8. Increased production of the ether-lipid platelet-activating factor in intestinal epithelial cells infected by Salmonella enteritidis.

    Science.gov (United States)

    Egea, Laia; Giménez, Rosa; Lúcia, David; Modolell, Ines; Badía, Josefa; Baldoma, Laura; Aguilar, Juan

    2008-05-01

    When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels. Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A(2), which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca(2+)-dependent.

  9. The Bacterial Species Campylobacter jejuni Induce Diverse Innate Immune Responses in Human and Avian Intestinal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Daniel A. John

    2017-09-01

    Full Text Available Campylobacter remain the major cause of human gastroenteritis in the Developed World causing a significant burden to health services. Campylobacter are pathogens in humans and chickens, although differences in mechanistic understanding are incomplete, in part because phenotypic strain diversity creates inconsistent findings. Here, we took Campylobacter jejuni isolates (n = 100 from multi-locus sequence typed collections to assess their pathogenic diversity, through their inflammatory, cytotoxicity, adhesion, invasion and signaling responses in a high-throughput model using avian and human intestinal epithelial cells. C. jejuni induced IL-8 and CXCLi1/2 in human and avian epithelial cells, respectively, in a MAP kinase-dependent manner. In contrast, IL-10 responses in both cell types were PI 3-kinase/Akt-dependent. C. jejuni strains showed diverse levels of invasion with high invasion dependent on MAP kinase signaling in both cell lines. C. jejuni induced diverse cytotoxic responses in both cell lines with cdt-positive isolates showing significantly higher toxicity. Blockade of endocytic pathways suggested that invasion by C. jejuni was clathrin- and dynamin-dependent but caveolae- independent in both cells. In contrast, IL-8 (and CXCLi1/2 production was dependent on clathrin, dynamin, and caveolae. This study is important because of its scale, and the data produced, suggesting that avian and human epithelial cells use similar innate immune pathways where the magnitude of the response is determined by the phenotypic diversity of the Campylobacter species.

  10. Thyroid hormone-regulated Wnt5a/Ror2 signaling is essential for dedifferentiation of larval epithelial cells into adult stem cells in the Xenopus laevis intestine.

    Directory of Open Access Journals (Sweden)

    Atsuko Ishizuya-Oka

    Full Text Available Amphibian intestinal remodeling, where thyroid hormone (T3 induces some larval epithelial cells to become adult stem cells analogous to the mammalian intestinal ones, serves as a unique model for studying how the adult stem cells are formed. To clarify its molecular mechanisms, we here investigated roles of non-canonical Wnt signaling in the larval-to-adult intestinal remodeling during Xenopus laevis metamorphosis.Our quantitative RT-PCR (qRT-PCR and immunohistochemical analyses indicated that the expressions of Wnt5a and its receptors, frizzled 2 (Fzd2 and receptor tyrosine kinase-like orphan receptor 2 (Ror2 are up-regulated by T3 and are spatiotemporally correlated with adult epithelial development in the X. laevis intestine. Notably, changes in morphology of larval absorptive epithelial cells expressing Ror2 coincide well with formation of the adult stem cells during metamorphosis. In addition, by using organ cultures of the tadpole intestine, we have experimentally shown that addition of exogenous Wnt5a protein to the culture medium causes morphological changes in the larval epithelium expressing Ror2 even in the absence of T3. In contrast, in the presence of T3 where the adult stem cells are formed in vitro, inhibition of endogenous Wnt5a by an anti-Wnt5a antibody suppressed the epithelial morphological changes, leading to the failure of stem cell formation.Our findings strongly suggest that the adult stem cells originate from the larval absorptive cells expressing Ror2, which require Wnt5a/Ror2 signaling for their dedifferentiation accompanied by changes in cell morphology.

  11. the intestinal expulsion of the roundworm Ascaris suum is associated with eosinophils, intra-epithelial T cells and decreased intestinal transit time.

    Science.gov (United States)

    Masure, Dries; Wang, Tao; Vlaminck, Johnny; Claerhoudt, Sarah; Chiers, Koen; Van den Broeck, Wim; Saunders, Jimmy; Vercruysse, Jozef; Geldhof, Peter

    2013-01-01

    Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections.

  12. the intestinal expulsion of the roundworm Ascaris suum is associated with eosinophils, intra-epithelial T cells and decreased intestinal transit time.

    Directory of Open Access Journals (Sweden)

    Dries Masure

    Full Text Available Ascaris lumbricoides remains the most common endoparasite in humans, yet there is still very little information available about the immunological principles of protection, especially those directed against larval stages. Due to the natural host-parasite relationship, pigs infected with A. suum make an excellent model to study the mechanisms of protection against this nematode. In pigs, a self-cure reaction eliminates most larvae from the small intestine between 14 and 21 days post infection. In this study, we investigated the mucosal immune response leading to the expulsion of A. suum and the contribution of the hepato-tracheal migration. Self-cure was independent of previous passage through the liver or lungs, as infection with lung stage larvae did not impair self-cure. When animals were infected with 14-day-old intestinal larvae, the larvae were being driven distally in the small intestine around 7 days post infection but by 18 days post infection they re-inhabited the proximal part of the small intestine, indicating that more developed larvae can counter the expulsion mechanism. Self-cure was consistently associated with eosinophilia and intra-epithelial T cells in the jejunum. Furthermore, we identified increased gut movement as a possible mechanism of self-cure as the small intestinal transit time was markedly decreased at the time of expulsion of the worms. Taken together, these results shed new light on the mechanisms of self-cure that occur during A. suum infections.

  13. Analysis of the human intestinal epithelial cell transcriptional response to Lactobacillus acidophilus, Lactobacillus salivarius, Bifidobacterium lactis and Escherichia coli

    DEFF Research Database (Denmark)

    Putaala, H; Barrangou, R; Leyer, G J

    2010-01-01

    a comparative analysis of the global in vitro transcriptional response of human intestinal epithelial cells to Lactobacillus acidophilus NCFM™, Lactobacillus salivarius Ls-33, Bifidobacterium animalis subsp. lactis 420, and enterohaemorrhagic Escherichia coli O157:H7 (EHEC). Interestingly, L. salivarius Ls-33......, fundamental differences were observed between the pathogenic and probiotic treatments in the Toll-like receptor pathway, especially for adapter molecules with a lowered level of transcriptional activation of MyD88, TRIF, IRAK1 and TRAF6 by probiotics compared to EHEC. The results in this study provide...

  14. Butyrate Produced by Commensal Bacteria Potentiates Phorbol Esters Induced AP-1 Response in Human Intestinal Epithelial Cells

    Science.gov (United States)

    Nepelska, Malgorzata; Cultrone, Antonietta; Béguet-Crespel, Fabienne; Le Roux, Karine; Doré, Joël; Arulampalam, Vermulugesan; Blottière, Hervé M.

    2012-01-01

    The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells. PMID:23300800

  15. Lipoteichoic Acid of Probiotic Lactobacillus plantarum Attenuates Poly I:C-Induced IL-8 Production in Porcine Intestinal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Kyoung Whun Kim

    2017-09-01

    Full Text Available Probiotics in livestock feed supplements are considered a replacement for antibiotics that enhance gastrointestinal immunity. Although bacterial cell wall components have been proposed to be associated with probiotic function, little evidence demonstrates that they are responsible for probiotic functions in livestock. The present study demonstrated that lipoteichoic acid (LTA of Lactobacillus plantarum (Lp.LTA confers anti-inflammatory responses in porcine intestinal epithelial cell line, IPEC-J2. A synthetic analog of viral double-stranded RNA, poly I:C, dose-dependently induced IL-8 production at the mRNA and protein levels in IPEC-J2 cells. Lp.LTA, but not lipoprotein or peptidoglycan from L. plantarum, exclusively suppressed poly I:C-induced IL-8 production. Compared with LTAs from other probiotic Lactobacillus strains including L. delbrueckii, L. sakei, and L. rhamnosus GG, Lp.LTA had higher potential to suppress poly I:C-induced IL-8 production. Dealanylated or deacylated Lp.LTA did not suppress poly I:C-induced IL-8 production, suggesting that D-alanine and lipid moieties in the Lp.LTA structure were responsible for the inhibition. Furthermore, Lp.LTA attenuated the phosphorylation of ERK and p38 kinase as well as the activation of NF-κB, resulting in decreased IL-8 production. Taken together, these results suggest that Lp.LTA acts as an effector molecule to inhibit viral pathogen-induced inflammatory responses in porcine intestinal epithelial cells.

  16. Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion.

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    Ferrero, Mariana C; Fossati, Carlos A; Rumbo, Martín; Baldi, Pablo C

    2012-10-01

    In spite of the frequent acquisition of Brucella infection by the oral route in humans, the interaction of the bacterium with cells of the intestinal mucosa has been poorly studied. Here, we show that different Brucella species can invade human colonic epithelial cell lines (Caco-2 and HT-29), in which only smooth species can replicate efficiently. Infection with smooth strains did not produce a significant cytotoxicity, while the rough strain RB51 was more cytotoxic. Infection of Caco-2 cells or HT-29 cells with either smooth or rough strains of Brucella did not result in an increased secretion of TNF-α, IL-1β, MCP-1, IL-10 or TGF-β as compared with uninfected controls, whereas all the infections induced the secretion of IL-8 and CCL20 by both cell types. The MCP-1 response to flagellin from Salmonella typhimurium was similar in Brucella-infected or uninfected cells, ruling out a bacterial inhibitory mechanism as a reason for the weak proinflammatory response. Infection did not modify ICAM-1 expression levels in Caco-2 cells, but increased them in HT-29 cells. These results suggest that Brucella induces only a weak proinflammatory response in gut epithelial cells, but produces a significant CCL20 secretion. The latter may be important for bacterial dissemination given the known ability of Brucella to survive in dendritic cells. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  17. Chromatin remodeling mediated by the FOXA1/A2 transcription factors activates CFTR expression in intestinal epithelial cells.

    Science.gov (United States)

    Kerschner, Jenny L; Gosalia, Nehal; Leir, Shih-Hsing; Harris, Ann

    2014-04-01

    The forkhead box A transcription factors, FOXA1 and FOXA2, function as pioneer factors to open condensed chromatin and facilitate binding of other proteins. We showed previously that these factors are key components of a transcriptional network that drives enhancer function at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in intestinal epithelial cells. The CFTR promoter apparently lacks tissue-specific regulatory elements and expression of the gene is controlled by multiple cis-acting elements, which coordinate gene expression in different cell types. Here we show that concurrent depletion of FOXA1 and FOXA2 represses CFTR expression and alters the three-dimensional architecture of the active locus by diminishing interactions between the promoter and intronic cis-acting elements. Reduction of FOXA1/A2 also modifies the enrichment profile of the active enhancer marks H3K27ac and H3K4me2 across the CFTR locus and alters chromatin accessibility at individual cis-elements. Moreover, loss of FOXA1/A2 suppresses the recruitment of other members of the transcriptional network including HNF1 and CDX2, to multiple cis-elements. These data reveal a complex molecular mechanism underlying the role of FOXA1/A2 in achieving high levels of CFTR expression in intestinal epithelial cells.

  18. Transcriptional regulation of the human Na+/H+ exchanger NHE3 by serotonin in intestinal epithelial cells

    International Nuclear Information System (INIS)

    Amin, Md Ruhul; Ghannad, Leda; Othman, Ahmad; Gill, Ravinder K.; Dudeja, Pradeep K.; Ramaswamy, Krishnamurthy; Malakooti, Jaleh

    2009-01-01

    Serotonin (5-HT) decreases NHE2 and NHE3 activities under acute conditions in human intestinal epithelial cells. Here, we have investigated the effects of 5-HT on expression of the human NHE3 gene and the mechanisms underlying its transcriptional regulation in differentiated C2BBe1 cells. Treatment of the human intestinal epithelial cell line, C2BBe1, with 5-HT (20 μM) resulted in a significant decrease in NHE3 mRNA and protein expression. In transient transfection studies, 5-HT repressed the NHE3 promoter activity by ∼55%. The repression of the NHE3 promoter activity in response to 5-HT was accompanied by reduced DNA-binding activity of transcription factors Sp1 and Sp3 to the NHE3 promoter without alteration in their nuclear levels. Pharmacological inhibitors of protein kinase C reversed the inhibitory effect of 5-HT on the promoter activity. Our data indicate that 5-HT suppresses the transcriptional activity of the NHE3 promoter and this effect may be mediated by PKCα and modulation of DNA-binding affinities of Sp1 and Sp3.

  19. Identification of a Predominantly Interferon-λ-Induced Transcriptional Profile in Murine Intestinal Epithelial Cells

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    Tharini A. Selvakumar

    2017-10-01

    Full Text Available Type I (α and β and type III (λ interferons (IFNs induce the expression of a large set of antiviral effector molecules via their respective surface membrane receptors. Whereas most cell types respond to type I IFN, type III IFN preferentially acts on epithelial cells and protects mucosal organs such as the lung and gastrointestinal tract. Despite the engagement of different receptor molecules, the type I and type III IFN-induced signaling cascade and upregulated gene profile is thought to be largely identical. Here, we comparatively analyzed the response of gut epithelial cells to IFN-β and IFN-λ2 and identified a set of genes predominantly induced by IFN-λ2. We confirm the influence of epithelial cell polarization for enhanced type III receptor expression and demonstrate the induction of predominantly IFN-λ2-induced genes in the gut epithelium in vivo. Our results suggest that IFN-λ2 targets the epithelium and induces genes to adjust the antiviral host response to the requirements at mucosal body sites.

  20. Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization.

    Science.gov (United States)

    McCall, Ingrid C; Betanzos, Abigail; Weber, Dominique A; Nava, Porfirio; Miller, Gary W; Parkos, Charles A

    2009-11-15

    Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

  1. Immunomodulatory Effects of Lactobacillus plantarum Lp62 on Intestinal Epithelial and Mononuclear Cells

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    Thalis Ferreira dos Santos

    2016-01-01

    Full Text Available Probiotic lactic acid bacteria are known for their ability to modulate the immune system. They have been shown to inhibit inflammation in experiments with animal models, cell culture, and clinical trials. The objective of this study was to elucidate the anti-inflammatory potential of Lactobacillus plantarum Lp62, isolated from cocoa fermentation, in a cell culture model. Lp62 inhibited IL-8 production by Salmonella Typhi-stimulated HT-29 cells and prevented the adhesion of pathogens to these epithelial cells. The probiotic strain was able to modulate TNF-α, IL1-β, and IL-17 secretion by J774 macrophages. J774 activation was reduced by coincubation with Lp62. PBMC culture showed significantly higher levels of CD4+CD25+ T lymphocytes following treatment with Lp62. Probiotics also induced increased IL-10 secretion by mononuclear cells. L. plantarum Lp62 was able to inhibit inflammatory stimulation in epithelial cells and macrophages and activated a tolerogenic profile in mononuclear cells of healthy donors. These results indicate this strain for a possible application in the treatment or prevention of inflammatory diseases.

  2. Fluorescently labeled methyl-beta-cyclodextrin enters intestinal epithelial Caco-2 cells by fluid-phase endocytosis.

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    Ferenc Fenyvesi

    Full Text Available Cyclodextrins are widely used excipients for increasing the bioavailability of poorly water-soluble drugs. Their effect on drug absorption in the gastrointestinal tract is explained by their solubility- and permeability-enhancement. The aims of this study were to investigate penetration properties of fluorescently labeled randomly methylated-beta-cyclodextrin (FITC-RAMEB on Caco-2 cell layer and examine the cellular entry of cyclodextrins on intestinal cells. The permeability of FITC-RAMEB through Caco-2 monolayers was very limited. Using this compound in 0.05 mM concentration the permeability coefficient was 3.35±1.29×10(-8 cm/s and its permeability did not change in the presence of 5 mM randomly methylated-beta-cyclodextrin. Despite of the low permeability, cellular accumulation of FITC-RAMEB in cytoplasmic vesicles was significant and showed strong time and concentration dependence, similar to the characteristics of the macropinocytosis marker Lucifer Yellow. The internalization process was fully inhibited at 0°C and it was drastically reduced at 37°C applying rottlerin, an inhibitor of macropinocytosis. Notably, FITC-RAMEB colocalized with the early endosome organizer Rab5a. These results have revealed that FITC-RAMEB is able to enter intestinal epithelial cells by fluid-phase endocytosis from the apical side. This mechanism can be an additional process which helps to overcome the intestinal barrier and contributes to the bioavailability enhancement of cyclodextrins.

  3. Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs.

    Science.gov (United States)

    Vlasova, Anastasia N; Paim, Francine C; Kandasamy, Sukumar; Alhamo, Moyasar A; Fischer, David D; Langel, Stephanie N; Deblais, Loic; Kumar, Anand; Chepngeno, Juliet; Shao, Lulu; Huang, Huang-Chi; Candelero-Rueda, Rosario A; Rajashekara, Gireesh; Saif, Linda J

    2017-01-01

    Malnutrition affects millions of children in developing countries, compromising immunity and contributing to increased rates of death from infectious diseases. Rotavirus is a major etiological agent of childhood diarrhea in developing countries, where malnutrition is prevalent. However, the interactions between the two and their combined effects on immune and intestinal functions are poorly understood. In this study, we used neonatal gnotobiotic (Gn) pigs transplanted with the fecal microbiota of a healthy 2-month-old infant (HIFM) and fed protein-deficient or -sufficient bovine milk diets. Protein deficiency induced hypoproteinemia, hypoalbuminemia, hypoglycemia, stunting, and generalized edema in Gn pigs, as observed in protein-malnourished children. Irrespective of the diet, human rotavirus (HRV) infection early, at HIFM posttransplantation day 3 (PTD3), resulted in adverse health effects and higher mortality rates (45 to 75%) than later HRV infection (PTD10). Protein malnutrition exacerbated HRV infection and affected the morphology and function of the small intestinal epithelial barrier. In pigs infected with HRV at PTD10, there was a uniform decrease in the function and/or frequencies of natural killer cells, plasmacytoid dendritic cells, and CD103 + and apoptotic mononuclear cells and altered gene expression profiles of intestinal epithelial cells (chromogranin A, mucin 2, proliferating cell nuclear antigen, SRY-Box 9, and villin). Thus, we have established the first HIFM-transplanted neonatal pig model that recapitulates major aspects of protein malnutrition in children and can be used to evaluate physiologically relevant interventions. Our findings provide an explanation of why nutrient-rich diets alone may lack efficacy in malnourished children. IMPORTANCE Malnutrition and rotavirus infection, prevalent in developing countries, individually and in combination, affect the health of millions of children, compromising their immunity and increasing the rates

  4. Selective non-steroidal glucocorticoid receptor agonists attenuate inflammation but do not impair intestinal epithelial cell restitution in vitro.

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    Kerstin C Reuter

    Full Text Available INTRODUCTION: Despite the excellent anti-inflammatory and immunosuppressive action of glucocorticoids (GCs, their use for the treatment of inflammatory bowel disease (IBD still carries significant risks in terms of frequently occurring severe side effects, such as the impairment of intestinal tissue repair. The recently-introduced selective glucocorticoid receptor (GR agonists (SEGRAs offer anti-inflammatory action comparable to that of common GCs, but with a reduced side effect profile. METHODS: The in vitro effects of the non-steroidal SEGRAs Compound A (CpdA and ZK216348, were investigated in intestinal epithelial cells and compared to those of Dexamethasone (Dex. GR translocation was shown by immunfluorescence and Western blot analysis. Trans-repressive effects were studied by means of NF-κB/p65 activity and IL-8 levels, trans-activation potency by reporter gene assay. Flow cytometry was used to assess apoptosis of cells exposed to SEGRAs. The effects on IEC-6 and HaCaT cell restitution were determined using an in vitro wound healing model, cell proliferation by BrdU assay. In addition, influences on the TGF-β- or EGF/ERK1/2/MAPK-pathway were evaluated by reporter gene assay, Western blot and qPCR analysis. RESULTS: Dex, CpdA and ZK216348 were found to be functional GR agonists. In terms of trans-repression, CpdA and ZK216348 effectively inhibited NF-κB activity and IL-8 secretion, but showed less trans-activation potency. Furthermore, unlike SEGRAs, Dex caused a dose-dependent inhibition of cell restitution with no effect on cell proliferation. These differences in epithelial restitution were TGF-β-independent but Dex inhibited the EGF/ERK1/2/MAPK-pathway important for intestinal epithelial wound healing by induction of MKP-1 and Annexin-1 which was not affected by CpdA or ZK216348. CONCLUSION: Collectively, our results indicate that, while their anti-inflammatory activity is comparable to Dex, SEGRAs show fewer side effects with

  5. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

    Science.gov (United States)

    Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-12-22

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

  6. Enterococcus faecium NCIMB 10415 Modulates Epithelial Integrity, Heat Shock Protein, and Proinflammatory Cytokine Response in Intestinal Cells

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    Shanti Klingspor

    2015-01-01

    Full Text Available Probiotics have shown positive effects on gastrointestinal diseases; they have barrier-modulating effects and change the inflammatory response towards pathogens in studies in vitro. The aim of this investigation has been to examine the response of intestinal epithelial cells to Enterococcus faecium NCIMB 10415 (E. faecium, a probiotic positively affecting diarrhea incidence in piglets, and two pathogenic Escherichia coli (E. coli strains, with specific focus on the probiotic modulation of the response to the pathogenic challenge. Porcine (IPEC-J2 and human (Caco-2 intestinal cells were incubated without bacteria (control, with E. faecium, with enteropathogenic (EPEC or enterotoxigenic E. coli (ETEC each alone or in combination with E. faecium. The ETEC strain decreased transepithelial resistance (TER and increased IL-8 mRNA and protein expression in both cell lines compared with control cells, an effect that could be prevented by pre- and coincubation with E. faecium. Similar effects were observed for the increased expression of heat shock protein 70 in Caco-2 cells. When the cells were challenged by the EPEC strain, no such pattern of changes could be observed. The reduced decrease in TER and the reduction of the proinflammatory and stress response of enterocytes following pathogenic challenge indicate the protective effect of the probiotic.

  7. Cellular mechanisms underlying the inhibitory effect of flufenamic acid on chloride secretion in human intestinal epithelial cells

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    Pawin Pongkorpsakol

    2017-06-01

    Full Text Available Intestinal Cl− secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl− secretion in human intestinal epithelial (T84 cells. FFA inhibited cAMP-dependent Cl− secretion in T84 cell monolayers with IC50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl− channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K+ channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca2+-dependent Cl− secretion with IC50 of ∼10 μM. FFA inhibited activities of Ca2+-activated Cl− channels and KCa3.1, a Ca2+-activated basolateral K+ channels, but had no effect on activities of Na+–K+–Cl− cotransporters and Na+–K+ ATPases. These results indicate that FFA inhibits both cAMP and Ca2+-dependent Cl− secretion by suppressing activities of both apical Cl− channels and basolateral K+ channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.

  8. Naturally Occurring Deletion Mutants of the Pig-Specific, Intestinal Crypt Epithelial Cell Protein CLCA4b without Apparent Phenotype.

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    Stephanie Plog

    Full Text Available The human CLCA4 (chloride channel regulator, calcium-activated modulates the intestinal phenotype of cystic fibrosis (CF patients via an as yet unknown pathway. With the generation of new porcine CF models, species-specific differences between human modifiers of CF and their porcine orthologs are considered critical for the translation of experimental data. Specifically, the porcine ortholog to the human CF modulator gene CLCA4 has recently been shown to be duplicated into two separate genes, CLCA4a and CLCA4b. Here, we characterize the duplication product, CLCA4b, in terms of its genomic structure, tissue and cellular expression patterns as well as its in vitro electrophysiological properties. The CLCA4b gene is a pig-specific duplication product of the CLCA4 ancestor and its protein is exclusively expressed in small and large intestinal crypt epithelial cells, a niche specifically occupied by no other porcine CLCA family member. Surprisingly, a unique deleterious mutation of the CLCA4b gene is spread among modern and ancient breeds in the pig population, but this mutation did not result in an apparent phenotype in homozygously affected animals. Electrophysiologically, neither the products of the wild type nor of the mutated CLCA4b genes were able to evoke a calcium-activated anion conductance, a consensus feature of other CLCA proteins. The apparently pig-specific duplication of the CLCA4 gene with unique expression of the CLCA4b protein variant in intestinal crypt epithelial cells where the porcine CFTR is also present raises the question of whether it may modulate the porcine CF phenotype. Moreover, the naturally occurring null variant of CLCA4b will be valuable for the understanding of CLCA protein function and their relevance in modulating the CF phenotype.

  9. A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide

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    Jonathan M. Williams

    2013-11-01

    The gut barrier, composed of a single layer of intestinal epithelial cells (IECs held together by tight junctions, prevents the entrance of harmful microorganisms, antigens and toxins from the gut lumen into the blood. Small intestinal homeostasis is normally maintained by the rate of shedding of senescent enterocytes from the villus tip exactly matching the rate of generation of new cells in the crypt. However, in various localized and systemic inflammatory conditions, intestinal homeostasis can be disturbed as a result of increased IEC shedding. Such pathological IEC shedding can cause transient gaps to develop in the epithelial barrier and result in increased intestinal permeability. Although pathological IEC shedding has been implicated in the pathogenesis of conditions such as inflammatory bowel disease, our understanding of the underlying mechanisms remains limited. We have therefore developed a murine model to study this phenomenon, because IEC shedding in this species is morphologically analogous to humans. IEC shedding was induced by systemic lipopolysaccharide (LPS administration in wild-type C57BL/6 mice, and in mice deficient in TNF-receptor 1 (Tnfr1−/−, Tnfr2 (Tnfr2−/−, nuclear factor kappa B1 (Nfκb1−/− or Nfĸb2 (Nfĸb2−/−. Apoptosis and cell shedding was quantified using immunohistochemistry for active caspase-3, and gut-to-circulation permeability was assessed by measuring plasma fluorescence following fluorescein-isothiocyanate–dextran gavage. LPS, at doses ≥0.125 mg/kg body weight, induced rapid villus IEC apoptosis, with peak cell shedding occurring at 1.5 hours after treatment. This coincided with significant villus shortening, fluid exudation into the gut lumen and diarrhea. A significant increase in gut-to-circulation permeability was observed at 5 hours. TNFR1 was essential for LPS-induced IEC apoptosis and shedding, and the fate of the IECs was also dependent on NFκB, with signaling via NFκB1 favoring cell survival and

  10. Cigarette smoke extract (CSE delays NOD2 expression and affects NOD2/RIPK2 interactions in intestinal epithelial cells.

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    Marian C Aldhous

    Full Text Available Genetic and environmental factors influence susceptibility to Crohn's disease (CD: NOD2 is the strongest individual genetic determinant and smoking the best-characterised environmental factor. Carriage of NOD2 mutations predispose to small-intestinal, stricturing CD, a phenotype also associated with smoking. We hypothesised that cigarette smoke extract (CSE altered NOD2 expression and function in intestinal epithelial cells.Intestinal epithelial cell-lines (SW480, HT29, HCT116 were stimulated with CSE and nicotine (to mimic smoking ±TNFα (to mimic inflammation. NOD2 expression was measured by qRT-PCR and western blotting; NOD2-RIPK2 interactions by co-immunoprecipitation (CoIP; nuclear NFκB-p65 by ELISA; NFκB activity by luciferase reporter assays and chemokines (CCL20, IL8 in culture supernatants by ELISA. In SW480 and HT29 cells the TNFα-induced NOD2 expression at 4 hours was reduced by CSE (p = 0.0226, a response that was dose-dependent (p = 0.003 and time-dependent (p = 0.0004. Similar effects of CSE on NOD2 expression were seen in cultured ileal biopsies from healthy individuals. In SW480 cells CSE reduced TNFα-induced NFκB-p65 translocation at 15 minutes post-stimulation, upstream of NOD2. Levels of the NOD2-RIPK2 complex were no different at 8 hours post-stimulation with combinations of CSE, nicotine and TNFα, but at 18 hours it was increased in cells stimulated with TNFα+CSE but decreased with TNFα alone (p = 0.0330; CSE reduced TNFα-induced NFκB activity (p = 0.0014 at the same time-point. At 24 hours, basal CCL20 and IL8 (p<0.001 for both and TNFα-induced CCL20 (p = 0.0330 production were decreased by CSE. CSE also reduced NOD2 expression, CCL20 and IL8 production seen with MDP-stimulation of SW480 cells pre-treated with combinations of TNFα and CSE.CSE delayed TNFα-induced NOD2 mRNA expression and was associated with abnormal NOD2/RIPK2 interaction, reduced NFκB activity and decreased chemokine

  11. Sugars increase non-heme iron bioavailability in human epithelial intestinal and liver cells.

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    Tatiana Christides

    Full Text Available Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55 increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

  12. Sugars increase non-heme iron bioavailability in human epithelial intestinal and liver cells.

    Science.gov (United States)

    Christides, Tatiana; Sharp, Paul

    2013-01-01

    Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55) increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl3 increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.

  13. Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells.

    Science.gov (United States)

    Ma'ayeh, Showgy Y; Liu, Jingyi; Peirasmaki, Dimitra; Hörnaeus, Katarina; Bergström Lind, Sara; Grabherr, Manfred; Bergquist, Jonas; Svärd, Staffan G

    2017-12-01

    Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known. To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs) when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively) interact with intestinal epithelial cells (IECs) in vitro. The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates). In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated) and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates) and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis. This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the importance of

  14. Characterization of the Giardia intestinalis secretome during interaction with human intestinal epithelial cells: The impact on host cells.

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    Showgy Y Ma'ayeh

    2017-12-01

    Full Text Available Giardia intestinalis is a non-invasive protozoan parasite that causes giardiasis in humans, the most common form of parasite-induced diarrhea. Disease mechanisms are not completely defined and very few virulence factors are known.To identify putative virulence factors and elucidate mechanistic pathways leading to disease, we have used proteomics to identify the major excretory-secretory products (ESPs when Giardia trophozoites of WB and GS isolates (assemblages A and B, respectively interact with intestinal epithelial cells (IECs in vitro.The main parts of the IEC and parasite secretomes are constitutively released proteins, the majority of which are associated with metabolism but several proteins are released in response to their interaction (87 and 41 WB and GS proteins, respectively, 76 and 45 human proteins in response to the respective isolates. In parasitized IECs, the secretome profile indicated effects on the cell actin cytoskeleton and the induction of immune responses whereas that of Giardia showed anti-oxidation, proteolysis (protease-associated and induction of encystation responses. The Giardia secretome also contained immunodominant and glycosylated proteins as well as new candidate virulence factors and assemblage-specific differences were identified. A minor part of Giardia ESPs had signal peptides (29% for both isolates and extracellular vesicles were detected in the ESPs fractions, suggesting alternative secretory pathways. Microscopic analyses showed ESPs binding to IECs and partial internalization. Parasite ESPs reduced ERK1/2 and P38 phosphorylation and NF-κB nuclear translocation. Giardia ESPs altered gene expression in IECs, with a transcriptional profile indicating recruitment of immune cells via chemokines, disturbances in glucose homeostasis, cholesterol and lipid metabolism, cell cycle and induction of apoptosis.This is the first study identifying Giardia ESPs and evaluating their effects on IECs. It highlights the

  15. Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

    Science.gov (United States)

    Claes, Ingmar; Tytgat, Hanne L. P.; Verhoeven, Tine L. A.; Marien, Eyra; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; de Vos, Willem M.; De Keersmaecker, Sigrid C. J.; Vanderleyden, Jos

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid. PMID:22020518

  16. Functional analysis of Lactobacillus rhamnosus GG pili in relation to adhesion and immunomodulatory interactions with intestinal epithelial cells.

    Science.gov (United States)

    Lebeer, Sarah; Claes, Ingmar; Tytgat, Hanne L P; Verhoeven, Tine L A; Marien, Eyra; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; Vos, Willem M de; Keersmaecker, Sigrid C J De; Vanderleyden, Jos

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid.

  17. Effects of Lactobacillus johnsonii and Lactobacillus reuteri on gut barrier function and heat shock proteins in intestinal porcine epithelial cells.

    Science.gov (United States)

    Liu, Hao-Yu; Roos, Stefan; Jonsson, Hans; Ahl, David; Dicksved, Johan; Lindberg, Jan Erik; Lundh, Torbjörn

    2015-04-01

    Heat shock proteins (HSPs) are a set of highly conserved proteins that can serve as intestinal gate keepers in gut homeostasis. Here, effects of a probiotic, Lactobacillus rhamnosus GG (LGG), and two novel porcine isolates, Lactobacillus johnsonii strain P47-HY and Lactobacillus reuteri strain P43-HUV, on cytoprotective HSP expression and gut barrier function, were investigated in a porcine IPEC-J2 intestinal epithelial cell line model. The IPEC-J2 cells polarized on a permeable filter exhibited villus-like cell phenotype with development of apical microvilli. Western blot analysis detected HSP expression in IPEC-J2 and revealed that L. johnsonii and L. reuteri strains were able to significantly induce HSP27, despite high basal expression in IPEC-J2, whereas LGG did not. For HSP72, only the supernatant of L. reuteri induced the expression, which was comparable to the heat shock treatment, which indicated that HSP72 expression was more stimulus specific. The protective effect of lactobacilli was further studied in IPEC-J2 under an enterotoxigenic Escherichia coli (ETEC) challenge. ETEC caused intestinal barrier destruction, as reflected by loss of cell-cell contact, reduced IPEC-J2 cell viability and transepithelial electrical resistance, and disruption of tight junction protein zonula occludens-1. In contrast, the L. reuteri treatment substantially counteracted these detrimental effects and preserved the barrier function. L. johnsonii and LGG also achieved barrier protection, partly by directly inhibiting ETEC attachment. Together, the results indicate that specific strains of Lactobacillus can enhance gut barrier function through cytoprotective HSP induction and fortify the cell protection against ETEC challenge through tight junction protein modulation and direct interaction with pathogens. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  18. Taste-signaling proteins are coexpressed in solitary intestinal epithelial cells.

    Science.gov (United States)

    Bezençon, Carole; le Coutre, Johannes; Damak, Sami

    2007-01-01

    The taste system, made up of taste receptor cells clustered in taste buds at the surface of the tongue and the soft palate, plays a key role in the decision to ingest or reject food and thereby is essential in protecting organisms against harmful toxins and in selecting the most appropriate nutrients. To determine if a similar chemosensory system exists in the gastrointestinal tract, we used immunohistochemistry and real-time polymerase chain reaction (PCR) to investigate which taste-signaling molecules are expressed in the intestinal mucosa. The PCR data showed that T1r1, T1r2, T1r3, alpha-gustducin, phospholipase Cbeta2 (PLCbeta2), and Trpm5 are expressed in the stomach, small intestine, and colon of mice and humans, with the exception of T1r2, which was not detected in the mouse and human stomach or in the mouse colon. Using transgenic mice expressing enhanced green fluorescent protein under the control of the Trpm5 promoter, we found colocalization of Trpm5 and alpha-gustducin in tufted cells at the surface epithelium of the colon, but these cells did not express T1r3 or PLCbeta2. In the duodenal glands, 43%, 33%, and 38% of Trpm5-expressing cells also express PLCbeta2, T1r3, or alpha-gustducin, respectively. The duodenal gland cells that coexpress PLCbeta2 and Trpm5 morphologically resemble enteroendocrine cells. We found a large degree of colocalization of Trpm5, alpha-gustducin, T1r1, and T1r3 in tufted cells of the duodenal villi, but these cells rarely expressed PLCbeta2. The data suggest that these duodenal cells are possibly involved in sensing amino acids.

  19. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells

    Energy Technology Data Exchange (ETDEWEB)

    Artursson, P.; Karlsson, J. (Uppsala Univ., (Sweden))

    1991-03-29

    Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10{sup {minus} 8} to 5 x 10{sup {minus} 5} cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10{sup {minus} 6} cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10{sup {minus} 6} cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10{sup {minus} 7} cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.

  20. Acrolein Disrupts Tight Junction Proteins and Causes Endoplasmic Reticulum Stress-Mediated Epithelial Cell Death Leading to Intestinal Barrier Dysfunction and Permeability.

    Science.gov (United States)

    Chen, Wei-Yang; Wang, Min; Zhang, Jingwen; Barve, Shirish S; McClain, Craig J; Joshi-Barve, Swati

    2017-12-01

    Increasing evidence suggests that environmental and dietary factors can affect intestinal epithelial integrity leading to gut permeability and bacterial translocation. Intestinal barrier dysfunction is a pathogenic process associated with many chronic disorders. Acrolein is an environmental and dietary pollutant and a lipid-derived endogenous metabolite. The impact of acrolein on the intestine has not been investigated before and is evaluated in this study, both in vitro and in vivo. Our data demonstrate that oral acrolein exposure in mice caused damage to the intestinal epithelial barrier, resulting in increased permeability and subsequently translocation of bacterial endotoxin-lipopolysaccharide into the blood. Similar results were seen in vitro using established Caco-2 cell monolayers wherein acrolein decreased barrier function and increased permeability. Acrolein also caused the down-regulation and/or redistribution of three representative tight junction proteins (ie, zonula occludens-1, Occludin, Claudin-1) that critically regulate epithelial paracellular permeability. In addition, acrolein induced endoplasmic reticulum stress-mediated death of epithelial cells, which is an important mechanism contributing to intestinal barrier damage/dysfunction, and gut permeability. Overall, we demonstrate that exposure to acrolein affects the intestinal epithelium by decrease/redistribution of tight junction proteins and endoplasmic reticulum stress-mediated epithelial cell death, thereby resulting in loss of barrier integrity and function. Our findings highlight the adverse consequences of environmental and dietary pollutants on intestinal barrier integrity/function with relevance to gut permeability and the development of disease. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

    Science.gov (United States)

    Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

    2014-12-01

    Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

  2. Effect of Hops Derived Prenylated Phenols on TNF-α Induced Barrier Dysfunction in Intestinal Epithelial Cells.

    Science.gov (United States)

    Luescher, Sandro; Urmann, Corinna; Butterweck, Veronika

    2017-04-28

    For the prenylated hops phenols 6- and 8-prenylnaringenin (1 and 2), xanthohumol (3), and isoxanthohumol (4), a variety of biological activities has been described. In the current study, a transwell based in vitro model using the human intestinal epithelial cell line Caco-2 was developed to assess potential beneficial effects of compounds 1-4 on TNF-α-induced impairment of tight junction (TJ) permeability. Transepithelial electrical resistance (TEER) was measured using the latest cellZScope online monitoring device. TNF-α treatment (25 ng/mL) induced a significant decrease in TEER values (204.71 ± 4.57 at 72 h) compared to that in control values (245.94 ± 1.68 at 72 h). To determine preventive effects on TNF-α-induced impairment of TJ permeability, 1-4 were added to the apical compartment of Caco-2 monolayers 1 h before TNF-α treatment; afterward, TNF-α was added to the basolateral compartment to induce TJ dysfunction and incubated for a further 72 h. Using this setting, only 1 and 2 prevented epithelial disruption induced by TNF-α. To evaluate restorative effects of 1-4, TNF-α was added to the basolateral compartment of Caco-2 cell monolayers. After 48 h of incubation, 1-4 were added to the apical side, and TEER values were monitored online for a further 72 h. Under these experimental conditions, only 2 restored TNF-α induced barrier dysfunction.

  3. Binding of Candida albicans to Human CEACAM1 and CEACAM6 Modulates the Inflammatory Response of Intestinal Epithelial Cells.

    Science.gov (United States)

    Klaile, Esther; Müller, Mario M; Schäfer, Miriam R; Clauder, Ann-Katrin; Feer, Sabina; Heyl, Kerstin A; Stock, Magdalena; Klassert, Tilman E; Zipfel, Peter F; Singer, Bernhard B; Slevogt, Hortense

    2017-03-14

    Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans ) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 cells in response to C. albicans In CEACAM1-competent cells, the addition of recombinant soluble CEACAM6 reduced the C. albicans -induced CXCL8 secretion. IMPORTANCE The present study demonstrates for the first time that fungal pathogens can be recognized by at least four members of the immunomodulatory CEACAM receptor family: CEACAM1, -3, -5, and -6. Three of the four receptors (i.e., CEACAM1, -5, and -6) are expressed in mucosal cells of the intestinal tract, where they are implicated in immunomodulation and control of tissue homeostasis. Importantly, the interaction of the major fungal pathogen in humans Candida albicans with CEACAM1 and CEACAM6 resulted in an altered epithelial immune response. With respect to the broad impact of CEACAM receptors on various aspects of the innate and the adaptive immune responses, in particular epithelial, neutrophil, and T cell behavior, understanding the role of CEACAMs in the host response to fungal pathogens might help to improve management of

  4. Knockout of MIMP protein in lactobacillus plantarum lost its regulation of intestinal permeability on NCM460 epithelial cells through the zonulin pathway.

    Science.gov (United States)

    Liu, Zhihua; Kang, Liang; Li, Chao; Tong, Chao; Huang, Meijin; Zhang, Xingwei; Huang, Nanqi; Moyer, Mary Pat; Qin, Huanlong; Wang, Jianping

    2014-10-03

    Previous studies indicated that the micro integral membrane protein located within the media place of the integral membrane protein of Lactobacillus plantarum CGMCC 1258 had protective effects against the intestinal epithelial injury. In our study, we mean to establish micro integral membrane protein -knockout Lactobacillus plantarum (LPKM) to investigate the change of its protective effects and verify the role of micro integral membrane protein on protection of normal intestinal barrier function. Binding assay and intestinal permeability were performed to verify the protective effects of micro integral membrane protein on intestinal permeability in vitro and in vivo. Molecular mechanism was also determined as the zonulin pathway. Clinical data were also collected for further verification of relationship between zonulin level and postoperative septicemia. LPKM got decreased inhibition of EPEC adhesion to NCM460 cells. LPKM had lower ability to alleviate the decrease of intestinal permeability induced by enteropathogenic-e.coli, and prevent enteropathogenic-e.coli -induced increase of zonulin expression. Overexpression of zonulin lowered the intestinal permeability regulated by Lactobacillus plantarum. There was a positive correlation between zonulin level and postoperative septicemia. Therefore, micro integral membrane protein could be necessary for the protective effects of Lactobacillus plantarum on intestinal barrier. MIMP might be a positive factor for Lactobacillus plantarum to protect the intestinal epithelial cells from injury, which could be related to the zonulin pathway.

  5. Toll-Like Receptor 2 Activation by beta 2 -> 1-Fructans Protects Barrier Function of T84 Human Intestinal Epithelial Cells in a Chain Length-Dependent Manner

    NARCIS (Netherlands)

    Vogt, Leonie M.; Meyer, Diederick; Pullens, Gerdie; Faas, Marijke M.; Venema, Koen; Ramasamy, Uttara; Schols, Henk A.; de Vos, Paul

    Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that beta 2 -> 1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2

  6. Conjugated primary bile salts reduce permeability of endotoxin through bacteria-stimulated intestinal epithelial cells and synergize with lecithin in suppression of inflammatory cytokine production

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Schaeckeler, Simone; Moser, Lydia

    2007-01-01

    Objective: Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent...

  7. Novel polyfucosylated N-linked glycopeptides with blood group A, H, X and Y determinants from human small intestinal epithelial cells

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Finne, J.; Breimer, M.E.; Hansson, G.C.; Karlsson, K.-A.; Leffler, H.; Halbeek, H. van

    1989-01-01

    A novel type of N-linked glycopeptides representing a major part of the glycans in human small intestinal epithelial cells from blood group A and O individuals were isolated by gel filtrations and affinity chromatography on concanavalin A-Sepharose and Bandeiraea simplicifolia lectin I-Sepharose.

  8. Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production

    DEFF Research Database (Denmark)

    Parlesak, Alexandr; Schaeckeler, S.; Moser, L.

    2007-01-01

    OBJECTIVE: Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent...

  9. The viability and intestinal epithelial cell adhesion of probiotic strain combination--in vitro study.

    Science.gov (United States)

    Piątek, Jacek; Gibas-Dorna, Magdalena; Olejnik, Anna; Krauss, Hanna; Wierzbicki, Krzysztof; Żukiewicz-Sobczak, Wioletta; Głowacki, Maciej

    2012-01-01

    To be effective, probiotic bacteria must exhibit a number of functional characteristics, including the resistance to gastric acidity and the ability to adhere to the intestinal epithelium. In this study, we examined in vitro the viability of lactic acid bacteria (LAB) combination after exposure to low pH, and the adhesion of LAB to Caco-2 cells during coincubation of 9 bacterial strains. To test bacterial viability, 6 commercially available products were incubated in 0.1 N HCl at pH 1.2 for 60 min. The greatest growth inhibition was noted for the non-capsulated product containing the Lactobacillus rhamnosus strain (log reduction of CFU = 6.4), and the best survival observed for the product containing 9 bacterial strains, equipped with a modern capsule made according to the Multi-Resistant Encapsulation technology (log reduction of CFU = 0.1). In the adhesion experiment, the combination of 9 bacterial strains was added to 17-day-old Caco-2 cell culture for 90 min. The greatest efficiency of adhesion was observed for the inoculum containing 5.5x10(8) CFU/mL/9.6 cm(2) of Caco-2 and the dose of probiotic bacteria of 190 cells per one Caco-2 cell. As a result, approximately 157 bacterial cells adhered to one Caco-2 cell. The results indicate that the combination of 9 bacterial strains in the examined product is characterized as highly adhesive.

  10. Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells.

    Science.gov (United States)

    Qiu, Yueqin; Ma, Xianyong; Yang, Xuefen; Wang, Li; Jiang, Zongyong

    2017-04-01

    Conflicting results have been reported that butyrate in normal piglets leads either to an increase or to a decrease of jejunal villus length, implying a possible effect on the proliferation of enterocytes. No definitive study was found for the biological effects of butyrate in porcine jejunal epithelial cells. The present study used IPEC-J2 cells, a non-transformed jejunal epithelial line to evaluate the direct effects of sodium butyrate on cell proliferation, cell cycle regulation, and apoptosis. Low concentrations (0.5 and 1 mM) of butyrate had no effect on cell proliferation. However, at 5 and 10 mM, sodium butyrate significantly decreased cell viability, accompanied by reduced levels of p-mTOR and PCNA protein. Sodium butyrate, in a dose-dependent manner, induced cell cycle arrest in G0/G1 phase and reduced the numbers of cells in S phase. In addition, relative expression of p21, p27, and pro-apoptosis bak genes, and protein levels of p21Waf1/Cip1, p27Kip1, cyclinD3, CDK4, and Cleave-caspase3 were increased by higher concentrations of sodium butyrate (1, 5, 10 mM), and the levels of cyclinD1 and CDK6 were reduced by 5 and 10 mM butyrate. Butyrate increased the phosphorylated form of the signaling molecule p38 and phosphorylated JNK. In conclusion, the present in vitro study indicated that sodium butyrate inhibited the proliferation of IPEC-J2 cells by inducing cell cycle arrest in the G0/G1 phase of cell cycles and by increasing apoptosis at high concentrations.

  11. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    International Nuclear Information System (INIS)

    Murano, Tatsuro; Okamoto, Ryuichi; Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Watanabe, Mamoru

    2014-01-01

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs

  12. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Murano, Tatsuro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Tsuchiya, Kiichiro; Nakamura, Tetsuya [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan)

    2014-01-17

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

  13. New insights into mycotoxin mixtures: The toxicity of low doses of Type B trichothecenes on intestinal epithelial cells is synergistic

    Energy Technology Data Exchange (ETDEWEB)

    Alassane-Kpembi, Imourana [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France); Institut des Sciences Biomédicales Appliquées, Cotonou, Bénin (Benin); Kolf-Clauw, Martine; Gauthier, Thierry; Abrami, Roberta [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France); Abiola, François A. [Institut des Sciences Biomédicales Appliquées, Cotonou, Bénin (Benin); Oswald, Isabelle P., E-mail: Isabelle.Oswald@toulouse.inra.fr [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France); Puel, Olivier [INRA, UMR 1331 Toxalim, Research Center in Food Toxicology, F-31027 Toulouse (France); Université de Toulouse, ENVT, INP, UMR 1331 Toxalim, F-31076 Toulouse (France)

    2013-10-01

    Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON < 15-ADON ≈ DON < NIV ≪ FX. Binary or ternary mixtures also showed a dose-dependent effect. At low concentrations (cytotoxic effect between 10 and 30–40%), mycotoxin combinations were synergistic; however DON–NIV–FX mixture showed antagonism. At higher concentrations (cytotoxic effect around 50%), the combinations had an additive or nearly additive effect. These results indicate that the simultaneous presence of low doses of mycotoxins in food commodities and diet may be more toxic than predicted from the mycotoxins alone. Considering the frequent co-occurrence of trichothecenes in the diet and the concentrations of toxins to which consumers are exposed, this synergy should be taken into account. - Highlights: • We assessed the individual and combined cytotoxicity of five trichothecenes. • The tested concentrations correspond to the French consumer exposure levels. • The type of interaction in combined cytotoxicity varied with the effect level. • Low doses of Type B trichothecenes induced synergistic

  14. Lutein Induces Autophagy via Beclin-1 Upregulation in IEC-6 Rat Intestinal Epithelial Cells.

    Science.gov (United States)

    Chang, Chi-Jen; Lin, Ji-Fan; Hsiao, Chien-Yu; Chang, Hsun-Hao; Li, Hsin-Ju; Chang, Hsun-Hsien; Lee, Gon-Ann; Hung, Chi-Feng

    2017-01-01

    Lutein is a carotenoid with anti-oxidant properties. Autophagy, an evolutionarily conserved catabolic cellular pathway for coping with stress conditions, is responsive to reactive oxygen species (ROS) and degrades damaged organelles. We previously demonstrated that lutein can induce anti-oxidant enzymes to relieve methotrexate-induced ROS stress. We therefore hypothesized that lutein, which activates ROS-scavenging enzymes, can also induce autophagy for cell survival. In this study, we demonstrated that lutein treatment attenuated the reduction in cell viability caused by H 2 O 2 . Lutein dose-dependently induced the processing of microtubule-associated protein light chain 3 (LC3)-II, an autophagy marker protein, and accumulation of LC3-positive puncta in rat intestinal IEC-6 cells. Furthermore, (a) direct observation of autophagosome formation through transmission electron microscopy, (b) upregulation of autophagy-related genes including ATG4A, ATG5, ATG7, ATG12, and beclin-1 (BENC1), and (c) increased BECN1/Bcl-2 ratio confirmed the induction of autophagy by lutein. The results revealed that bafilomycin-A1-induced inhibition of autophagy reduced cell viability and increased apoptosis in lutein-treated cells, indicating a protective role of lutein-induced autophagy. Lutein treatment also activated adenosine monophosphate-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK), and p-38, but had no effects on the induction of extracellular signal-related kinase or inhibition of mTOR; however, the inhibition of activated AMPK, JNK, or p-38 did not attenuate lutein-induced autophagy. Finally, increased BECN1 expression levels were detected in lutein-treated cells, and BECN1 knockdown abolished autophagy induction. These results suggest that lutein-induced autophagy was mediated by the upregulation of BECN1 in IEC-6 cells. We are the first to demonstrate that lutein induces autophagy. Elevated autophagy in lutein-treated IEC-6 cells may have a protective role

  15. Inflammatory bowel diseases influence major histocompatibility complex class I (MHC I) and II compartments in intestinal epithelial cells.

    Science.gov (United States)

    Bär, F; Sina, C; Hundorfean, G; Pagel, R; Lehnert, H; Fellermann, K; Büning, J

    2013-05-01

    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. © 2012 British Society for Immunology.

  16. Up-regulation of intestinal epithelial cell derived IL-7 expression by keratinocyte growth factor through STAT1/IRF-1, IRF-2 pathway.

    Directory of Open Access Journals (Sweden)

    Yu-Jiao Cai

    Full Text Available BACKGROUND: Epithelial cells(EC-derived interleukin-7 (IL-7 plays a crucial role in control of development and homeostasis of neighboring intraepithelial lymphocytes (IEL, and keratinocyte growth factor (KGF exerts protective effects on intestinal epithelial cells and up-regulates EC-derived IL-7 expression through KGFR pathway. This study was to further investigate the molecular mechanism involved in the regulation of IL-7 expression by KGF in the intestine. METHODS: Intestinal epithelial cells (LoVo cells and adult C57BL/6J mice were treated with KGF. Epithelial cell proliferation was studied by flow cytometry for BrdU-incorporation and by immunohistochemistry for PCNA staining. Western blot was used to detect the changes of expression of P-Tyr-STAT1, STAT1, and IL-7 by inhibiting STAT1. Alterations of nuclear extracts and total proteins of IRF-1, IRF-2 and IL-7 following IRF-1 and IRF-2 RNA interference with KGF treatment were also measured with western blot. Moreover, IL-7 mRNA expressions were also detected by Real-time PCR and IL-7 protein level in culture supernatants was measured by enzyme linked immunosorbent assay(ELISA. RESULTS: KGF administration significantly increased LoVo cell proliferation and also increased intestinal wet weight, villus height, crypt depth and crypt cell proliferation in mice. KGF treatment led to increased levels of P-Tyr-STAT1, RAPA and AG490 both blocked P-Tyr-STAT1 and IL-7 expression in LoVo cells. IRF-1 and IRF-2 expression in vivo and in vitro were also up-regulated by KGF, and IL-7 expression was decreased after IRF-1 and IRF-2 expression was silenced by interfering RNA, respectively. CONCLUSION: KGF could up-regulate IL-7 expression through the STAT1/IRF-1, IRF-2 signaling pathway, which is a new insight in potential effects of KGF on the intestinal mucosal immune system.

  17. Heat shock protein 90β inhibits apoptosis of intestinal epithelial cells induced by hypoxia through stabilizing phosphorylated Akt

    Directory of Open Access Journals (Sweden)

    Shuai Zhang

    2013-01-01

    Full Text Available Intestinal epithelial cell (IEC apoptosis induced by hypoxiacompromise intestinal epithelium barrier function. Both Akt andHsp90 have cytoprotective function. However, the specific roleof Akt and Hsp90β in IEC apoptosis induced by hypoxia has notbeen explored. We confirmed that hypoxia-induced apoptosiswas reduced by Hsp90β overexpression but enhanced bydecreasing Hsp90β expression. Hsp90β overexpressionenhanced BAD phosphorylation and thus reduced mitochondrialrelease of cytochrome C. Reducing Hsp90β expression hadopposite effects. The protective effect of Hsp90β againstapoptosis was negated by LY294002, an Akt inhibitor. Furtherstudy showed that Akt phosphorylation was enhanced byHsp90β, which was not due to the activation of upstream PI3Kand PDK1 but because of stabilization of pAkt via directinteraction between Hsp90β and pAkt. These results demonstratethat Hsp90β may play a significant role in protecting IECs fromhypoxia-induced apoptosis via stabilizing pAkt to phosphorylateBAD and reduce cytochrome C release. [BMB Reports 2013;46(1: 47-52

  18. The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport.

    Science.gov (United States)

    Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

    2009-06-01

    The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.

  19. Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth

    NARCIS (Netherlands)

    Aust, Gabriela; Kerner, Christiane; Gonsior, Susann; Sittig, Doreen; Schneider, Hartmut; Buske, Peter; Scholz, Markus; Dietrich, Norman; Oldenburg, Sindy; Karpus, Olga N.; Galle, Jörg; Amasheh, Salah; Hamann, Jörg

    2013-01-01

    Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein-coupled receptor CD97 shows an expression

  20. Administration of Protein kinase D1 induce an immunomodulatory effect on lipopolysaccharide-induced intestinal inflammation in a co-culture model of intestinal epithelial Caco-2 cells and RAW 264.7 macrophage cells

    DEFF Research Database (Denmark)

    Nielsen, Ditte Søvsø Gundelund; Fredborg, Marlene; Andersen, Vibeke

    2017-01-01

    the effects of human PKD1 in relation to intestinal inflammation, using a co-culture model of intestinal epithelial Caco-2 cells and RAW264.7 macrophages. An inflammatory response was induced in the macrophages by lipopolysaccharide (LPS), upregulating the expression of tumour necrosis factor alpha (TNF......-α), interleukin- (IL-) 1β, and IL-6 besides increasing the secretion of TNF-α protein. The effect of administering PKD1 to Caco-2 was evaluated in relation to both amelioration of inflammation and the ability to suppress inflammation initiation. Administration of PKD1 (10–100 ng/ml) following induction...

  1. Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate

    Directory of Open Access Journals (Sweden)

    Ju-Ri Sim

    2018-01-01

    Full Text Available Short-chain fatty acids (SCFAs, such as acetate, butyrate, and propionate, modulate immune responses in the gut. However, the effect of SCFAs on mucosal vaccine-induced immune cell migration is poorly understood. Here, we investigated whether SCFAs modulate chemokine expression induced by the killed whole-cell oral cholera vaccine, Shanchol™, in human intestinal epithelial cells. Shanchol™ induced expression of CCL2, CCL5, CCL20, and CXCL10 at the mRNA level, but not at the protein level. Interestingly, CCL20 secretion was substantially increased by co-stimulation with Shanchol™ and butyrate, while neither acetate nor propionate showed such effect. Enhanced CCL20 secretion was associated with GPR109A activation, and histone deacetylase (HDAC inhibition. In addition, co-treatment with Shanchol™ and butyrate synergistically increased the secretion of adenosine triphosphate (ATP. Moreover, CCL20 secretion was decreased by inhibiting the extracellular ATP receptor P2X7. However, neither inflammasomes nor caspases were involved in CCL20 production. The culture supernatant of cells treated with Shanchol™ and butyrate augmented human immature dendritic cell migration. Collectively, these results suggest that butyrate enhances Shanchol™-induced CCL20 production in human intestinal epithelial cells via HDAC inhibition and ATP-P2X7 signaling by activating GPR109A. These effects potentially enhance the mucosal immune responses in the gut induced by this oral cholera vaccine.

  2. Killed Whole-Cell Oral Cholera Vaccine Induces CCL20 Secretion by Human Intestinal Epithelial Cells in the Presence of the Short-Chain Fatty Acid, Butyrate.

    Science.gov (United States)

    Sim, Ju-Ri; Kang, Seok-Seong; Lee, Daesang; Yun, Cheol-Heui; Han, Seung Hyun

    2018-01-01

    Short-chain fatty acids (SCFAs), such as acetate, butyrate, and propionate, modulate immune responses in the gut. However, the effect of SCFAs on mucosal vaccine-induced immune cell migration is poorly understood. Here, we investigated whether SCFAs modulate chemokine expression induced by the killed whole-cell oral cholera vaccine, Shanchol™, in human intestinal epithelial cells. Shanchol™ induced expression of CCL2, CCL5, CCL20, and CXCL10 at the mRNA level, but not at the protein level. Interestingly, CCL20 secretion was substantially increased by co-stimulation with Shanchol™ and butyrate, while neither acetate nor propionate showed such effect. Enhanced CCL20 secretion was associated with GPR109A activation, and histone deacetylase (HDAC) inhibition. In addition, co-treatment with Shanchol™ and butyrate synergistically increased the secretion of adenosine triphosphate (ATP). Moreover, CCL20 secretion was decreased by inhibiting the extracellular ATP receptor P2X7. However, neither inflammasomes nor caspases were involved in CCL20 production. The culture supernatant of cells treated with Shanchol™ and butyrate augmented human immature dendritic cell migration. Collectively, these results suggest that butyrate enhances Shanchol™-induced CCL20 production in human intestinal epithelial cells via HDAC inhibition and ATP-P2X7 signaling by activating GPR109A. These effects potentially enhance the mucosal immune responses in the gut induced by this oral cholera vaccine.

  3. Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

    Science.gov (United States)

    Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

    2016-01-01

    High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

  4. Effects of lactoferrin on the production of interferon-λ by the human intestinal epithelial cell line HT-29.

    Science.gov (United States)

    Shin, Kouichirou; Oda, Hirotsugu; Wakabayashi, Hiroyuki; Yamauchi, Koji; Abe, Fumiaki

    2017-02-01

    We examined the in-vitro effects of bovine lactoferrin (LF) on the production of interferon-λ (IFN-λ), an antiviral cytokine important for the defense of enterocytes, using the human intestinal epithelial cell line HT-29. HT-29 cell cultures were treated with LF for 1 h, and the cultures were stimulated with polyinosinic-polycytidylic acid (poly I:C). LF increased the concentration of IFN-λ in the culture supernatant after stimulation in a dose-dependent manner. A similar increase in the concentration of IFN-λ was observed in the supernatant of cells washed between treatment with LF and stimulation with poly I:C. At 6 and 24 h after stimulation with poly I:C (early and late phases, respectively) treated cultures contained significantly higher concentrations of IFN-λ1 in the culture supernatant, and significantly higher IFN-λ1 and IFN-λ2 mRNA levels, than controls. These results suggest that LF activates the innate cellular immunity of the enterocytes to double-stranded RNA and increases the production of IFN-λ.

  5. Immunomodulation and signaling mechanism of Lactobacillus rhamnosus GG and its components on porcine intestinal epithelial cells stimulated by lipopolysaccharide.

    Science.gov (United States)

    Gao, Kan; Wang, Chong; Liu, Li; Dou, Xiaoxiao; Liu, Jianxin; Yuan, Lijuan; Zhang, Wenming; Wang, Haifeng

    2017-10-01

    This study aimed to evaluate the immunomodulatory effects and signaling mechanisms of Lactobacillus rhamnosus GG (LGG) and its components [surface-layer protein (SLP), DNA, exopolysaccharides, and CpG oligodeoxynucleotides] on lipopolysaccharide (LPS)-stimulated porcine intestinal epithelial cell (IEC) IPEC-J2. The mRNA expressions of inflammatory cytokines and Toll-like receptors (TLRs) were measured by quantitative real-time polymerase chain reaction. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling was detected by western blot and immunofluorescence. Pretreatment of IPEC-J2 cells with LGG, SLP, or exopolysaccharides significantly alleviated LPS-induced inflammatory cytokines and TLR activation at mRNA level. LGG, SLP, and exopolysaccharides also attenuated LPS-induced MAPK and NF-κB signaling activations. CpG oligodeoxynucleotides significantly increased the interleukin 12, tumor necrosis factor α, and TLR9 mRNA levels and enhanced NF-κB signaling activation in LPS-stimulated cells. LGG had immunomodulatory effects on LPS-induced porcine IECs by modulating TLR expressions and inhibiting MAPK and NF-κB signaling to decrease inflammatory cytokine expressions. Components of LGG exerted immunomodulatory effects on porcine IECs, especially immunostimulatory CpG oligodeoxynucleotides. Copyright © 2015. Published by Elsevier B.V.

  6. The prevention of radiation-induced DNA damage and apoptosis in human intestinal epithelial cells by salvianic acid A

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    Yanjun Zhang

    2014-07-01

    Full Text Available The topic of radiation always provokes public debate, and the uses of radiation for therapeutic and other purposes have always been associated with some anxiety. Salvia miltiorrhiza Bunge has been widely used for the treatment of various diseases including cerebrovascular diseases, coronary artery diseases, and myocardial infarction. Salvianolic acid A (SAA d (+-(3,4-dihydroxyphenyl lactic acid is the principal effective, watersoluble constituent of Salvia miltiorrhiza Bunge. In our present study, radiation-induced DNA damage and apoptosis in human intestinal epithelial cells (HIEC in the presence and absence of SAA were examined. We investigated the effects of SAA on ROS formation and the activity of enzymatic antioxidants (SOD, the lipid peroxidative index and the levels of non-enzymatic antioxidant (GSH. Finally, we investigated whether the reduction of radiation-induced cell death caused by SAA might be related to mitochondria-dependent apoptosis. Present findings indicate that SAA is a promising radioprotective agent with a strong antioxidant activity. SAA exerted its protective action on the proliferative activity of HIEC cells as evidenced by decreased cytotoxicity after exposure to γ-radiation. It is possible that SAA achieved its radioprotective action, at least in part, by enhancing DNA repair and the activity of antioxidant enzymes, by scavenging ROS and by inhibiting the mitochondria-dependent apoptotic pathway.

  7. Intestinal Epithelial Cell-Intrinsic Deletion of Setd7 Identifies Role for Developmental Pathways in Immunity to Helminth Infection

    Science.gov (United States)

    Chenery, Alistair L.; Redpath, Stephen A.; Braam, Mitchell J.; Perona-Wright, Georgia

    2016-01-01

    The intestine is a common site for a variety of pathogenic infections. Helminth infections continue to be major causes of disease worldwide, and are a significant burden on health care systems. Lysine methyltransferases are part of a family of novel attractive targets for drug discovery. SETD7 is a member of the Suppressor of variegation 3-9-Enhancer of zeste-Trithorax (SET) domain-containing family of lysine methyltransferases, and has been shown to methylate and alter the function of a wide variety of proteins in vitro. A few of these putative methylation targets have been shown to be important in resistance against pathogens. We therefore sought to study the role of SETD7 during parasitic infections. We find that Setd7 -/- mice display increased resistance to infection with the helminth Trichuris muris but not Heligmosomoides polygyrus bakeri. Resistance to T. muris relies on an appropriate type 2 immune response that in turn prompts intestinal epithelial cells (IECs) to alter differentiation and proliferation kinetics. Here we show that SETD7 does not affect immune cell responses during infection. Instead, we found that IEC-specific deletion of Setd7 renders mice resistant to T. muris by controlling IEC turnover, an important aspect of anti-helminth immune responses. We further show that SETD7 controls IEC turnover by modulating developmental signaling pathways such as Hippo/YAP and Wnt/β-Catenin. We show that the Hippo pathway specifically is relevant during T. muris infection as verteporfin (a YAP inhibitor) treated mice became susceptible to T. muris. We conclude that SETD7 plays an important role in IEC biology during infection. PMID:27598373

  8. Effect of metals on β-actin and total protein synthesis in cultured human intestinal epithelial cells.

    Science.gov (United States)

    Calabro, Anthony R; Gazarian, Dmitry I; Barile, Frank A

    2011-01-01

    As an important structural protein, β-actin is associated with anchoring of tight junctions (TJs) to the cell scaffold. Caco-2 cells, an immortal intestinal epithelial cell line, rely on β-actin to form intact monolayers with high transepithelial electrical resistance in cell culture inserts. We examined the effect of six metals on expression of β-actin mRNA and β-actin synthesis, on total and net production of newly synthesized proteins, on paracellular transport of TJ markers, and on cell viability in confluent monolayers. [(3)H]-glycine and [(3)H]-tyrosine were used as indicators of newly synthesized proteins in the absence or presence of increasing concentrations of arsenic, cadmium, copper, manganese, mercury and nickel. The monolayers were exposed to 24-h single exposures as well as continuous daily repeated doses of metals for 48-h and 96-h. Results suggest that decreases in newly synthesized proteins, in which β-actin represents about 10%, correlated with 2- to 5-fold higher expression of β-actin mRNA for the higher concentrations of metals. Interestingly, IC(50)s calculated for each chemical for 24-h acute and 48- and 96-h repeated dosing experiments, using the MTT viability assay and paracellular permeability markers, decreased newly synthesized and total proteins to 10% and 40% of control, respectively. Overall, the results indicate that, at equivalent concentrations, the metals affect β-actin mRNA and newly synthesized proteins before cell viability and paracellular permeability are compromised. Consequently the results help in elucidating mechanisms of metal cytotoxicity that lead to understanding the relationship between tight junction integrity, paracellular transport, and cell viability. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria.

    LENUS (Irish Health Repository)

    Sibartie, Shomik

    2009-01-01

    BACKGROUND: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays. RESULTS: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion. CONCLUSION: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

  10. Effect of Utipro(®) (containing gelatin-xyloglucan) against Escherichia coli invasion of intestinal epithelial cells: results of an in vitro study.

    Science.gov (United States)

    de Servi, Barbara; Ranzini, Francesco; Piqué, Nuria

    2016-05-01

    To evaluate whether Utipro(®), a natural product approved to prevent urinary tract infections, protects intestinal epithelial cells from Escherichia coli adherence/intracellular invasion in vitro. Caco-2 and CacoGoblet(TM) cells were treated with Utipro (1.5 to 10 mg/ml) or untreated (controls). E. coli adherence/intracellular invasion was evaluated by Trans-Epithelial Electrical Resistance, Lucifer Yellow assay and microbial counts. Utipro was noncytotoxic. Utipro 5 and 10 mg/ml protected cell tight junctions (mean ± SD Trans-Epithelial Electrical Resistance [Ω × cm(2)] 66.83 ± 0.29 and 71.33 ± 0.29, respectively), and protected cells from E. coli intracellular invasion (mean ± SD reductions in total bacteria counts [Log10] 0.9 ± 0.06 and 2.1 ± 0.56, respectively). Results indicate that Utipro creates a protective physical barrier on intestinal epithelial cells in vitro which reduces the settling of E. coli reservoirs. These results constitute the first step in the demonstration of the efficacy of Utipro to prevent urinary tract infections. Further research is needed in in vivo models and clinical trials.

  11. Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2)

    DEFF Research Database (Denmark)

    Boyd, Mette; Hansen, Morten; Jensen, Tine G K

    2010-01-01

    , revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing...

  12. Anti-Inflammatory Effects of the Nicotinergic Peptides SLURP-1 and SLURP-2 on Human Intestinal Epithelial Cells and Immunocytes

    Directory of Open Access Journals (Sweden)

    Alex I. Chernyavsky

    2014-01-01

    Full Text Available A search for novel and more efficient therapeutic modalities of inflammatory bowel disease (IBD is one of the most important tasks of contemporary medicine. The anti-inflammatory action of nicotine in IBD might be therapeutic, but its toxicity due to off-target and nonreceptor effects limited its use and prompted a search for nontoxic nicotinergic drugs. We tested the hypothesis that SLURP-1 and -2—the physiological nicotinergic substances produced by the human intestinal epithelial cells (IEC and immunocytes—can mimic the anti-inflammatory effects of nicotine. We used human CCL-241 enterocytes, CCL-248 colonocytes, CCRF-CEM T-cells, and U937 macrophages. SLURP-1 diminished the TLR9-dependent secretion of IL-8 by CCL-241, and IFNγ-induced upregulation of ICAM-1 in both IEC types. rSLURP-2 inhibited IL-1β-induced secretion of IL-6 and TLR4- and TLR9-dependent induction of CXCL10 and IL-8, respectively, in CCL-241. rSLURP-1 decreased production of TNFα by T-cells, downregulated IL-1β and IL-6 secretion by macrophages, and moderately upregulated IL-10 production by both types of immunocytes. SLURP-2 downregulated TNFα and IFNγR in T-cells and reduced IL-6 production by macrophages. Combining both SLURPs amplified their anti-inflammatory effects. Learning the pharmacology of SLURP-1 and -2 actions on enterocytes, colonocytes, T cells, and macrophages may help develop novel effective treatments of IBD.

  13. Real-time monitoring of trans-epithelial electrical resistance in cultured intestinal epithelial cells: the barrier protection of water-soluble dietary fiber.

    Science.gov (United States)

    Majima, Atsushi; Handa, Osamu; Naito, Yuji; Suyama, Yosuke; Onozawa, Yuriko; Higashimura, Yasuki; Mizushima, Katsura; Morita, Mayuko; Uehara, Yukiko; Horie, Hideki; Iida, Takaya; Fukui, Akifumu; Dohi, Osamu; Okayama, Tetsuya; Yoshida, Naohisa; Kamada, Kazuhiro; Katada, Kazuhiro; Uchiyama, Kazuhiko; Ishikawa, Takeshi; Takagi, Tomohisa; Konishi, Hideyuki; Yasukawa, Zenta; Tokunaga, Makoto; Okubo, Tsutomu; Itoh, Yoshito

    2017-03-01

    In this study we aimed to verify a real-time trans-epithelial electrical resistance (TEER) monitoring system in a Caco-2 monolayer and to investigate the therapeutic effect of partially hydrolyzed guar gum (PHGG), a dietary fiber, against interferon (IFN)-γ-induced intestinal barrier dysfunction using this monitoring system. We measured TEER using a real-time monitoring system and evaluated epithelial paracellular permeability using fluorescein isothiocyanate-conjugated dextran (4 kDa; FD4) in Caco-2 monolayers treated with IFN-γ for 48 h. The expression and distribution of tight junction (TJ)-associated proteins, ZO-1 and occludin, were analyzed by Western blot and immunocytochemistry, respectively. In some experiments PHGG was added prior to IFN-γ treatment in order to investigate its protective effect on barrier function. IFN-γ treatment significantly decreased TEER and increased FD4 flux across Caco-2 monolayers, indicating a great influence of IFN-γ on the intestinal epithelial paracellular permeability. In contrast, the pretreatment of PHGG significantly reduced the IFN-γ-induced increment of FD4 flux without affecting TEER. Neither IFN-γ nor PHGG treatment affected the expressions of TJ-associated proteins, while immunocytochemistry showed that IFN-γ-induced redistribution of occludin was clearly restored by PHGG. Real-time TEER monitoring enabled us to evaluate the dynamic changes of intestinal epithelial barrier function. PHGG may have a protective effect against IFN-γ-induced barrier dysfunction by attenuating the paracellular hyperpermeability; thus, its promotion as a functional food is anticipated. © 2017 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  14. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease.

    Directory of Open Access Journals (Sweden)

    Gangwei Ou

    Full Text Available BACKGROUND: Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s to induce interleukin-8 (IL-8 production by human intestinal epithelial cells. The aim was to identify the secreted component(s of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV. METHODOLOGY/PRINCIPAL FINDINGS: Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-alpha (TNF-alpha secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.

  15. Fermented milk containing Lactobacillus GG alleviated DSS-induced colitis in mice and activated epidermal growth factor receptor and Akt signaling in intestinal epithelial cells

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    Kazutoyo Yoda

    2012-06-01

    Full Text Available Lactobacillus rhamnosus GG was assessed for its ability to alleviate DSS-induced colitis in mice and activate epidermal growth factor receptor and Akt signaling in intestinal epithelial cells. In this study mice were treated with DSS to induce colitis and they were given Lactobacillus GG fermented milk to assess the effect of probiotic on colitis. Lactobacillus GG fermented milk significantly reduced the colitis associated changes suggesting a protective effect against DSS induced colitis.

  16. (Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: Presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit

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    Marxer, A.; Stieger, B.; Quaroni, A.; Kashgarian, M.; Hauri, H.P. (Univ. of Basel (Switzerland))

    1989-09-01

    The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

  17. Intestinal Microbial Dysbiosis and Colonic Epithelial Cell Hyperproliferation by Dietary α-Mangostin is Independent of Mouse Strain

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    Fabiola Gutierrez-Orozco

    2015-01-01

    Full Text Available Beverages and supplements prepared from mangosteen fruit are claimed to support gut health and immunity, despite the absence of supporting evidence from clinical trials. We recently reported that α-mangostin (α-MG, the most abundant xanthone in mangosteen fruit, altered the intestinal microbiome, promoted dysbiosis, and exacerbated colitis in C57BL/6J mice. The objective of this study was to determine whether induction of dysbiosis by dietary α-MG is limited to the C57BL/6J strain or represents a more generic response to chronic intake of the xanthone on the gut microbiota of mice. C3H, Balb/c, Nude FoxN1nu, and C57BL/6J mice, each demonstrating unique microbiomes, were fed standard diet or diet containing 0.1% α-MG for four weeks. Dietary α-MG significantly altered the cecal and colonic microbiota in all four strains of mice, promoting a reduction in generally assumed beneficial bacterial groups while increasing the abundance of pathogenic bacteria. Consumption of α-MG was associated with reduced abundance of Firmicutes and increased abundance of Proteobacteria. The abundance of Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae was reduced in α-MG-fed mice, while that of Enterobacteriaceae and Enterococcaceae was increased. Dietary α-MG also was associated with increased proliferation of colonic epithelial cells, infiltration of immune cells, infiltration of immune cells and increased fluid content in stool. These results suggest that ingestion of pharmacologic doses of xanthones in mangosteen-containing supplements may adversely alter the gut microbiota and should be used with caution.

  18. Intestinal Microbial Dysbiosis and Colonic Epithelial Cell Hyperproliferation by Dietary α-Mangostin is Independent of Mouse Strain

    Science.gov (United States)

    Gutierrez-Orozco, Fabiola; Thomas-Ahner, Jennifer M.; Galley, Jeffrey D.; Bailey, Michael T.; Clinton, Steven K.; Lesinski, Gregory B.; Failla, Mark L.

    2015-01-01

    Beverages and supplements prepared from mangosteen fruit are claimed to support gut health and immunity, despite the absence of supporting evidence from clinical trials. We recently reported that α-mangostin (α-MG), the most abundant xanthone in mangosteen fruit, altered the intestinal microbiome, promoted dysbiosis, and exacerbated colitis in C57BL/6J mice. The objective of this study was to determine whether induction of dysbiosis by dietary α-MG is limited to the C57BL/6J strain or represents a more generic response to chronic intake of the xanthone on the gut microbiota of mice. C3H, Balb/c, Nude FoxN1nu, and C57BL/6J mice, each demonstrating unique microbiomes, were fed standard diet or diet containing 0.1% α-MG for four weeks. Dietary α-MG significantly altered the cecal and colonic microbiota in all four strains of mice, promoting a reduction in generally assumed beneficial bacterial groups while increasing the abundance of pathogenic bacteria. Consumption of α-MG was associated with reduced abundance of Firmicutes and increased abundance of Proteobacteria. The abundance of Lachnospiraceae, Ruminococcaceae, and Lactobacillaceae was reduced in α-MG-fed mice, while that of Enterobacteriaceae and Enterococcaceae was increased. Dietary α-MG also was associated with increased proliferation of colonic epithelial cells, infiltration of immune cells, infiltration of immune cells and increased fluid content in stool. These results suggest that ingestion of pharmacologic doses of xanthones in mangosteen-containing supplements may adversely alter the gut microbiota and should be used with caution. PMID:25621505

  19. Gut microbiota regulates NKG2D ligand expression on intestinal epithelial cells

    DEFF Research Database (Denmark)

    Hansen, Camilla Hartmann Friis; Holm, Thomas L.; Krych, Lukasz

    2013-01-01

    expression on small IECs. Germ-free and ampicillin-treated mice were shown to have a significant increase in NKG2D ligand expression. Interestingly, vancomycin treatment, which propagated the bacterium Akkermansia muciniphila and reduced the level of IFN-¿ and IL-15 in the intestine, decreased the NKG2D...... that the constitutive levels of NKG2D ligand expression on IECs are regulated by microbial signaling in the gut and further disfavor the intuitive notion that IEC NKG2D ligand expression is caused by low-grade immune reaction against commensal bacteria. It is more likely that constitutively high IEC NKG2D ligand...

  20. Effects of ethanol and acetaldehyde on tight junction integrity: in vitro study in a three dimensional intestinal epithelial cell culture model.

    Directory of Open Access Journals (Sweden)

    Elhaseen Elamin

    Full Text Available BACKGROUND: Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. METHODS AND FINDINGS: Caco-2 cells were grown in a basement membrane matrix (Matrigel™ to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10-40 mM or acetaldehyde (25-200 µM for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. CONCLUSIONS: These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in

  1. Goblet Cell Derived RELM-β Recruits CD4+ T Cells during Infectious Colitis to Promote Protective Intestinal Epithelial Cell Proliferation.

    Science.gov (United States)

    Bergstrom, Kirk S B; Morampudi, Vijay; Chan, Justin M; Bhinder, Ganive; Lau, Jennifer; Yang, Hyungjun; Ma, Caixia; Huang, Tina; Ryz, Natasha; Sham, Ho Pan; Zarepour, Maryam; Zaph, Colby; Artis, David; Nair, Meera; Vallance, Bruce A

    2015-08-01

    Enterohemorrhagic Escherichia coli and related food and waterborne pathogens pose significant threats to human health. These attaching/effacing microbes infect the apical surface of intestinal epithelial cells (IEC), causing severe diarrheal disease. Colonizing the intestinal luminal surface helps segregate these microbes from most host inflammatory responses. Based on studies using Citrobacter rodentium, a related mouse pathogen, we speculate that hosts rely on immune-mediated changes in IEC, including goblet cells to defend against these pathogens. These changes include a CD4+ T cell-dependent increase in IEC proliferation to replace infected IEC, as well as altered production of the goblet cell-derived mucin Muc2. Another goblet cell mediator, REsistin-Like Molecule (RELM)-β is strongly induced within goblet cells during C. rodentium infection, and was detected in the stool as well as serum. Despite its dramatic induction, RELM-β's role in host defense is unclear. Thus, wildtype and RELM-β gene deficient mice (Retnlb-/-) were orally infected with C. rodentium. While their C. rodentium burdens were only modestly elevated, infected Retnlb-/- mice suffered increased mortality and mucosal ulceration due to deep pathogen penetration of colonic crypts. Immunostaining for Ki67 and BrDU revealed Retnlb-/- mice were significantly impaired in infection-induced IEC hyper-proliferation. Interestingly, exposure to RELM-β did not directly increase IEC proliferation, rather RELM-β acted as a CD4+ T cell chemoattractant. Correspondingly, Retnlb-/- mice showed impaired CD4+ T cell recruitment to their infected colons, along with reduced production of interleukin (IL)-22, a multifunctional cytokine that directly increased IEC proliferation. Enema delivery of RELM-β to Retnlb-/- mice restored CD4+ T cell recruitment, concurrently increasing IL-22 levels and IEC proliferation, while reducing mucosal pathology. These findings demonstrate that RELM-β and goblet cells play an

  2. Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors

    International Nuclear Information System (INIS)

    Voisin, Laure; Basik, Mark; Meloche, Sylvain; Julien, Catherine; Duhamel, Stéphanie; Gopalbhai, Kailesh; Claveau, Isabelle; Saba-El-Leil, Marc K; Rodrigue-Gervais, Ian Gaël; Gaboury, Louis; Lamarre, Daniel

    2008-01-01

    The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells. We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells

  3. [Absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro].

    Science.gov (United States)

    Wang, Fu-Rong; Yang, Xiu-Wei

    2017-08-01

    Tongmai formula (TMF) is a drug combination of three components including Puerariae Lobatae Radix [roots of Pueraria lobata], Salviae Miltiorrhizae Radix (roots of Salvia miltiorrhiza) and Chuanxiong Rhizoma (rhizomes of Ligusticum chuanxiong) in a weight ratio of 1∶1∶1. The absorption and transport of isoflavonoid compounds from Tongmai formula across human intestinal epithelial (Caco-2) cells in vitro were studied in this paper. The assay isoflavonoid compounds include daidzein, formononetin, 5-hydroxylononin, ononin, daidzin, 3'-methoxypuerarin, genistin, puerarin, formononetin-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, formononetin-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, lanceolarin, kakkanin, daidzein-7,4'-di-O-β-D-glucopyranoside, mirificin, 3'-hydroxypuerarin, 3'-methoxydaidzin, formononetin-8-C-β-D-xylopyranosyl-(1→6)-O-β-D-glucopyranoside, genistein-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, genistein-7-O-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside (ambocin), 3'-hydroxymirificin, 6″-O-β-D-xylosylpuerarin, biochanin A-8-C-β-D-apiofuranosyl-(1→6)-O-β-D-glucopyranoside, 3'-methoxydaidzein-7,4'-di-O-β-D-glucopyranoside, daidzein-7-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside, and daidzein-7-O-α-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside. By using human Caco-2 monolayer as an intestinal epithelial cell model in vitro, the permeability of above-mentioned 25 isoflavonoids in TMF were studied from the apical (AP) side to basolateral (BL) side or from the BL side to AP side. The assay compounds were determined by reversed phased high-performance liquid chromatography (HPLC) coupled with UV detector. Transport parameters and apparent permeability coefficients (Papp) were then calculated and and compared with those of propranolol and atenolol, which are the transcellular transport marker and as a control substance for high and poor permeability, respectively. The Papp values of daidzein and

  4. The proliferative status of intestinal epithelial clonogenic cells: sensitivity to S phase specific cytotoxic agents.

    Science.gov (United States)

    Boarder, T A; Blackett, N M

    1976-11-01

    High concentrations of tritiated thymidine and cytosine arabinoside (Ara-C) have been used to selectively kill cells in the crypts of Lieberkuhn that are synthesizing DNA. The effect of these agents on the number of regenerating microcolonies seen 3 1/2 days after a range of radiation doses indicates that a majority of the clonogenic cells are proliferating rapidly and that the slowly proliferating cells at the base of the crypt do not represent the whole clonogenic population.

  5. miR-122 targets NOD2 to decrease intestinal epithelial cell injury in Crohn’s disease

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu; Wang, Chengxiao; Liu, Ying; Tang, Liwei; Zheng, Mingxia [Department of Pediatrics, Jiangwan Hospital of Shanghai, Shanghai 200434 (China); Xu, Chundi [Department of Pediatrics, Ruijin affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, 200025 (China); Song, Jian, E-mail: jiansongkxy@126.com [Department of Gastroenterology, Jiangwan Hospital of Shanghai, Shanghai 200434 (China); Meng, Xiaochun [Department of Pediatrics, Jiangwan Hospital of Shanghai, Shanghai 200434 (China)

    2013-08-16

    Highlights: •NOD2 is a target gene of miR-122. •miR-122 inhibits LPS-induced apoptosis by suppressing NOD2 in HT-29 cells. •miR-122 reduces the expression of pro-inflammatory cytokines (TNF-α and IFN-γ). •miR-122 promotes the release of anti-inflammatory cytokines (IL-4 and IL-10). •NF-κB signaling pathway is involved in inflammatory response induced by LPS. -- Abstract: Crohn’s disease (CD) is one of the two major types of inflammatory bowel disease (IBD) thought to be caused by genetic and environmental factors. Recently, miR-122 was found to be deregulated in association with CD progression. However, the underlying molecular mechanisms remain unclear. In the present study, the gene nucleotide-binding oligomerization domain 2 (NOD2/CARD15), which is strongly associated with susceptibility to CD, was identified as a functional target of miR-122. MiR-122 inhibited LPS-induced apoptosis by suppressing NOD2 in HT-29 cells. NOD2 interaction with LPS initiates signal transduction mechanisms resulting in the activation of nuclear factor κB (NF-κB) and the stimulation of downstream pro-inflammatory events. The activation of NF-κB was inhibited in LPS-stimulated HT-29 cells pretreated with miR-122 precursor or NOD2 shRNA. The expression of the pro-inflammatory cytokines TNF-α and IFN-γ was significantly decreased, whereas therelease of the anti-inflammatory cytokines IL-4 and IL-10 was increased in LPS-stimulated HT-29 cells pretreated with miR-122 precursor, NOD2 shRNA or the NF-κB inhibitor QNZ. Taken together, these results indicate that miR-122 and its target gene NOD2 may play an important role in the injury of intestinal epithelial cells induced by LPS.

  6. Autonomous cure of damaged human intestinal epithelial cells by TLR2 and TLR4-dependent production of IL-22 in response to Spirulina polysaccharides.

    Science.gov (United States)

    Tominaga, Akira; Konishi, Yuko; Taguchi, Takahiro; Fukuoka, Satoshi; Kawaguchi, Tokuichi; Noda, Tetsuo; Shimizu, Keiji

    2013-12-01

    In order to analyze the damage of human epithelial cells, we used human quasi-normal FPCK-1-1 cells derived from a colonic polyp in a patient with familial adenomatous polyposis as a monolayer, which is co-cultured with peptidoglycan (PGN)-stimulated THP-1 cells. Co-cultured FPCK-1-1 cells showed a decreased transepithelial electrical resistance (TER) and the lower level of claudin-2. When Spirulina complex polysaccharides were added one day before the start of the co-culture, there was no decrease of TER and claudin-2 (early phase damage). In contrast, when Spirulina complex polysaccharides were added to FPCK-1-1 cells after the level of TER had decreased, there was no recovery at the level of claudin-2, though the TER level recovered (late phase damage). The mucosa reconstitution is suggested to be involved in the recovery from the damaged status. Interestingly, autonomous recovery of FPCK-1-1 cells from both the early and late phase damage requires the production of IL-22, because anti-IL-22 antibodies inhibited recovery in these cases. Antibodies against either TLR2 or TLR4 inhibited the production of IL-22 from FPCK-1-1 colon epithelial cells, suggesting that signals through TLR2 and TLR4 are necessary for autonomous recovery of FPCK-1-1 colon epithelial cells by producing IL-22. In conclusion, we have established a useful model for the study of intestinal damage and recovery using human colon epithelial cells and our data suggest that damage to human colon epithelial cells can, at least in part, be recovered by the autonomous production of IL-22 in response to Spirulina complex polysaccharides. © 2013.

  7. Deficiency of Protein Tyrosine Phosphatase Non-Receptor Type 2 in Intestinal Epithelial Cells Has No Appreciable Impact on Dextran Sulphate Sodium Colitis Severity But Promotes Wound Healing.

    Science.gov (United States)

    Kasper, Stephanie H; Spalinger, Marianne R; Leonardi, Irina; Gerstgrasser, Alexandra; Raselli, Tina; Gottier, Claudia; Atrott, Kirstin; Frey-Wagner, Isabelle; Fischbeck-Terhalle, Anne; Rogler, Gerhard; Scharl, Michael

    2016-01-01

    The protein tyrosine phosphatase non-receptor type 2 (PTPN2) is known to mediate susceptibility to inflammatory bowel diseases. Cell culture experiments suggest that PTPN2 influences barrier function, autophagy and secretion of pro-inflammatory cytokines. PTPN2 knockout mice die a few weeks after birth due to systemic inflammation, emphasizing the importance of this phosphatase in inflammatory processes. The aim of this study was to investigate the role of PTPN2 in colon epithelial cells by performing dextran sulphate sodium (DSS)-induced colitis in PTPN2xVilCre mice. Acute colitis was induced by administering 2.5 or 2% DSS for 7 days and chronic colitis by 4 cycles of treatment using 1% DSS. Body weight of mice was measured regularly and colonoscopy was done at the end of the experiments. Mice were sacrificed afterwards and colon specimens were obtained for H&E staining. For analysis of wound healing, mechanical wounds were introduced during endoscopy and wound closure assessed by daily colonoscopy. Although colonoscopy and weight development suggested changes in colitis severity, the lack of any influence of PTPN2 deficiency on histological scoring for inflammation severity after acute or chronic DSS colitis indicates that colitis severity is not influenced by epithelial-specific loss of PTPN2. Chronic colitis induced the development of aberrant crypt foci more frequently in PTPN2xVilCre mice compared to their wild type littermates. On the other hand, loss of PTPN2-induced enhanced epithelial cell proliferation and promoted wound closure. Loss of PTPN2 in intestinal epithelial cells (IECs) has no significant influence on inflammation in DSS colitis. Obviously, loss of PTPN2 in IECs can be compensated in vivo, thereby suppressing a phenotype. This lack of a colitis-phenotype might be due to enhanced epithelial cell proliferation and subsequent increased wound-healing capacity of the epithelial layer. © 2016 S. Karger AG, Basel.

  8. Proteomic View of the Crosstalk between Lactobacillus mucosae and Intestinal Epithelial Cells in Co-culture Revealed by Q Exactive-Based Quantitative Proteomics

    Directory of Open Access Journals (Sweden)

    Edward Alain B. Pajarillo

    2017-12-01

    Full Text Available Lactobacilli are bacteria that are beneficial to host health, but information on communication between Lactobacilli and host cells in the intestine is lacking. In this study, we examined the proteomes of the Lactobacillus mucosae strain LM1, as a model of beneficial bacteria, and the intestinal porcine epithelial cell line (IPEC-J2 after co-culture. Label-free proteomics demonstrated the high-throughput capability of the technique, and robust characterization of the functional profiles and changes in the bacteria and intestinal cells was achieved in pure and mixed cultures. After co-culture, we identified totals of 376 and 653 differentially expressed proteins in the LM1 and IPEC-J2 proteomes, respectively. The major proteomic changes in the LM1 strain occurred in the functional categories of transcription, general function, and translation, whereas those in IPEC-J2 cells involved metabolic and cellular processes, and cellular component organization/biogenesis. Among them, elongation factor Tu, glyceraldehyde 3-phosphate dehydrogenase, and phosphocarrier protein HPr, which are known to be involved in bacterial adhesion, were upregulated in LM1. In contrast, proteins involved in tight junction assembly, actin organization, and genetic information processing (i.e., histones and signaling pathways were significantly upregulated in IPEC-J2 cells. Furthermore, we identified functional pathways that are possibly involved in host–microbe crosstalk and response. These findings will provide novel insights into host–bacteria communication and the molecular mechanism of probiotic establishment in the intestine.

  9. Proteomic View of the Crosstalk betweenLactobacillus mucosaeand Intestinal Epithelial Cells in Co-culture Revealed by Q Exactive-Based Quantitative Proteomics.

    Science.gov (United States)

    Pajarillo, Edward Alain B; Kim, Sang Hoon; Valeriano, Valerie Diane; Lee, Ji Yoon; Kang, Dae-Kyung

    2017-01-01

    Lactobacilli are bacteria that are beneficial to host health, but information on communication between Lactobacilli and host cells in the intestine is lacking. In this study, we examined the proteomes of the Lactobacillus mucosae strain LM1, as a model of beneficial bacteria, and the intestinal porcine epithelial cell line (IPEC-J2) after co-culture. Label-free proteomics demonstrated the high-throughput capability of the technique, and robust characterization of the functional profiles and changes in the bacteria and intestinal cells was achieved in pure and mixed cultures. After co-culture, we identified totals of 376 and 653 differentially expressed proteins in the LM1 and IPEC-J2 proteomes, respectively. The major proteomic changes in the LM1 strain occurred in the functional categories of transcription, general function, and translation, whereas those in IPEC-J2 cells involved metabolic and cellular processes, and cellular component organization/biogenesis. Among them, elongation factor Tu, glyceraldehyde 3-phosphate dehydrogenase, and phosphocarrier protein HPr, which are known to be involved in bacterial adhesion, were upregulated in LM1. In contrast, proteins involved in tight junction assembly, actin organization, and genetic information processing (i.e., histones and signaling pathways) were significantly upregulated in IPEC-J2 cells. Furthermore, we identified functional pathways that are possibly involved in host-microbe crosstalk and response. These findings will provide novel insights into host-bacteria communication and the molecular mechanism of probiotic establishment in the intestine.

  10. EspP, a Type V-secreted serine protease of enterohaemorrhagic Escherichia coli O157:H7, influences intestinal colonization of calves and adherence to bovine primary intestinal epithelial cells.

    Science.gov (United States)

    Dziva, Francis; Mahajan, Arvind; Cameron, Pamela; Currie, Carol; McKendrick, Iain J; Wallis, Timothy S; Smith, David G E; Stevens, Mark P

    2007-06-01

    Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of zoonotic diarrhoeal pathogens of worldwide importance. Cattle are a key reservoir; however the molecular mechanisms that promote persistent colonization of the bovine intestines by EHEC are ill-defined. The large plasmid of EHEC O157:H7 encodes several putative virulence factors. Here, it is reported that the pO157-encoded Type V-secreted serine protease EspP influences the intestinal colonization of calves. To dissect the basis of attenuation, a bovine primary rectal epithelial cell line was developed. Adherence of E. coli O157:H7 to such cells was significantly impaired by espP mutation but restored upon addition of highly purified exogenous EspP. Data of this study add to the growing body of evidence that cytotoxins facilitate intestinal colonization by EHEC.

  11. CDX2 Stimulates the Proliferation of Porcine Intestinal Epithelial Cells by Activating the mTORC1 and Wnt/β-Catenin Signaling Pathways.

    Science.gov (United States)

    Fan, Hong-Bo; Zhai, Zhen-Ya; Li, Xiang-Guang; Gao, Chun-Qi; Yan, Hui-Chao; Chen, Zhe-Sheng; Wang, Xiu-Qi

    2017-11-18

    Caudal type homeobox 2 (CDX2) is expressed in intestinal epithelial cells and plays a role in gut development and homeostasis by regulating cell proliferation. However, whether CDX2 cooperates with the mammalian target of rapamycin complex 1 (mTORC1) and Wnt/β-catenin signaling pathways to stimulate cell proliferation remains unknown. The objective of this study was to investigate the effect of CDX2 on the proliferation of porcine jejunum epithelial cells (IPEC-J2) and the correlation between CDX2, the mTORC1 and Wnt/β-catenin signaling pathways. CDX2 overexpression and knockdown cell culture models were established to explore the regulation of CDX2 on both pathways. Pathway-specific antagonists were used to verify the effects. The results showed that CDX2 overexpression increased IPEC-J2 cell proliferation and activated both the mTORC1 and Wnt/β-catenin pathways, and that CDX2 knockdown decreased cell proliferation and inhibited both pathways. Furthermore, the mTORC1 and Wnt/β-catenin pathway-specific antagonist rapamycin and XAV939 (3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)]-4H -thiopyrano[4,3-d]pyrimidin-4-one) both suppressed the proliferation of IPEC-J2 cells overexpressing CDX2, and that the combination of rapamycin and XAV939 had an additive effect. Regardless of whether the cells were treated with rapamycin or XAV939 alone or in combination, both mTORC1 and Wnt/β-catenin pathways were down-regulated, accompanied by a decrease in CDX2 expression. Taken together, our data indicate that CDX2 stimulates porcine intestinal epithelial cell proliferation by activating the mTORC1 and Wnt/β-catenin signaling pathways.

  12. Effects of dietary soyabean meal, inulin and oxytetracycline on intestinal microbiota and epithelial cell stress, apoptosis and proliferation in the teleost Atlantic salmon (Salmo salar L.).

    Science.gov (United States)

    Bakke-McKellep, Anne Marie; Penn, Michael H; Salas, Patricia Mora; Refstie, Ståle; Sperstad, Sigmund; Landsverk, Thor; Ringø, Einar; Krogdahl, Ashild

    2007-04-01

    Soyabean meal (SBM)-induced enteritis in the distal intestine of the teleost Atlantic salmon (Salmo salar L.) and other salmonids may be considered a model for diet-related mucosal disorders in other animals and man. The role of the intestinal microbiota in its pathogenesis was explored. Compared to diets containing fishmeal (FM) as the sole protein source, responses to extracted SBM or the prebiotic inulin, with or without oxytetracycline (OTC) inclusion, were studied following a 3-week feeding trial. Intestinal microbiota, organosomatic indices and histology, as well as immunohistochemical detection of proliferating cell nuclear antigen (PCNA), heat shock protein 70 (HSP70) and caspase-3-positive cells in the distal intestine, were studied. Distal intestine somatic indices (DISI) were higher in inulin and lower in SBM compared to FM-fed fish. The low DISI caused by SBM corresponded with histological changes, neither of which was affected by OTC, despite a significant decrease in adherent bacteria count. Image analysis of PCNA-stained sections showed a significant increase in the proliferative compartment length in SBM-fed fish, accompanied by apparent increases in reactivity to HSP70 and caspase-3 along the mucosal folds, indicating induction of cellular repair and apoptosis, respectively. Fish fed the SBM diet had higher total number as well as a more diverse population composition of adherent bacteria in the distal intestine. Thus SBM-induced enteritis is accompanied by induction of distal intestinal epithelial cell protective responses and changes in microbiota. Putative involvement of bacteria in the inflammatory response merits further investigation.

  13. A defect in epithelial barrier integrity is not required for a systemic response to bacterial antigens or intestinal injury in T cell receptor-alpha gene-deficient mice.

    Science.gov (United States)

    Sydora, Beate C; Tavernini, Michele M; Doyle, Jason; Fedorak, Richard N

    2006-08-01

    Genetically induced disruption of the intestinal epithelial barrier leads to development of intestinal inflammation. In the interleukin-10 gene-deficient inflammatory bowel disease (IBD) mouse model, for instance, a primary defect in intestinal epithelial integrity occurs before the development of enterocolitis. In humans, a causal role for epithelial barrier disruption is still controversial. Although studies with first-degree relatives of IBD patients suggests an underlying role of impaired barrier function, a primary epithelial barrier defect in IBD patients has not been confirmed. The purpose of this article is to examine whether a primary epithelial barrier disruption is a prerequisite for the development of intestinal inflammation or whether intestinal inflammation can develop in the absence of epithelial disruption. We examined the intestinal epithelial integrity of the T cell receptor (TCR)-alpha gene-deficient mouse model of IBD. In vivo colonic permeability, determined by mannitol transmural flux, was assessed in 6-week-, 12-week-, and 25-week-old TCR-alpha gene-deficient and wild-type control mice using a single-pass perfusion technique. Mice were scored for intestinal histological injury and intestinal cytokine levels measured in organ cultures. Systemic responses to bacterial antigens were determined through 48-h spleen cell cultures stimulated with sonicate derived from endogenous bacterial strains. In contrast with previous findings in the interleukin-10 gene-deficient IBD model, TCR-alpha gene-deficient mice did not demonstrate evidence of primary intestinal epithelial barrier disruption at any age, despite developing a moderate to severe colitis within 12 weeks. A rise in intestinal interferon (IFN)-gamma levels preceded the onset of mucosal inflammation and then correlated closely with the degree of intestinal inflammation and injury. Spleen cells from TCR-alpha gene-deficient mice released IFN-gamma in response to stimulation with endogenous

  14. Activation of intestinal epithelial Stat3 orchestrates tissue defense during gastrointestinal infection.

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    Nadine Wittkopf

    Full Text Available Gastrointestinal infections with EHEC and EPEC are responsible for outbreaks of diarrheal diseases and represent a global health problem. Innate first-line-defense mechanisms such as production of mucus and antimicrobial peptides by intestinal epithelial cells are of utmost importance for host control of gastrointestinal infections. For the first time, we directly demonstrate a critical role for Stat3 activation in intestinal epithelial cells upon infection of mice with Citrobacter rodentium - a murine pathogen that mimics human infections with attaching and effacing Escherichia coli. C. rodentium induced transcription of IL-6 and IL-22 in gut samples of mice and was associated with activation of the transcription factor Stat3 in intestinal epithelial cells. C. rodentium infection induced expression of several antimicrobial peptides such as RegIIIγ and Pla2g2a in the intestine which was critically dependent on Stat3 activation. Consequently, mice with specific deletion of Stat3 in intestinal epithelial cells showed increased susceptibility to C. rodentium infection as indicated by high bacterial load, severe gut inflammation, pronounced intestinal epithelial cell death and dissemination of bacteria to distant organs. Together, our data implicate an essential role for Stat3 activation in intestinal epithelial cells during C. rodentium infection. Stat3 concerts the host response to bacterial infection by controlling bacterial growth and suppression of apoptosis to maintain intestinal epithelial barrier function.

  15. Fish oil enhances recovery of intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplant.

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    Qiurong Li

    Full Text Available BACKGROUND: The intestinal chronic rejection (CR is the major limitation to long-term survival of transplanted organs. This study aimed to investigate the interaction between intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplantation, and to find out whether fish oil enhances recovery of intestinal microbiota and epithelial integrity. METHODS/PRINCIPAL FINDINGS: The luminal and mucosal microbiota composition of CR rats were characterized by DGGE analysis at 190 days after intestinal transplant. The specific bacterial species were determined by sequence analysis. Furthermore, changes in the localization of intestinal TJ proteins were examined by immunofluorescent staining. PCR-DGGE analysis revealed that gut microbiota in CR rats had a shift towards Escherichia coli, Bacteroides spp and Clostridium spp and a decrease in the abundance of Lactobacillales bacteria in the intestines. Fish oil supplementation could enhance the recovery of gut microbiota, showing a significant decrease of gut bacterial proportions of E. coli and Bacteroides spp and an increase of Lactobacillales spp. In addition, CR rats showed pronounced alteration of tight junction, depicted by marked changes in epithelial cell ultrastructure and redistribution of occuldin and claudins as well as disruption in TJ barrier function. Fish oil administration ameliorated disruption of epithelial integrity in CR, which was associated with an improvement of the mucosal structure leading to improved tight junctions. CONCLUSIONS/SIGNIFICANCE: Our study have presented novel evidence that fish oil is involved in the maintenance of epithelial TJ integrity and recovery of gut microbiota, which may have therapeutic potential against CR in intestinal transplantation.

  16. Atg9 antagonizes TOR signaling to regulate intestinal cell growth and epithelial homeostasis in Drosophila.

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    Wen, Jung-Kun; Wang, Yi-Ting; Chan, Chih-Chiang; Hsieh, Cheng-Wen; Liao, Hsiao-Man; Hung, Chin-Chun; Chen, Guang-Chao

    2017-11-16

    Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.

  17. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

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    Woodfint, Rachel M; Chen, Paula R; Ahn, Jinsoo; Suh, Yeunsu; Hwang, Seongsoo; Lee, Sang Suk; Lee, Kichoon

    2017-01-19

    Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 ( MUC2 ) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis -acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  18. Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells

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    Rachel M. Woodfint

    2017-01-01

    Full Text Available Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2 expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2, GATA binding protein 4 (GATA4, hepatocyte nuclear factor 4 α (HNF4A, and transcription factor 4 (TCF4 that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

  19. Disruption of Escherichia coli Nissle 1917 K5 capsule biosynthesis, through loss of distinct kfi genes, modulates interaction with intestinal epithelial cells and impact on cell health.

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    Jonathan Nzakizwanayo

    Full Text Available Escherichia coli Nissle 1917 (EcN is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants, prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB, or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC, were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

  20. Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

    Science.gov (United States)

    Nair Gourikutty Sajay, Bhuvanendran; Yin, Chiam Su; Ramadan, Qasem

    2017-12-01

    In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5-3 µm were used and a shear stress of ~0.03 dyne cm-2 was created by applying a low flow rate of 20 nl s-1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

  1. Surface Proteome Analysis of a Natural Isolate of Lactococcus lactis Reveals the Presence of Pili Able to Bind Human Intestinal Epithelial Cells*

    Science.gov (United States)

    Meyrand, Mickael; Guillot, Alain; Goin, Mélodie; Furlan, Sylviane; Armalyte, Julija; Kulakauskas, Saulius; Cortes-Perez, Naima G.; Thomas, Ginette; Chat, Sophie; Péchoux, Christine; Dupres, Vincent; Hols, Pascal; Dufrêne, Yves F.; Trugnan, Germain; Chapot-Chartier, Marie-Pierre

    2013-01-01

    Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells

  2. Interleukin-7 produced by intestinal epithelial cells in response to Citrobacter rodentium infection plays a major role in innate immunity against this pathogen.

    Science.gov (United States)

    Zhang, Wei; Du, Jiang-Yuan; Yu, Qing; Jin, Jun-O

    2015-08-01

    Interleukin-7 (IL-7) engages multiple mechanisms to overcome chronic viral infections, but the role of IL-7 in bacterial infections, especially enteric bacterial infections, remains unclear. Here we characterized the previously unexplored role of IL-7 in the innate immune response to the attaching and effacing bacterium Citrobacter rodentium. C. rodentium infection induced IL-7 production from intestinal epithelial cells (IECs). IL-7 production from IECs in response to C. rodentium was dependent on gamma interferon (IFN-γ)-producing NK1.1(+) cells and IL-12. Treatment with anti-IL-7Rα antibody during C. rodentium infection resulted in a higher bacterial burden, enhanced intestinal damage, and greater weight loss and mortality than observed with the control IgG treatment. IEC-produced IL-7 was only essential for protective immunity against C. rodentium during the first 6 days after infection. An impaired bacterial clearance upon IL-7Rα blockade was associated with a significant decrease in macrophage accumulation and activation in the colon. Moreover, C. rodentium-induced expansion and activation of intestinal CD4(+) lymphoid tissue inducer (LTi) cells was completely abrogated by IL-7Rα blockade. Collectively, these data demonstrate that IL-7 is produced by IECs in response to C. rodentium infection and plays a critical role in the protective immunity against this intestinal attaching and effacing bacterium. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  3. Chitin-binding domains of Escherichia coli ChiA mediate interactions with intestinal epithelial cells in mice with colitis.

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    Low, Daren; Tran, Hoa T; Lee, In-Ah; Dreux, Nicolas; Kamba, Alan; Reinecker, Hans-Christian; Darfeuille-Michaud, Arlette; Barnich, Nicolas; Mizoguchi, Emiko

    2013-09-01

    Inducible chitinase 3-like-1 is expressed by intestinal epithelial cells (IECs) and adheres to bacteria under conditions of inflammation. We performed a structure-function analysis of the chitin-binding domains encoded by the chiA gene, which mediates the pathogenic effects of adherent invasive Escherichia coli (AIEC). We created AIEC (strain LF82) with deletion of chiA (LF82-ΔchiA) or that expressed chiA with specific mutations. We investigated the effects of infecting different IEC lines with these bacteria compared with nonpathogenic E coli; chitinase activities were measured using the colloidal chitin-azure method. Colitis was induced in C57/Bl6 mice by administration of dextran sodium sulfate, and mice were given 10(8) bacteria for 15 consecutive days by gavage. Stool/tissue samples were collected and analyzed. LF82-ΔchiA had significantly less adhesion to IEC lines than LF82. Complementation of LF82-ΔchiA with the LF82 chiA gene, but not chiA from nonpathogenic (K12) E coli, increased adhesion. We identified 5 specific polymorphisms in the chitin-binding domain of LF82 chiA (at amino acids 362, 370, 378, 388, and 548) that differ from chiA of K12 and were required for LF82 to interact directly with IECs. This interaction was mediated by an N-glycosylated asparagine in chitinase 3-like-1 (amino acid 68) on IECs. Mice infected with LF82, or LF82-ΔchiA complemented with LF82 chiA, developed more severe colitis after administration of dextran sodium sulfate than mice infected with LF82-ΔchiA or LF82 that expressed mutant forms of chiA. AIEC adheres to an N-glycosylated chitinase 3-like-1 on IECs via the chitin-binding domain of chiA. This mechanism promotes the pathogenic effects of AIEC in mice with colitis. Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.

  4. Intestinal Epithelial Sirtuin 1 Regulates Intestinal Inflammation During Aging in Mice by Altering the Intestinal Microbiota.

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    Wellman, Alicia S; Metukuri, Mallikarjuna R; Kazgan, Nevzat; Xu, Xiaojiang; Xu, Qing; Ren, Natalie S X; Czopik, Agnieszka; Shanahan, Michael T; Kang, Ashley; Chen, Willa; Azcarate-Peril, M Andrea; Gulati, Ajay S; Fargo, David C; Guarente, Leonard; Li, Xiaoling

    2017-09-01

    Intestinal epithelial homeostasis is maintained by complex interactions among epithelial cells, commensal gut microorganisms, and immune cells. Disruption of this homeostasis is associated with disorders such as inflammatory bowel disease (IBD), but the mechanisms of this process are not clear. We investigated how Sirtuin 1 (SIRT1), a conserved mammalian NAD + -dependent protein deacetylase, senses environmental stress to alter intestinal integrity. We performed studies of mice with disruption of Sirt1 specifically in the intestinal epithelium (SIRT1 iKO, villin-Cre+, Sirt1 flox/flox mice) and control mice (villin-Cre-, Sirt1 flox/flox ) on a C57BL/6 background. Acute colitis was induced in some mice by addition of 2.5% dextran sodium sulfate to drinking water for 5-9 consecutive days. Some mice were given antibiotics via their drinking water for 4 weeks to deplete their microbiota. Some mice were fed with a cholestyramine-containing diet for 7 days to sequester their bile acids. Feces were collected and proportions of microbiota were analyzed by 16S rRNA amplicon sequencing and quantitative PCR. Intestines were collected from mice and gene expression profiles were compared by microarray and quantitative PCR analyses. We compared levels of specific mRNAs between colon tissues from age-matched patients with ulcerative colitis (n=10) vs without IBD (n=8, controls). Mice with intestinal deletion of SIRT1 (SIRT1 iKO) had abnormal activation of Paneth cells starting at the age of 5-8 months, with increased activation of NF-κB, stress pathways, and spontaneous inflammation at 22-24 months of age, compared with control mice. SIRT1 iKO mice also had altered fecal microbiota starting at 4-6 months of age compared with control mice, in part because of altered bile acid metabolism. Moreover, SIRT1 iKO mice with defective gut microbiota developed more severe colitis than control mice. Intestinal tissues from patients with ulcerative colitis expressed significantly lower

  5. Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

    NARCIS (Netherlands)

    Lebeer, S.; Claes, I.J.; Tytgat, H.L.P.; Verhoeven, T.L.A.; Marien, E.; Ossowski, von I.; Reunanen, J.; Palva, A.; Vos, de W.M.; Keersmaecker, de S.C.; Vanderleyden, J.

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a

  6. Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

    Science.gov (United States)

    Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

    2018-02-01

    Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Amplification loop of the inflammatory process is induced by P2X7R activation in intestinal epithelial cells in response to neutrophil transepithelial migration.

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    Cesaro, Annabelle; Brest, Patrick; Hofman, Véronique; Hébuterne, Xavier; Wildman, Scott; Ferrua, Bernard; Marchetti, Sandrine; Doglio, Alain; Vouret-Craviari, Valérie; Galland, Franck; Naquet, Philippe; Mograbi, Baharia; Unwin, Robert; Hofman, Paul

    2010-07-01

    Inflammatory bowel diseases (IBD) are characterized during their active phase by polymorphonuclear leukocyte (PMNL) transepithelial migration. The efflux of PMNL into the mucosa is associated with the production of proinflammatory cytokines and the release of ATP from damaged and necrotic cells. The expression and function of purinergic P2X(7) receptor (P2X(7)R) in intestinal epithelial cells (IEC) and its potential role in the "cross talk" between IEC and PMNL have not been explored. The aims of the present study were 1) to examine P2X(7)R expression in IEC (T84 cells) and in human intestinal biopsies; 2) to detect any changes in P2X(7)R expression in T84 cells during PMNL transepithelial migration, and during the active and quiescent phases of IBD; and 3) to test whether P2X(7)R stimulation in T84 monolayers can induce caspase-1 activation and IL-1beta release by IEC. We found that a functional ATP-sensitive P2X(7)R is constitutively expressed at the apical surface of IEC T84 cells. PMNL transmigration regulates dynamically P2X(7)R expression and alters its distribution from the apical to basolateral surface of IEC during the early phase of PMNL transepithelial migration in vitro. P2X(7)R expression was weak in intestinal biopsies obtained during the active phase of IBD. We show that activation of epithelial P2X(7)R is mandatory for PMNL-induced caspase-1 activation and IL-1beta release by IEC. Overall, these changes in P2X(7)R function may serve to tailor the intensity of the inflammatory response and to prevent IL-1beta overproduction and inflammatory disease.

  8. Effect of Saccharomyces cerevisiae var. Boulardii and β-galactomannan oligosaccharide on porcine intestinal epithelial and dendritic cells challenged in vitro with Escherichia coli F4 (K88

    Directory of Open Access Journals (Sweden)

    Badia Roger

    2012-01-01

    Full Text Available Abstract Probiotic and prebiotics, often called "immune-enhancing" feed additives, are believed to deal with pathogens, preventing the need of an immune response and reducing tissue damage. In this study, we investigated if a recently developed β-galactomannan (βGM had a similar protective role compared to Saccharomyces cerevisiae var. Boulardii (Scb, a proven probiotic, in the context of enterotoxigenic Escherichia coli (ETEC infection. ETEC causes inflammation, diarrhea and intestinal damage in piglets, resulting in large economic loses worldwide. We observed that Scb and βGM products inhibited in vitro adhesion of ETEC on cell surface of porcine intestinal IPI-2I cells. Our data showed that Scb and βGM decreased the mRNA ETEC-induced gene expression of pro-inflammatory cytokines TNF-α, IL-6, GM-CSF and chemokines CCL2, CCL20 and CXCL8 on intestinal IPI-2I. Furthermore, we investigated the putative immunomodulatory role of Scb and βGM on porcine monocyte-derived dendritic cells (DCs per se and under infection conditions. We observed a slight up-regulation of mRNA for TNF-α and CCR7 receptor after co-incubation of DC with Scb and βGM. However, no differences were found in DC activation upon ETEC infection and Scb or βGM co-culture. Therefore, our results indicate that, similar to probiotic Scb, prebiotic βGM may protect intestinal epithelial cells against intestinal pathogens. Finally, although these products may modulate DC activation, their effect under ETEC challenge conditions remains to be elucidated.

  9. Giardia duodenalis assemblage-specific induction of apoptosis and tight junction disruption in human intestinal epithelial cells: effects of mixed infections.

    Science.gov (United States)

    Koh, Wan Hon; Geurden, Thomas; Paget, Tim; O'Handley, Ryan; Steuart, Robert F; Thompson, R C Andrew; Buret, Andre G

    2013-04-01

    In view of the interest in genotype-specific pathogenesis in Giardia duodenalis , the aim of the present study was to examine the effects of infection with different, or mixed, G. duodenalis assemblages on the integrity of human intestinal epithelia. To that end, human epithelial cells (HCT-8) were cultured and exposed to different G. duodenalis assemblages (A, B, and E) or a combination of these assemblages. Epithelial disruption and apoptosis were evaluated by fluorescent microscopy and apoptotic oligonucleosome quantification. The results indicate that infection with trophozoites disrupts epithelial tight junctions and induces varying degrees of enterocyte apoptosis, depending on the infecting assemblage. All disruptions were caspase-3 dependent and were more pronounced when caused by a non-host specific assemblage. Furthermore, infections by isolates in combination with isolates from another assemblage enhanced the epithelial disruption and apoptosis. Further studies in vitro and in vivo are required to confirm the mechanisms of enhanced pathogenicity of mixed or non-host specific (or both) G. duodenalis infections. Findings in the present study point to the potential pathogenic importance of intra-species polyparasitism in giardiasis.

  10. Remodeling of Tight Junctions and Enhancement of Barrier Integrity of the CACO-2 Intestinal Epithelial Cell Layer by Micronutrients

    Science.gov (United States)

    Valenzano, Mary Carmen; DiGuilio, Katherine; Mercado, Joanna; Teter, Mimi; To, Julie; Ferraro, Brendan; Mixson, Brittany; Manley, Isabel; Baker, Valerissa; Moore, Beverley A.; Wertheimer, Joshua; Mullin, James M.

    2015-01-01

    The micronutrients zinc, quercetin, butyrate, indole and berberine were evaluated for their ability to induce remodeling of epithelial tight junctions (TJs) and enhance barrier integrity in the CACO-2 gastrointestinal epithelial cell culture model. All five of these chemically very diverse micronutrients increased transepithelial electrical resistance (Rt) significantly, but only berberine also improved barrier integrity to the non-electrolyte D-mannitol. Increases of Rt as much as 200% of untreated controls were observed. Each of the five micronutrients also induced unique, signature-like changes in TJ protein composition, suggesting multiple pathways (and TJ arrangements) by which TJ barrier function can be enhanced. Decreases in abundance by as much as 90% were observed for claudin-2, and increases of over 300% could be seen for claudins -5 and -7. The exact effects of the micronutrients on barrier integrity and TJ protein composition were found to be highly dependent on the degree of differentiation of the cell layer at the time it was exposed to the micronutrient. The substratum to which the epithelial layer adheres was also found to regulate the response of the cell layer to the micronutrient. The implications of these findings for therapeutically decreasing morbidity in Inflammatory Bowel Disease are discussed. PMID:26226276

  11. Influence of dietary ingredients on in vitro inflammatory response of intestinal porcine epithelial cells challenged by an enterotoxigenic Escherichia coli (K88).

    Science.gov (United States)

    Hermes, Rafael G; Manzanilla, Edgar G; Martín-Orúe, Susana M; Pérez, José F; Klasing, Kirk C

    2011-12-01

    Enterotoxigenic Escherichia coli (ETEC) K88 is the main bacterial cause of diarrhea in piglets around weaning and the adhesion of ETEC to the intestinal mucosa is a prerequisite step for its colonization. In this study, the adhesion of a fimbriated ETEC and a non-fimbriated E. coli (NFEC) to the intestinal cells and the activation of the innate immune system were evaluated using a porcine intestinal epithelial cell line (IPEC-J2). The impact of several feedstuffs (wheat bran (WB); casein glycomacropeptide (CGMP); mannan-oligosaccharides (MOS); locust bean extract (LB) and Aspergillus oryzae fermentation extract (AO)) on ETEC attachment and the inflammatory response were also studied. The gene expression of TLR-4; TLR-5; IL-1β; IL-8; IL-10 and TNF-α were quantified using Cyclophilin-A, as a reference gene, and related to a non-challenged treatment. The fimbriated strain was markedly better than the non-fimbriated strain at adherence to intestinal cells and inducing an inflammatory response. All the feedstuffs studied were able to reduce the adhesion of ETEC, with the greatest decrease with CGMP or MOS at highest concentration. Regarding the inflammatory response, the highest dose of WB promoted the lowest relative expression of cytokines and chemokines. All tested feedstuffs were able to reduce the adhesion of ETEC to IPEC-J2 and interfere on the innate inflammatory response; however WB should be further studied according to the beneficial results on the intestinal inflammatory process evidenced in this study. 2011 Elsevier Ltd. All rights reserved.

  12. The epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells

    OpenAIRE

    Hu, Weiwei; Zhu, Liqi; Yang, Xing; Lin, Jian; Yang, Qian

    2016-01-01

    Transmissible gastroenteritis virus (TGEV), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. The porcine intestinal epithelium is the target of TGEV infection, but the mechanisms that TGEV disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. We not only found that TGEV infection stimulates F-actin to gather at the cell membrane but the disruption of F-actin inhibits TGEV entry as well. Cofilin is involved in F-actin reorganization...

  13. Effect of cholera toxin on cAMP levels and Na/sup +/ influx in isolated intestinal epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hyun, C.S.; Kimmich, G.A.

    1982-09-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 ..mu..g/ml cholera toxin (CT) at 37/sup 0/C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na/sup +/ influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na/sup +/ entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na/sup +/ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na/sup +/ during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na/sup +/ influx suggest that the reactivation of the Na/sup +/ transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP.

  14. Effect of cholera toxin on cAMP levels and Na+ influx in isolated intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.; Kimmich, G.A.

    1982-01-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 μg/ml cholera toxin (CT) at 37 0 C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na + influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na + entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na + influx suggest that the reactivation of the Na + transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP

  15. Anaerobic conditions promote expression of Sfp fimbriae and adherence of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:NM to human intestinal epithelial cells.

    Science.gov (United States)

    Müsken, Anne; Bielaszewska, Martina; Greune, Lilo; Schweppe, Christian H; Müthing, Johannes; Schmidt, Herbert; Schmidt, M Alexander; Karch, Helge; Zhang, Wenlan

    2008-02-01

    The sfp gene cluster, unique to sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM strains, encodes fimbriae that mediate mannose-resistant hemagglutination in laboratory E. coli strains but are not expressed in wild-type SF EHEC O157:NM strains under standard laboratory conditions. We investigated whether Sfp fimbriae are expressed under conditions that mimic the intestinal environment and whether they contribute to the adherence of SF EHEC O157:NM strains to human intestinal epithelial cells. The transcription of sfpA (encoding the major fimbrial subunit) was upregulated in all strains investigated, and all expressed SfpA and possessed fimbriae that reacted with an anti-SfpA antibody when the strains were grown on solid media under anaerobic conditions. Sfp expression was absent under aerobic conditions and in liquid media. Sfp upregulation under anaerobic conditions was significantly higher on blood agar and a medium simulating the colonic environment than on a medium simulating the ileal environment (P Sfp fimbriae in SF E. coli O157:NM strains correlates with increased adherence to Caco-2 and HCT-8 cells. Our data indicate that the expression of Sfp fimbriae in SF E. coli O157:NM strains is induced under conditions resembling those of the natural site of infection and that Sfp fimbriae may contribute to the adherence of the organisms to human intestinal epithelium.

  16. L-arginine stimulates CAT-1-mediated arginine uptake and regulation of inducible nitric oxide synthase for the growth of chick intestinal epithelial cells.

    Science.gov (United States)

    Yuan, Chao; Zhang, Xiaoyun; He, Qiang; Li, Junming; Lu, Jianjun; Zou, Xiaoting

    2015-01-01

    L-arginine (L-Arg) uptake is mediated by members of cationic amino acid transporter (CAT) family and may coincide with the induction of nitric oxide synthases (NOS). The present study was conducted to investigate the extracellular concentrations of L-Arg regulating the CAT-1, CAT-4 and inducible NOS (iNOS) in chick intestinal epithelial cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's medium containing 10, 100, 200, 400, or 600 μM L-Arg. Cell viability, nitric oxide (NO) concentrations, uptake and metabolism of L-[3H]-Arg as well as expression of CAT-1, CAT-4, and iNOS were determined. Our results showed that L-Arg enhances cell growth with a maximal response at 10-400 μM. Addition of 100, 200, or 400 μM L-Arg increased the L-[3H]-Arg uptake, which was associated with greater conversion of L-[3H]-citrulline and NO production in comparison with 10 μM L-Arg group. Increasing extracellular concentrations of L-Arg from 10 to 400 μM dose dependently increased the levels of CAT-1 mRNA and protein, while no effect on CAT-4 mRNA abundance was found. Furthermore, supplementation of 100, 200, or 400 μM L-Arg upregulated the expression of iNOS mRNA, and the relative protein levels for iNOS in 200 and 400 μM L-Arg groups were higher than those in 10 and 100 μM L-Arg groups. Collectively, we conclude that the CAT-1 isoform plays a role in L-Arg uptake, and L-Arg-mediated elevation of NO via iNOS promotes the growth of chick intestinal epithelial cells.

  17. Prior lactose glycation of caseinate via the Maillard reaction affects in vitro activities of the pepsin-trypsin digest toward intestinal epithelial cells.

    Science.gov (United States)

    Wang, X P; Zhao, X H

    2017-07-01

    The well-known Maillard reaction in milk occurs between lactose and milk proteins during thermal treatment, and its effects on milk nutrition and safety have been well studied. A lactose-glycated caseinate was prepared via this reaction and digested using 2 digestive proteases, pepsin and trypsin. The glycated caseinate digest was assessed for its in vitro activities on rat intestinal epithelial cells in terms of growth proliferation, anti-apoptotic effect, and differentiation induction using caseinate digest as reference, to verify potential effects of the Maillard reaction on these activities of caseinate digest to the cells. Two digests had proliferative and anti-apoptotic effects, and reached the highest effects at 0.02 g/L of digest concentration with treatment time of 24 h. In comparison with caseinate digest, glycated caseinate digest always showed weaker proliferative (5.3-14.2%) and anti-apoptotic (5.9-39.0%) effects, and was more toxic to the cells at 0.5 g/L of digest concentration with treatment time of 48 h. However, glycated caseinate digest at 2 incubation times of 4 to 7 d showed differentiation induction higher than caseinate digest, as it could confer the cells with increased activities in lactase (16.3-26.6%), sucrase (22.4-31.2%), and alkaline phosphatase (17.4-24.8%). Transmission electron microscopy observation results also confirmed higher differentiation induction of glycated caseinate digest. Amino acid loss and lactose glycation partially contributed to these decreased and enhanced activities of glycated caseinate digest, respectively. The Maillard reaction of caseinate and lactose is thus shown in this study to have effects on the activities of caseinate digest to intestinal epithelial cells. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  18. Differential effect of immune cells on non-pathogenic Gram-negative bacteria-induced nuclear factor-kappaB activation and pro-inflammatory gene expression in intestinal epithelial cells

    DEFF Research Database (Denmark)

    Haller, D.; Holt, L.; Parlesak, Alexandr

    2004-01-01

    We have previously shown that non-pathogenic Gram negative bacteria induce RelA phosphorylation, nuclear factor (NF)-kappaB transcriptional activity and pro-inflammatory gene expression in intestinal epithelial cells (IEC) in vivo and in vitro. In this study, we investigated the molecular mechanism......, PBMC from patients with active ulcerative colitis and Crohn's disease differentially trigger epithelial cell activation in response to E. coli and E. coli-derived LPS. In conclusion, this study provides evidence for a differential regulation of non-pathogenic Gram-negative bacteria-induced NF......-kappaB signalling and IL-8 gene expression in IEC cocultured with immune cells and suggests the presence of mechanisms that assure hyporesponsiveness of the intestinal epithelium to certain commensally enteric bacteria....

  19. Aminopeptidase-N-independent entry of porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells.

    Science.gov (United States)

    Ji, Chun-Miao; Wang, Bin; Zhou, Jiyong; Huang, Yao-Wei

    2018-04-01

    A monkey cell line Vero (ATCC CCL-81) is commonly used for porcine epidemic diarrhea virus (PEDV) propagation in vitro. However, it is still controversial whether the porcine aminopeptidase N (pAPN) counterpart on Vero cells (Vero-APN) confers PEDV entry. We found that endogenous expression of Vero-APN was undetectable in the mRNA and the protein levels in Vero cells. We cloned the partial Vero-APN gene (3340-bp) containing exons 1 to 9 from cellular DNA and subsequently generated two APN-knockout Vero cell lines by CRISPR/Cas9 approach. PEDV infection of two APN-knockout Vero cells had the same efficiency as the Vero cells with or without neuraminidase treatment. A Vero cells stably expressing pAPN did not increase PEDV production. SiRNA-knockdown of pAPN in porcine jejunum epithelial cells had no effects on PEDV infection. The results suggest that there exists an additional cellular receptor on Vero or porcine jejunal cells independent of APN for PEDV entry. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Expression ofIfnlr1on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

    Science.gov (United States)

    Baldridge, Megan T; Lee, Sanghyun; Brown, Judy J; McAllister, Nicole; Urbanek, Kelly; Dermody, Terence S; Nice, Timothy J; Virgin, Herbert W

    2017-04-01

    Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1 -deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1 -null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity. IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified

  1. Bacterial viability differentially influences the immunomodulatory capabilities of potential host-derived probiotics in the intestinal epithelial cells of Atlantic cod Gadus morhua.

    Science.gov (United States)

    Lazado, C C; Caipang, C M A

    2014-04-01

    This study explored the effect of heat inactivation on the immunomodulatory capabilities of two potential host-derived probiotics (GP21 and GP12) on the intestinal epithelial cells (IEPC) derived from Atlantic cod. The cells were isolated from the four segments of the gut, namely anterior intestine (AI), mid-intestine (MI), posterior intestine (PI) and rectum (RC). The IEPC cultures were exposed to live or heat-inactivated form of GP21 and GP12 for 24 h. The expression profiles of bacterial defence genes and cytokine genes in the probiotics-exposed IEPCs showed differential patterns. Heat inactivation did not drastically affect the immunomodulatory properties of the probiotics, and this was explicitly typified by the stimulated expression of g-type lysozyme, hepcidin, transferrin and metallothionein in both forms of the bacteria. There was no distinct expression pattern of the interleukin genes during bacterial exposure. This was in contrast to the chemokines where the expression of these genes in IEPCs was down-regulated upon exposure to the heat-inactivated probiotics. Although heat inactivation did not drastically affect the immunomodulatory capabilities of the probiotics, the live form elicited higher immune responses in the IEPCs in most cases. This study showed that bacterial viability was a contributing influence, but not a major limiting factor on the immune-related functions of the host-derived probiotics in vitro. GP21 and GP12 are beneficial host-derived bacteria and could be utilized as candidate probiotics in cod aquaculture. © 2013 The Society for Applied Microbiology.

  2. Glycomacropeptide Reduces Intestinal Epithelial Cell Barrier Dysfunction and Adhesion of Entero-Hemorrhagic and Entero-Pathogenic Escherichia coli in Vitro

    Directory of Open Access Journals (Sweden)

    Shane Feeney

    2017-10-01

    Full Text Available In recent years, the potential of glycosylated food components to positively influence health has received considerable attention. Milk is a rich source of biologically active glycoconjugates which are associated with antimicrobial, immunomodulatory, anti-adhesion, anti-inflammatory and prebiotic properties. Glycomacropeptide (GMP is the C-terminal portion of kappa-casein that is released from whey during cheese-making by the action of chymosin. Many of the biological properties associated with GMP, such as anti-adhesion, have been linked with the carbohydrate portion of the protein. In this study, we investigated the ability of GMP to inhibit the adhesion of a variety of pathogenic Escherichia coli strains to HT-29 and Caco-2 intestinal cell lines, given the importance of E. coli in causing bacterial gastroenteritis. GMP significantly reduced pathogen adhesion, albeit with a high degree of species specificity toward enteropathogenic E. coli (EPEC strains O125:H32 and O111:H2 and enterohemorrhagic E. coli (EHEC strain 12900 O157:H7. The anti-adhesive effect resulted from the interaction of GMP with the E. coli cells and was also dependent on GMP concentration. Pre-incubation of intestinal Caco-2 cells with GMP reduced pathogen translocation as represented by a decrease in transepithelial electrical resistance (TEER. Thus, GMP is an effective in-vitro inhibitor of adhesion and epithelial injury caused by E. coli and may have potential as a biofunctional ingredient in foods to improve gastrointestinal health.

  3. Reovirus intermediate subviral particles constitute a strategy to infect intestinal epithelial cells by exploiting TGF-β dependent pro-survival signaling.

    Science.gov (United States)

    Stanifer, Megan L; Rippert, Anja; Kazakov, Alexander; Willemsen, Joschka; Bucher, Delia; Bender, Silke; Bartenschlager, Ralf; Binder, Marco; Boulant, Steeve

    2016-12-01

    Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF-β acting as a pro-survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular-death pathways. © 2016 John Wiley & Sons Ltd.

  4. Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin Isomers and their Antioxidative Effects in Human Intestinal Epithelial Caco-2 Cells.

    Science.gov (United States)

    Yang, Cheng; Zhang, Hua; Liu, Ronghua; Zhu, Honghui; Zhang, Lianfu; Tsao, Rong

    2017-11-29

    The bioaccessibility, bioavailability, and antioxidative activities of three astaxanthin geometric isomers were investigated using an in vitro digestion model and human intestinal Caco-2 cells. This study demonstrated that the trans-cis isomerization of all-E-astaxanthin and the cis-trans isomerization of Z-astaxanthins could happen both during in vitro gastrointestinal digestion and cellular uptake processes. 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-E-astaxanthins during in vitro digestion, and 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins. These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than all-E-astaxanthin in reported studies. All three astaxanthin isomers were effective in maintaining cellular redox homeostasis as seen in the antioxidant enzyme (CAT, SOD) activities ; 9Z- and 13Z- astaxanthins exhibited a higher protective effect than all-E-astaxanthin against oxidative stress as demonstrated by the lower cellular uptake of Z-astaxanthins and lower secretion and gene expression of the pro-inflammatory cytokine IL-8 in Caco-2 cells treated with H 2 O 2 . We conclude, for the first time, that Z-astaxanthin isomers may play a more important role in preventing oxidative stress induced intestinal diseases.

  5. Isolation and gene expression profiling of intestinal epithelial cells: crypt isolation by calcium chelation from in vivo samples.

    LENUS (Irish Health Repository)

    Balfe, Aine

    2018-01-01

    The epithelial layer within the colon represents a physical barrier between the luminal contents and its underlying mucosa. It plays a pivotal role in mucosal homeostasis, and both tolerance and anti-pathogenic immune responses. Identifying signals of inflammation initiation and responses to stimuli from within the epithelial layer is critical to understanding the molecular pathways underlying disease pathology. This study validated a method to isolate and analyze epithelial populations, enabling investigations of epithelial function and response in a variety of disease setting.

  6. Active Hexose Correlated Compound (AHCC) promotes an intestinal immune response in BALB/c mice and in primary intestinal epithelial cell culture involving toll-like receptors TLR-2 and TLR-4.

    Science.gov (United States)

    Mallet, Jean-François; Graham, Émilie; Ritz, Barry W; Homma, Kohei; Matar, Chantal

    2016-02-01

    Active Hexose Correlated Compound (AHCC(®)) is a cultured mushroom extract that is commercially available and promoted for immune support. Available data suggest that AHCC supplementation affects immune cell populations and immune outcomes, including natural killer cell response to infection. The mechanism by which AHCC exerts its effects is not well understood. The present work aimed to characterize the immunomodulatory activity of AHCC in the gut and to study the effects of AHCC on toll-like receptor (TLR) signaling in intestinal epithelial cells (IECs). BALB/c mice were fed AHCC by gavage. In vivo activities were assessed by immunohistochemistry and cytokine production. The effects of AHCC on ex vivo primary cell culture from IECs were examined after challenge with LPS or E. coli alone or in the presence of anti-TLR-2 and TLR-4 blocking antibodies. Feeding AHCC resulted in increased IgA+ cells in the intestine and increased sIgA, IL-10, and IFN-γ in the intestinal fluid. In IECs, contact with AHCC increased IL-6 production but not to the pro-inflammatory level of positive controls, LPS and E. coli. Blocking TLR-2 and TLR-4 reduced the induction of IL-6 by AHCC, suggesting that these innate receptors are involved in generating the immune response of IECs to AHCC. AHCC may play a role in the orchestration of immune response and the maintenance of immune homeostasis in part by priming the TLR-2 and TLR-4 gate at the intestinal epithelium. Such a response is likely due to the recognition of non-pathogenic food-associated molecular patterns (FAMPs) such as those found associated with other mushroom or yeast-derived compounds.

  7. Anti-inflammatory properties of an extract of M. ilicifolia in the human intestinal epithelial Caco-2 cell line.

    Science.gov (United States)

    Wonfor, Ruth; Natoli, Manuela; Parveen, Ifat; Beckman, Manfred; Nash, Robert; Nash, Deborah

    2017-09-14

    Maytenus ilicifolia is a Celastracea plant used in traditional medicine to alleviate digestive tract inflammatory disorders. We investigated anti-inflammatory properties of M. ilicifolia crude extract towards Caco-2 cell line, as a model of Toll-like Receptor 2 (TLR-2) inflammatory pathway. Toxicity was assessed following culture of Caco-2 with M. ilicifolia, using apparent cell permeability and trans-epithelial electric resistance. Anti-inflammatory properties of M. ilicifolia were assessed through IL-8 secretion and TLR-2 associated gene expression of Caco-2 cells with or without an LTA challenge. M. ilicifolia was not toxic to Caco-2 cells. M. ilicifolia down-regulated TLR2 expression with and without LTA challenge but had no effect on other genes. Following LTA challenge of Caco-2 cells, 100 and 200µg/mL M. ilicifolia abrogated IL-8 secretion. We provide preliminary data for some M. ilicifolia anti-inflammatory properties. Further research must establish the full extent and mode of action on particular inflammatory pathways. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  8. Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.

    Science.gov (United States)

    Mills, Dominic C; Gundogdu, Ozan; Elmi, Abdi; Bajaj-Elliott, Mona; Taylor, Peter W; Wren, Brendan W; Dorrell, Nick

    2012-05-01

    Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

  9. Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hans Linde Nielsen

    Full Text Available Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05, by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001, suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6 cm/s in control, P<0.05 but showed no difference in permeability for 4 kDa FITC-dextran (FD-4. The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and

  10. Mast Cell Tryptase Reduces Junctional Adhesion Molecule-A (JAM-A) Expression in Intestinal Epithelial Cells: Implications for the Mechanisms of Barrier Dysfunction in Irritable Bowel Syndrome.

    LENUS (Irish Health Repository)

    Wilcz-Villega, Ewa M

    2013-07-01

    The objective of this study was to investigate how mast cell tryptase may influence intestinal permeability and tight junction (TJ) proteins in vitro and explore translation to irritable bowel syndrome (IBS).

  11. Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

    DEFF Research Database (Denmark)

    López-Posadas, Rocío; Becker, Christoph; Günther, Claudia

    2016-01-01

    Although defects in intestinal barrier function are a key pathogenic factor in patients with inflammatory bowel diseases (IBDs), the molecular pathways driving disease-specific alterations of intestinal epithelial cells (IECs) are largely unknown. Here, we addressed this issue by characterizing t...

  12. Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures

    Directory of Open Access Journals (Sweden)

    Mainil Jacques

    2005-12-01

    Full Text Available Abstract Background Cultures of enterocytes and colonocytes represent valuable tools to study growth and differentiation of epithelial cells. In vitro models may be used to evaluate passage or toxicity of drugs, interactions of enteropathogenes bacteria strains with intestinal epithelium and other physiologic or pathologic phenomenon involving the digestive tract. Results Cultures of bovine colonocytes and jejunocytes were obtained from organoid-enriched preparations, using a combination of enzymatic and mechanical disruption of the intestine epithelium, followed by an isopicnic centrifugation discarding most single cells. Confluent cell monolayers arising from plated organoids exhibited epithelium typical features, such as the pavement-like structure, the presence of apical microvilli and tight junctions. Accordingly, cells expressed several markers of enterocyte brush border (i.e. maltase, alkaline phosphatase and fatty acid binding protein as well as an epithelial cytoskeleton component (cytokeratin 18. However, enterocyte primocultures were also positive for the vimentin immunostaining (mesenchyme marker. Vimentin expression studies showed that this gene is constitutively expressed in bovine enterocytes. Comparison of the vimentin expression profile with the pattern of brush border enzymes activities, suggested that the decrease of cell differentiation level observed during the enterocyte isolation procedure and early passages of the primoculture could result from a post-transcriptional de-repression of vimentin synthesis. The low differentiation level of bovine enterocytes in vitro could partly be counteracted adding butyrate (1–2 mM or using a glucose-deprived culture medium. Conclusion The present study describes several complementary approaches to characterize bovine primary cultures of intestinal cells. Cultured cells kept their morphologic and functional characteristics during several generations.

  13. In Vitro Evaluation of Swine-Derived Lactobacillus reuteri: Probiotic Properties and Effects on Intestinal Porcine Epithelial Cells Challenged with Enterotoxigenic Escherichia coli K88.

    Science.gov (United States)

    Wang, Zhilin; Wang, Li; Chen, Zhuang; Ma, Xianyong; Yang, Xuefen; Zhang, Jian; Jiang, Zongyong

    2016-06-28

    Probiotics are considered as the best effective alternatives to antibiotics. The aim of this study was to characterize the probiotic potential of lactobacilli for use in swine farming by using in vitro evaluation methods. A total of 106 lactic acid bacterial isolates, originating from porcine feces, were first screened for the capacity to survive stresses considered important for putative probiotic strains. Sixteen isolates showed notable acid and bile resistance, antibacterial activity, and adherence to intestinal porcine epithelial cells (IPEC-1). One isolate, LR1, identified as Lactobacillus reuteri, was selected for extensive study of its probiotic and functional properties in IPEC-1 cell models. L. reuteri LR1 exhibited good adhesion to IPEC-1 cells and could inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells. L. reuteri LR1 could also modulate transcript and protein expression of cytokines involved in inflammation in IPEC-1 cells; the Lactobacillus strain inhibited the ETEC-induced expression of proinflammatory transcripts (IL-6 and TNF-α) and protein (IL-6), and increased the level of anti-inflammatory cytokine (IL-10). Measurement of the permeation of FD-4 showed that L. reuteri LR1 could maintain barrier integrity in monolayer IPEC-1 cells exposed to ETEC. Immunolocalization experiments showed L. reuteri LR1 could also prevent ETEC-induced tight junction ZO-1 disruption. Together, these results indicate that L. reuteri LR1 exhibits desirable probiotic properties and could be a potential probiotic for use in swine production.

  14. Characterizing microbiota-independent effects of oligosaccharides on intestinal epithelial cells: insight into the role of structure and size : Structure-activity relationships of non-digestible oligosaccharides.

    Science.gov (United States)

    Akbari, Peyman; Fink-Gremmels, Johanna; Willems, Rianne H A M; Difilippo, Elisabetta; Schols, Henk A; Schoterman, Margriet H C; Garssen, Johan; Braber, Saskia

    2017-08-01

    The direct effects of galacto-oligosaccharides (GOS), including Vivinal ® GOS syrup (VGOS) and purified Vivinal ® GOS (PGOS), on the epithelial integrity and corresponding interleukin-8 (IL-8/CXCL8) release were examined in a Caco-2 cell model for intestinal barrier dysfunction. To investigate structure-activity relationships, the effects of individual DP fractions of VGOS were evaluated. Moreover, the obtained results with GOS were compared with Caco-2 monolayers incubated with fructo-oligosaccharides (FOS) and inulin. Caco-2 monolayers were pretreated (24 h) with or without specific oligosaccharides or DP fractions of VGOS (DP2 to DP6) before being exposed for 12 or 24 h to the fungal toxin deoxynivalenol (DON). Transepithelial electrical resistance and lucifer yellow permeability were measured to investigate barrier integrity. A calcium switch assay was used to study the reassembly of tight junction proteins. Release of CXCL8, a typical marker for inflammation, was quantified by ELISA. In comparison with PGOS, FOS and inulin, VGOS showed the most pronounced protective effect on the DON-induced impairment of the monolayer integrity, acceleration of the tight junction reassembly and the subsequent CXCL8 release. DP2 and DP3 in concentrations occurring in VGOS prevented the DON-induced epithelial barrier disruption, which could be related to their high prevalence in VGOS. However, no effects of the separate DP GOS fractions were observed on CXCL8 release. This comparative study demonstrates the direct, microbiota-independent effects of oligosaccharides on the intestinal barrier function and shows the differences between individual galacto- and fructo-oligosaccharides. This microbiota-independent effect of oligosaccharides depends on the oligosaccharide structure, DP length and concentration.

  15. Campylobacter jejuni induces an anti-inflammatory response in human intestinal epithelial cells through activation of phosphatidylinositol 3-kinase/Akt pathway

    DEFF Research Database (Denmark)

    Li, Yiping; Vegge, Christina S.; Brøndsted, Lone

    2011-01-01

    to activate phosphatidylinositol 3-kinase (PI3K)/Akt pathway and induce pro-inflammatory interleukin-8(IL-8) as well as anti-inflammatory cytokine IL-10 in human intestinal epithelial cell line Colo 205. The signalling pathways PI3K/Akt and mitogen-activated protein (MAP)kinases ERK and p38 were involved in C....... jejuni-induced IL-8 and IL-10 expression. Inhibition of PI3K resulted in augmentation of C. jejuni-induced IL-8 production, concomitant with down-regulation of IL-10 mRNA, indicating an anti-inflammatory response was activated and associated with the activation of P13K/Akt. Similar effect was observed...... for cytolethal distending toxin (CDT) deficient mutants. Moreover, we demonstrated that heat-killed bacteria were able to induce IL-8 and IL-10 expression to a lower level than live bacteria. We therefore conclude that C. jejuni activate a PI3K/Akt-dependent anti-inflammatory pathway in human intestinal...

  16. Transmissible gastroenteritis virus nsp7 protein localized in the cytoplasm down-regulates interleukin 8 expression in porcine intestinal epithelial cell.

    Science.gov (United States)

    Zhang, Q; Huang, J L; Liang, Y B; He, Y P; Tong, D W; Xu, X G

    2018-01-01

    Transmissible gastroenteritis virus (TGEV) is an important pathogen in swine that is responsible for substantial economic losses. Previous studies suggest that the TGEV non-structural protein 7 (nsp7) plays an important role in the viral assembly process. However, the subcellular localization and other functions of the TGEV nsp7 protein are still unclear. In this study we have examined the subcellular localization and other functions of TGEV nsp7 protein through analysis of its effects on cell growth, cell cycle progression, interleukin 8 (IL-8) expression, and NF-κB activation. Our results showed that the nsp7 protein is localized in the cytoplasm and has no effect on intestinal epithelial cells (IECs) growth, cell cycle, and cyclin A expression. Further studies showed that TGEV nsp7 protein had no effect on GRP78 expression, could not induce endoplasmic reticulum (ER) stress and activate NF-κB activity. Interestingly, the IECs expressing nsp7 protein secreted lower levels of IL-8 than control cells. This is the first report to demonstrate the subcellular localization and novel functions of TGEV nsp7 protein. These findings provide novel information about the function of the poorly characterized TGEV non-structural protein 7.

  17. Lactobacillus bulgaricus Prevents Intestinal Epithelial Cell Injury Caused by Enterobacter sakazakii-Induced Nitric Oxide both In Vitro and in the Newborn Rat Model of Necrotizing Enterocolitis▿

    Science.gov (United States)

    Hunter, Catherine J.; Williams, Monica; Petrosyan, Mikael; Guner, Yigit; Mittal, Rahul; Mock, Dennis; Upperman, Jeffrey S.; Ford, Henri R.; Prasadarao, Nemani V.

    2009-01-01

    Enterobacter sakazakii is an emerging pathogen that has been associated with outbreaks of necrotizing enterocolitis (NEC) as well as infant sepsis and meningitis. Our previous studies demonstrated that E. sakazakii induces NEC in a newborn rat model by inducing enterocyte apoptosis. However, the mechanisms responsible for enterocyte apoptosis are not known. Here we demonstrate that E. sakazakii induces significant production of nitric oxide (NO) in rat intestinal epithelial cells (IEC-6) upon infection. The elevated production of NO, which is due to increased expression of inducible NO synthase, is responsible for apoptosis of IEC-6 cells. Notably, pretreatment of IEC-6 cells with Lactobacillus bulgaricus (ATCC 12278) attenuated the upregulation of NO production and thereby protected the cells from E. sakazakii-induced apoptosis. Furthermore, pretreatment with L. bulgaricus promoted the integrity of enterocytes both in vitro and in the infant rat model of NEC, even after challenge with E. sakazakii. Infection of IEC-6 cells with E. sakazakii upregulated several genes related to apoptosis, cytokine production, and various signaling pathways, as demonstrated by rat gene array analysis, and this upregulation was subdued by pretreatment with L. bulgaricus. In agreement with these data, L. bulgaricus pretreatment protected newborn rats infected with E. sakazakii from developing NEC, resulting in improved survival. PMID:19075027

  18. Epithelial adhesion molecules and the regulation of intestinal homeostasis during neutrophil transepithelial migration

    Science.gov (United States)

    Sumagin, Ronen; Parkos, Charles A

    2014-01-01

    Epithelial adhesion molecules play essential roles in regulating cellular function and maintaining mucosal tissue homeostasis. Some form epithelial junctional complexes to provide structural support for epithelial monolayers and act as a selectively permeable barrier separating luminal contents from the surrounding tissue. Others serve as docking structures for invading viruses and bacteria, while also regulating the immune response. They can either obstruct or serve as footholds for the immune cells recruited to mucosal surfaces. Currently, it is well appreciated that adhesion molecules collectively serve as environmental cue sensors and trigger signaling events to regulate epithelial function through their association with the cell cytoskeleton and various intracellular adapter proteins. Immune cells, particularly neutrophils (PMN) during transepithelial migration (TEM), can modulate adhesion molecule expression, conformation, and distribution, significantly impacting epithelial function and tissue homeostasis. This review discusses the roles of key intestinal epithelial adhesion molecules in regulating PMN trafficking and outlines the potential consequences on epithelial function. PMID:25838976

  19. Regulators of Intestinal Epithelial Migration in Sepsis.

    Science.gov (United States)

    Meng, Mei; Klingensmith, Nathan J; Liang, Zhe; Lyons, John D; Fay, Katherine T; Chen, Ching-Wen; Ford, Mandy L; Coopersmith, Craig M

    2018-02-08

    The gut is a continuously renewing organ, with cell proliferation, migration and death occurring rapidly under basal conditions. Since the impact of critical illness on cell movement from crypt base to villus tip is poorly understood, the purpose of this study was to determine how sepsis alters enterocyte migration. Wild type, transgenic and knockout mice were injected with 5-bromo-2'deoxyuridine (BrdU) to label cells in S phase before and after the onset of cecal ligation and puncture and were sacrificed at pre-determined endpoints to determine distance proliferating cells migrated up the crypt-villus unit. Enterocyte migration rate was decreased from 24-96 hours following sepsis. BrdU was not detectable on villi 6 days after sham laparotomy, meaning all cells had migrated the length of the gut and been exfoliated into its lumen. However, BrdU positive cells were detectable on villi 10 days after sepsis. Multiple components of gut integrity altered enterocyte migration. Sepsis decreased crypt proliferation, which further slowed enterocyte transit as mice injected with BrdU after the onset of sepsis (decreased proliferation) had slower migration than mice injected with BrdU prior to the onset of sepsis (normal proliferation). Decreasing intestinal apoptosis via gut-specific overexpression of Bcl-2 prevented sepsis-induced slowing of enterocyte migration. In contrast, worsened intestinal hyperpermeability by genetic deletion of JAM-A increased enterocyte migration. Sepsis therefore significantly slows enterocyte migration, and intestinal proliferation, apoptosis and permeability all affect migration time, which can potentially be targeted both genetically and pharmacologically.

  20. The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

    International Nuclear Information System (INIS)

    Kim, Han-Soo; Kim, Ju Won; Gang, Jingu; Wen, Jing; Koh, Sang Seok; Koh, Jong Sung; Chung, Hyun-Ho; Song, Si Young

    2006-01-01

    LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21 CIP1/WAF1 , RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21 CIP1/WAF1 and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21 CIP1/WAF1 and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells

  1. In vitro evaluation of Lactobacillus crispatus K313 and K243: high-adhesion activity and anti-inflammatory effect on Salmonella braenderup infected intestinal epithelial cell.

    Science.gov (United States)

    Sun, Zhilan; Huang, Lihua; Kong, Jian; Hu, Shumin; Zhang, Xiaowei; Kong, Wentao

    2012-09-14

    Currently, there is an increasing interest in the use of probiotics as an alternative strategy to antimicrobial compounds. In this study, two high adhesive strains Lactobacillus crispatus K313 adhering to HT-29 cells as well as Lb. crispatus K243 adhering to collagen type IV were isolated from chicken intestines. SDS-PAGE analysis revealed the presence of the potential S-proteins SlpA and SlpB in Lb. crispatus K243 and K313. SlpA and SlpB, rich in hydrophobic amino acids, were proved to be involved in adhering to collagen type IV and HT-29 cells, respectively, based on the LiCl treatment assay. After removal of S-proteins, the viability and tolerance of the two Lb. crispatus strains to simulated gastric and small intestinal juice were reduced, indicating the protective role of S-proteins against the hostile environments. Lb. crispatus K313 exhibited the stronger autoaggregation ability and inhibitive activity against Salmonella braenderup H9812 adhesion to HT-29 cells than the strain K243. To elucidate the inhibitive mechanism, cultured epithelial cells were exposed with Lb. crispatus strains, and followed by a challenge with S. braenderup H9812. The pro-inflammatory signaling factors (IL-8, CXCL1 and CCL20) from HT-29 were detected by real-time PCR technology. The results showed that both of Lb. crispatus strains down-regulated the transcription level of those pro-inflammatory genes induced by S. braenderup H9812 by 36.2-58.8%. ELISA analysis was further confirmed that Lb. crispatus K243 and K313 inhibited the IL-8 secretion triggered by S. braenderup H9812 by 32.8% and 47.0%, indicating that the two isolates could attenuate the pro-inflammatory signaling induced by S. braenderup H9812, and have the potential application in clinical practice to prevent diarrhea. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. The epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells.

    Science.gov (United States)

    Hu, Weiwei; Zhu, Liqi; Yang, Xing; Lin, Jian; Yang, Qian

    2016-03-15

    Transmissible gastroenteritis virus (TGEV), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. The porcine intestinal epithelium is the target of TGEV infection, but the mechanisms that TGEV disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. We not only found that TGEV infection stimulates F-actin to gather at the cell membrane but the disruption of F-actin inhibits TGEV entry as well. Cofilin is involved in F-actin reorganization and TGEV entry. The TGEV spike protein is capable of binding with EGFR, activating the downstream phosphoinositide-3 kinase (PI3K), then causing the phosphorylation of cofilin and F-actin polymerization via Rac1/Cdc42 GTPases. Inhibition of EGFR and PI3K decreases the entry of TGEV. EGFR is also the upstream activator of mitogen-activated protein kinase (MAPK) signaling pathways that is involved in F-actin reorganization. Additionally, lipid rafts act as signal platforms for the EGFR-associated signaling cascade and correlate with the adhesion of TGEV. In conlusion, these results provide valuable data of the mechanisms which are responsible for the TGEV pathogenesis and may lead to the development of new methods about controlling TGEV.

  3. Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium.

    Science.gov (United States)

    Arce, C; Ramírez-Boo, M; Lucena, C; Garrido, J J

    2010-03-01

    The innate immune system has the basic function of identifying and eradicating microbial invaders and alerting the adaptative immune system to their presence. In this study, the porcine intestinal innate immune response was evaluated by analysing the expression of TLRs, cytokines and chemokines in two porcine epithelial cell lines from different regions: IPEC-J2 (jejunum) and IPI-2I (ileum). Both cells lines were stimulated with 1microg of LPS from Salmonella typhimurium. RNA was collected at 30min, 1, 2, 3 and 4h after treatment. Expression of TLR-1, -2, -3, -4, -6, -8, -9, -10, TNF-alpha, IL-1beta, -8 and MCP-1 was quantified relative to the quantity of Cyclophilin-A mRNA using real-time quantitative PCR (RTQ-PCR). The results obtained show up differences in the gene expression between both cell lines IPEC-J2 and IPI-2I as response to LPS from S. typhimurium during the activation time, which may suggest an in vivo variability in the innate immune response against pathogens in different regions of the host's gut. 2008 Elsevier Ltd. All rights reserved.

  4. Polyphosphate kinases modulate Campylobacter jejuni outer membrane constituents and alter its capacity to invade and survive in intestinal epithelial cells in vitro

    Science.gov (United States)

    Pina-Mimbela, Ruby; Madrid, Jesús Arcos; Kumar, Anand; Torrelles, Jordi B; Rajashekara, Gireesh

    2015-01-01

    Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection. PMID:26714783

  5. Polyphosphate kinases modulate Campylobacter jejuni outer membrane constituents and alter its capacity to invade and survive in intestinal epithelial cells in vitro.

    Science.gov (United States)

    Pina-Mimbela, Ruby; Madrid, Jesús Arcos; Kumar, Anand; Torrelles, Jordi B; Rajashekara, Gireesh

    2015-12-30

    Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Polyphosphate kinases 1 and 2 (PPK1 and PPK2) regulate several cellular processes, including the biosynthesis of the bacterial cell wall. Despite their importance, whether PPK1 and PPK2 modulate the composition of C. jejuni outer membrane constituents (OMCs) and consequently impact its interaction with host cells remains unknown. Our comparative analysis between C. jejuni wild type, Δppk1, and Δppk2 strains showed qualitative and quantitative differences in the total OMC composition among these strains. Importantly, these OMC variations observed on the C. jejuni polyphosphate kinase mutants are directly related to their capacity to invade, survive, and alter the immune response of intestinal epithelial cells in vitro. Specifically, sub-fractionation of the C. jejuni OMC indicated that OMC proteins are uniquely associated with bacterial invasion, whereas C. jejuni OMC proteins, lipids, and lipoglycans are all associated with C. jejuni intracellular survival. This study provides new insights regarding the function of polyphosphate kinases and their role in C. jejuni infection.

  6. Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1

    International Nuclear Information System (INIS)

    Sugi, Yutaka; Takahashi, Kyoko; Nakano, Kou; Hosono, Akira; Kaminogawa, Shuichi

    2011-01-01

    Highlights: → Transcriptional activation of the Tollitip gene is higher in IECs than in monocytes. → Nt -194/-186 region acts as a cis-element and is recognized by Elf-1. → Elf-1 suppresses Tollip gene transcription in monocytes but not in IECs. → O-GlcNAc modification is necessary for nuclear translocation of Elf-1. → O-GlcNAcylation-dependent nuclear translocation of Elf-1 is impaired in IECs. -- Abstract: Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

  7. Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1

    Energy Technology Data Exchange (ETDEWEB)

    Sugi, Yutaka [College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-0880 (Japan); Takahashi, Kyoko, E-mail: ktaka@brs.nihon-u.ac.jp [College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-0880 (Japan); Nakano, Kou; Hosono, Akira; Kaminogawa, Shuichi [College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-0880 (Japan)

    2011-09-09

    Highlights: {yields} Transcriptional activation of the Tollitip gene is higher in IECs than in monocytes. {yields} Nt -194/-186 region acts as a cis-element and is recognized by Elf-1. {yields} Elf-1 suppresses Tollip gene transcription in monocytes but not in IECs. {yields} O-GlcNAc modification is necessary for nuclear translocation of Elf-1. {yields} O-GlcNAcylation-dependent nuclear translocation of Elf-1 is impaired in IECs. -- Abstract: Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

  8. The impact of dietary fibers on dendritic cell responses in vitro is dependent on the differential effects of the fibers on intestinal epithelial cells

    NARCIS (Netherlands)

    Bermudez-Brito, M.; Sahasrabudhe, N.M.; Rösch, C.; Schols, H.A.; Faas, M.M.; Vos, de P.

    2015-01-01

    Scope In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied. Methods and results The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This

  9. The impact of dietary fibers on dendritic cell responses IN VITRO is dependent on the differential effects of the fibers on intestinal epithelial cells

    NARCIS (Netherlands)

    Bermudez-Brito, Miriam; Sahasrabudhe, Neha M.; Rosch, Christiane; Schols, Henk A.; Faas, Marijke M.; de Vos, Paul

    Scope: In the present study, the direct interaction of commonly consumed fibers with epithelial or dendritic cells (DCs) was studied. Methods and results: The fibers were characterized for their sugar composition and chain length profile. When in direct contact, fibers activate DCs only mildly. This

  10. Intestinal epithelial organoids fuse to form self-organizing tubes in floating collagen gels

    NARCIS (Netherlands)

    Sachs, Norman; Tsukamoto, Yoshiyuki; Kujala, Pekka; Peters, Peter J.; Clevers, Hans

    2017-01-01

    Multiple recent examples highlight how stem cells can self-organize in vitro to establish organoids that closely resemble their in vivo counterparts. Single Lgr5+ mouse intestinal stem cells can be cultured under defined conditions forming ever-expanding epithelial organoids that retain cell

  11. Propofol Does Not Reduce Pyroptosis of Enterocytes and Intestinal Epithelial Injury After Lipopolysaccharide Challenge.

    Science.gov (United States)

    Zhang, Xu-Yu; Chen, Xi; Zhang, Hu-Fei; Guan, Su; Wen, Shi-Hong; Huang, Wen-Qi; Liu, Zi-Meng

    2018-01-01

    To date, mechanisms of sepsis-induced intestinal epithelial injury are not well known. P2X7 receptor (P2X7R) regulates pyroptosis of lymphocytes, and propofol is usually used for sedation in septic patients. We aimed to determine the occurrence of enterocyte pyroptosis mediated by P2X7R and to explore the effects of propofol on pyroptosis and intestinal epithelial injury after lipopolysaccharide (LPS) challenge. A novel regimen of LPS challenge was applied in vitro and in vivo. Inhibitors of P2X7R (A438079) and NLRP3 inflammasome (MCC950), and different doses of propofol were administered. The caspase-1 expression, caspase-3 expression, caspase-11 expression, P2X7R expression and NLRP3 expression, extracellular ATP concentration and YO-PRO-1 uptake, and cytotoxicity and HMGB1 concentration were detected to evaluate enterocyte pyroptosis in cultured cells and intestinal epithelial tissues. Chiu's score, diamine oxidase and villus length were used to evaluate intestinal epithelial injury. Moreover, survival analysis was performed. LPS challenge activated caspase-11 expression and P2X7R expression, enhanced ATP concentration and YO-PRO-1 uptake, and led to increased cytotoxicity and HMGB1 concentration. Subsequently, LPS resulted in intestinal epithelial damage, as evidenced by increased levels of Chiu's score and diamine oxidase, and shorter villus length and high mortality of animals. A438079, but not MCC950, significantly relieved LPS-induced enterocyte pyroptosis and intestinal epithelial injury. Importantly, propofol did not confer the protective effects on enterocyte pyroptosis and intestinal epithelia although it markedly decreased P2X7R expression. LPS attack leads to activation of caspase-11/P2X7R and pyroptosis of enterocytes. Propofol does not reduce LPS-induced pyroptosis and intestinal epithelial injury, although it inhibits P2X7R upregulation.

  12. Neutrophil Interactions with Epithelial Expressed ICAM-1 Enhances Intestinal Mucosal Wound Healing

    Science.gov (United States)

    Sumagin, R; Brazil, JC; Nava, P; Nishio, H; Alam, A; Luissint, AC; Weber, DA; Neish, AS; Nusrat, A; Parkos, CA

    2015-01-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. While epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 plays an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing. PMID:26732677

  13. Neutrophil interactions with epithelial-expressed ICAM-1 enhances intestinal mucosal wound healing.

    Science.gov (United States)

    Sumagin, R; Brazil, J C; Nava, P; Nishio, H; Alam, A; Luissint, A C; Weber, D A; Neish, A S; Nusrat, A; Parkos, C A

    2016-09-01

    A characteristic feature of gastrointestinal tract inflammatory disorders, such as inflammatory bowel disease, is polymorphonuclear neutrophil (PMN) transepithelial migration (TEM) and accumulation in the gut lumen. PMN accumulation within the intestinal mucosa contributes to tissue injury. Although epithelial infiltration by large numbers of PMNs results in mucosal injury, we found that PMN interactions with luminal epithelial membrane receptors may also play a role in wound healing. Intercellular adhesion molecule-1 (ICAM-1) is a PMN ligand that is upregulated on apical surfaces of intestinal epithelial cells under inflammatory conditions. In our study, increased expression of ICAM-1 resulted in enhanced PMN binding to the apical epithelium, which was associated with reduced PMN apoptosis. Following TEM, PMN adhesion to ICAM-1 resulted in activation of Akt and β-catenin signaling, increased epithelial-cell proliferation, and wound healing. Such responses were ICAM-1 dependent as engagement of epithelial ICAM-1 by antibody-mediated cross-linking yielded similar results. Furthermore, using an in-vivo biopsy-based, colonic-mucosal-injury model, we demonstrated epithelial ICAM-1 has an important role in activation of epithelial Akt and β-catenin signaling and wound healing. These findings suggest that post-migrated PMNs within the intestinal lumen can regulate epithelial homeostasis, thereby identifying ICAM-1 as a potential therapeutic target for promoting mucosal wound healing.

  14. Recent Advances in Intestinal Stem Cells.

    Science.gov (United States)

    McCabe, Laura R; Parameswaran, Narayanan

    2017-09-01

    The intestine is a dynamic organ with rapid stem cell division generating epithelial cells that mature and apoptose in 3-5 days. Rapid turnover maintains the epithelial barrier and homeostasis. Current insights on intestinal stem cells (ISCs) and their regulation are discussed here. The Lgr5+ ISCs maintain intestinal homeostasis by dividing asymmetrically, but also divide symmetrically to extinguish or replace ISCs. Following radiation or mucosal injury, reserve BMI1+ ISCs as well as other crypt cells can de-differentiate into Lgr5+ ISCs. ISC niche cells, including Paneth, immune and myofibroblast cells secrete factors that regulate ISC proliferation. Finally, several studies indicate that the microbiome metabolites regulate ISC growth. ISC cells can be plastic and integrate a complexity of environmental/niche cues to trigger or suppress proliferation as needed.

  15. Protective effects of transforming growth factor β2 in intestinal epithelial cells by regulation of proteins associated with stress and endotoxin responses.

    Directory of Open Access Journals (Sweden)

    Duc Ninh Nguyen

    Full Text Available Transforming growth factor (TGF-β2 is an important anti-inflammatory protein in milk and colostrum. TGF-β2 supplementation appears to reduce gut inflammatory diseases in early life, such as necrotizing enterocolitis (NEC in young mice. However, the molecular mechanisms by which TGF-β2 protects immature intestinal epithelial cells (IECs remain to be more clearly elucidated before interventions in infants can be considered. Porcine IECs PsIc1 were treated with TGF-β2 and/or lipopolysaccharide (LPS, and changes in the cellular proteome were subsequently analyzed using two-dimensional gel electrophoresis-MS and LC-MS-based proteomics. TGF-β2 alone induced the differential expression of 13 proteins and the majority of the identified proteins were associated with stress responses, TGF-β and Toll-like receptor 4 signaling cascades. In particular, a series of heat shock proteins had similar differential trends as previously shown in the intestine of NEC-resistant preterm pigs and young mice. Furthermore, LC-MS-based proteomics and Western blot analyses revealed 20 differentially expressed proteins following treatment with TGF-β2 in LPS-challenged IECs. Thirteen of these proteins were associated with stress response pathways, among which five proteins were altered by LPS and restored by TGF-β2, whereas six were differentially expressed only by TGF-β2 in LPS-challenged IECs. Based on previously reported biological functions, these patterns indicate the anti-stress and anti-inflammatory effects of TGF-β2 in IECs. We conclude that TGF-β2 of dietary or endogenous origin may regulate the IEC responses against LPS stimuli, thereby supporting cellular homeostasis and innate immunity in response to bacterial colonization, and the first enteral feeding in early life.

  16. Coronaviruses in polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J. W.; Bekker, C. P.; Voorhout, W. F.; Horzinek, M. C.; van der Ende, A.; Strous, G. J.; Rottier, P. J.

    1995-01-01

    Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable

  17. Preventive Effect of TU-100 on a Type-2 Model of Colitis in Mice: Possible Involvement of Enhancing Adrenomedullin in Intestinal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Atsushi Kaneko

    2013-01-01

    Full Text Available Purpose. Crohn's disease (CD and ulcerative colitis (UC, the two major forms of inflammatory bowel disease (IBD, have histopathologically and immunologically different characteristics. We previously reported that a traditional Japanese medicine, daikenchuto (TU-100, ameliorated a trinitrobenzenesulfonic acid- (TNBS- induced type-1 model colitis exhibiting histopathological features of CD through adrenomedullin (ADM enhancement. Our current aims were to examine whether TU-100 ameliorates a type-2 model colitis that histologically resembles UC and identify the active ingredients. Methods. TU-100 was administered orally to mice with oxazolone- (OXN- induced type-2 model colitis. The morbidity was evaluated by body weight loss and the macroscopic score of colonic lesions. ADM was quantified using an EIA kit. Results. TU-100 prevented weight loss and colon ulceration. ADM production by intestinal epithelial cells was increased by TU-100 addition. Screening to identify active ingredients showed that [6]-shogaol and hydroxy α-sanshool enhanced ADM production. Conclusions. TU-100 exerted a protective effect in OXN-induced type-2 model colitis, indicating that TU-100 may be a beneficial agent for treatment of UC.

  18. Polarized secretion of IL-18 by IEC-18 intestinal epithelial cells: effect of probiotic Lactobacillus casei/paracasei

    Czech Academy of Sciences Publication Activity Database

    Kolínská, Jiřina; Zákostelecká, Marie; Lisá, Věra; Kozáková, Hana; Dvorak, B.

    2008-01-01

    Roč. 64, č. 4 (2008), s. 338-338 ISSN 1138-7548. [Meeting European Intestinal Transport Group /22./. 07.09.2008-10.09.2008, Pamplona] R&D Projects: GA MŠk(CZ) 1P05ME783 Grant - others:NIH(US) HD-47237 Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * cytokine IL-18 * probiotic Subject RIV: CE - Biochemistry

  19. Epithelial Cell Cultures

    Directory of Open Access Journals (Sweden)

    Imran S. Chaudhry

    2011-01-01

    Full Text Available The biological effects of only a finite number of tobacco toxins have been studied. Here, we describe exposure of cultures of human bronchial epithelial cells to low concentrations of tobacco carcinogens: nickel sulphate, benzo(bfluoranthene, N-nitrosodiethylamine, and 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. After a 24-hour exposure, EGFR was expressed in cell membrane and cytoplasm, BCL-2 was expressed only in the irregular nuclei of large atypical cells, MKI67 was expressed in nuclei with no staining in larger cells, cytoplasmic BIRC5 with stronger nuclear staining was seen in large atypical cells, and nuclear TP53 was strongly expressed in all cells. After only a 24-hour exposure, cells exhibited atypical nuclear and cytoplasmic features. After a 48-hour exposure, EGFR staining was localized to the nucleus, BCL-2 was slightly decreased in intensity, BIRC5 was localized to the cytoplasm, and TP53 staining was increased in small and large cells. BCL2L1 was expressed in both the cytoplasm and nuclei of cells at 24- and 48-hour exposures. We illustrate that short-termexposure of a bronchial epithelial cell line to smoking-equivalent concentrations of tobacco carcinogens alters the expression of key proliferation regulatory genes, EGFR, BCL-2, BCL2L1, BIRC5, TP53, and MKI67, similar to that reported in biopsy specimens of pulmonary epithelium described to be preneoplastic lesions.

  20. Forkhead box transcription factors Foxa1 and Foxa2 are important regulators of Muc2 mucin expression in intestinal epithelial cells

    NARCIS (Netherlands)

    van der Sluis, Maria; Vincent, Audrey; Bouma, Janneke; Korteland-van Male, Anita; van Goudoever, Johannes B.; Renes, Ingrid B.; van Seuningen, Isabelle

    2008-01-01

    The mucin Muc2 is the main component of the intestinal mucus layer and thus plays important roles in intestinal protection. Therefore, it is important to understand its regulation during goblet cell differentiation. Foxa1 and Foxa2 forkhead box transcription factors (TFs) participate in

  1. MEK/ERK pathway activation by insulin receptor isoform alteration is associated with the abnormal proliferation and differentiation of intestinal epithelial cells in diabetic mice.

    Science.gov (United States)

    Ouyang, Hui; Yang, Hong-Sheng; Yu, Tao; Shan, Ti-Dong; Li, Jie-Yao; Huang, Can-Ze; Zhong, Wa; Xia, Zhong-Sheng; Chen, Qi-Kui

    2016-02-01

    In previous studies, we have reported the abnormal proliferation and differentiation of intestinal epithelial cells (IECs) in diabetes mellitus (DM) mice. The insulin receptor (IR) and its downstream mitogen-activated protein kinase kinase (MAPKK also known as MEK)/extracellular-regulated protein kinase (ERK) pathway is a classic pathway associated with cell proliferation and differentiation. The purpose of the present study is to investigate the role of the MEK/ERK pathway in abnormal proliferation and differentiation of IECs in DM mice. DM mouse models were induced by intraperitoneal injection of streptozotocin. The expression levels of the IR and its isoforms in IECs of DM mice and in IEC-6 cells were investigated. To ensure that the downstream pathways were monitored, QPCR and Western blotting were performed to detect the expression levels of MEK1/2, ERK1/2, PI3K, and Akt. Moreover, siRNA for IR-A and U0126, a specific inhibitor of MEK, were used to further investigate the relationship between the IR/MEK/ERK pathway and abnormal proliferation and differentiation of IECs in DM mice. In DM mice, excessive proliferation, disturbed differentiation, and a high ratio of IR-A/IR-B were detected in IECs. The expression levels of MEK1, MEK2, and ERK1/2 and their phosphorylated proteins in DM mice were significantly higher than those in the control group (P < 0.05), which could be offset by using siRNA for IR-A. The abnormal proliferation and differentiation of IECs in DM mice were normalized after the in vivo administration of U0126. The abnormal proliferation and differentiation of IECs in DM mice are associated with high IR-A/IR-B ratio and increased IR/MEK/ERK pathway activity.

  2. EPS-SJ exopolisaccharide produced by the strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 is involved in adhesion to epithelial intestinal cells and decrease on E. coli association to Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Milica eZivkovic

    2016-03-01

    Full Text Available The aim of this study was to determine the role of an exopolysaccharide produced by natural dairy isolate Lactobacillus paracasei subsp. paracasei BGSJ2-8, in the adhesion to intestinal epithelial cells and a decrease in E. coli’s association with Caco-2 cells. Annotation of the BGSJ2-8 genome showed the presence of a gene cluster, epsSJ, which encodes the biosynthesis of the strain-specific exopolysaccharide EPS-SJ, detected as two fractions (P1 and P2 by size exclusion chromatography (SEC coupled with multi-angle laser light scattering (MALLS detection. SEC-MALLS analysis revealed that an EPS-SJ‒ mutant (EPS7, obtained by insertion mutagenesis of the glps_2198 gene encoding primary glycosyltransferase does not produce the P2 fraction of EPS-SJ. Transmission electron microscopy showed that EPS7 mutant has a thinner cell wall compared to the EPS-SJ+ strain BGSJ2-83 (a plasmid free-derivative of BGSJ2-8. Interestingly, strain BGSJ2-83 showed higher adhesion to Caco-2 epithelial intestinal cell line than the EPS7 mutant. Accordingly, BGSJ2-83 effectively reduced E. coli ATCC25922’s association with Caco-2 cells, while EPS7 did not show statistically significant differences. In addition, the effect of EPS-SJ on the proliferation of lymphocytes in gastrointestinal associated lymphoid tissue (GALT was tested and the results showed that the reduction of GALT lymphocyte proliferation was higher by BGSJ2-83 than by the mutant. To the best of our knowledge this is the first report indicating that the presence of EPS (EPS-SJ on the surface of lactobacilli can improve communication between bacteria and intestinal epithelium, implying its possible role in gut colonization.

  3. NOD-Like Receptors in Intestinal Homeostasis and Epithelial Tissue Repair

    Science.gov (United States)

    Parlato, Marianna; Yeretssian, Garabet

    2014-01-01

    The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease. PMID:24886810

  4. Bacterial Signaling at the Intestinal Epithelial Interface in Inflammation and Cancer

    Directory of Open Access Journals (Sweden)

    Olivia I. Coleman

    2018-01-01

    Full Text Available The gastrointestinal (GI tract provides a compartmentalized interface with an enormous repertoire of immune and metabolic activities, where the multicellular structure of the mucosa has acquired mechanisms to sense luminal factors, such as nutrients, microbes, and a variety of host-derived and microbial metabolites. The GI tract is colonized by a complex ecosystem of microorganisms, which have developed a highly coevolved relationship with the host’s cellular and immune system. Intestinal epithelial pattern recognition receptors (PRRs substantially contribute to tissue homeostasis and immune surveillance. The role of bacteria-derived signals in intestinal epithelial homeostasis and repair has been addressed in mouse models deficient in PRRs and signaling adaptors. While critical for host physiology and the fortification of barrier function, the intestinal microbiota poses a considerable health challenge. Accumulating evidence indicates that dysbiosis is associated with the pathogenesis of numerous GI tract diseases, including inflammatory bowel diseases (IBD and colorectal cancer (CRC. Aberrant signal integration at the epithelial cell level contributes to such diseases. An increased understanding of bacterial-specific structure recognition and signaling mechanisms at the intestinal epithelial interface is of great importance in the translation to future treatment strategies. In this review, we summarize the growing understanding of the regulation and function of the intestinal epithelial barrier, and discuss microbial signaling in the dynamic host–microbe mutualism in both health and disease.

  5. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen Lillelund

    2002-01-01

    by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial...... cells by performing reverse transcriptase-polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one - SART3 - was identified as a marker for human colonic crypts....

  6. Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

    Science.gov (United States)

    Ude, Victor C; Brown, David M; Viale, Luca; Kanase, Nilesh; Stone, Vicki; Johnston, Helinor J

    2017-08-23

    Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm 2 Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm 2 for CuO NMs, and 4.25 μg/cm 2 for copper sulphate (CuSO 4 ), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO 4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO 4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO 4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (P app ); a measure of barrier permeability to CuO NMs. For all experiments, CuSO 4 was used as an ionic control. CuO NMs and CuSO 4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO 4

  7. Selective adhesion of intestinal epithelial cells on patterned films with amine functionalities formed by plasma enhanced chemical vapor deposition

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung Seop; Choi, Changrok; Kim, Soo Heon; Choi, Kun oh [Department of Physics, Brain Korea 21 Physics Research Division and Institute of Basic Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of); Kim, Jeong Min [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook University, Yongin 448-701 (Korea, Republic of); Kim, Hong Ja [Department of Internal Medicine, Dankook University College of Medicine, Cheonan 330-715 (Korea, Republic of); Yeo, Sanghak [R and D Center, ELBIO Incorporation, 426-5 Gasan-dong Geumchun-gu, Seoul (Korea, Republic of); Park, Heonyong [Department of Molecular Biology and Institute of Nanosensor and Biotechnology, BK21 Graduate Program for RNA Biology, Dankook University, Yongin 448-701 (Korea, Republic of); Jung, Donggeun, E-mail: djung@skku.ac.kr [Department of Physics, Brain Korea 21 Physics Research Division and Institute of Basic Science, Sungkyunkwan University, Suwon 440-746 (Korea, Republic of)

    2010-11-01

    Control of cell adhesion to surfaces is important to develop analytical tools in the areas of biomedical engineering. To control cell adhesiveness of the surface, we constructed a variety of plasma polymerized hexamethyldisiloxane (PPHMDSO) thin films deposited at the plasma power range of 10-100 W by plasma enhanced chemical vapor deposition (PECVD). The PPHMDSO film that was formed at 10 W was revealed to be resistant to cell adhesion. The resistance to cell adhesion is closely related to physicochemical properties of the film. Atomic force microscopic data show an increase in surface roughness from 0.52 nm to 0.74 nm with increasing plasma power. From Fourier transform infrared (FT-IR) absorption spectroscopy data, it was also determined that the methyl (-CH{sub 3}) peak intensity increases with increasing plasma power, whereas the hydroxyl (-OH) peak decreases. X-ray photoelectron spectroscopy data reveal an increase in C-O bonding with increasing plasma power. These results suggest that C-O bonding and hydroxyl (-OH) and methyl (-CH{sub 3}) functional groups play a critical part in cell adhesion. Furthermore, to enhance a diversity of film surface, we accumulated the patterned plasma polymerized ethylenediamine (PPEDA) thin film on the top of the PPHMDSO thin film. The PPEDA film is established to be strongly cell-adherent. This patterned two-layer film stacking method can be used to form the selectively limited cell-adhesive PPEDA spots over the adhesion-resistant surface.

  8. ER Stress Causes Rapid Loss of Intestinal Epithelial Stemness through Activation of the Unfolded Protein Response

    Directory of Open Access Journals (Sweden)

    Jarom Heijmans

    2013-04-01

    Full Text Available Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER stress and activity of the unfolded protein response (UPR are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.

  9. Hypo-responsiveness of interleukin-8 production in human embryonic epithelial intestine 407 cells independent of NF-κB pathway: New lessons from endotoxin and ribotoxic deoxynivalenol

    International Nuclear Information System (INIS)

    Moon, Yuseok; Yang, Hyun; Park, Seung-Hwan

    2008-01-01

    Mucosal epithelium senses external toxic insults and transmits the danger signals into the epithelial cells in order to activate a broad range of inflammatory responses. However, pre-exposure to the commensal endotoxins can induce inflammatory tolerance and maintain the homeostasis without excessive immune responses. We recently reported that ribotoxin deoxynivalenol (DON) and its derivatives elicited the pro-inflammatory response as the mucosal insults in human epithelial cells. Taking the knowledge into consideration, we tested the hypothesis that endotoxin pre-exposure can attenuate ribotoxin-induced epithelial interleukin-8 (IL-8) production via a tolerance mechanism. Pre-exposure to endotoxin repressed IL-8 release and its gene expression. However, inflammatory tolerance was not mediated by the attenuated NF-κB activation which has been generally recognized as the major mediator of LPS-mediated toll-like receptor (TLR) signaling pathway. Instead, pre-exposure to endotoxin was observed to trigger the delayed induction of peroxisome proliferator-activated receptor gamma (PPAR-γ) which contributed to the diminished IL-8 production in the human epithelial cells. Moreover, endogenous PPAR-γ agonist suppressed toxicant-mediated interleukin-8 production and IL-8 mRNA stability. Taken together, endotoxin induced hypo-production of pro-inflammatory cytokine IL-8 in the human epithelial cells, which was associated with the delayed activation of PPAR-γ expression by pre-existing endotoxin

  10. Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2

    DEFF Research Database (Denmark)

    Boyd, Mette; Coskun, Mehmet; Lilje, Berit

    2014-01-01

    The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated...... genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52...... promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers....

  11. Gut microbial colonization orchestrates TLR2 expression, signaling and epithelial proliferation in the small intestinal mucosa.

    Directory of Open Access Journals (Sweden)

    Nives Hörmann

    Full Text Available The gut microbiota is an environmental factor that determines renewal of the intestinal epithelium and remodeling of the intestinal mucosa. At present, it is not resolved if components of the gut microbiota can augment innate immune sensing in the intestinal epithelium via the up-regulation of Toll-like receptors (TLRs. Here, we report that colonization of germ-free (GF Swiss Webster mice with a complex gut microbiota augments expression of TLR2. The microbiota-dependent up-regulation of components of the TLR2 signaling complex could be reversed by a 7 day broad-spectrum antibiotic treatment. TLR2 downstream signaling via the mitogen-activated protein kinase (ERK1/2 and protein-kinase B (AKT induced by bacterial TLR2 agonists resulted in increased proliferation of the small intestinal epithelial cell line MODE-K. Mice that were colonized from birth with a normal gut microbiota (conventionally-raised; CONV-R showed signs of increased small intestinal renewal and apoptosis compared with GF controls as indicated by elevated mRNA levels of the proliferation markers Ki67 and Cyclin D1, elevated transcripts of the apoptosis marker Caspase-3 and increased numbers of TUNEL-positive cells per intestinal villus structure. In accordance, TLR2-deficient mice showed reduced proliferation and reduced apoptosis. Our findings suggest that a tuned proliferation response of epithelial cells following microbial colonization could aid to protect the host from its microbial colonizers and increase intestinal surface area.

  12. Almond milk fermented with different potentially probiotic bacteria improves iron uptake by intestinal epithelial (Caco-2 cells

    Directory of Open Access Journals (Sweden)

    Neus Bernat

    2015-04-01

    Full Text Available New fermented almond milks were developed, using different potentially probiotic bacteria, in order to meet the current demand for healthy, versatile non-dairy products. An in vitro digestion/Caco-2 cell model was used to evaluate the effect of both non-fermented and fermented almond milks on the mitochondrial enzymatic activities of enterocytes. Moreover, macrophages were challenged with the in-vitro digested samples and the production of pro-inflammatory biomarkers TNF-a and IL-6 was quantified. Enzymatic activities of cell cultures seemed to be stimulated by the exposure to both fermented and non-fermented almond milks. Both biomarkers decreased (p< 0.05 in fermented almond milks with either B. bifidum or B. longum. Results showed that fermented almond products favored the energetic metabolism of enterocytes and had a lower inflammatory response than non-fermented almond milk, suggesting its benefits for the management of allergies/intolerances. Moreover, the fermentation process enhanced the uptake of iron by Caco-2 cells, especially when using L. rhamnosus and either B. bifidum or B. longum as starters, thus improving the product bioactivity. Therefore, new non-dairy fermented products with functional properties were developed, which might be positioned as alternatives to cow-milk products for sensitized groups of population (allergic and/or intolerant to cow milk or anemic population, among others.

  13. Cellular cross talk in the small intestinal mucosa: postnatal lymphocytic immigration elicits a specific epithelial transcriptional response

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Maltesen, Henrik R; Balmer, Sophie

    2008-01-01

    During the early postnatal period lymphocytes migrate into the mouse small intestine. Migrating infiltrative lymphocytes have the potential to affect the epithelial cells via secreted cytokines. Such cross talk can result in the elicitation of an epithelial transcriptional response. Knowledge about...

  14. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  15. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Science.gov (United States)

    Bhargava, Amol; Cotton, James A; Dixon, Brent R; Gedamu, Lashitew; Yates, Robin M; Buret, Andre G

    2015-01-01

    Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates) trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate) trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1), suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK). Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  16. An in vitro study on bacterial growth interactions and intestinal epithelial cell adhesion characteristics of probiotic combinations.

    Science.gov (United States)

    Moussavi, Mahta; Adams, Michelle Catherine

    2010-05-01

    The aims of this study were to examine long-term growth interactions of five probiotic strains (Lactobacillus casei 01, Lactobacillus plantarum HA8, Lactobacillus rhamnosus GG, Lactobacillus reuteri ATCC 55730 and Bifidobacterium lactis Bb12) either alone or in combination with Propionibacterium jensenii 702 in a co-culture system and to determine their adhesion ability to human colon adenocarcinoma cell line Caco-2. Growth patterns of probiotic Lactobacillus strains were not considerably affected by the presence of P. jensenii 702, whereas lactobacilli exerted a strong antagonistic action against P. jensenii 702. In the co-culture of Bif. lactis Bb12 and P. jensenii 702, a significant synergistic influence on growth of both bacteria was observed (P strains were tested in combination, there was evidence of an associated effect on percentage adherence. However, in most cases these differences were not statistically significant (P casei 01 and Lb. rhamnosus GG both decreased significantly in the presence of P. jensenii 702 compared to their adhesion levels when alone (P survival and percentage adhesion of some probiotic strains may be influenced by the presence of other strains and this should be considered when formulating in the probiotic products.

  17. Surfactant Proteins-A and -D Attenuate LPS-Induced Apoptosis in Primary Intestinal Epithelial Cells (IECs).

    Science.gov (United States)

    Zhang, Linlin; Meng, Qinghe; Yepuri, Natesh; Wang, Guirong; Xi, Xiuming; Cooney, Robert N

    2018-01-01

    SP-A/D KO mice with sepsis demonstrate more severe lung, kidney, and gut injury/apoptosis than WT controls. We hypothesize SP-A and SP-D directly regulate lipopolysaccharide (LPS)-induced P38 mitogen-activated protein kinase (MAPK) activation and gut apoptosis during sepsis. Primary IECs were established from SP-A/D KO or C57BL/6 WT mice, stimulated with LPS and harvested at 24 h. IECs from WT mice were treated with SP-A, SP-D, or vehicle for 20 h, then LPS for 24 h. Apoptosis, cleaved caspase-3 levels and the ratio of BAX/Bcl-2 were assayed. The role of P38 MAPK was examined using the P38 MAPK-agonist U46619 and inhibitor SB203580 in LPS-treated cells. p-P38 MAPK/t-P38 MAPK, TLR4, and CD14 were measured by Western Blot. LPS-induced apoptosis, caspase-3 levels, BAX/Bcl-2, and p-P38/t-P38 MAPK were increased in SP-A/D KO IECs. SP-A and SP-D attenuate LPS-induced increase in apoptosis, cleaved caspase-3, BAX/Bcl-2, and p-P38/t-P38 MAPK in WT IECs. U46619 increased apoptosis, caspase-3, and BAX/Bcl-2 in IECs which was attenuated by SP-A/D. SB203580 attenuates the LPS-induced increase in apoptosis, caspase-3, and BAX/Bcl-2 in WT IECs. Addition of SP-A or SP-D to SB203580 completely ameliorates LPS-induced apoptosis. The LPS-induced increase in TLR4 and CD14 expression is greater in IECs from SP-A/D KO mice and treatment of WT IECs with SP-A or SP-D prevents the LPS-induced increase in TLR4 and CD14. SP-A and SP-D attenuate LPS-induced increases in apoptosis, caspase-3, and BAX/Bcl-2 in IECs. Attenuation of LPS-induced activation of TLR4 and P38 MAPK signaling pathways represents potential mechanisms for the protective effects of SP-A/D on apoptosis.

  18. The role of nitric oxide in intestinal epithelial injury and restitution in neonatal necrotizing enterocolitis.

    Science.gov (United States)

    Chokshi, Nikunj K; Guner, Yigit S; Hunter, Catherine J; Upperman, Jeffrey S; Grishin, Anatoly; Ford, Henri R

    2008-04-01

    Necrotizing enterocolitis (NEC) is the most common life-threatening gastrointestinal disease encountered in the premature infant. Although the inciting events leading to NEC remain elusive, various risk factors, including prematurity, hypoxemia, formula feeding, and intestinal ischemia, have been implicated in the pathogenesis of NEC. Data from our laboratory and others suggest that NEC evolves from disruption of the intestinal epithelial barrier, as a result of a combination of local and systemic insults. We postulate that nitric oxide (NO), an important second messenger and inflammatory mediator, plays a key role in intestinal barrier failure seen in NEC. Nitric oxide and its reactive nitrogen derivative, peroxynitrite, may affect gut barrier permeability by inducing enterocyte apoptosis (programmed cell death) and necrosis, or by altering tight junctions or gap junctions that normally play a key role in maintaining epithelial monolayer integrity. Intrinsic mechanisms that serve to restore monolayer integrity following epithelial injury include enterocyte proliferation, epithelial restitution via enterocyte migration, and re-establishment of cell contacts. This review focuses on the biology of NO and the mechanisms by which it promotes epithelial injury while concurrently disrupting the intrinsic repair mechanisms.

  19. Epithelial cell polarity, stem cells and cancer

    DEFF Research Database (Denmark)

    Martin-Belmonte, Fernando; Perez-Moreno, Mirna

    2011-01-01

    After years of extensive scientific discovery much has been learned about the networks that regulate epithelial homeostasis. Loss of expression or functional activity of cell adhesion and cell polarity proteins (including the PAR, crumbs (CRB) and scribble (SCRIB) complexes) is intricately related......, deregulation of adhesion and polarity proteins can cause misoriented cell divisions and increased self-renewal of adult epithelial stem cells. In this Review, we highlight some advances in the understanding of how loss of epithelial cell polarity contributes to tumorigenesis....

  20. Outer Membrane Vesicles from the Probiotic Escherichia coli Nissle 1917 and the Commensal ECOR12 Enter Intestinal Epithelial Cells via Clathrin-Dependent Endocytosis and Elicit Differential Effects on DNA Damage.

    Directory of Open Access Journals (Sweden)

    María-Alexandra Cañas

    Full Text Available Interactions between intestinal microbiota and the human host are complex. The gut mucosal surface is covered by a mucin layer that prevents bacteria from accessing the epithelial cells. Thus, the crosstalk between microbiota and the host mainly rely on secreted factors that can go through the mucus layer and reach the epithelium. In this context, vesicles released by commensal strains are seen as key players in signaling processes in the intestinal mucosa. Studies with Gram-negative pathogens showed that outer membrane vesicles (OMVs are internalized into the host cell by endocytosis, but the entry mechanism for microbiota-derived vesicles is unknown. Escherichia coli strains are found as part of normal human gut microbiota. In this work, we elucidate the pathway that mediate internalization of OMVs from the probiotic E.coli Nissle 1917 (EcN and the commensal ECOR12 strains in several human intestinal epithelial cell lines. Time course measurement of fluorescence and microscopy analysis performed with rhodamine B-R18-labeled OMVs in the presence of endocytosis inhibitors showed that OMVs from these strains enter epithelial cells via clathrin-mediated endocytosis. Vesicles use the same endocytosis pathway in polarized epithelial monolayers. Internalized OMVs are sorted to lysosomal compartments as shown by their colocalization with clathrin and specific markers of endosomes and lysosomes. OMVs from both strains did not affect cell viability, but reduce proliferation of HT-29 cells. Labeling of 8-oxo-dG adducts in DNA revealed that neither OMVs from EcN nor from ECOR12 promoted oxidative DNA damage. In contrast, flow cytometry analysis of phosphorylated γH2AX evidenced that OMVs from the probiotic EcN significantly produced more double strand breaks in DNA than ECOR12 OMVs. The EcN genotoxic effects have been attributed to the synthesis of colibactin. However, it is not known how colibactin is exported and delivered into host cells. Whether

  1. Exogenous HIV-1 Nef upsets the IFN-γ-induced impairment of human intestinal epithelial integrity.

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    Maria Giovanna Quaranta

    Full Text Available The mucosal tissues play a central role in the transmission of HIV-1 infection as well as in the pathogenesis of AIDS. Despite several clinical studies reported intestinal dysfunction during HIV infection, the mechanisms underlying HIV-induced impairments of mucosal epithelial barrier are still unclear. It has been postulated that HIV-1 alters enterocytic function and HIV-1 proteins have been detected in several cell types of the intestinal mucosa. In the present study, we analyzed the effect of the accessory HIV-1 Nef protein on human epithelial cell line.We used unstimulated or IFN-γ-stimulated Caco-2 cells, as a model for homeostatic and inflamed gastrointestinal tracts, respectively. We investigated the effect of exogenous recombinant Nef on monolayer integrity analyzing its uptake, transepithelial electrical resistance, permeability to FITC-dextran and the expression of tight junction proteins. Moreover, we measured the induction of proinflammatory mediators. Exogenous Nef was taken up by Caco-2 cells, increased intestinal epithelial permeability and upset the IFN-γ-induced reduction of transepithelial resistance, interfering with tight junction protein expression. Moreover, Nef inhibited IFN-γ-induced apoptosis and up-regulated TNF-α, IL-6 and MIP-3α production by Caco-2 cells while down-regulated IL-10 production. The simultaneous exposure of Caco-2 cells to Nef and IFN-γ did not affect cytokine secretion respect to untreated cells. Finally, we found that Nef counteracted the IFN-γ induced arachidonic acid cascade.Our findings suggest that exogenous Nef, perturbing the IFN-γ-induced impairment of intestinal epithelial cells, could prolong cell survival, thus allowing for accumulation of viral particles. Our results may improve the understanding of AIDS pathogenesis, supporting the discovery of new therapeutic interventions.

  2. Leukotriene D4 activates distinct G-proteins in intestinal epithelial cells to regulate stress fibre formation and to generate intracellular Ca2+ mobilisation and ERK1/2 activation

    International Nuclear Information System (INIS)

    Nielsen, Christian Kamp; Massoumi, Ramin; Sonnerlind, Maria; Sjoelander, Anita

    2005-01-01

    We have shown that the pro-inflammatory mediator LTD 4 , via its G-protein-coupled receptor CysLT 1 , signals through both pertussis-toxin-sensitive and -insensitive G-proteins to induce various cellular responses. To further characterise the initial step of the different signalling pathways emanating from the CysLT 1 receptor, we transfected intestinal epithelial cells, Int 407, with different mini vectors that each express a specific inhibitory peptide directed against a unique α subunit of a specific heterotrimeric G-protein. Our results revealed that LTD 4 -induced stress fibre formation is inhibited approximately 80% by a vector expressing an inhibitory peptide against the pertussis-toxin-insensitive Gα 12 -protein in intestinal epithelial Int 407 cells. Control experiments revealed that the LPA-induced stress fibre formation, mediated via the Gα 12 -protein in other cell types, was blocked by the same peptide in intestinal Int 407 cells. Furthermore, the CysLT 1 -receptor-mediated calcium signal and activation of the proliferative ERK1/2 kinase are blocked in cells transfected with a vector expressing an inhibitory peptide against the Gα i3 -protein, whereas in cells transfected with an empty ECFP-vector or vectors expressing inhibitory peptides against the Gα i1-2 -, Gα 12 -, Gα R -proteins these signals are not significantly affected. Consequently, the CysLT 1 receptor has the capacity to activate at least two distinctly different heterotrimeric G-proteins that transduce activation of unique downstream cellular events

  3. Integrins and epithelial cell polarity.

    Science.gov (United States)

    Lee, Jessica L; Streuli, Charles H

    2014-08-01

    Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell-matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical-basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. © 2014. Published by The Company of Biologists Ltd.

  4. Effects of the ionising radiations on the structure and the function of the intestinal epithelial cell; Effets des rayonnements ionisants sur la structure et la fonction de la cellule epitheliale intestinale

    Energy Technology Data Exchange (ETDEWEB)

    Haton, C

    2005-06-15

    The intestinal mucosa is a particularly radio-sensitive tissue and damage may occur following either accidental or therapeutic exposure. the deleterious actions of ionizing radiation are linked to the formation of sometimes overwhelming quantities of reactive oxygen species (R.O.S.). Production of R.O.S. is both direct and indirect from the secondary effects of irradiation. A better comprehension of the underlying mechanisms of injury will lead to more adapted therapeutic approaches to limit the harmful effects of irradiation. The homeostasis of the intestinal epithelium is regulated by three factors: proliferation, apoptosis and differentiation. these three factors were studied using the cell model, HT29, in order to analyze modulations of this balance after irradiation. our results, in agreement with other data, showed the establishment of mitotic delay. This arrest of proliferation was followed by apoptosis to be the major mechanism leading to cell death in this model. thus, for the first time, we have shown that irradiated intestinal epithelial cells preserve their capacity to differentiate. This indicates, although indirectly, that intestinal cells have and preserve an intrinsic capacity restore a functional epithelium. R.O.S. are considered as intermediates between the physical nature of radiations and biological responses. It seems essential to understand anti-oxidant mechanisms used by the cell for defence against the deleterious effects of R.O.S post exposure. This study of several anti-oxidant defence mechanisms of intestinal mucosa, was carried out in vivo in the mouse at different times following abdominal irradiation. We observed an early mitochondrial response in the hours following irradiation revealing this organelle as a particular target. We demonstrated a strong alteration of anti-oxidant capacity as revealed by a decrease in S.O.D.s, catalase and an increase of the G.P.X.s and M.T.s. A part of these modifications appeared to depend on an

  5. Cathelicidin-WA Improves Intestinal Epithelial Barrier Function and Enhances Host Defense against Enterohemorrhagic Escherichia coli O157:H7 Infection.

    Science.gov (United States)

    Yi, Hongbo; Hu, Wangyang; Chen, Shan; Lu, Zeqing; Wang, Yizhen

    2017-02-15

    Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines, leading to many intestinal diseases. Cathelicidin peptides suppress intestinal inflammation and improve intestinal epithelial barrier function independently of their antimicrobial activity. In this study, we investigated the effects of Cathelicidin-WA (CWA) on intestinal epithelial barrier function, as well as the underlying mechanism, by using enterohemorrhagic Escherichia coli (EHEC)-infected mice and intestinal epithelial cells. The results showed that CWA attenuated EHEC-induced clinical symptoms and intestinal colitis, as did enrofloxacin (Enro). CWA decreased IL-6 production in the serum, jejunum, and colon of EHEC-infected mice. Additionally, CWA alleviated the EHEC-induced disruption of mucin-2 and goblet cells in the intestine. Interestingly, CWA increased the mucus layer thickness, which was associated with increasing expression of trefoil factor 3, in the jejunum of EHEC-infected mice. CWA increased the expression of tight junction proteins in the jejunum of EHEC-infected mice. Using intestinal epithelial cells and a Rac1 inhibitor in vitro, we demonstrated that the CWA-mediated increases in the tight junction proteins might depend on the Rac1 pathway. Furthermore, CWA improved the microbiota and short-chain fatty acid concentrations in the cecum of EHEC-infected mice. Although Enro and CWA had similar effects on intestinal inflammation, CWA was superior to Enro with regard to improving intestinal epithelial barrier and microbiota in the intestine. In conclusion, CWA attenuated EHEC-induced inflammation, intestinal epithelial barrier damage, and microbiota disruption in the intestine of mice, suggesting that CWA may be an effective therapy for many intestinal diseases. Copyright © 2017 by The American Association of Immunologists, Inc.

  6. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses.

    Science.gov (United States)

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka; Marycz, Krzysztof

    2017-08-03

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)-the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs' and IECs' morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome.

  7. Spirulina platensis Improves Mitochondrial Function Impaired by Elevated Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells (ASCs) and Intestinal Epithelial Cells (IECs), and Enhances Insulin Sensitivity in Equine Metabolic Syndrome (EMS) Horses

    Science.gov (United States)

    Nawrocka, Daria; Kornicka, Katarzyna; Śmieszek, Agnieszka

    2017-01-01

    Equine Metabolic Syndrome (EMS) is a steadily growing life-threatening endocrine disorder linked to insulin resistance, oxidative stress, and systemic inflammation. Inflammatory microenvironment of adipose tissue constitutes the direct tissue milieu for various cell populations, including adipose-derived mesenchymal stromal cells (ASCs), widely considered as a potential therapeutic cell source in the course of the treatment of metabolic disorders. Moreover, elevated oxidative stress induces inflammation in intestinal epithelial cells (IECs)—the first-line cells exposed to dietary compounds. In the conducted research, we showed that in vitro application of Spirulina platensis contributes to the restoration of ASCs’ and IECs’ morphology and function through the reduction of cellular oxidative stress and inflammation. Enhanced viability, suppressed senescence, and improved proliferation of ASCs and IECs isolated from metabolic syndrome-affected individuals were evident following exposition to Spirulina. A protective effect of the investigated extract against mitochondrial dysfunction and degeneration was also observed. Moreover, our data demonstrate that Spirulina extract effectively suppressed LPS-induced inflammatory responses in macrophages. In vivo studies showed that horses fed with a diet based on Spirulina platensis supplementation lost weight and their insulin sensitivity improved. Thus, our results indicate the engagement of Spirulina platensis nourishing as an interesting alternative approach for supporting the conventional treatment of equine metabolic syndrome. PMID:28771165

  8. Evolutionary insights into postembryonic development of adult intestinal stem cells

    Directory of Open Access Journals (Sweden)

    Ishizuya-Oka Atsuko

    2011-11-01

    Full Text Available Abstract In the adult vertebrate intestine, multi-potent stem cells continuously generate all of the epithelial cells throughout the adulthood. While it has long been known that the frog intestine is formed via the development of adult intestinal stem cells during thyroid hormone (TH-dependent metamorphosis, the basic structure of the adult intestine is formed by birth in mammals and it is unclear if the subsequent maturation of the intestine involves any changes in the intestinal stem cells. Two recent papers showing that B lymphocyte-induced maturation protein 1 (Blimp1 regulates postnatal epithelial stem cell reprogramming during mouse intestinal maturation support the model that adult intestinal stem cells are developed during postembryonic development in mammals, in a TH-dependent process similar to intestinal remodeling during amphibian metamorphosis. Since the formation of the adult intestine in both mammals and amphibians is closely associated with the adaptation from aquatic to terrestrial life during the peak of endogenous TH levels, the molecular mechanisms by which the adult stem cells are developed are likely evolutionally conserved.

  9. Bovine dairy complex lipids improve in vitro measures of small intestinal epithelial barrier integrity.

    Science.gov (United States)

    Anderson, Rachel C; MacGibbon, Alastair K H; Haggarty, Neill; Armstrong, Kelly M; Roy, Nicole C

    2018-01-01

    Appropriate intestinal barrier maturation is essential for absorbing nutrients and preventing pathogens and toxins from entering the body. Compared to breast-fed infants, formula-fed infants are more susceptible to barrier dysfunction-associated illnesses. In infant formula dairy lipids are usually replaced with plant lipids. We hypothesised that dairy complex lipids improve in vitro intestinal epithelial barrier integrity. We tested milkfat high in conjugated linoleic acid, beta serum (SureStart™Lipid100), beta serum concentrate (BSC) and a ganglioside-rich fraction (G600). Using Caco-2 cells as a model of the human small intestinal epithelium, we analysed the effects of the ingredients on trans-epithelial electrical resistance (TEER), mannitol flux, and tight junction protein co-localisation. BSC induced a dose-dependent improvement in TEER across unchallenged cell layers, maintained the co-localisation of tight junction proteins in TNFα-challenged cells with increased permeability, and mitigated the TEER-reducing effects of lipopolysaccharide (LPS). G600 also increased TEER across healthy and LPS-challenged cells, but it did not alter the co-location of tight junction proteins in TNFα-challenged cells. SureStart™Lipid100 had similar TEER-increasing effects to BSC when added at twice the concentration (similar lipid concentration). Ultimately, this research aims to contribute to the development of infant formulas supplemented with dairy complex lipids that support infant intestinal barrier maturation.

  10. Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells.

    Science.gov (United States)

    Shao, Yuxin; Wolf, Patricia G; Guo, Shuangshuang; Guo, Yuming; Gaskins, H Rex; Zhang, Bingkun

    2017-05-01

    Zinc plays an important role in maintaining intestinal barrier function as well as modulating cellular signaling recognition and protein kinase activities. The phosphatidylinositol 3-kinase (PI3K) cascade has been demonstrated to affect intercellular integrity and tight junction (TJ) proteins. The current study investigated the hypothesis that zinc regulates intestinal intercellular junction integrity through the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. A transwell model of Caco-2 cell was incubated with 0, 50 and 100 μM of zinc at various time points. Transepithelial electrical resistance (TEER), paracellular permeability, TJ proteins, cell proliferation, differentiation and cell damage were measured. Compared with controls, 50 and 100 μM of zinc increased cell growth at 6, 12 and 24 h and the expression of proliferating cell nuclear antigen at 24 h. Zinc (100 μM) significantly elevated TEER at 6-24 h and reduced TJ permeability at 24 h, accompanied by the up-regulation of alkaline phosphatase (AP) activity and zonula occludens (ZO)-1 expression. In addition, zinc (100 μM) affected the PI3K/AKT/mTOR pathway by stimulating phosphorylation of AKT and the downstream target mTOR. Inhibition of PI3K signaling by LY294002 counteracted zinc promotion, as shown by a decrease in AP activity, TEER, the abundance of ZO-1 and phosphorylation of AKT and mTOR. Additionally, TJ permeability and the expression of caspase-3 and LC3II (markers of cell damage) were increased by addition of PI3K inhibitor. In conclusion, the activation of PI3K/AKT/mTOR signaling by zinc is involved in improving intestinal barrier function by enhancing cell differentiation and expression of TJ protein ZO-1. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Curcumin Anti-Apoptotic Action in a Model of Intestinal Epithelial Inflammatory Damage.

    Science.gov (United States)

    Loganes, Claudia; Lega, Sara; Bramuzzo, Matteo; Vecchi Brumatti, Liza; Piscianz, Elisa; Valencic, Erica; Tommasini, Alberto; Marcuzzi, Annalisa

    2017-06-06

    The purpose of this study is to determine if a preventive treatment with curcumin can protect intestinal epithelial cells from inflammatory damage induced by IFNγ. To achieve this goal we have used a human intestinal epithelial cell line (HT29) treated with IFNγ to undergo apoptotic changes that can reproduce the damage of intestinal epithelia exposed to inflammatory cytokines. In this model, we measured the effect of curcumin (curcuminoid from Curcuma Longa ) added as a pre-treatment at different time intervals before stimulation with IFNγ. Curcumin administration to HT29 culture before the inflammatory stimulus IFNγ reduced the cell apoptosis rate. This effect gradually declined with the reduction of the curcumin pre-incubation time. This anti-apoptotic action by curcumin pre-treatment was paralleled by a reduction of secreted IL7 in the HT29 culture media, while there was no relevant change in the other cytokine levels. Even though curcumin pre-administration did not impact the activation of the NF-κB pathway, a slight effect on the phosphorylation of proteins in this inflammatory signaling pathway was observed. In conclusion, curcumin pre-treatment can protect intestinal cells from inflammatory damage. These results can be the basis for studying the preventive role of curcumin in inflammatory bowel diseases.

  12. Modification in oxidative stress, inflammation, and lipoprotein assembly in response to hepatocyte nuclear factor 4alpha knockdown in intestinal epithelial cells.

    Science.gov (United States)

    Marcil, Valérie; Seidman, Ernest; Sinnett, Daniel; Boudreau, François; Gendron, Fernand-Pierre; Beaulieu, Jean-François; Ménard, Daniel; Precourt, Louis-Philippe; Amre, Devendra; Levy, Emile

    2010-12-24

    Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor mainly expressed in the liver, intestine, kidney, and pancreas. Many of its hepatic and pancreatic functions have been described, but limited information is available on its role in the gastrointestinal tract. The objectives of this study were to evaluate the anti-inflammatory and antioxidant functions of HNF4α as well as its implication in intestinal lipid transport and metabolism. To this end, the HNF4A gene was knocked down by transfecting Caco-2 cells with a pGFP-V-RS lentiviral vector containing an shRNA against HNF4α. Inactivation of HNF4α in Caco-2 cells resulted in the following: (a) an increase in oxidative stress as demonstrated by the levels of malondialdehyde and conjugated dienes; (b) a reduction in secondary endogenous antioxidants (catalase, glutathione peroxidase, and heme oxygenase-1); (c) a lower protein expression of nuclear factor erythroid 2-related factor that controls the antioxidant response elements-regulated antioxidant enzymes; (d) an accentuation of cellular inflammatory activation as shown by levels of nuclear factor-κB, interleukin-6, interleukin-8, and leukotriene B4; (e) a decrease in the output of high density lipoproteins and of their anti-inflammatory and anti-oxidative components apolipoproteins (apo) A-I and A-IV; (f) a diminution in cellular lipid transport revealed by a lower cellular secretion of chylomicrons and their apoB-48 moiety; and (g) alterations in the transcription factors sterol regulatory element-binding protein 2, peroxisome proliferator-activated receptor α, and liver X receptor α and β. In conclusion, HNF4α appears to play a key role in intestinal lipid metabolism as well as intestinal anti-oxidative and anti-inflammatory defense mechanisms.

  13. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  14. Identification of multi-drug resistant Pseudomonas aeruginosa clinical isolates that are highly disruptive to the intestinal epithelial barrier

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    Shevchenko Olga

    2006-06-01

    Full Text Available Abstract Background Multi-drug resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. In this study, we focused on the virulence of multi-drug resistant clinical strains P. aeruginosa against the intestinal epithelial barrier, since P. aeruginosa can cause lethal sepsis from within the intestinal tract of critically ill and immuno-compromised patients via mechanisms involving disruption of epithelial barrier function. Methods We screened consecutively isolated multi-drug resistant P. aeruginosa clinical strains for their ability to disrupt the integrity of human cultured intestinal epithelial cells (Caco-2 and correlated these finding to related virulence phenotypes such as adhesiveness, motility, biofilm formation, and cytotoxicity. Results Results demonstrated that the majority of the multi-drug resistant P. aeruginosa clinical strains were attenuated in their ability to disrupt the barrier function of cultured intestinal epithelial cells. Three distinct genotypes were found that displayed an extreme epithelial barrier-disrupting phenotype. These strains were characterized and found to harbor the exoU gene and to display high swimming motility and adhesiveness. Conclusion These data suggest that detailed phenotypic analysis of the behavior of multi-drug resistant P. aeruginosa against the intestinal epithelium has the potential to identify strains most likely to place patients at risk for lethal gut-derived sepsis. Surveillance of colonizing strains of P. aeruginosa in critically ill patients beyond antibiotic sensitivity is warranted.

  15. Increased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters.

    Science.gov (United States)

    Liu, Julia J; Wong, Karen; Thiesen, Aducio L; Mah, Stephanie J; Dieleman, Levinus A; Claggett, Brian; Saltzman, John R; Fedorak, Richard N

    2011-06-01

    Epithelial gaps created by shedding of epithelial cells in the small intestine can be visualized by using confocal laser endomicroscopy (CLE). The density of epithelial gaps in the small bowels of patients with inflammatory bowel disease (IBD) and controls without IBD is unknown. To determine whether the epithelial gap density in patients with IBD is different from that in controls. Prospective, controlled, cohort study. A tertiary-care referral center. This study involved patients with IBD and control patients without IBD undergoing colonoscopy. Probe-based CLE (pCLE) was used to image the terminal ileum. The primary outcome of the study was gap density, defined as the total number of gaps per 1000 cells counted in adequately imaged villi by using pCLE. The pCLE images were blindly reviewed, and the number of epithelial gaps and cells were manually counted. The secondary outcomes were correlation of gap density with disease activity, location, and severity of clinical disease. There were 30 controls and 28 patients with IBD. Of the patients with IBD, 16 had Crohn's disease, and 12 had ulcerative colitis. The median epithelial gap densities for controls and patients with IBD were 18 and 61 gaps/1000 cells, respectively (P < .001). Gap density did not correlate with disease activity. Patients with ulcerative pan-colitis tended toward gap densities lower than those of patients with limited colitis (32 versus 97 gaps/1000 cells, P = .06). Patients with IBD with severe clinical disease also had lower median gap densities (37 vs 90 gaps/1000 cells, P = .04). A single-center study. The epithelial gap density was significantly increased in patients with IBD compared with controls. ( NCT00988273.). Copyright © 2011. Published by Mosby, Inc.

  16. Mucosal pathobiology and molecular signature of epithelial barrier dysfunction in the small intestine in irritable bowel syndrome.

    Science.gov (United States)

    González-Castro, Ana M; Martínez, Cristina; Salvo-Romero, Eloísa; Fortea, Marina; Pardo-Camacho, Cristina; Pérez-Berezo, Teresa; Alonso-Cotoner, Carmen; Santos, Javier; Vicario, María

    2017-01-01

    Irritable bowel syndrome (IBS) is one of the most prevalent gastrointestinal disorders in developed countries. Its etiology remains unknown; however, a common finding, regardless of IBS subtype, is the presence of altered intestinal barrier. In fact, signaling and location of cell-to-cell adhesion proteins, in connection with increased immune activity, seem abnormal in the intestinal epithelium of IBS patients. Despite that most research is performed on distal segments of the intestine, altered permeability has been reported in both, the small and the large bowel of all IBS subtypes. The small intestine carries out digestion and nutrient absorption and is also the site where the majority of immune responses to luminal antigens takes place. In fact, the upper intestine is more exposed to environmental antigens than the colon and is also a site of symptom generation. Recent studies have revealed small intestinal structural alterations of the epithelial barrier and mucosal immune activation in association with intestinal dysfunction, suggesting the commitment of the intestine as a whole in the pathogenesis of IBS. This review summarizes the most recent findings on mucosal barrier alterations and its relationship to symptoms arising from the small intestine in IBS, including epithelial structural abnormalities, mucosal immune activation, and microbial dysbiosis, further supporting the hypothesis of an organic origin of IBS. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  17. MicroRNA-122a Regulates Zonulin by Targeting EGFR in Intestinal Epithelial Dysfunction

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    Bin Zhang

    2017-06-01

    Full Text Available Background/Aims: This study aimed to investigate the role of microRNA (miR-122a in regulating zonulin during the modulation of intestinal barrier. Methods: Zonulin proteins and their target gene expression were analyzed in miR-122a-overexpressing cell lines and in the target gene of epidermal growth factor receptor (EGFR. An mmu-miR-122a intestinal epithelial conditional transgenic (miR-122a-TG mouse model was established to investigate EGFR and zonulin expression. MiR-122a was also detected in the clinical specimens of inflammatory bowel disease. Results: EGFR was identified as a target gene of miR-122a. The expression level of miR-122a was positively correlated with that of zonulin. The expression level of zonulin was significantly increased, whereas the expression level of EGFR was significantly decreased in the miR-122a-TG mice and in the corresponding primary epithelial culture (P < 0.05. These results were consistent with the data of the clinical specimens. Conclusions: miR-122a could be a positive factor of zonulin by targeting EGFR, which increased the intestinal epithelial permeability in vivo and in vitro.

  18. MicroRNA-122a Regulates Zonulin by Targeting EGFR in Intestinal Epithelial Dysfunction.

    Science.gov (United States)

    Zhang, Bin; Tian, Yinghai; Jiang, Ping; Jiang, Yanqiong; Li, Chao; Liu, Ting; Zhou, Rujian; Yang, Ning; Zhou, Xinke; Liu, Zhihua

    2017-01-01

    This study aimed to investigate the role of microRNA (miR)-122a in regulating zonulin during the modulation of intestinal barrier. Zonulin proteins and their target gene expression were analyzed in miR-122a-overexpressing cell lines and in the target gene of epidermal growth factor receptor (EGFR). An mmu-miR-122a intestinal epithelial conditional transgenic (miR-122a-TG) mouse model was established to investigate EGFR and zonulin expression. MiR-122a was also detected in the clinical specimens of inflammatory bowel disease. EGFR was identified as a target gene of miR-122a. The expression level of miR-122a was positively correlated with that of zonulin. The expression level of zonulin was significantly increased, whereas the expression level of EGFR was significantly decreased in the miR-122a-TG mice and in the corresponding primary epithelial culture (P zonulin by targeting EGFR, which increased the intestinal epithelial permeability in vivo and in vitro. © 2017 The Author(s). Published by S. Karger AG, Basel.

  19. Lactobacillus protects the integrity of intestinal epithelial barrier damaged by pathogenic bacteria

    Directory of Open Access Journals (Sweden)

    Qinghua eYu

    2015-03-01

    Full Text Available Pathogens invade intestinal mucosal barrier through phagocytosis of antigen presenting cells (dendritic cell, microfold cells, or through the invasion into the intestinal epithelial directly. Some pathogens could damage the cell junction between epithelial cells and use the paracellular pathway as an entrance to invade. Moreover, some Lactobacillus could inhibit the adhesion of the pathogens and protect the integrity of the cell junction and mucosal barrier. This research focused on the potential therapeutic effect of Lactobacillus fructosus (L. fructosus C2 to attenuate ETEC K88 or S. typhimurium SL1344 induced changes to mucosal barrier. The results demonstrated that treatment of polarized Caco-2 cells with L. fructosus C2 reduced the permeation of dextran, and expression of IL-8, p-ERK and p-JNK when cells were infected with pathogenic bacteria. The findings indicated that L. fructosus C2 exerted a protective effect against the damage to the integrity of Caco-2 cells by ETEC or S. typhimurium infection.

  20. Effects of small intestinal submucosa content on the adhesion and proliferation of retinal pigment epithelial cells on SIS-PLGA films.

    Science.gov (United States)

    Lee, Ga Young; Kang, Su Ji; Lee, So Jin; Song, Jeong Eun; Joo, Choun-Ki; Lee, Dongwon; Khang, Gilson

    2017-01-01

    The retinal pigment epithelium (RPE) plays a critical role in the maintenance of the normal functions of the retina, particularly the photoreceptors. RPE dysfunction, vision loss and degeneration have been implicated as the cause of many retinal diseases, including retinitis pigmentosa and age-related macular degeneration (AMD). To overcome such disorders, tissue engineering could offer useful strategies, using biodegradable polymeric films to replace diseased or lost RPE. Synthetic/natural hybrid films have been studied as a temporary substrate for growing RPEs in biological implantations. In this study, we prepared small intestinal submucosa (SIS)-poly(lactic-co-glycolic) (PLGA) hybrid films and seeded human RPE cells (ARPE-19 cells) onto the film surface. We investigated the film suitability for RPE cell proliferation by MTT assay. The morphology of cellular adhesion on the film was confirmed by scanning electron microscopy (SEM). Reverse transcription-polymerase chain reaction (RT-PCR) and 3-amino-9-ethylcarbazole (AEC) staining were performed to examine mRNA expression and to compare cell proliferation on the films, using cytokeratin as a marker of RPE. Conclusively, we confirmed the higher cell survival rate and much stronger phenotype expression of RPEs on SIS-PLGA films compared to pure PLGA films. These results demonstrated the potential application of SIS-PLGA films in tissue-engineering strategies. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  1. Soluble Dietary Fiber Ameliorates Radiation-Induced Intestinal Epithelial-to-Mesenchymal Transition and Fibrosis.

    Science.gov (United States)

    Yang, Jianbo; Ding, Chao; Dai, Xujie; Lv, Tengfei; Xie, Tingbing; Zhang, Tenghui; Gao, Wen; Gong, Jianfeng; Zhu, Weiming; Li, Ning; Li, Jieshou

    2017-11-01

    Intestinal fibrosis is a late complication of pelvic radiotherapy. Epithelial-to-mesenchymal transition (EMT) plays an important role in tissue fibrosis. The aim of this study was to examine the effect of soluble dietary fiber on radiation-induced intestinal EMT and fibrosis in a mouse model. Apple pectin (4% wt/wt in drinking water) was administered to wild-type and pVillin-Cre-EGFP transgenic mice with intestinal fibrosis induced by a single dose of abdominal irradiation of 10 Gy. The effects of pectin on intestinal EMT and fibrosis, gut microbiota, and short-chain fatty acid (SCFA) concentration were evaluated. Intestinal fibrosis in late radiation enteropathy showed increased submucosal thickness and subepithelial collagen deposition. Enhanced green fluorescent protein (EGFP) + /vimentin + and EGFP + /α-smooth muscle actin (SMA) + coexpressing cells were most clearly observed at 2 weeks after irradiation and gradually decreased at 4 and 12 weeks. Pectin significantly attenuated the thickness of submucosa and collagen deposition at 12 weeks (24.3 vs 27.6 µm in the pectin + radiation-treated group compared with radiation-alone group, respectively, P soluble dietary fiber pectin protected the terminal ileum against radiation-induced fibrosis. This effect might be mediated by altered SCFA concentration in the intestinal lumen and reduced EMT in the ileal epithelium.

  2. Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J. W.; Bekker, C. P.; Voorhout, W. F.; Strous, G. J.; van der Ende, A.; Rottier, P. J.

    1994-01-01

    The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were

  3. Preterm infant gut microbiota affects intestinal epithelial development in a humanized microbiome gnotobiotic mouse model.

    Science.gov (United States)

    Yu, Yueyue; Lu, Lei; Sun, Jun; Petrof, Elaine O; Claud, Erika C

    2016-09-01

    Development of the infant small intestine is influenced by bacterial colonization. To promote establishment of optimal microbial communities in preterm infants, knowledge of the beneficial functions of the early gut microbiota on intestinal development is needed. The purpose of this study was to investigate the impact of early preterm infant microbiota on host gut development using a gnotobiotic mouse model. Histological assessment of intestinal development was performed. The differentiation of four epithelial cell lineages (enterocytes, goblet cells, Paneth cells, enteroendocrine cells) and tight junction (TJ) formation was examined. Using weight gain as a surrogate marker for health, we found that early microbiota from a preterm infant with normal weight gain (MPI-H) induced increased villus height and crypt depth, increased cell proliferation, increased numbers of goblet cells and Paneth cells, and enhanced TJs compared with the changes induced by early microbiota from a poor weight gain preterm infant (MPI-L). Laser capture microdissection (LCM) plus qRT-PCR further revealed, in MPI-H mice, a higher expression of stem cell marker Lgr5 and Paneth cell markers Lyz1 and Cryptdin5 in crypt populations, along with higher expression of the goblet cell and mature enterocyte marker Muc3 in villus populations. In contrast, MPI-L microbiota failed to induce the aforementioned changes and presented intestinal characteristics comparable to a germ-free host. Our data demonstrate that microbial communities have differential effects on intestinal development. Future studies to identify pioneer settlers in neonatal microbial communities necessary to induce maturation may provide new insights for preterm infant microbial ecosystem therapeutics. Copyright © 2016 the American Physiological Society.

  4. Regulation of the epithelial Ca2+ channels in small intestine as studied by quantitative mRNA detection.

    NARCIS (Netherlands)

    Abel, M. van; Hoenderop, J.G.J.; Kemp, J.W.C.M. van der; Leeuwen, J.P.P.M. van; Bindels, R.J.M.

    2003-01-01

    The epithelial Ca2+ channels TRPV5 and TRPV6 are localized to the brush border membrane of intestinal cells and constitute the postulated rate-limiting entry step of active Ca2+ absorption. The aim of the present study was to investigate the hormonal regulation of these channels. To this end, the

  5. Rhubarb Supplementation Promotes Intestinal Mucosal Innate Immune Homeostasis through Modulating Intestinal Epithelial Microbiota in Goat Kids.

    Science.gov (United States)

    Jiao, Jinzhen; Wu, Jian; Wang, Min; Zhou, Chuanshe; Zhong, Rongzhen; Tan, Zhiliang

    2018-01-31

    The abuse and misuse of antibiotics in livestock production pose a potential health risk globally. Rhubarb can serve as a potential alternative to antibiotics, and several studies have looked into its anticancer, antitumor, and anti-inflammatory properties. The aim of this study was to test the effects of rhubarb supplementation to the diet of young ruminants on innate immune function and epithelial microbiota in the small intestine. Goat kids were fed with a control diet supplemented with or without rhubarb (1.25% DM) and were slaughtered at days 50 and 60 of age. Results showed that the supplementation of rhubarb increased ileal villus height (P = 0.036), increased jejujal and ileal anti-inflammatory IL-10 production (P immune function were accompanied by shifts in ileal epithelial bacterial ecosystem in favor of Blautia, Clostridium, Lactobacillus, and Pseudomonas, and with a decline in the relative abundance of Staphylococcus (P immune homeostasis by modulating intestinal epithelial microbiota during the early stages of animal development.

  6. Relaxation of DNA supercoiling leads to increased invasion of epithelial cells and protein secretion by Campylobacter jejuni

    Science.gov (United States)

    Invasion of intestinal epithelial cells by Campylobacter jejuni is a critical step during infection of the human intestine by this important human pathogen. In this study we investigated the role played by DNA supercoiling in the regulation of invasion of epithelial cells and the mechanism by which ...

  7. Synthesis of protein in intestinal cells exposed to cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, J.W.; Berg, W.D. Jr.; Coppenhaver, D.H.

    1987-11-01

    The mechanism by which cyclic adenosine monophosphate (AMP), formed by intestinal epithelial cells in response to cholera toxin, ultimately results in alterations in water and electrolyte transport is poorly understood. Several studies have indicated that inhibitors of transcription or translation block much of the transport of ions and water in the intestine and edema formation in tissue elicited by cholera toxin. Data presented in this study confirmed the inhibitory effects of cycloheximide on cholera toxin-induced fluid accumulation in the rabbit intestinal loop model. Neither cycloheximide nor actinomycin D altered the amount of cyclic AMP that accumulated in intestinal cells and Chinese hamster ovary cells exposed to cholera toxin. An increase in (/sup 3/H) leucine incorporation was readily demonstrable in intestinal epithelial cells from rabbits challenged with Vibrio cholerae. Similarly, intestinal epithelial cells incubated with cholera toxin for 4 hr synthesized substantially more protein than controls as determined by relative incorporation of (/sup 35/S) methionine. Most of the new protein synthesized in response to cholera toxin was membrane associated and of high molecular weight. The possible significance of the toxin-induced protein relative to cholera pathogenesis was discussed.

  8. Synthesis of protein in intestinal cells exposed to cholera toxin

    International Nuclear Information System (INIS)

    Peterson, J.W.; Berg, W.D. Jr.; Coppenhaver, D.H.

    1987-01-01

    The mechanism by which cyclic adenosine monophosphate (AMP), formed by intestinal epithelial cells in response to cholera toxin, ultimately results in alterations in water and electrolyte transport is poorly understood. Several studies have indicated that inhibitors of transcription or translation block much of the transport of ions and water in the intestine and edema formation in tissue elicited by cholera toxin. Data presented in this study confirmed the inhibitory effects of cycloheximide on cholera toxin-induced fluid accumulation in the rabbit intestinal loop model. Neither cycloheximide nor actinomycin D altered the amount of cyclic AMP that accumulated in intestinal cells and Chinese hamster ovary cells exposed to cholera toxin. An increase in [ 3 H] leucine incorporation was readily demonstrable in intestinal epithelial cells from rabbits challenged with Vibrio cholerae. Similarly, intestinal epithelial cells incubated with cholera toxin for 4 hr synthesized substantially more protein than controls as determined by relative incorporation of [ 35 S] methionine. Most of the new protein synthesized in response to cholera toxin was membrane associated and of high molecular weight. The possible significance of the toxin-induced protein relative to cholera pathogenesis was discussed

  9. Differential Influence of Inositol Hexaphosphate on the Expression of Genes Encoding TGF-β Isoforms and Their Receptors in Intestinal Epithelial Cells Stimulated with Proinflammatory Agents

    Directory of Open Access Journals (Sweden)

    Małgorzata Kapral

    2013-01-01

    Full Text Available Transforming growth factor β (TGF-β is a multifunctional cytokine recognized as an important regulator of inflammatory responses. The effect of inositol hexaphosphate (IP6, a naturally occurring phytochemical, on the mRNA expression of TGF-β1, TGF-β2, TGF-β3 and TβRI, TβRII, and TβRIII receptors stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium and IL-1β in intestinal cells Caco-2 for 3 and 12 h was investigated. Real-time qRT-PCR was used to validate mRNAs level of examined genes. Bacterial endotoxin promoted differential expression of TGF-βs and their receptors in a time-dependent manner. IL-1β upregulated mRNA levels of all TGF-βs and receptors at both 3 h and 12 h. IP6 elicited the opposed to LPS effect by increasing downregulated transcription of the examined genes and suppressing the expression of TGF-β1 at 12 h. IP6 counteracted the stimulatory effect of IL-1β on TGF-β1 and receptors expression by decreasing their mRNA levels. IP6 enhanced LPS- and IL-1β-stimulated mRNA expression of TGF-β2 and -β3. Based on these studies it may be concluded that IP6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe-induced infection/inflammation by modulating the expression of genes encoding TGF-βs and their receptors at transcriptional level.

  10. Epithelial structure and function in the hen lower intestine

    DEFF Research Database (Denmark)

    Laverty, G.; Elbrønd, Vibeke Sødring; Árnason, Sigvatur S.

    2006-01-01

    In birds, transport processes in the lower intestine mediate absorption of ions, water and a variety of organic substrates, including significant amounts of glucose, amino acids derived from protein associated with urate spheres, and short-chain fatty acids derived from fermentation processes....... These transport pathways contribute to both osmoregulation and energy homeostasis. Although birds lack a urinary bladder, evidence has shown that ureteral urine, entering the distal lower intestine, is forced into the colon, caecae and even distal portions of the small intestine. Further, substrates also enter......, by resalination of low-salt hens, or by aldosterone administration to high-salt-acclimated birds. In the coprodeum, the changes in transport are paralleled by extensive remodelling of the mucosal surface, with low-salt acclimation increasing cell numbers, microvillus density and length and the proportion...

  11. Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

    DEFF Research Database (Denmark)

    Olsen, Line; Bressendorff, Simon; Troelsen, Jesper T

    2005-01-01

    The intestinal alkaline phosphatase gene (ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the en...

  12. MDR1 is Related to Intestinal Epithelial Injury Induced by Acetylsalicylic Acid

    Directory of Open Access Journals (Sweden)

    Munehiro Kugai

    2013-10-01

    Full Text Available Background/Aims: Although the cytotoxicity of aspirin against the intestinal epithelium is a major clinical problem, little is known about its pathogenesis. We assessed the involvement of Multi Drug Resistance (MDR 1 in intestinal epithelial cell injury caused by aspirin using MDR1 gene-transfected Caco2 cells. Methods: Caco2 cells were treated with various concentrations of aspirin for 24 h. After treatment of Caco2 cells with verapamil, a specific inhibitor of MDR1, we assessed the extent of cell injury using a WST-8 assay at 24 h after aspirin-stimulation. We performed the same procedure in MDR1 gene-transfected Caco2 cells. To determine the function of MDR1 in the metabolism of aspirin, flux study was performed using 14C-labeled aspirin. Results: The level of aspirin-induced cell injury was higher in verapamil-treated Caco2 cells than in control cells and was less serious in MDR1-transfected Caco2 cells than in control vector-transfected cells. The efflux of 14C-labeled aspirin was higher in verapamil-treated Caco2 cells than in control cells. Conclusion: These data suggest that aspirin effux occurs through the MDR1 transporter and that the MDR1 transporter is involved in the pathogenesis of aspirin-induced cell injury.

  13. Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells.

    Science.gov (United States)

    Beltrán, Ana R; Carraro-Lacroix, Luciene R; Bezerra, Camila N A; Cornejo, Marcelo; Norambuena, Katrina; Toledo, Fernando; Araos, Joaquín; Pardo, Fabián; Leiva, Andrea; Sanhueza, Carlos; Malnic, Gerhard; Sobrevia, Luis; Ramírez, Marco A

    2015-01-01

    The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl-release in intestinal cells, including the human colonic carcinoma T84 cell line, involving increased cGMP and membrane alkalization due to reduced Na+/H+ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pHi), and NHE1, NHE2, and NHE4 are expressed in T84 cells, we characterized the STa role as modulator of these exchangers. pHi was assayed by the NH4Cl pulse technique and measured by fluorescence microscopy in BCECF-preloaded cells. pHi recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H+ efflux (JH+) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and JH+ (~63%), without altering basal pHi (range 7.144-7.172). STa did not alter ßi value in a range of 1.6 pHi units. The dpHi/dt and JH+ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa-decreased dpHi/dt and JH+ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T84 cells with STa results in reduced NHE4 activity leading to a lower capacity of pHi recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human

  14. Glucose stimulates intestinal epithelial crypt proliferation by modulating cellular energy metabolism.

    Science.gov (United States)

    Zhou, Weinan; Ramachandran, Deepti; Mansouri, Abdelhak; Dailey, Megan J

    2018-04-01

    The intestinal epithelium plays an essential role in nutrient absorption, hormone release, and barrier function. Maintenance of the epithelium is driven by continuous cell renewal by stem cells located in the intestinal crypts. The amount and type of diet influence this process and result in changes in the size and cellular make-up of the tissue. The mechanism underlying the nutrient-driven changes in proliferation is not known, but may involve a shift in intracellular metabolism that allows for more nutrients to be used to manufacture new cells. We hypothesized that nutrient availability drives changes in cellular energy metabolism of small intestinal epithelial crypts that could contribute to increases in crypt proliferation. We utilized primary small intestinal epithelial crypts from C57BL/6J mice to study (1) the effect of glucose on crypt proliferation and (2) the effect of glucose on crypt metabolism using an extracellular flux analyzer for real-time metabolic measurements. We found that glucose increased both crypt proliferation and glycolysis, and the glycolytic pathway inhibitor 2-deoxy-d-glucose (2-DG) attenuated glucose-induced crypt proliferation. Glucose did not enhance glucose oxidation, but did increase the maximum mitochondrial respiratory capacity, which may contribute to glucose-induced increases in proliferation. Glucose activated Akt/HIF-1α signaling pathway, which might be at least in part responsible for glucose-induced glycolysis and cell proliferation. These results suggest that high glucose availability induces an increase in crypt proliferation by inducing an increase in glycolysis with no change in glucose oxidation. © 2017 Wiley Periodicals, Inc.

  15. Boswellia serrata Preserves Intestinal Epithelial Barrier from Oxidative and Inflammatory Damage.

    Directory of Open Access Journals (Sweden)

    Daniela Catanzaro

    Full Text Available Aminosalicylates, corticosteroids and immunosuppressants are currently the therapeutic choices in inflammatory bowel diseases (IBD, however, with limited remission and often serious side effects. Meanwhile complementary and alternative medicine (CAM use is increasing, particularly herbal medicine. Boswellia serrata is a traditional Ayurvedic remedy with anti-inflammatory properties, of interest for its usefulness in IBDs. The mechanism of this pharmacological potential of Boswellia serrata was investigated in colonic epithelial cell monolayers exposed to H2O2 or INF-γ+TNF-α, chosen as in vitro experimental model of intestinal inflammation. The barrier function was evaluated by the transepithelial electrical resistance (TEER and paracellular permeability assay, and by the tight junction proteins (zonula occludens-1, ZO-1 and occludin immunofluorescence. The expression of phosphorylated NF-κB and reactive oxygen species (ROS generation were determined by immunoblot and cytofluorimetric assay, respectively. Boswellia serrata oleo-gum extract (BSE and its pure derivative acetyl-11-keto-β-boswellic acid (AKBA, were tested at 0.1-10 μg/ml and 0.027 μg/ml, respectively. BSE and AKBA safety was demonstrated by no alteration of intestinal cell viability and barrier function and integrity biomarkers. H2O2 or INF-γ+TNF-α treatment of Caco-2 cell monolayers significantly reduced TEER, increased paracellular permeability and caused the disassembly of tight junction proteins occludin and ZO-1. BSE and AKBA pretreatment significantly prevented functional and morphological alterations and also the NF-κB phosphorylation induced by the inflammatory stimuli. At the same concentrations BSE and AKBA counteracted the increase of ROS caused by H2O2 exposure. Data showed the positive correlation of the antioxidant activity with the mechanism involved in the physiologic maintenance of the integrity and function of the intestinal epithelium. This study

  16. Mechanisms of decreased intestinal epithelial proliferation and increased apoptosis in murine acute lung injury.

    Science.gov (United States)

    Husain, Kareem D; Stromberg, Paul E; Woolsey, Cheryl A; Turnbull, Isaiah R; Dunne, W Michael; Javadi, Pardis; Buchman, Timothy G; Karl, Irene E; Hotchkiss, Richard S; Coopersmith, Craig M

    2005-10-01

    The aim of this study was to determine the effects of acute lung injury on the gut epithelium and examine mechanisms underlying changes in crypt proliferation and apoptosis. The relationship between severity and timing of lung injury to intestinal pathology was also examined. Randomized, controlled study. University research laboratory. Genetically inbred mice. Following induction of acute lung injury, gut epithelial proliferation and apoptosis were assessed in a) C3H/HeN wild-type and C3H/HeJ mice, which lack functional Toll-like receptor 4 (n = 17); b) C57Bl/6 mice that received monoclonal anti-tumor necrosis factor-alpha or control antibody (n = 22); and c) C57Bl/6 wild-type and transgenic mice that overexpress Bcl-2 in their gut epithelium (n = 21). Intestinal epithelial proliferation and death were also examined in animals with differing degrees of lung inflammation (n = 24) as well as in a time course analysis following a fixed injury (n = 18). Acute lung injury caused decreased proliferation and increased apoptosis in crypt epithelial cells in all animals studied. C3H/HeJ mice had higher levels of proliferation than C3H/HeN animals without additional changes in apoptosis. Anti-tumor necrosis factor-alpha antibody had no effect on gut epithelial proliferation or death. Overexpression of Bcl-2 did not change proliferation despite decreasing gut apoptosis. Proliferation and apoptosis were not correlated to severity of lung injury, as gut alterations were lost in mice with more severe acute lung injury. Changes in both gut epithelial proliferation and death were apparent within 12 hrs, but proliferation was decreased 36 hrs following acute lung injury while apoptosis returned to normal. Acute lung injury causes disparate effects on crypt proliferation and apoptosis, which occur, at least in part, through differing mechanisms involving Toll-like receptor 4 and Bcl-2. Severity of lung injury does not correlate with perturbations in proliferation or death in the

  17. Radioprotection of intestinal crypt cells by cox-inhibitors

    International Nuclear Information System (INIS)

    Bisnar, Paul O.; Dones, Rosa Angela S.A.; Serna, Paulene-Ver A.; Deocaris, Chester C.; Guttierez, Kalangitan V.; Deocaris, Custer C.

    2006-01-01

    The regulation of tissue homeostasis in the gastrointestinal epithelium after epithelial injury focuses on the prostaglandins(PGs) as its major mediators. The two cyclooxygenase isoforms, cox-1 and cox-2, catalyze synthesis of PGs. Cox-1 is the predominant cyclooxygenase isoform found in the normal intestine. In contrast, cox-2 is present at low levels in normal intestine but is elevated at sites of inflammation, and in adenomas and carcinomas. To study the effects of various commercially-available cox-inhibitors (Ketorolac: cox-1 selective; Celecoxib: cox-2 selective; and Indocid: cox-1/2 non-selective), we determine mouse crypt epithelial cell fate after genotoxic injury with whole-body gamma-ray exposure at 15 Gy. Intestinal tissues of mice treated with cox-2 inhibitors that showed invariable apoptotic event, however, have increased occurrence of regenerating cells. Our results suggest a potential application of cox-2 selective inhibitors as radioprotective agent for normal cells after radiotherapy. (Author)

  18. Culturing intestinal stem cells: applications for colorectal cancer research

    Directory of Open Access Journals (Sweden)

    Masayuki eFujii

    2014-06-01

    Full Text Available Recent advance of sequencing technology has revealed genetic alterations in colorectal cancer. The biological function of recurrently mutated genes has been intensively investigated through mouse genetic models and colorectal cancer cell lines. Although these experimental models may not fully reflect biological traits of human intestinal epithelium, they provided insights into the understanding of intestinal stem cell self-renewal, leading to the development of novel human intestinal organoid culture system. Intestinal organoid culture enabled to expand normal or tumor epithelial cells in vitro retaining their stem cell self-renewal and multiple differentiation. Gene manipulation of these cultured cells may provide an attractive tool for investigating genetic events involved in colorectal carcinogenesis.

  19. Hypoxia Inducible Factor (HIF Hydroxylases as Regulators of Intestinal Epithelial Barrier FunctionSummary

    Directory of Open Access Journals (Sweden)

    Mario C. Manresa

    2017-05-01

    Full Text Available Human health is dependent on the ability of the body to extract nutrients, fluids, and oxygen from the external environment while at the same time maintaining a state of internal sterility. Therefore, the cell layers that cover the surface areas of the body such as the lung, skin, and gastrointestinal mucosa provide vital semipermeable barriers that allow the transport of essential nutrients, fluid, and waste products, while at the same time keeping the internal compartments free of microbial organisms. These epithelial surfaces are highly specialized and differ in their anatomic structure depending on their location to provide appropriate and effective site-specific barrier function. Given this important role, it is not surprising that significant disease often is associated with alterations in epithelial barrier function. Examples of such diseases include inflammatory bowel disease, chronic obstructive pulmonary disease, and atopic dermatitis. These chronic inflammatory disorders often are characterized by diminished tissue oxygen levels (hypoxia. Hypoxia triggers an adaptive transcriptional response governed by hypoxia-inducible factors (HIFs, which are repressed by a family of oxygen-sensing HIF hydroxylases. Here, we review recent evidence suggesting that pharmacologic hydroxylase inhibition may be of therapeutic benefit in inflammatory bowel disease through the promotion of intestinal epithelial barrier function through both HIF-dependent and HIF-independent mechanisms. Keywords: Epithelial Barrier, Inflammatory Bowel Disease, Hypoxia, Hypoxia-Inducible Factor (HIF Hydroxylases

  20. A transferrin-like GPI-linked iron-binding protein in detergent-insoluble noncaveolar microdomains at the apical surface of fetal intestinal epithelial cells

    DEFF Research Database (Denmark)

    Danielsen, E M; van Deurs, B

    1995-01-01

    A GPI-anchored 80-kD protein was found to be the major component of detergent-insoluble complexes, prepared from fetal porcine small intestine, constituting about 25% of the total amount of protein. An antibody was raised to the 80-kD protein, and by immunogold electron microscopy of ultracryosec......A GPI-anchored 80-kD protein was found to be the major component of detergent-insoluble complexes, prepared from fetal porcine small intestine, constituting about 25% of the total amount of protein. An antibody was raised to the 80-kD protein, and by immunogold electron microscopy...

  1. Nitric oxide (NO) production in mammalian non-tumorigenic epithelial cells of the small intestine and macrophages induced by individual strains of lactobacilli and bifidobacteria

    DEFF Research Database (Denmark)

    Pipenbaher, Natasa; Møller, Peter Lange; Dolinsek, Jan

    2009-01-01

    and absence of interferon gamma (INF-¿). Production of NO in intestinal epithelium was stimulated by individual strains of lactobacilli without INF-¿ priming. While none of the tested bifidobacteria were capable of inducing NO production, most constitutively secreted NO. Most tested strains induced...

  2. Target of rapamycin (TOR) signaling controls epithelial morphogenesis in the vertebrate intestine.

    Science.gov (United States)

    Makky, Khadijah; Tekiela, Jackie; Mayer, Alan N

    2007-03-15

    The target of rapamycin (TOR) signaling pathway regulates cell growth and proliferation, however the extent to which TOR signaling mediates particular organogenesis programs remains to be determined. Here we report an examination of TOR signaling during zebrafish development, using a combination of small molecule treatment and morpholino-mediated gene knockdown. First, we amplified and sequenced the full-length cDNA for the zebrafish TOR ortholog (ztor). By in situ hybridization, we found that ztor is expressed ubiquitously in the early embryo, but displays a dynamic pattern in the gut between 48 and 72 h post-fertilization (hpf). Treatment of zebrafish embryos with rapamycin induced only a mild general developmental delay up to 72 hpf, but digestive tract development became arrested at the primitive gut tube stage. Rapamycin inhibited intestinal epithelial growth, morphogenesis and differentiation. Using morpholino-mediated gene knockdown of TOR pathway components, we show that this effect is mediated specifically by the rapamycin-sensitive TOR complex 1 (TORC1). Thus, in addition to regulating cell growth and proliferation, TOR signaling controls the developmental program guiding epithelial morphogenesis in the vertebrate intestine.

  3. Low uptake of silica nanoparticles in Caco-2 intestinal epithelial barriers.

    Science.gov (United States)

    Ye, Dong; Bramini, Mattia; Hristov, Delyan R; Wan, Sha; Salvati, Anna; Åberg, Christoffer; Dawson, Kenneth A

    2017-01-01

    Cellular barriers, such as the skin, the lung epithelium or the intestinal epithelium, constitute one of the first obstacles facing nanomedicines or other nanoparticles entering organisms. It is thus important to assess the capacity of nanoparticles to enter and transport across such barriers. In this work, Caco-2 intestinal epithelial cells were used as a well-established model for the intestinal barrier, and the uptake, trafficking and translocation of model silica nanoparticles of different sizes were investigated using a combination of imaging, flow cytometry and transport studies. Compared to typical observations in standard cell lines commonly used for in vitro studies, silica nanoparticle uptake into well-developed Caco-2 cellular barriers was found to be very low. Instead, nanoparticle association to the apical outer membrane was substantial and these particles could easily be misinterpreted as internalised in the absence of imaging. Passage of nanoparticles through the barrier was very limited, suggesting that the low amount of internalised nanoparticles was due to reduced uptake into cells, rather than a considerable transport through them.

  4. Does small intestinal atresia affect epithelial protein expression in human newborns?

    NARCIS (Netherlands)

    Schaart, Maaike W.; Yamanouchi, Takeshi; van Nispen, Danielle J. P. M.; Raatgeep, Rolien H. C.; van Goudoever, Johannes B.; de Krijger, Ronald R.; Tibboel, Dick; Einerhand, Alexandra W. C.; Renes, Ingrid B.

    2006-01-01

    Bowel segments distal to a congenital intestinal obstruction have been suggested to be immature. In other words, luminal components such as amniotic fluid (before birth) and/or enteral nutrition (after birth) may be required to activate intestinal epithelial protein expression, thereby influencing

  5. EICOSAPENTAENOIC ACID ENHANCES HEATSTROKE-IMPAIRED INTESTINAL EPITHELIAL BARRIER FUNCTION IN RATS.

    Science.gov (United States)

    Xiao, Guizhen; Yuan, Fangfang; Geng, Yan; Qiu, Xiaowen; Liu, Zhifeng; Lu, Jiefu; Tang, Liqun; Zhang, Yali; Su, Lei

    2015-10-01

    Dysfunction of the intestinal barrier plays an important role in the pathological process of heatstroke. Omega-3 (or n-3) polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), help protect the intestinal mucosal barrier. This study assessed if pretreating rats with EPA or DHA could alleviate heat stress-induced damage to the intestinal barrier caused by experimental heatstroke. Male Wistar rats were pregavaged with either EPA, DHA, corn oil, or normal saline (all 1 g/kg) for 21 days before the heatstroke experiment (control rats were not exposed to heat). Experimental rats were exposed to an ambient temperature of 37°C and 60% humidity to induce heatstroke, and then they were allowed to recover at room temperature after rapid cooling. Survival time of rats was monitored after heatstroke. Horseradish peroxidase flux from the gut lumen and the level of plasma D-lactate were measured to analyze intestinal permeability at 6 h after heatstroke. Plasma endotoxin levels were determined using a limulus amoebocyte lysate assay. Expressions of the tight junction (TJ) proteins occludin and ZO-1 were analyzed by Western blot and localized by immunofluorescence microscopy. Tight junction protein morphology was observed by transmission electron microscopy. Fatty acids of ileal mucosa were analyzed using gas chromatography-mass selective detector. Eicosapentaenoic acid significantly increased survival time after heatstroke. Eicosapentaenoic acid significantly decreased intestinal permeability and plasma endotoxin levels. Eicosapentaenoic acid effectively attenuated the heatstroke-induced disruption of the intestinal structure and improved the histology score, whereas DHA was less effective, and corn oil was ineffective. Pretreatment with EPA also increased expression of occludin and ZO-1 to effectively prevent TJ disruption. Eicosapentaenoic acid pretreatment enriched itself in the membrane of intestinal cells. Our results

  6. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    Chronic inflammatory diseases of the intestinal tract are associated with an increased risk of colorectal cancer. As an example ulcerative colitis (UC) is associated with a production of reactive oxygen species (ROS), including nitrogen monoxide (NO), which is produced in high amounts by inducibl...... nitrogen oxide synthase (iNOS). NO as well as other ROS are potential DNA damaging agents. The aim was to determine the effect of long-term cytokine exposure on NO formation and DNA damage in epithelial cells....

  7. Activated endothelial cells elicit paracrine induction of epithelial chloride secretion. 6-Keto-PGF1alpha is an epithelial secretagogue.

    Science.gov (United States)

    Blume, E D; Taylor, C T; Lennon, P F; Stahl, G L; Colgan, S P

    1998-09-15

    Endothelial cells play a central role in the coordination of the inflammatory response. In mucosal tissue, such as the lung and intestine, endothelia are anatomically positioned in close proximity to epithelia, providing the potential for cell-cell crosstalk. Thus, in this study endothelial-epithelial biochemical crosstalk pathways were studied using a human intestinal crypt cell line (T84) grown in noncontact coculture with human umbilical vein endothelia. Exposure of such cocultures to endothelial-specific agonists (LPS) resulted in activation of epithelial electrogenic Cl- secretion and vectorial fluid transport. Subsequent experiments revealed that in response to diverse stimuli (LPS, IL-1alpha, TNF-alpha, hypoxia), endothelia produce and secrete a small, stable epithelial secretagogue into conditioned media supernatants. Further experiments identified this secretagogue as 6-keto-PGF1alpha, a stable hydrolysis product of prostacyclin (PGI2). Results obtained with synthetic prostanoids indicated that 6-keto-PGF1alpha (EC50 = 80 nM) and PGI2 stable analogues (EC50 = 280 nM) activate the same basolaterally polarized, Ca2+-coupled epithelial receptor. In summary, these findings reveal a previously unappreciated 6-keto-PGF1alpha receptor on intestinal epithelia, the ligation of which results in activation of electrogenic Cl- secretion. In addition, these data reveal a novel action for the prostacyclin hydrolysis product 6-keto-PGF1alpha and provide a potential endothelial- epithelial crosstalk pathway in mucosal tissue.

  8. Neuropeptide Y (NPY) promotes inflammation-induced tumorigenesis by enhancing epithelial cell proliferation.

    Science.gov (United States)

    Jeppsson, Sabrina; Srinivasan, Shanthi; Chandrasekharan, Bindu

    2017-02-01

    We have demonstrated that neuropeptide Y (NPY), abundantly produced by enteric neurons, is an important regulator of intestinal inflammation. However, the role of NPY in the progression of chronic inflammation to tumorigenesis is unknown. We investigated whether NPY could modulate epithelial cell proliferation and apoptosis, and thus regulate tumorigenesis. Repeated cycles of dextran sodium sulfate (DSS) were used to model inflammation-induced tumorigenesis in wild-type (WT) and NPY knockout (NPY -/- ) mice. Intestinal epithelial cell lines (T84) were used to assess the effects of NPY (0.1 µM) on epithelial proliferation and apoptosis in vitro. DSS-WT mice exhibited enhanced intestinal inflammation, polyp size, and polyp number (7.5 ± 0.8) compared with DSS-NPY -/- mice (4 ± 0.5, P inflammation-induced tumorigenesis by NPY-epithelial cross talk as mediated by activation of PI3-K signaling and downregulation of miR-375. Our work exemplifies a novel role of neuropeptide Y (NPY) in regulating inflammation-induced tumorigenesis via two modalities: first by enhanced proliferation (PI3-K/pAkt), and second by downregulation of microRNA-375 (miR-375)-dependent apoptosis in intestinal epithelial cells. Our data establish the existence of a microRNA-mediated cross talk between enteric neurons producing NPY and intestinal epithelial cells, and the potential of neuropeptide-regulated miRNAs as potential therapeutic molecules for the management of inflammation-associated tumors in the gut.

  9. Loss of Sonic hedgehog leads to alterations in intestinal secretory cell maturation and autophagy.

    Directory of Open Access Journals (Sweden)

    Jessica Gagné-Sansfaçon

    Full Text Available BACKGROUND: Intestinal epithelial cells express the Sonic and Indian hedgehog ligands. Despite the strong interest in gut hedgehog signaling in GI diseases, no studies have specifically addressed the singular role of intestinal epithelial cell Sonic hedgehog signaling. The aim of this study was to investigate the specific role of Sonic hedgehog in adult ileal epithelial homeostasis. METHODOLOGY/PRINCIPAL FINDINGS: A Sonic hedgehog intestinal epithelial conditional knockout mouse model was generated. Assessment of ileal histological abnormalities, crypt epithelial cell proliferation, epithelial cell fate, junctional proteins, signaling pathways, as well as ultrastructural analysis of intracellular organelles were performed in control and mutant mice. Mice lacking intestinal epithelial Sonic Hedgehog displayed decreased ileal crypt/villus length, decreased crypt proliferation as well as a decrease in the number of ileal mucin-secreting goblet cells and antimicrobial peptide-secreting Paneth cells during adult life. These secretory cells also exhibited disruption of their secretory products in mutant mice. Ultrastructural microscopy analysis revealed a dilated ER lumen in secretory cells. This phenotype was also associated with a decrease in autophagy. CONCLUSIONS/SIGNIFICANCE: Altogether, these findings indicate that the loss of Sonic hedgehog can lead to ileal secretory cell modifications indicative of endoplasmic reticulum stress, accompanied by a significant reduction in autophagy.

  10. Commensal Bacteria and Epithelial Cross Talk in the Developing Intestine

    OpenAIRE

    Rautava, Samuli; Walker, W. Allan

    2007-01-01

    Indigenous intestinal microbes have co-evolved with the intestinal immune system to form a symbiotic ecosystem. In the postnatal period, intestinal microbes provide the developing gut with stimuli that are necessary for healthy maturation of the intestinal immune system. Cross talk between the host and commensal microbes is an essential component of gut homeostasis mechanisms also in later life. During recent years, innovative research has shed light on the molecular mechanisms of these inter...

  11. Radiation biology of human mammary epithelial cells

    International Nuclear Information System (INIS)

    Smith, H.S.; Yang, T.C.; Stampfer, M.R.; Hackett, A.J.

    1982-01-01

    Techniques have been developed for growing mass cultures of normal mammary epithelial cells (from reduction mammoplasties) and, most recently, for growing mammary epithelial cells in a highly efficient clonal assay. The availability of this clonal assay has enabled us to examine the dose-response curves for x rays

  12. Glycocalyx of lung epithelial cells.

    Science.gov (United States)

    Martins, Maria de Fátima; Bairos, Vasco A

    2002-01-01

    Due to their diversity and external location on cell membranes, glycans, as glycocalyx components, are key elements in eukaryotic cell, tissue, and organ homeostasis. Although information on the lung glycocalyx is scarce, this article aims to review, discuss, and summarize what is known about bronchoalveolar glycocalyx composition, mainly the sialic acids. It was deemed relevant, however, to make a brief introductory overview of the cell glycocalyx and its particular development in epithelial cells. After that, follows a summary of the evolution of the knowledge regarding the bronchoalveolar glycocalyx composition throughout the years, particularly its morphological features. Since sialic acids are located terminally on the bronchoalveolar lining cells' glycocalyx and play crucial roles, we focused mainly on the existing lung histochemical and biochemical data of these sugar residues, as well as their evolution throughout lung development. The functions of the lung glycocalyx sialic acids are discussed and interpretations of their roles analyzed, including those related to the negative overall superficial shield provided by these molecules. The increasing presence of these sugar residues throughout postnatal lung development should be regarded as pivotal in the development and maintenance of a dynamic bronchoalveolar architecture, supporting the normal histophysiology of the respiratory system. The case for a profound knowledge of lung glycocalyx--given its potential to provide answers to serious clinical problems--is made with particular reference to cystic fibrosis. Finally, concluding remarks and perspectives for future research in this field are put forth.

  13. Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine

    Science.gov (United States)

    Vyas, Dinesh; Robertson, Charles M; Stromberg, Paul E; Martin, James R.; Dunne, W. Michael; Houchen, Courtney W; Barrett, Terrence A; Ayala, Alfred; Perl, Mario; Buchman, Timothy G; Coopersmith, Craig M

    2007-01-01

    Gut epithelial apoptosis is involved in the pathophysiology of multiple diseases. This study characterized intestinal apoptosis in three mechanistically distinct injuries with different kinetics of cell death. FVB/N mice were subjected to gamma radiation, Pseudomonas aeruginosa pneumonia or injection of monoclonal anti-CD3 antibody and sacrificed 4, 12, or 24 hours post-injury (n=10/time point). Apoptosis was quantified in the jejunum by hematoxylin and eosin (H&E), active caspase-3, terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling (TUNEL), in situ oligoligation reaction (ISOL,) cytokeratin 18, and annexin V staining. Reproducible results were obtained only for H&E, active caspase-3, TUNEL and ISOL, which were quantified and compared against each other for each injury at each time point. Kinetics of injury were different with early apoptosis highest following radiation, late apoptosis highest following anti CD3, and more consistent levels following pneumonia. ISOL was the most consistent stain and was always statistically indistinguishable from at least 2 stains. In contrast, active caspase-3 demonstrated lower levels of apoptosis, while the TUNEL assay had higher levels of apoptosis in the most severely injured intestine regardless of mechanism of injury. H&E was a statistical outlier more commonly than any other stain. This suggests that regardless of mechanism or kinetics of injury, ISOL correlates to other quantification methods of detecting gut epithelial apoptosis more than any other method studied and compares favorably to other commonly accepted techniques of quantifying apoptosis in a large intestinal cross sectional by balancing sensitivity and specificity across a range of times and levels of death. PMID:17357092

  14. Imbalance of gut microbiome and intestinal epithelial barrier dysfunction in patients with high blood pressure.

    Science.gov (United States)

    Kim, Seungbum; Goel, Ruby; Kumar, Ashok; Qi, Yanfei; Lobaton, Gil; Hosaka, Koji; Mohammed, Mohammed; Handberg, Eileen M; Richards, Elaine M; Pepine, Carl J; Raizada, Mohan K

    2018-03-30

    Recent evidence indicates a link between gut pathology and microbiome with hypertension (HTN) in animal models. However, whether this association exists in humans is unknown. Thus, our objectives in the present study were to test the hypotheses that high blood pressure (BP) patients have distinct gut microbiomes and that gut-epithelial barrier function markers and microbiome composition could predict systolic BP (SBP). Fecal samples, analyzed by shotgun metagenomics, displayed taxonomic and functional changes, including altered butyrate production between patients with high BP and reference subjects. Significant increases in plasma of intestinal fatty acid binding protein (I-FABP), lipopolysaccharide (LPS), and augmented gut-targetting proinflammatory T helper 17 (Th17) cells in high BP patients demonstrated increased intestinal inflammation and permeability. Zonulin, a gut epithelial tight junction protein regulator, was markedly elevated, further supporting gut barrier dysfunction in high BP. Zonulin strongly correlated with SBP (R 2 = 0.5301, P <0.0001). Two models predicting SBP were built using stepwise linear regression analysis of microbiome data and circulating markers of gut health, and validated in a separate cohort by prediction of SBP from zonulin in plasma (R 2 = 0.4608, P <0.0001). The mouse model of HTN, chronic angiotensin II (Ang II) infusion, was used to confirm the effects of butyrate and gut barrier function on the cardiovascular system and BP. These results support our conclusion that intestinal barrier dysfunction and microbiome function are linked to HTN in humans. They suggest that manipulation of gut microbiome and its barrier functions could be the new therapeutic and diagnostic avenues for HTN. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  15. Epithelial Cells in Urine: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... page: https://medlineplus.gov/labtests/epithelialcellsinurine.html Epithelial Cells in Urine To use the sharing features on ... page, please enable JavaScript. What is an Epithelial Cells in Urine Test? Epithelial cells are a type ...

  16. How Shigella Utilizes Ca2+ Jagged Edge Signals during Invasion of Epithelial Cells

    OpenAIRE

    Bonnet, Mariette; Tran Van Nhieu, Guy

    2016-01-01

    Shigella, the causative agent of bacillary dysentery invades intestinal epithelial cells using a type III secretion system (T3SS). Through the injection of type III effectors, Shigella manipulates the actin cytoskeleton to induce its internalization in epithelial cells. At early invasion stages, Shigella induces atypical Ca2+ responses confined at entry sites allowing local cytoskeletal remodeling for bacteria engulfment. Global Ca2+ increase in the cell triggers the opening of connexin hemic...

  17. Apical trafficking in epithelial cells: signals, clusters and motors.

    Science.gov (United States)

    Weisz, Ora A; Rodriguez-Boulan, Enrique

    2009-12-01

    In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases.

  18. γδ T cells in homeostasis and host defence of epithelial barrier tissues.

    Science.gov (United States)

    Nielsen, Morten M; Witherden, Deborah A; Havran, Wendy L

    2017-12-01

    Epithelial surfaces line the body and provide a crucial interface between the body and the external environment. Tissue-resident epithelial γδ T cells represent a major T cell population in the epithelial tissues and are ideally positioned to carry out barrier surveillance and aid in tissue homeostasis and repair. In this Review, we focus on the intraepithelial γδ T cell compartment of the two largest epithelial tissues in the body - namely, the epidermis and the intestine - and provide a comprehensive overview of the crucial contributions of intraepithelial γδ T cells to tissue integrity and repair, host homeostasis and protection in the context of the symbiotic relationship with the microbiome and during pathogen clearance. Finally, we describe epithelium-specific butyrophilin-like molecules and briefly review their emerging role in selectively shaping and regulating epidermal and intestinal γδ T cell repertoires.

  19. G protein-coupled receptor 120 (GPR120) transcription in intestinal epithelial cells is significantly affected by bacteria belonging to the Bacteroides, Proteobacteria, and Firmicutes phyla

    DEFF Research Database (Denmark)

    Fredborg, Marlene; Theil, Peter Kappel; Jensen, Bent Borg

    2012-01-01

    examined using real-time reverse transcriptase polymerase chain reaction. The abundance of GLP-1 mRNA decreased when cells were exposed to 4 of the 12 supernatants (P ≤ 0.05) compared with cells without bacteria added. Supernatants from 8 of the 12 bacteria analyzed increased the mRNA level of GPR120 (P...

  20. DNA repair in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Lechner, J.F.; Grafstrom, R.C.; Harris, C.C.

    1982-01-01

    The purpose of this investigation was to compare the response of human cell types (bronchial epithelial cells and fibroblasts and skin fibroblasts) to various DNA damaging agents. Repair of DNA single strand breaks (SSB) induced by 5 krads of X-ray was similar for all cell types; approximately 90% of the DNA SSB were rejoined within one hour. During excision repair of DNA damage from u.v.-radiation, the frequencies of DNA SSB as estimated by the alkaline elution technique, were similar in all cell types. Repair replication as measured by BND cellulose chromatography was also similar in epithelial and fibroblastic cells after u.v.-irradiation. Similar levels of SSB were also observed in epithelial and fibroblastic cells after exposure to chemical carcinogens: 7,12-dimethylbenz[a]anthracene; benzo[a]pyrene diol epoxide (BPDE); or N-methyl-N-nitro-N-nitrosoguanidine. Significant repair replication of BPDE-induced DNA damage was detected in both bronchial epithelial and fibroblastic cells, although the level in fibroblasts was approximately 40% of that in epithelial cells. The pulmonary carcinogen asbestos did not damage DNA. DNA-protein crosslinks induced by formaldehyde were rapidly removed in bronchial cells. Further, epithelial and fibroblastic cells, which were incubated with formaldehyde and the polymerase inhibitor combination of cytosine arabinoside and hydroxyurea, accumulated DNA SSB at approximately equal frequencies. These results should provide a useful background for further investigations of the response of human bronchial cells to various DNA damaging agents

  1. RHOA GTPase Controls YAP-Mediated EREG Signaling in Small Intestinal Stem Cell Maintenance

    Directory of Open Access Journals (Sweden)

    Ming Liu

    2017-12-01

    Full Text Available Summary: RHOA, a founding member of the Rho GTPase family, is critical for actomyosin dynamics, polarity, and morphogenesis in response to developmental cues, mechanical stress, and inflammation. In murine small intestinal epithelium, inducible RHOA deletion causes a loss of epithelial polarity, with disrupted villi and crypt organization. In the intestinal crypts, RHOA deficiency results in reduced cell proliferation, increased apoptosis, and a loss of intestinal stem cells (ISCs that mimic effects of radiation damage. Mechanistically, RHOA loss reduces YAP signaling of the Hippo pathway and affects YAP effector epiregulin (EREG expression in the crypts. Expression of an active YAP (S112A mutant rescues ISC marker expression, ISC regeneration, and ISC-associated Wnt signaling, but not defective epithelial polarity, in RhoA knockout mice, implicating YAP in RHOA-regulated ISC function. EREG treatment or active β-catenin Catnblox(ex3 mutant expression rescues the RhoA KO ISC phenotypes. Thus, RHOA controls YAP-EREG signaling to regulate intestinal homeostasis and ISC regeneration. : In this article, Zheng and colleagues show that inducible RHOA deletion in mice causes defects in intestine epithelial polarity and deficiencies in intestinal stem cell proliferation, survival, and regeneration. They further demonstrate by genetic rescues that RHOA controls a YAP-EREG axis to mediate canonical Wnt signaling, intestinal stem cell function, and intestinal homeostasis. Keywords: mouse model, intestinal stem cell, regeneration, Rho GTPase, RhoA, Hippo signaling, YAP, Wnt signaling

  2. Circadian regulation of epithelial functions in the intestine

    Czech Academy of Sciences Publication Activity Database

    Pácha, Jiří; Sumová, Alena

    2013-01-01

    Roč. 208, č. 1 (2013), s. 11-24 ISSN 1748-1708 R&D Projects: GA ČR(CZ) GAP303/10/0969; GA ČR(CZ) GAP303/11/0668 Institutional support: RVO:67985823 Keywords : circadian rhythms * intestine * colon * proliferation * digestion * intestinal transport Subject RIV: ED - Physiology Impact factor: 4.251, year: 2013

  3. Investigating the Transport Dynamics of Anthocyanins from Unprocessed Fruit and Processed Fruit Juice from Sour Cherry (Prunus cerasus L.) across Intestinal Epithelial Cells

    NARCIS (Netherlands)

    Toydemir, G.; Boyacioglu, D.; Capanoglu, E.; Meer, van der I.M.; Tomassen, M.M.M.; Hall, R.D.; Mes, J.J.; Beekwilder, J.

    2013-01-01

    Anthocyanins can contribute to human health through preventing a variety of diseases. The uptake of these compounds from food and the parameters determining uptake efficiency within the human body are still poorly understood. Here we have employed a Caco-2 cell based system to investigate the

  4. Intestinal epithelial barrier function and tight junction proteins with heat and exercise

    DEFF Research Database (Denmark)

    Dokladny, Karol; Zuhl, Micah N; Moseley, Pope L

    2016-01-01

    (passive hyperthermia) heat stress on tight junction barrier function in in vitro and in vivo (animals and humans) models. Our secondary focus is to review changes in tight junction proteins in response to exercise or hyperthermic conditions. Finally, we discuss some pharmacological or nutritional...... interventions that may affect the cellular mechanisms involved in maintaining homeostasis of the intestinal epithelial tight junction barrier during heat stress or exercise....... permeation of luminal antigens, endotoxins, and bacteria into the blood stream. Various substances and conditions have been shown to affect the maintenance of the intestinal epithelial tight junction barrier. The primary focus of the present review is to analyze the effects of exertional or nonexertional...

  5. Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors

    OpenAIRE

    Saba-El-Leil Marc K; Claveau Isabelle; Gopalbhai Kailesh; Duhamel Stéphanie; Julien Catherine; Voisin Laure; Rodrigue-Gervais Ian; Gaboury Louis; Lamarre Daniel; Basik Mark; Meloche Sylvain

    2008-01-01

    Abstract Background The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. Methods Wild type and constitutively active forms of MEK1 and ME...

  6. Protective effects of ψ taraxasterol 3-O-myristate and arnidiol 3-O-myristate isolated from Calendula officinalis on epithelial intestinal barrier.

    Science.gov (United States)

    Dall'Acqua, Stefano; Catanzaro, Daniela; Cocetta, Veronica; Igl, Nadine; Ragazzi, Eugenio; Giron, Maria Cecilia; Cecconello, Laura; Montopoli, Monica

    2016-03-01

    The triterpene esters ᴪ taraxasterol-3-O-myristate (1) and arnidiol-3-O-myristate (2) were tested for their ability to protect epithelial intestinal barrier in an in vitro model. Their effects on ROS production and on trans-epithelial resistance were investigated on CaCo-2 cell monolayers both in basal and stress-induced conditions. Both compounds were able to modulate the stress damage induced by H2O2 and INFγ+TNFα, showing a potential use as model compounds for the study of new therapeutic agents for intestinal inflammations. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    Science.gov (United States)

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-08

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. TTC7A mutations disrupt intestinal epithelial apicobasal polarity

    NARCIS (Netherlands)

    Bigorgne, Amélie E; Farin, Henner F; Lemoine, Roxane; Mahlaoui, Nizar; Lambert, Nathalie; Gil, Marine; Schulz, Ansgar; Philippet, Pierre; Schlesser, Patrick; Abrahamsen, Tore G; Oymar, Knut; Davies, E Graham; Ellingsen, Christian Lycke; Leteurtre, Emmanuelle; Moreau-Massart, Brigitte; Berrebi, Dominique; Bole-Feysot, Christine; Nischke, Patrick; Brousse, Nicole; Fischer, Alain; Clevers, Hans; de Saint Basile, Geneviève

    Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and

  9. Differential expression of P-type ATPases in intestinal epithelial cells: Identification of putative new atp1a1 splice-variant

    International Nuclear Information System (INIS)

    Rocafull, Miguel A.; Thomas, Luz E.; Barrera, Girolamo J.; Castillo, Jesus R. del

    2010-01-01

    P-type ATPases are membrane proteins that couple ATP hydrolysis with cation transport across the membrane. Ten different subtypes have been described. In mammalia, 15 genes of P-type ATPases from subtypes II-A, II-B and II-C, that transport low-atomic-weight cations (Ca 2+ , Na + , K + and H + ), have been reported. They include reticulum and plasma-membrane Ca 2+ -ATPases, Na + /K + -ATPase and H + /K + -ATPases. Enterocytes and colonocytes show functional differences, which seem to be partially due to the differential expression of P-type ATPases. These enzymes have 9 structural motifs, being the phosphorylation (E) and the Mg 2+ ATP-binding (H) motifs the most preserved. These structural characteristics permitted developing a Multiplex-Nested-PCR (MN-PCR) for the simultaneous identification of different P-type ATPases. Thus, using MN-PCR, seven different cDNAs were cloned from enterocytes and colonocytes, including SERCA3, SERCA2, Na + /K + -ATPase α1-isoform, H + /K + -ATPase α2-isoform, PMCA1, PMCA4 and a cDNA-fragment that seems to be a new cassette-type splice-variant of the atp1a1 gen. PMCA4 in enterocytes and H + /K + -ATPase α2-isoform in colonocytes were differentially expressed. This cell-specific expression pattern is related with the distinctive enterocyte and colonocyte functions.

  10. Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development.

    Science.gov (United States)

    Schlieve, Christopher R; Mojica, Salvador Garcia; Holoyda, Kathleen A; Hou, Xiaogang; Fowler, Kathryn L; Grikscheit, Tracy C

    2016-01-01

    Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations. VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration. Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2. Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal

  11. Ketogenesis contributes to intestinal cell differentiation.

    Science.gov (United States)

    Wang, Qingding; Zhou, Yuning; Rychahou, Piotr; Fan, Teresa W-M; Lane, Andrew N; Weiss, Heidi L; Evers, B Mark

    2017-03-01

    The intestinal epithelium undergoes a continual process of proliferation, differentiation and apoptosis. Previously, we have shown that the PI3K/Akt/mTOR pathway has a critical role in intestinal homeostasis. However, the downstream targets mediating the effects of mTOR in intestinal cells are not known. Here, we show that the ketone body β-hydroxybutyrate (βHB), an endogenous inhibitor of histone deacetylases (HDACs) induces intestinal cell differentiation as noted by the increased expression of differentiation markers (Mucin2 (MUC2), lysozyme, IAP, sucrase-isomaltase, KRT20, villin, Caudal-related homeobox transcription factor 2 (CDX2) and p21 Waf1 ). Conversely, knockdown of the ketogenic mitochondrial enzyme hydroxymethylglutaryl CoA synthase 2 (HMGCS2) attenuated spontaneous differentiation in the human colon cancer cell line Caco-2. Overexpression of HMGCS2, which we found is localized specifically in the more differentiated portions of the intestinal mucosa, increased the expression of CDX2, thus further suggesting the contributory role of HMGCS2 in intestinal differentiation. In addition, mice fed a ketogenic diet demonstrated increased differentiation of intestinal cells as noted by an increase in the enterocyte, goblet and Paneth cell lineages. Moreover, we showed that either knockdown of mTOR or inhibition of mTORC1 with rapamycin increases the expression of HMGCS2 in intestinal cells in vitro and in vivo, suggesting a possible cross-talk between mTOR and HMGCS2/βHB signaling in intestinal cells. In contrast, treatment of intestinal cells with βHB or feeding mice with a ketogenic diet inhibits mTOR signaling in intestinal cells. Together, we provide evidence showing that HMGCS2/βHB contributes to intestinal cell differentiation. Our results suggest that mTOR acts cooperatively with HMGCS2/βHB to maintain intestinal homeostasis.

  12. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches

    DEFF Research Database (Denmark)

    Giannakis, Marios; Stappenbeck, Thaddeus S; Mills, Jason C

    2006-01-01

    We have sequenced 36,641 expressed sequence tags from laser capture microdissected adult mouse gastric and small intestinal epithelial progenitors, obtaining 4031 and 3324 unique transcripts, respectively. Using Gene Ontology (GO) terms, each data set was compared with cDNA libraries from intact ...

  13. Interpreting heterogeneity in intestinal tuft cell structure and function.

    Science.gov (United States)

    Banerjee, Amrita; McKinley, Eliot T; von Moltke, Jakob; Coffey, Robert J; Lau, Ken S

    2018-05-01

    Intestinal tuft cells are a morphologically unique cell type, best characterized by striking microvilli that form an apical tuft. These cells represent approximately 0.5% of gut epithelial cells depending on location. While they are known to express chemosensory receptors, their function has remained unclear. Recently, numerous groups have revealed startling insights into intestinal tuft cell biology. Here, we review the latest developments in understanding this peculiar cell type's structure and function. Recent advances in volumetric microscopy have begun to elucidate tuft cell ultrastructure with respect to its cellular neighbors. Moreover, single-cell approaches have revealed greater diversity in the tuft cell population than previously appreciated and uncovered novel markers to characterize this heterogeneity. Finally, advanced model systems have revealed tuft cells' roles in mucosal healing and orchestrating type 2 immunity against eukaryotic infection. While much remains unknown about intestinal tuft cells, these critical advances have illuminated the physiological importance of these previously understudied cells and provided experimentally tractable tools to interrogate this rare cell population. Tuft cells act as luminal sensors, linking the luminal microbiome to the host immune system, which may make them a potent clinical target for modulating host response to a variety of acute or chronic immune-driven conditions.

  14. Coronavirus infection of polarized epithelial cells

    NARCIS (Netherlands)

    Rossen, J W; Horzinek, M C; Rottier, P J

    1995-01-01

    Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

  15. Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

    DEFF Research Database (Denmark)

    Claesson, Mogens Helweg; Ropke, C

    1990-01-01

    We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu....... It is concluded that the epithelial stromal cells of the thymus, by acting as veto cells, may be responsible for the negative intrathymic selection of self-reactive thymocytes leading to elimination of the vast majority of immature thymic lymphocytes....

  16. Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor

    Science.gov (United States)

    Yoda, Kazutoyo; Miyazawa, Kenji; Hosoda, Masataka; Hiramatsu, Masaru; Yan, Fang; He, Fang

    2014-01-01

    Background Fermented milk is considered one of the best sources for efficient consumption of probiotic strains by hosts to promote good health. The purpose of this study was to investigate the effects of orally administering LGG-fermented milk (LGG milk) on intestinal inflammation and injury and to study the mechanisms of LGG milk's action. Methods LGG milk and non-LGG-fermented milk (non-LGG milk) were administered through gavage to mice before and during dextran sodium sulfate (DSS)-induced intestinal injury and colitis. Inflammatory/injury score and colon length were assessed. Intestinal epithelial cells were treated with the soluble fraction of LGG milk to detect its effects on the epidermal growth factor receptor (EGFR) and its down stream target, Akt activation, cytokine-induced apoptosis, and hydrogen peroxide (H2O2)-induced disruption of tight junctions. Results LGG milk treatment significantly reduced DSS-induced colonic inflammation and injury, and colon shortening in mice, compared to that in non-LGG milk-treated and untreated mice. The soluble fraction of LGG milk, but not non-LGG milk, stimulated activation of EGFR and Akt in a concentration-dependent manner, suppressed cytokine-induced apoptosis, and attenuated H2O2-induced disruption of tight junction complex in the intestinal epithelial cells. These effects of LGG milk were blocked by the EGFR kinase inhibitor. LGG milk, but not non-LGG milk, contained two soluble proteins, p40 and p75, which have been reported to promote survival and growth of intestinal epithelial cells through activation of EGFR. Depletion of p40 and p75 from LGG milk abolished the effects of LGG milk on prevention of cytokine-induced apoptosis and H2O2-induced disruption of tight junctions. Conclusions These results suggest that LGG milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins. PMID:23468308

  17. Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor.

    Science.gov (United States)

    Yoda, Kazutoyo; Miyazawa, Kenji; Hosoda, Masataka; Hiramatsu, Masaru; Yan, Fang; He, Fang

    2014-02-01

    Fermented milk is considered one of the best sources for efficient consumption of probiotic strains by hosts to promote good health. The purpose of this study was to investigate the effects of orally administering LGG-fermented milk (LGG milk) on intestinal inflammation and injury and to study the mechanisms of LGG milk's action. LGG milk and non-LGG-fermented milk (non-LGG milk) were administered through gavage to mice before and during dextran sodium sulfate (DSS)-induced intestinal injury and colitis. Inflammatory/injury score and colon length were assessed. Intestinal epithelial cells were treated with the soluble fraction of LGG milk to detect its effects on the epidermal growth factor receptor (EGFR) and its downstream target, Akt activation, cytokine-induced apoptosis, and hydrogen peroxide (H2O2)-induced disruption of tight junctions. LGG milk treatment significantly reduced DSS-induced colonic inflammation and injury, and colon shortening in mice, compared to that in non-LGG milk-treated and -untreated mice. The soluble fraction of LGG milk, but not non-LGG milk, stimulated the activation of EGFR and Akt in a concentration-dependent manner, suppressed cytokine-induced apoptosis, and attenuated H2O2-induced disruption of tight junction complex in the intestinal epithelial cells. These effects of LGG milk were blocked by the EGFR kinase inhibitor. LGG milk, but not non-LGG milk, contained two soluble proteins, p40 and p75, that have been reported to promote survival and growth of intestinal epithelial cells through the activation of EGFR. Depletion of p40 and p75 from LGG milk abolished the effects of LGG milk on prevention of cytokine-induced apoptosis and H2O2-induced disruption of tight junctions. These results suggest that LGG milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins.

  18. Epithelial structure and function in the hen lower intestine

    DEFF Research Database (Denmark)

    Laverty, G.; Elbrønd, Vibeke Sødring; Árnason, Sigvatur S.

    2006-01-01

    In birds, transport processes in the lower intestine mediate absorption of ions, water and a variety of organic substrates, including significant amounts of glucose, amino acids derived from protein associated with urate spheres, and short-chain fatty acids derived from fermentation processes. Th...

  19. Heterogeneity of limbal basal epithelial progenitor cells.

    Science.gov (United States)

    Hayashida, Yasutaka; Li, Wei; Chen, Ying-Ting; He, Hua; Chen, Szu-yu; Kheirkah, Ahmad; Zhu, Ying-Tien; Matsumoto, Yukihiro; Tseng, Scheffer C G

    2010-11-01

    Although corneal epithelial stem cells (SCs) are located at the limbus between the cornea and the conjunctiva, not all limbal basal epithelial cells are SCs. Using 2 dispase digestions to remove different amounts of limbal basal epithelial cells for cross-sections, flat mounts, and cytospin preparations, double immunostaining to pancytokeratins (PCK) and vimentin (Vim) identified 3 p63+ epithelial progenitors such as PCK-/Vim+, PCK/Vim, and PCK-/Vim+ and 1 p63+ mesenchymal cell, PCK-/Vim+. PCK-/Vim- progenitors had the smallest cell size were 10-20 times more enriched on collagen I-coated dishes in the 5-minute rapid adherent fraction that contained the highest percentage of p63+ cells but the lowest percentage of cytokeratin12+ cells, and gave rise to high Ki67 labeling and vivid clonal growth. In contrast, PCK+/Vim+ and PCK+/Vim- progenitors were found more in the slow-adherent fraction and yielded poor clonal growth. PCK/Vim progenitors and clusters of PCK-/Vim+ mesenchymal cells, which were neither melanocytes nor Langerhans cells, were located in the limbal basal region. Therefore, differential expression of PCK and Vim helps identify small PCK-/Vim- cells as the most likely candidate for SCs among a hierarchy of heterogeneous limbal basal progenitors, and their close association with PCK-/Vim+ presumed "niche" cells.

  20. Interleukin-8, CXCL1, and MicroRNA miR-146a Responses to Probiotic Escherichia coli Nissle 1917 and Enteropathogenic E. coli in Human Intestinal Epithelial T84 and Monocytic THP-1 Cells after Apical or Basolateral Infection.

    Science.gov (United States)

    Sabharwal, Harshana; Cichon, Christoph; Ölschläger, Tobias A; Sonnenborn, Ulrich; Schmidt, M Alexander

    2016-09-01

    Bacterium-host interactions in the gut proceed via directly contacted epithelial cells, the host's immune system, and a plethora of bacterial factors. Here we characterized and compared exemplary cytokine and microRNA (miRNA) responses of human epithelial and THP-1 cells toward the prototype enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6) and the probiotic strain Escherichia coli Nissle 1917 (EcN) (O6:K5:H1). Human T84 and THP-1 cells were used as cell culture-based model systems for epithelial and monocytic cells. Polarized T84 monolayers were infected apically or basolaterally. Bacterial challenges from the basolateral side resulted in more pronounced cytokine and miRNA responses than those observed for apical side infections. Interestingly, the probiotic EcN also caused a pronounced transcriptional increase of proinflammatory CXCL1 and interleukin-8 (IL-8) levels when human T84 epithelial cells were infected from the basolateral side. miR-146a, which is known to regulate adaptor molecules in Toll-like receptor (TLR)/NF-κB signaling, was found to be differentially regulated in THP-1 cells between probiotic and pathogenic bacteria. To assess the roles of flagella and flagellin, we employed several flagellin mutants of EcN. EcN flagellin mutants induced reduced IL-8 as well as CXCL1 responses in T84 cells, suggesting that flagellin is an inducer of this cytokine response. Following infection with an EPEC type 3 secretion system (T3SS) mutant, we observed increased IL-8 and CXCL1 transcription in T84 and THP-1 cells compared to that in wild-type EPEC. This study emphasizes the differential induction of miR-146a by pathogenic and probiotic E. coli strains in epithelial and immune cells as well as a loss of probiotic properties in EcN interacting with cells from the basolateral side. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  1. Development of Functional Microfold (M Cells from Intestinal Stem Cells in Primary Human Enteroids.

    Directory of Open Access Journals (Sweden)

    Joshua D Rouch

    Full Text Available Intestinal microfold (M cells are specialized epithelial cells that act as gatekeepers of luminal antigens in the intestinal tract. They play a critical role in the intestinal mucosal immune response through transport of viruses, bacteria and other particles and antigens across the epithelium to immune cells within Peyer's patch regions and other mucosal sites. Recent studies in mice have demonstrated that M cells are generated from Lgr5+ intestinal stem cells (ISCs, and that infection with Salmonella enterica serovar Typhimurium increases M cell formation. However, it is not known whether and how these findings apply to primary human small intestinal epithelium propagated in an in vitro setting.Human intestinal crypts were grown as monolayers with growth factors and treated with recombinant RANKL, and assessed for mRNA transcripts, immunofluorescence and uptake of microparticles and S. Typhimurium.Functional M cells were generated by short-term culture of freshly isolated human intestinal crypts in a dose- and time-dependent fashion. RANKL stimulation of the monolayer cultures caused dramatic induction of the M cell-specific markers, SPIB, and Glycoprotein-2 (GP2 in a process primed by canonical WNT signaling. Confocal microscopy demonstrated a pseudopod phenotype of GP2-positive M cells that preferentially take up microparticles. Furthermore, infection of the M cell-enriched cultures with the M cell-tropic enteric pathogen, S. Typhimurium, led to preferential association of the bacteria with M cells, particularly at lower inoculum sizes. Larger inocula caused rapid induction of M cells.Human intestinal crypts containing ISCs can be cultured and differentiate into an epithelial layer with functional M cells with characteristic morphological and functional properties. This study is the first to demonstrate that M cells can be induced to form from primary human intestinal epithelium, and that S. Typhimurium preferentially infect these cells in an

  2. BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation

    Science.gov (United States)

    Reddy, Vishruth K.; Short, Sarah P.; Barrett, Caitlyn W.; Mittal, Mukul K.; Keating, Cody E.; Thompson, Joshua J.; Harris, Elizabeth I.; Revetta, Frank; Bader, David M.; Brand, Thomas; Washington, M. Kay; Williams, Christopher S.

    2016-01-01

    Blood Vessel Epicardial Substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves−/− mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wildtype (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves−/− mice. To examine stem cell function after BVES deletion, we employed ex vivo 3D-enteroid cultures. Bves−/− enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar “CBC” and “+4” stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves−/− enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves−/− mice demonstrated significantly greater small intestinal crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves−/− mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. PMID:26891025

  3. Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

    DEFF Research Database (Denmark)

    Sakamori, Ryotaro; Das, Soumyashree; Yu, Shiyan

    2012-01-01

    The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases...... reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells...... activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data...

  4. Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines.

    Science.gov (United States)

    Drago, Sandro; El Asmar, Ramzi; Di Pierro, Mariarosaria; Grazia Clemente, Maria; Tripathi, Amit; Sapone, Anna; Thakar, Manjusha; Iacono, Giuseppe; Carroccio, Antonio; D'Agate, Cinzia; Not, Tarcisio; Zampini, Lucia; Catassi, Carlo; Fasano, Alessio

    2006-04-01

    Little is known about the interaction of gliadin with intestinal epithelial cells and the mechanism(s) through which gliadin crosses the intestinal epithelial barrier. We investigated whether gliadin has any immediate effect on zonulin release and signaling. Both ex vivo human small intestines and intestinal cell monolayers were exposed to gliadin, and zonulin release and changes in paracellular permeability were monitored in the presence and absence of zonulin antagonism. Zonulin binding, cytoskeletal rearrangement, and zonula occludens-1 (ZO-1) redistribution were evaluated by immunofluorescence microscopy. Tight junction occludin and ZO-1 gene expression was evaluated by real-time polymerase chain reaction (PCR). When exposed to gliadin, zonulin receptor-positive IEC6 and Caco2 cells released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell cytoskeleton, loss of occludin-ZO1 protein-protein interaction, and increased monolayer permeability. Pretreatment with the zonulin antagonist FZI/0 blocked these changes without affecting zonulin release. When exposed to luminal gliadin, intestinal biopsies from celiac patients in remission expressed a sustained luminal zonulin release and increase in intestinal permeability that was blocked by FZI/0 pretreatment. Conversely, biopsies from non-celiac patients demonstrated a limited, transient zonulin release which was paralleled by an increase in intestinal permeability that never reached the level of permeability seen in celiac disease (CD) tissues. Chronic gliadin exposure caused down-regulation of both ZO-1 and occludin gene expression. Based on our results, we concluded that gliadin activates zonulin signaling irrespective of the genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules.

  5. Media from macrophages co-incubated with Enterococcus faecalis induces epithelial cell monolayer reassembly and altered cell morphology.

    Directory of Open Access Journals (Sweden)

    Natalia Belogortseva

    Full Text Available Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE and serine protease (SprE leads to resultant condition media (CM capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis.

  6. Media from macrophages co-incubated with Enterococcus faecalis induces epithelial cell monolayer reassembly and altered cell morphology.

    Science.gov (United States)

    Belogortseva, Natalia; Krezalek, Monika; Guyton, Kristina; Labno, Christine; Poroyko, Valeriy; Zaborina, Olga; Alverdy, John C

    2017-01-01

    Signal exchange between intestinal epithelial cells, microbes and local immune cells is an important mechanism of intestinal homeostasis. Given that intestinal macrophages are in close proximity to both the intestinal epithelium and the microbiota, their pathologic interactions may result in epithelial damage. The present study demonstrates that co-incubation of murine macrophages with E. faecalis strains producing gelatinase (GelE) and serine protease (SprE) leads to resultant condition media (CM) capable of inducing reassembly of primary colonic epithelial cell monolayers. Following the conditioned media (CM) exposure, some epithelial cells are shed whereas adherent cells are observed to undergo dissolution of cell-cell junctions and morphologic transformation with actin cytoskeleton reorganization resulting in flattened and elongated shapes. These cells exhibit marked filamentous filopodia and lamellipodia formation. Cellular reorganization is not observed when epithelial monolayers are exposed to: CM from macrophages co-incubated with E. faecalis GelE/SprE-deficient mutants, CM from macrophages alone, or E. faecalis (GelE/SprE) alone. Flow cytometry analysis reveals increased expression of CD24 and CD44 in cells treated with macrophage/E. faecalis CM. This finding in combination with the appearance colony formation in matrigel demonstrate that the cells treated with macrophage/E. faecalis CM contain a higher proportion progenitor cells compared to untreated control. Taken together, these findings provide evidence for a triangulated molecular dialogue between E. faecalis, macrophages and colonic epithelial cells, which may have important implications for conditions in the gut that involve inflammation, injury or tumorigenesis.

  7. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    Science.gov (United States)

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  8. Intestinal stem cells and the colorectal cancer microenvironment.

    Science.gov (United States)

    Ong, Bryan A; Vega, Kenneth J; Houchen, Courtney W

    2014-02-28

    Colorectal cancer (CRC) remains a highly fatal condition in part due to its resilience to treatment and its propensity to spread beyond the site of primary occurrence. One possible avenue for cancer to escape eradication is via stem-like cancer cells that, through phenotypic heterogeneity, are more resilient than other tumor constituents and are key contributors to cancer growth and metastasis. These proliferative tumor cells are theorized to possess many properties akin to normal intestinal stem cells. Not only do these CRC "stem" cells demonstrate similar restorative ability, they also share many cell pathways and surface markers in common, as well as respond to the same key niche stimuli. With the improvement of techniques for epithelial stem cell identification, our understanding of CRC behavior is also evolving. Emerging evidence about cellular plasticity and epithelial mesenchymal transition are shedding light onto metastatic CRC processes and are also challenging fundamental concepts about unidirectional epithelial proliferation. This review aims to reappraise evidence supporting the existence and behavior of CRC stem cells, their relationship to normal stem cells, and their possible dependence on the stem cell niche.

  9. Oral activated charcoal adsorbent (AST-120) ameliorates chronic kidney disease-induced intestinal epithelial barrier disruption.

    Science.gov (United States)

    Vaziri, Nosratola D; Yuan, Jun; Khazaeli, Mahyar; Masuda, Yuichi; Ichii, Hirohito; Liu, Shuman

    2013-01-01

    Chronic kidney disease (CKD) impairs intestinal barrier function which by allowing influx of noxious products causes systemic inflammation. We have recently shown that intestinal barrier dysfunction in CKD is due to degradation of epithelial tight junction (TJ) which is, in part, mediated by influx of urea and its conversion to ammonia by microbial urease. We hypothesized that by adsorbing urea and urea-derived ammonia, oral activated charcoal (AST-120) may ameliorate CKD-induced intestinal epithelial barrier disruption and systemic inflammation. Rats were randomized to the CKD or control groups. The CKD group was fed a chow containing 0.7% adenine for 2 weeks. They were then randomized to receive a chow with or without AST-120 (4 g/kg/day) for 2 weeks. Rats consuming regular diet served as controls. Animals were then euthanized, colons were removed and processed for Western blot and immunohistology, and plasma was used to measure endotoxin and oxidative and inflammatory markers. Compared with the controls, the untreated CKD rats showed elevated plasma endotoxin, IL-6, TNF-α, MCP-1, CINC-3, L-selectin, ICAM-1, and malondialdehyde, and depletions of colonic epithelial TJ proteins, claudin-1, occludin, and ZO1. Administration of AST-120 resulted in partial restoration of the epithelial TJ proteins and reduction in plasma endotoxin and markers of oxidative stress and inflammation. CKD animals exhibited depletion of the key protein constituents of the colonic epithelial TJ which was associated with systemic inflammation, oxidative stress and endotoxemia. Administration of AST-120 attenuated uremia-induced disruption of colonic epithelial TJ and the associated endotoxemia, oxidative stress and inflammation. Copyright © 2013 S. Karger AG, Basel.

  10. Cellular Heterogeneity in the Mouse Esophagus Implicates the Presence of a Nonquiescent Epithelial Stem Cell Population

    Directory of Open Access Journals (Sweden)

    Aaron D. DeWard

    2014-10-01

    Full Text Available Because the esophageal epithelium lacks a defined stem cell niche, it is unclear whether all basal epithelial cells in the adult esophagus are functionally equivalent. In this study, we showed that basal cells in the mouse esophagus contained a heterogeneous population of epithelial cells, similar to other rapidly cycling tissues such as the intestine or skin. Using a combination of cell-surface markers, we separated primary esophageal tissue into distinct cell populations that harbored differences in stem cell potential. We also used an in vitro 3D organoid assay to demonstrate that Sox2, Wnt, and bone morphogenetic protein signaling regulate esophageal self-renewal. Finally, we labeled proliferating basal epithelial cells in vivo to show differing cell-cycle profiles and proliferation kinetics. Based on our results, we propose that a nonquiescent stem cell population resides in the basal epithelium of the mouse esophagus.

  11. Effect of Psychoneural Factors on Intestinal Epithelial Function

    Directory of Open Access Journals (Sweden)

    M Cecilia Berin

    1997-01-01

    Full Text Available Stress has been associated with abnormal gastrointestinal function, including diarrhea and abdominal pain, and stress-associated gastric ulceration has frequently been documented. Stress can also exacerbate ongoing pathophysiology and often precedes relapses in patients with inflammatory bowel disease or irritable bowel syndrome. The relatively new field of psychoneuroimmunology is involved with the elucidation of mechanisms that explain the link between the central nervous system and immune-mediated pathophysiology. Recent progress examining the interaction among the nervous system, the immune system and the epithelium of the intestine is discussed, and the evidence for central nervous sysytem control of this interaction is examined.

  12. Damaged intestinal epithelial integrity linked to microbial translocation in pathogenic simian immunodeficiency virus infections.

    Directory of Open Access Journals (Sweden)

    Jacob D Estes

    Full Text Available The chronic phase of HIV infection is marked by pathological activation of the immune system, the extent of which better predicts disease progression than either plasma viral load or CD4(+ T cell count. Recently, translocation of microbial products from the gastrointestinal tract has been proposed as an underlying cause of this immune activation, based on indirect evidence including the detection of microbial products and specific immune responses in the plasma of chronically HIV-infected humans or SIV-infected Asian macaques. We analyzed tissues from SIV-infected rhesus macaques (RMs to provide direct in situ evidence for translocation of microbial constituents from the lumen of the intestine into the lamina propria and to draining and peripheral lymph nodes and liver, accompanied by local immune responses in affected tissues. In chronically SIV-infected RMs this translocation is associated with breakdown of the integrity of the epithelial barrier of the gastrointestinal (GI tract and apparent inability of lamina propria macrophages to effectively phagocytose translocated microbial constituents. By contrast, in the chronic phase of SIV infection in sooty mangabeys, we found no evidence of epithelial barrier breakdown, no increased microbial translocation and no pathological immune activation. Because immune activation is characteristic of the chronic phase of progressive HIV/SIV infections, these findings suggest that increased microbial translocation from the GI tract, in excess of capacity to clear the translocated microbial constituents, helps drive pathological immune activation. Novel therapeutic approaches to inhibit microbial translocation and/or attenuate chronic immune activation in HIV-infected individuals may complement treatments aimed at direct suppression of viral replication.

  13. Intestinal lineage commitment of embryonic stem cells.

    Science.gov (United States)

    Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles

    2011-01-01

    Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue. Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  14. Membrane lipidome of an epithelial cell line

    DEFF Research Database (Denmark)

    Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

    2011-01-01

    Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to glycosphingolipid, together with an increase in plasmalogen, phosphatidylethanolamine, and cholesterol content, whereas the opposite changes took place during an epithelial-to-mesenchymal transition. Moreover, during polarization, the sphingolipids became longer, more saturated, and more hydroxylated as required...

  15. Protective effect of intestinal trefoil factor on injury of intestinal epithelial tight junction induced by platelet activating factor.

    Science.gov (United States)

    Xu, Ling-fen; Teng, Xu; Guo, Jing; Sun, Mei

    2012-02-01

    Intestinal barrier dysfunction plays an important role in the pathogenesis of inflammatory bowel disease (IBD). To evaluate the effect of intestinal trefoil factor (ITF) on increased intestinal permeability and its association with tight junction proteins, an in vitro intestinal epithelia barrier model was established with Caco-2 cells and treated with platelet-activating factor (PAF). We found that exposing cells to 0.3 M ITF (30 min before or 30 min after PAF treatment) attenuated the PAF-induced changes in transepithelial electrical resistance and Lucifer yellow flux. A quantitative RT-PCR and western blot analysis revealed that ITF suppressed PAF-induced downregulation of tight junction proteins claudin-1 and ZO-1 expression; furthermore, an abnormal localization and distribution of these proteins was inhibited, as assessed by immunofluorescence staining. These results suggest that ITF decreases mucosal permeability and shows potential as a therapy for treating IBD.

  16. Oral Administration of Probiotics Increases Paneth Cells and Intestinal Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Silvia I. Cazorla

    2018-04-01

    Full Text Available The huge amount of intestinal bacteria represents a continuing threat to the intestinal barrier. To meet this challenge, gut epithelial cells produce antimicrobial peptides (AMP that act at the forefront of innate immunity. We explore whether this antimicrobial activity and Paneth cells, the main intestinal cell responsible of AMP production, are influenced by probiotics administration, to avoid the imbalance of intestinal microbiota and preserve intestinal barrier. Administration of Lactobacillus casei CRL 431 (Lc 431 and L. paracasei CNCM I-1518 (Lp 1518 to 42 days old mice, increases the number of Paneth cells on small intestine, and the antimicrobial activity against the pathogens Staphylococcus aureus and Salmonella Typhimurium in the intestinal fluids. Specifically, strong damage of the bacterial cell with leakage of cytoplasmic content, and cellular fragmentation were observed in S. Typhimurium and S. aureus. Even more important, probiotics increase the antimicrobial activity of the intestinal fluids at the different ages, from weaning (21 days old to old age (180 days old. Intestinal antimicrobial activity stimulated by oral probiotics, do not influence significantly the composition of total anaerobic bacteria, lactobacilli and enterobacteria in the large intestine, at any age analyzed. This result, together with the antimicrobial activity observed against the same probiotic bacteria; endorse the regular consumption of probiotics without adverse effect on the intestinal homeostasis in healthy individuals. We demonstrate that oral probiotics increase intestinal antimicrobial activity and Paneth cells in order to strengthen epithelial barrier against pathogens. This effect would be another important mechanism by which probiotics protect the host mainly against infectious diseases.

  17. Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers.

    Directory of Open Access Journals (Sweden)

    Judit Váradi

    Full Text Available Alpha-melanocyte-stimulating hormone (α-MSH is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines.

  18. Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers

    Science.gov (United States)

    Váradi, Judit; Harazin, András; Fenyvesi, Ferenc; Réti-Nagy, Katalin; Gogolák, Péter; Vámosi, György; Bácskay, Ildikó; Fehér, Pálma; Ujhelyi, Zoltán; Vasvári, Gábor; Róka, Eszter; Haines, David; Deli, Mária A.; Vecsernyés, Miklós

    2017-01-01

    Alpha-melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB) was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines. PMID:28103316

  19. In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes

    Directory of Open Access Journals (Sweden)

    Hornsey Mark A

    2006-05-01

    Full Text Available Abstract Background Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. Results The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8 and mesenchymal (smooth muscle actin markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase, goblet cells (Periodic Acid Schiff positive, enteroendocrine cells (chromogranin A and Paneth cells (lysozyme. Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. Conclusion We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up

  20. Intestinal epithelial apoptosis initiates gross bowel necrosis in an experimental rat model of neonatal necrotizing enterocolitis.

    Science.gov (United States)

    Jilling, Tamas; Lu, Jing; Jackson, Michele; Caplan, Michael S

    2004-04-01

    The histopathology of necrotizing enterocolitis (NEC) is characterized by destruction of the mucosal layer in initial stages and by transmural necrosis of the intestinal wall in advanced stages of the disease. To test the hypothesis that enhanced epithelial apoptosis is an initial event underlying the gross histologic changes, we analyzed epithelial apoptosis and tissue morphology in an animal model of NEC and evaluated the effect of caspase inhibition on the incidence of experimental NEC in this model. Apoptosis was analyzed with terminal deoxynucleotidyltransferase-mediated dUTP-FITC nick end labeling (TUNEL) staining in intestinal sections and by measuring caspase 3 activity from intestinal lysates of neonatal rats subjected to formula feeding and cold/asphyxia stress (FFCAS) and from mother-fed (MF) controls. Morphologic evaluation was based on hematoxylin and eosin staining of intestinal sections. FFCAS resulted in histologic changes consistent with NEC, which were absent from MF animals. FFCAS was also associated with a significantly increased rate of nuclear DNA fragmentation in the small intestinal epithelium compared with MF. Elevated tissue caspase 3 activity confirmed the presence of apoptosis in samples with increased DNA fragmentation. Analysis of the coincidence of morphologic damage and apoptosis in corresponding tissue sections indicated that apoptosis precedes gross morphologic changes in this model. Furthermore, supplementation of formula with 8 boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, significantly reduced the incidences of apoptosis and experimental NEC. These findings indicate that in neonatal rats FFCAS induces epithelial apoptosis that serves as an underlying cause for subsequent gross tissue necrosis.

  1. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    Science.gov (United States)

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  2. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    Directory of Open Access Journals (Sweden)

    Dismuke Adria D

    2009-07-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  3. Lentiviral-Mediated Transgene Expression Can Potentiate Intestinal Mesenchymal-Epithelial Signaling

    Directory of Open Access Journals (Sweden)

    Kohn Aimee

    2009-01-01

    Full Text Available Abstract Mesenchymal-epithelial signaling is essential for the development of many organs and is often disrupted in disease. In this study, we demonstrate the use of lentiviral-mediated transgene delivery as an effective approach for ectopic transgene expression and an alternative to generation of transgenic animals. One benefit to this approach is that it can be used independently or in conjunction with established transgenic or knockout animals for studying modulation of mesenchymal-epithelial interactions. To display the power of this approach, we explored ectopic expression of a Wnt ligand in the mouse intestinal mesenchyme and demonstrate its functional influence on the adjacent epithelium. Our findings highlight the efficient use of lentiviral-mediated transgene expression for modulating mesenchymal-epithelial interactions in vivo.

  4. Inhibition of Epithelial TNF-α Receptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

    Science.gov (United States)

    Zhou, Zijuan; Wang, Liang; Feng, Panpan; Yin, Lianhong; Wang, Chen; Zhi, Shengxu; Dong, Jianyi; Wang, Jingyu; Lin, Yuan; Chen, Dapeng; Xiong, Yongjian; Peng, Jinyong

    2017-01-01

    Activation of the TNF-α receptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

  5. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells

    Directory of Open Access Journals (Sweden)

    Park Tae

    2008-12-01

    Full Text Available Abstract Background Acinetobacter baumannii is a nosocomial pathogen of increasing importance, but the pathogenic mechanism of this microorganism has not been fully explored. This study investigated the potential of A. baumannii to invade epithelial cells and determined the role of A. baumannii outer membrane protein A (AbOmpA in interactions with epithelial cells. Results A. baumannii invaded epithelial cells by a zipper-like mechanism, which is associated with microfilament- and microtubule-dependent uptake mechanisms. Internalized bacteria were located in the membrane-bound vacuoles. Pretreatment of recombinant AbOmpA significantly inhibited the adherence to and invasion of A. baumannii in epithelial cells. Cell invasion of isogenic AbOmpA- mutant significantly decreased as compared with wild-type bacteria. In a murine pneumonia model, wild-type bacteria exhibited a severe lung pathology and induced a high bacterial burden in blood, whereas AbOmpA- mutant was rarely detected in blood. Conclusion A. baumannii adheres to and invades epithelial cells. AbOmpA plays a major role in the interactions with epithelial cells. These findings contribute to the understanding of A. baumannii pathogenesis in the early stage of bacterial infection.

  6. Live Faecalibacterium prausnitzii in an apical anaerobic model of the intestinal epithelial barrier

    NARCIS (Netherlands)

    Ulluwishewa, D.; Anderson, R.C.; Young, W.; McNabb, W.C.; Baarlen, van P.; Moughan, P.J.; Wells, J.M.; Roy, N.C.

    2015-01-01

    Faecalibacterium prausnitzii, an abundant member of the human commensal microbiota, has been proposed to have a protective role in the intestine. However, it is an obligate anaerobe, difficult to co-culture in viable form with oxygen-requiring intestinal cells. To overcome this limitation, a unique

  7. Mucosal innate immune cells regulate both gut homeostasis and intestinal inflammation.

    Science.gov (United States)

    Kurashima, Yosuke; Goto, Yoshiyuki; Kiyono, Hiroshi

    2013-12-01

    Continuous exposure of intestinal mucosal surfaces to diverse microorganisms and their metabolites reflects the biological necessity for a multifaceted, integrated epithelial and immune cell-mediated regulatory system. The development and function of the host cells responsible for the barrier function of the intestinal surface (e.g., M cells, Paneth cells, goblet cells, and columnar epithelial cells) are strictly regulated through both positive and negative stimulation by the luminal microbiota. Stimulation by damage-associated molecular patterns and commensal bacteria-derived microbe-associated molecular patterns provokes the assembly of inflammasomes, which are involved in maintaining the integrity of the intestinal epithelium. Mucosal immune cells located beneath the epithelium play critical roles in regulating both the mucosal barrier and the relative composition of the luminal microbiota. Innate lymphoid cells and mast cells, in particular, orchestrate the mucosal regulatory system to create a mutually beneficial environment for both the host and the microbiota. Disruption of mucosal homeostasis causes intestinal inflammation such as that seen in inflammatory bowel disease. Here, we review the recent research on the biological interplay among the luminal microbiota, epithelial cells, and mucosal innate immune cells in both healthy and pathological conditions. © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Intestinal Stem Cell Niche Insights Gathered from Both In Vivo and Novel In Vitro Models

    Directory of Open Access Journals (Sweden)

    Nikolce Gjorevski

    2017-01-01

    Full Text Available Intestinal stem cells are located at the base of the crypts and are surrounded by a complex structure called niche. This environment is composed mainly of epithelial cells and stroma which provides signals that govern cell maintenance, proliferation, and differentiation. Understanding how the niche regulates stem cell fate by controlling developmental signaling pathways will help us to define how stem cells choose between self-renewal and differentiation and how they maintain their undifferentiated state. Tractable in vitro assay systems, which reflect the complexity of the in vivo situation but provide higher level of control, would likely be crucial in identifying new players and mechanisms controlling stem cell function. Knowledge of the intestinal stem cell niche gathered from both in vivo and novel in vitro models may help us improve therapies for tumorigenesis and intestinal damage and make autologous intestinal transplants a feasible clinical practice.

  9. Intestinal endocrine cells in radiation enteritis

    International Nuclear Information System (INIS)

    Pietroletti, R.; Blaauwgeers, J.L.; Taat, C.W.; Simi, M.; Brummelkamp, W.H.; Becker, A.E.

    1989-01-01

    In this study, the intestinal endocrine cells were investigated in 13 surgical specimens affected by radiation enteritis. Endocrine cells were studied by means of Grimelius' silver staining and immunostaining for chromogranin, a general marker of endocrine cells. Positively stained cells were quantified by counting their number per unit length of muscularis mucosa. Results in radiation enteritis were compared with matched control specimens by using Student's t test. Chromogranin immunostaining showed a statistically significant increase of endocrine cells in radiation enteritis specimens compared with controls both in small and large intestine (ileum, 67.5 +/- 23.5 cells per unit length of muscularis mucosa in radiation enteritis versus 17.0 +/- 6.1 in controls; colon, 40.9 +/- 13.7 cells per unit length of muscularis mucosa in radiation enteritis versus 9.5 +/- 4.1 in controls--p less than 0.005 in both instances). Increase of endocrine cells was demonstrated also by Grimelius' staining; however, without reaching statistical significance. It is not clear whether or not the increase of endocrine cells in radiation enteritis reported in this study is caused by a hyperplastic response or by a sparing phenomenon. We should consider that increased endocrine cells, when abnormally secreting their products, may be involved in some of the clinical features of radiation enteropathy. In addition, as intestinal endocrine cells produce trophic substances to the intestine, their increase could be responsible for the raised risk of developing carcinoma of the intestine in long standing radiation enteritis

  10. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  11. Development of Thymic Epithelial Cells

    DEFF Research Database (Denmark)

    Ulyanchenko, Svetlana; Vaidya, Harsh J.; O'Neill, Kathy E.

    2016-01-01

    The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell-mediated imm......The thymus is the primary lymphoid organ in which the T cell repertoire is generated. The complex cellularity of this organ is uniquely designed to facilitate T cell development: defects in thymus development or function can cause immunodeficiencies ranging from the absence of T cell......-mediated immunity to broad-spectrum autoimmune disease. Peak thymus size and output occurs early in life, after which the thymus undergoes a natural process of involution. This results in the progressive loss of functional thymus tissue and correspondingly in decreased production of new naïve T cells with age...... - contributing to the diminished capacity of the aged immune system to adequately respond to new antigenic challenge. Age-related thymic involutions, together with the thymic involutions associated with cytotoxic therapies (e.g., radio- or chemotherapy), have raised interest in development of clinically useful...

  12. Epithelial Cell-Neutrophil Interactions in the Alimentary Tract: A Complex Dialog in Mucosal Surveillance and Inflammation

    Directory of Open Access Journals (Sweden)

    Sean P. Colgan

    2002-01-01

    Full Text Available Inflammatory diseases of mucosal organs as diverse as the lung, kidney, and intestine, inevitably require the intimate interactions of neutrophils with columnar epithelia. The physiologic consequences of such interactions often determine endpoint organ function, and for this reason, much recent interest has developed in identifying mechanisms and novel targets for the treatment of mucosal inflammation. Elegant in vitro model systems incorporating purified human neutrophils and human epithelial cells grown in physiologic orientations have aided in discovery of new and insightful pathways to define basic inflammatory pathways. Here, we will review the recent literature regarding the interactions between columnar epithelial cells and neutrophils, with an emphasis on intestinal epithelial cells, structural aspects of neutrophil transepithelial migration, molecular determinants of neutrophil-epithelial cell interactions, as well as modulation of these pathways. These recent studies highlight the dynamic nature of these pathways and lend insight into the complexity of treating mucosal inflammation.

  13. The intestinal tuft cell nanostructure in 3D.

    Science.gov (United States)

    Hoover, Ben; Baena, Valentina; Kaelberer, Melanie M; Getaneh, Feven; Chinchilla, Skarleth; Bohórquez, Diego V

    2017-05-10

    Once referred to as "peculiar," tuft cells are enigmatic epithelial cells. Here, we reasoned that future functional studies could be derived from a complete account of the tuft cell ultrastructure. We identified and documented the volumetric ultrastructure at nanometer resolution (4-5 nm/pixel) of specific intestinal tuft cells. The techniques used were Serial Block-Face (SBF) and Automated Tape-collecting Ultra-Microtome (ATUM) Scanning Electron Microscopy (SEM). Our results exposed a short (~15 µm) basal cytoplasmic process devoid of secretory vesicles. Volume rendering of serial sections unveiled several thin cytospinules (~1 µm). These cytospinules project from the tuft cell into the nuclei of neighboring epithelial cells. Volume rendering also revealed within the tuft cell an elegant network of interconnected tubules. The network forms a passage from the base of the microvilli to the rough endoplasmic reticulum. Based on their location and microanatomy, the tuft cells' cytospinules, and tubular network, might facilitate the exchange of molecular cargo with nuclei of neighboring cells, and the gut lumen.

  14. The interplay between Entamoeba and enteropathogenic bacteria modulates epithelial cell damage.

    Directory of Open Access Journals (Sweden)

    José Manuel Galván-Moroyoqui

    Full Text Available BACKGROUND: Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba-bacteria interactions remain largely unexamined. METHODOLOGY: Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC, Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified. PRINCIPAL FINDINGS: E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica. CONCLUSIONS: Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage. SIGNIFICANCE

  15. Traction forces exerted by epithelial cell sheets

    International Nuclear Information System (INIS)

    Saez, A; Anon, E; Ghibaudo, M; Di Meglio, J-M; Hersen, P; Ladoux, B; Du Roure, O; Silberzan, P; Buguin, A

    2010-01-01

    Whereas the adhesion and migration of individual cells have been well described in terms of physical forces, the mechanics of multicellular assemblies is still poorly understood. Here, we study the behavior of epithelial cells cultured on microfabricated substrates designed to measure cell-to-substrate interactions. These substrates are covered by a dense array of flexible micropillars whose deflection enables us to measure traction forces. They are obtained by lithography and soft replica molding. The pillar deflection is measured by video microscopy and images are analyzed with home-made multiple particle tracking software. First, we have characterized the temporal and spatial distributions of traction forces of cellular assemblies of various sizes. The mechanical force balance within epithelial cell sheets shows that the forces exerted by neighboring cells strongly depend on their relative position in the monolayer: the largest deformations are always localized at the edge of the islands of cells in the active areas of cell protrusions. The average traction stress rapidly decreases from its maximum value at the edge but remains much larger than the inherent noise due to the force resolution of our pillar tracking software, indicating an important mechanical activity inside epithelial cell islands. Moreover, these traction forces vary linearly with the rigidity of the substrate over about two decades, suggesting that cells exert a given amount of deformation rather than a force. Finally, we engineer micropatterned substrates supporting pillars with anisotropic stiffness. On such substrates cellular growth is aligned with respect to the stiffest direction in correlation with the magnitude of the applied traction forces.

  16. Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis.

    Science.gov (United States)

    Perrone, Erin E; Jung, Enjae; Breed, Elise; Dominguez, Jessica A; Liang, Zhe; Clark, Andrew T; Dunne, W Michael; Burd, Eileen M; Coopersmith, Craig M

    2012-07-01

    Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia-induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus-induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid-/- mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand-/- mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

  17. Epithelial structure and function in the hen lower intestine

    DEFF Research Database (Denmark)

    Laverty, G.; Elbrønd, Vibeke Sødring; Árnason, Sigvatur S.

    2006-01-01

    in turn varies inversely with dietary salt content. Numerous studies have shown that following acclimation to low-salt diets, sodium-linked organic substrate co-transporters are largely suppressed, while electrogenic sodium channels (ENaCs) are induced. These changes can be manipulated, at least in part......, by resalination of low-salt hens, or by aldosterone administration to high-salt-acclimated birds. In the coprodeum, the changes in transport are paralleled by extensive remodelling of the mucosal surface, with low-salt acclimation increasing cell numbers, microvillus density and length and the proportion...

  18. Development of microfluidic cell culture devices towards an in vitro human intestinal barrier model

    DEFF Research Database (Denmark)

    Tan, Hsih-Yin

    Existing in vitro models of the human intestine such as the established epithelial cell line, Caco-2, cultured on porous membranes have been extensively used for assessing and predicting permeability and absorption of oral drugs in the pharmaceutical industries. However, such in vitro human intes...

  19. The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells.

    Science.gov (United States)

    Schewe, Matthias; Sacchetti, Andrea; Schmitt, Mark; Fodde, Riccardo

    2017-11-20

    The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberkühn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate 1 . Throughout the small intestine, Lgr5 + stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5 + stem cells through several key ligands as Wnt3, EGF, and Dll1 2 . When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine 3 . Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5 + and Paneth cells. Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5 + cells to associate and form organoids when co-cultured. This approach, here referred to as "organoid reconstitution assay" (ORA), allows the genetic and biochemical modification of Paneth or Lgr5 + stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

  20. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change in the transc......The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change...

  1. Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

    Science.gov (United States)

    Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

    2006-02-17

    Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

  2. Inhalation of methane preserves the epithelial barrier during ischemia and reperfusion in the rat small intestine.

    Science.gov (United States)

    Mészáros, András T; Büki, Tamás; Fazekas, Borbála; Tuboly, Eszter; Horváth, Kitti; Poles, Marietta Z; Szűcs, Szilárd; Varga, Gabriella; Kaszaki, József; Boros, Mihály

    2017-06-01

    Methane is part of the gaseous environment of the intestinal lumen. The purpose of this study was to elucidate the bioactivity of exogenous methane on the intestinal barrier function in an antigen-independent model of acute inflammation. Anesthetized rats underwent sham operation or 45-min occlusion of the superior mesenteric artery. A normoxic methane (2.2%)-air mixture was inhaled for 15 min at the end of ischemia and at the beginning of a 60-min or 180-min reperfusion. The integrity of the epithelial barrier of the ileum was assessed by determining the lumen-to-blood clearance of fluorescent dextran, while microvascular permeability changes were detected by the Evans blue technique. Tissue levels of superoxide, nitrotyrosine, myeloperoxidase, and endothelin-1 were measured, the superficial mucosal damage was visualized and quantified, and the serosal microcirculation and mesenteric flow was recorded. Erythrocyte deformability and aggregation were tested in vitro. Reperfusion significantly increased epithelial permeability, worsened macro- and microcirculation, increased the production of proinflammatory mediators, and resulted in a rapid loss of the epithelium. Exogenous normoxic methane inhalation maintained the superficial mucosal structure, decreased epithelial permeability, and improved local microcirculation, with a decrease in reactive oxygen and nitrogen species generation. Both the deformability and aggregation of erythrocytes improved with incubation of methane. Normoxic methane decreases the signs of oxidative and nitrosative stress, improves tissue microcirculation, and thus appears to modulate the ischemia-reperfusion-induced epithelial permeability changes. These findings suggest that the administration of exogenous methane may be a useful strategy for maintaining the integrity of the mucosa sustaining an oxido-reductive attack. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Generation of Mouse Lung Epithelial Cells.

    Science.gov (United States)

    Kasinski, Andrea L; Slack, Frank J

    2013-08-05

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of Kras LSL-G12D/+ ; p53 LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra -G12D and p53 R172 . While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  4. Epithelial cell proliferation arrest induced by lactate and acetate from Lactobacillus casei and Bifidobacterium breve.

    Directory of Open Access Journals (Sweden)

    Takahiro Matsuki

    Full Text Available In an attempt to identify and characterize how symbiotic bacteria of the gut microbiota affect the molecular and cellular mechanisms of epithelial homeostasis, intestinal epithelial cells were co-cultured with either Lactobacillus or Bifidobacterium as bona fide symbionts to examine potential gene modulations. In addition to genes involved in the innate immune response, genes encoding check-point molecules controlling the cell cycle were among the most modulated in the course of these interactions. In the m-ICcl2 murine cell line, genes encoding cyclin E1 and cyclin D1 were strongly down regulated by L. casei and B. breve respectively. Cell proliferation arrest was accordingly confirmed. Short chain fatty acids (SCFA were the effectors of this modulation, alone or in conjunction with the acidic pH they generated. These results demonstrate that the production of SCFAs, a characteristic of these symbiotic microorganisms, is potentially an essential regulatory effector of epithelial proliferation in the gut.

  5. Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Millaku, Agron, E-mail: agron.mi@hotmail.com [Limnos-Company for Applied Ecology Ltd, Podlimbarskega 31, 1000 Ljubljana (Slovenia); Drobne, Damjana [University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana (Slovenia); Centre of Excellence, Advanced Materials and Technologies for the Future (CO NAMASTE), Jamova cesta 39, 1000 Ljubljana (Slovenia); Centre of Excellence, Nanoscience and Nanotechnology (Nanocentre), Jamova cesta 39, 1000 Ljubljana (Slovenia); Torkar, Matjaz [Institute of Metals and T