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Sample records for polarized human intestinal

  1. A novel method for the culture and polarized stimulation of human intestinal mucosa explants.

    PubMed

    Tsilingiri, Katerina; Sonzogni, Angelica; Caprioli, Flavio; Rescigno, Maria

    2013-05-01

    Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food. Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out. To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina

  2. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

  3. Ricin crosses polarized human intestinal cells and intestines of ricin-gavaged mice without evident damage and then disseminates to mouse kidneys.

    PubMed

    Flora, Alyssa D; Teel, Louise D; Smith, Mark A; Sinclair, James F; Melton-Celsa, Angela R; O'Brien, Alison D

    2013-01-01

    Ricin is a potent toxin found in the beans of Ricinus communis and is often lethal for animals and humans when aerosolized or injected and causes significant morbidity and occasional death when ingested. Ricin has been proposed as a bioweapon because of its lethal properties, environmental stability, and accessibility. In oral intoxication, the process by which the toxin transits across intestinal mucosa is not completely understood. To address this question, we assessed the impact of ricin on the gastrointestinal tract and organs of mice after dissemination of toxin from the gut. We first showed that ricin adhered in a specific pattern to human small bowel intestinal sections, the site within the mouse gut in which a variable degree of damage has been reported by others. We then monitored the movement of ricin across polarized human HCT-8 intestinal monolayers grown in transwell inserts and in HCT-8 cell organoids. We observed that, in both systems, ricin trafficked through the cells without apparent damage until 24 hours post intoxication. We delivered a lethal dose of purified fluorescently-labeled ricin to mice by oral gavage and followed transit of the toxin from the gastrointestinal tracts to the internal organs by in vivo imaging of whole animals over time and ex vivo imaging of organs at various time points. In addition, we harvested organs from unlabeled ricin-gavaged mice and assessed them for the presence of ricin and for histological damage. Finally, we compared serum chemistry values from buffer-treated versus ricin-intoxicated animals. We conclude that ricin transverses human intestinal cells and mouse intestinal cells in situ prior to any indication of enterocyte damage and that ricin rapidly reaches the kidneys of intoxicated mice. We also propose that mice intoxicated orally with ricin likely die from distributive shock.

  4. Expression and polarization of intercellular adhesion molecule-1 on human intestinal epithelia: consequences for CD11b/CD18-mediated interactions with neutrophils.

    PubMed Central

    Parkos, C. A.; Colgan, S. P.; Diamond, M. S.; Nusrat, A.; Liang, T. W.; Springer, T. A.; Madara, J. L.

    1996-01-01

    BACKGROUND: Epithelial dysfunction and patient symptoms in inflammatory intestinal diseases such as ulcerative colitis and Crohn's disease correlate with migration of neutrophils (PMN) across the intestinal epithelium. In vitro modeling of PMN transepithelial migration has revealed distinct differences from transendothelial migration. By using polarized monolayers of human intestinal epithelia (T84), PMN transepithelial migration has been shown to be dependent on the leukocyte integrin CD11b/CD18 (Mac-1), but not on CD11a/CD18 (LFA-1). Since intercellular adhesion molecule-I (ICAM-1) is an important endothelial counterreceptor for these integrins, its expression in intestinal epithelia and role in PMN-intestinal epithelial interactions was investigated. MATERIALS AND METHODS: A panel of antibodies against different domains of ICAM-1, polarized monolayers of human intestinal epithelia (T84), and natural human colonic epithelia were used to examine the polarity of epithelial ICAM-1 surface expression and the functional role of ICAM-1 in neutrophil-intestinal epithelial adhesive interactions. RESULTS: While no surface expression of ICAM-1 was detected on unstimulated T84 cells, interferon-gamma (IFN gamma) elicited a marked expression of ICAM-1 that selectively polarized to the apical epithelial membrane. Similarly, apically restricted surface expression of ICAM-1 was detected in natural human colonic epithelium only in association with active inflammation. With or without IFN gamma pre-exposure, physiologically directed (basolateral-to-apical) transepithelial migration of PMN was unaffected by blocking monoclonal antibodies (mAbs) to ICAM-1. In contrast, PMN migration across IFN gamma-stimulated monolayers in the reverse (apical-to-basolateral) direction was inhibited by anti-ICAM-1 antibodies. Adhesion studies revealed that T84 cells adhered selectively to purified CD11b/CD18 and such adherence, with or without IFN gamma pre-exposure, was unaffected by ICAM-1 m

  5. Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic

    PubMed Central

    1992-01-01

    The massive secretion of salt and water in cholera-induced diarrhea involves binding of cholera toxin (CT) to ganglioside GM1 in the apical membrane of intestinal epithelial cells, translocation of the enzymatically active A1-peptide across the membrane, and subsequent activation of adenylate cyclase located on the cytoplasmic surface of the basolateral membrane. Studies on nonpolarized cells show that CT is internalized by receptor-mediated endocytosis, and that the A1-subunit may remain membrane associated. To test the hypothesis that toxin action in polarized cells may involve intracellular movement of toxin- containing membranes, monolayers of the polarized intestinal epithelial cell line T84 were mounted in modified Ussing chambers and the response to CT was examined. Apical CT at 37 degrees C elicited a short circuit current (Isc: 48 +/- 2.1 microA/cm2; half-maximal effective dose, ED50 integral of 0.5 nM) after a lag of 33 +/- 2 min which bidirectional 22Na+ and 36Cl- flux studies showed to be due to electrogenic Cl- secretion. The time course of the CT-induced Isc response paralleled the time course of cAMP generation. The dose response to basolateral toxin at 37 degrees C was identical to that of apical CT but lag times (24 +/- 2 min) and initial rates were significantly less. At 20 degrees C, the Isc response to apical CT was more strongly inhibited (30-50%) than the response to basolateral CT, even though translocation occurred in both cases as evidenced by the formation of A1-peptide. A functional rhodamine-labeled CT-analogue applied apically or basolaterally at 20 degrees C was visualized only within endocytic vesicles close to apical or basolateral membranes, whereas movement into deeper apical structures was detected at 37 degrees C. At 15 degrees C, in contrast, reduction to the A1-peptide was completely inhibited and both apical and basolateral CT failed to stimulate Isc although Isc responses to 1 nM vasoactive intestinal peptide, 10 micro

  6. Intermediate Filaments and Polarization in the Intestinal Epithelium

    PubMed Central

    Coch, Richard A.; Leube, Rudolf E.

    2016-01-01

    The cytoplasmic intermediate filament cytoskeleton provides a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. This is particularly apparent in the intestinal epithelium, in which the intermediate filament network is localized below the apical terminal web region and is anchored to the apical junction complex. This arrangement is conserved from the nematode Caenorhabditis elegans to humans. The review summarizes compositional, morphological and functional features of the polarized intermediate filament cytoskeleton in intestinal cells of nematodes and mammals. We emphasize the cross talk of intermediate filaments with the actin- and tubulin-based cytoskeleton. Possible links of the intermediate filament system to the distribution of apical membrane proteins and the cell polarity complex are highlighted. Finally, we discuss how these properties relate to the establishment and maintenance of polarity in the intestine. PMID:27429003

  7. Fluorescence polarization studies of rat intestinal microvillus membranes.

    PubMed Central

    Schachter, D; Shinitzky, M

    1977-01-01

    Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin. PMID:14174

  8. Afa/Dr diffusely adhering Escherichia coli C1845 infection promotes selective injuries in the junctional domain of polarized human intestinal Caco-2/TC7 cells.

    PubMed

    Peiffer, I; Blanc-Potard, A B; Bernet-Camard, M F; Guignot, J; Barbat, A; Servin, A L

    2000-06-01

    The Afa/Dr diffusely adhering Escherichia coli (DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [(3)H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinant E. coli strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhesin-CD55 interaction is not sufficient for the induction of structural and functional TJ lesions. Moreover, using the actin filament-stabilizing agent Jasplakinolide, we demonstrate that the C1845-induced functional alterations in TJs are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845.

  9. [Optical properties of human normal small intestine tissue with theoretical model of optics about biological tissues at Ar+ laser and 532 nm laser and their linearly polarized laser irradiation in vitro].

    PubMed

    Wei, Hua-jiang; Xing, Da; Wu, Guo-yong; Jin, Ying; Gu, Huai-min

    2004-05-01

    A double-integrating-spheres system, basic principle of measuring technology of ray radiation, and optical model of biological tissues were used for the study. Optical properties of human normal small intestine tissue at 476.5, 488, 496.5, 514.5 and 532 nm laser and their linearly polarized laser irradiation were studied. The results of measurement showed that the total attenuation coefficient and scattering coefficient of the tissue at these wavelengths of laser and their linearly polarized laser irradiation increased with decreasing wavelengths. And obviously there was a distinction at 514.5 to 532 nm wavelength between lasers and their linearly polarized laser irradiation. Absorption coefficient of tissue at these wavelengths of laser and their linearly polarized laser irradiation increased with decreasing wavelengths. Absorption coefficient of tissue at 514.5 to 532 nm wavelength of laser was obviously decreasing, which was independent of these wavelengths of laser or their linearly polarized laser irradiation. Mean cosine of scattering of tissue at these wavelengths of laser and their linearly polarized laser irradiation also increased with decreasing wavelengths. But penetration depth of tissue at these wavelengths of laser and their linearly polarized laser irradiation also increased with increasing of wavelengths. Refractive index of tissue between these wavelengths of laser was within 1.38 to 1.48. Absorption coefficient, scattering coefficient, total attenuation coefficient, effective attenuation coefficients of tissue in Kubelka-Munk two-flux model at the same wavelength of laser and their linearly polarized laser irradiation showed no prominent distinction (P>0.01). Absorption coefficient, scattering coefficient, total attenuation coefficient, effective attenuation coefficients of tissue in Kubelka-Munk two-flux model at different wavelength of laser and their linearly polarized laser irradiation showed obvious distinction. Optical properties of tissue

  10. Structural and functional lesions in brush border of human polarized intestinal Caco-2/TC7 cells infected by members of the Afa/Dr diffusely adhering family of Escherichia coli.

    PubMed

    Peiffer, I; Guignot, J; Barbat, A; Carnoy, C; Moseley, S L; Nowicki, B J; Servin, A L; Bernet-Camard, M F

    2000-10-01

    Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.

  11. Polarizing intestinal epithelial cells electrically through Ror2

    PubMed Central

    Cao, Lin; McCaig, Colin D.; Scott, Roderick H.; Zhao, Siwei; Milne, Gillian; Clevers, Hans; Zhao, Min; Pu, Jin

    2014-01-01

    ABSTRACT The apicobasal polarity of enterocytes determines where the brush border membrane (apical membrane) will form, but how this apical membrane faces the lumen is not well understood. The electrical signal across the epithelium could serve as a coordinating cue, orienting and polarizing enterocytes. Here, we show that applying a physiological electric field to intestinal epithelial cells, to mimic the natural electric field created by the transepithelial potential difference, polarized phosphorylation of the actin-binding protein ezrin, increased expression of intestinal alkaline phosphatase (ALPI, a differentiation marker) and remodeled the actin cytoskeleton selectively on the cathode side. In addition, an applied electric field also activated ERK1/2 and LKB1 (also known as STK11), key molecules in apical membrane formation. Disruption of the tyrosine protein kinase transmembrane receptor Ror2 suppressed activation of ERK1/2 and LKB1 significantly, and subsequently inhibited apical membrane formation in enterocytes. Our findings indicate that the endogenous electric field created by the transepithelial potential difference might act as an essential coordinating signal for apical membrane formation at a tissue level, through activation of LKB1 mediated by Ror2–ERK signaling. PMID:24928904

  12. Polarization-sensitive multispectral tissue characterization for optimizing intestinal anastomosis

    NASA Astrophysics Data System (ADS)

    Cha, Jaepyeong; Triana, Brian; Shademan, Azad; Krieger, Axel; Kim, Peter C. W.; Kang, Jin U.

    2014-03-01

    A novel imaging system that recommends potential suture placement for anastomosis to surgeons is developed. This is achieved by a multispectral imaging system coupled with polarizers and image analysis software. We performed preliminary imaging of ex vivo porcine intestine to evaluate the system. Vulnerable tissue regions including blood vessels were successfully identified and segmented. Thickness of different tissue areas is visualized. Strategies towards optimal points for suture placements have been discussed. Preliminary data suggest our imaging platform and analysis algorithm may be useful in avoiding blood vessels, identifying optimal regions for suture placements to perform safer operations in possibly reduced time.

  13. Human intestinal capillariasis in Thailand

    PubMed Central

    Saichua, Prasert; Nithikathkul, Choosak; Kaewpitoon, Natthawut

    2008-01-01

    Intestinal capillariasis caused by Capillaria philippinensis appeared first in the Philippines and subsequently in Thailand, Japan, Iran, Egypt and Taiwan; major outbreaks have occurred in the Philippines and Thailand. This article reviews the epidemiology, history and sources of C. philippinensis infection in Thailand. The annual epidemiological surveillance reports indicated that 82 accumulated cases of intestinal capillariasis were found in Thailand from 1994-2006. That made Thailand a Capillaria-prevalent area. Sisaket, in northeast Thailand, was the first province which has reported intestinal capillariasis. Moreover, Buri Ram presented a high prevalence of intestinal capillariasis, totaling 24 cases from 1994-2006. About half of all cases have consumed raw or undercooked fish. However, even if the numbers of the intestinal capillariasis cases in Thailand is reduced, C. philippinensis infection cases are still reported. The improvement of personal hygiene, specifically avoiding consumption of undercooked fish and promoting a health education campaign are required. These strategies may minimize or eliminate C. philippinensis infection in Thailand. PMID:18203280

  14. Recruitment of CD55 and CD66e brush border-associated glycosylphosphatidylinositol-anchored proteins by members of the Afa/Dr diffusely adhering family of Escherichia coli that infect the human polarized intestinal Caco-2/TC7 cells.

    PubMed

    Guignot, J; Peiffer, I; Bernet-Camard, M F; Lublin, D M; Carnoy, C; Moseley, S L; Servin, A L

    2000-06-01

    The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

  15. The Contributions of Human Mini-Intestines to the Study of Intestinal Physiology and Pathophysiology.

    PubMed

    Yu, Huimin; Hasan, Nesrin M; In, Julie G; Estes, Mary K; Kovbasnjuk, Olga; Zachos, Nicholas C; Donowitz, Mark

    2017-02-10

    The lack of accessibility to normal and diseased human intestine and the inability to separate the different functional compartments of the intestine even when tissue could be obtained have held back the understanding of human intestinal physiology. Clevers and his associates identified intestinal stem cells and established conditions to grow "mini-intestines" ex vivo in differentiated and undifferentiated conditions. This pioneering work has made a new model of the human intestine available and has begun making contributions to the understanding of human intestinal transport in normal physiologic conditions and the pathophysiology of intestinal diseases. However, this model is reductionist and lacks many of the complexities of normal intestine. Consequently, it is not yet possible to predict how great the advances using this model will be for understanding human physiology and pathophysiology, nor how the model will be modified to include multiple other intestinal cell types and physical forces necessary to more closely approximate normal intestine. This review describes recent studies using mini-intestines, which have readdressed previously established models of normal intestinal transport physiology and newly examined intestinal pathophysiology. The emphasis is on studies with human enteroids grown either as three-dimensional spheroids or two-dimensional monolayers. In addition, comments are provided on mouse studies in cases when human studies have not yet been described.

  16. A geometric description of human intestine.

    PubMed

    Coşkun, Ihsaniye; Yildiz, Hüseyin; Arslan, Kadri; Yildiz, Bahri

    2007-01-01

    Mathematical models of natural phenomena play a central role in the physical sciences. Moreover, modeling of the organs draws from some beautiful areas of mathematics, such as nonlinear dynamics, multiscale transforms and stability analysis. In this study, a geometric recognition of the separate intestine sections (duodenum, jejunum, ileum, cecum and colon) of the human is presented. The human intestine was considered a tubular shape along a special curve and two male Turkish men were used for the modeling study. The length (cm) and diameter (mm) of the intestines were measured with a digital compass and formulated. These models were compared with their original photographs. It has been concluded that the geometric modeling and experimental work were consistent. These kinds of organ modeling techniques will also profit to medical lecturers to show 3-D figures to their students.

  17. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.

    PubMed

    Grant, Christa N; Mojica, Salvador Garcia; Sala, Frederic G; Hill, J Ryan; Levin, Daniel E; Speer, Allison L; Barthel, Erik R; Shimada, Hiroyuki; Zachos, Nicholas C; Grikscheit, Tracy C

    2015-04-15

    Short bowel syndrome (SBS) is a devastating condition in which insufficient small intestinal surface area results in malnutrition and dependence on intravenous parenteral nutrition. There is an increasing incidence of SBS, particularly in premature babies and newborns with congenital intestinal anomalies. Tissue-engineered small intestine (TESI) offers a therapeutic alternative to the current standard treatment, intestinal transplantation, and has the potential to solve its biggest challenges, namely donor shortage and life-long immunosuppression. We have previously demonstrated that TESI can be generated from mouse and human small intestine and histologically replicates key components of native intestine. We hypothesized that TESI also recapitulates native small intestine function. Organoid units were generated from mouse or human donor intestine and implanted into genetically identical or immunodeficient host mice. After 4 wk, TESI was harvested and either fixed and paraffin embedded or immediately subjected to assays to illustrate function. We demonstrated that both mouse and human tissue-engineered small intestine grew into an appropriately polarized sphere of intact epithelium facing a lumen, contiguous with supporting mesenchyme, muscle, and stem/progenitor cells. The epithelium demonstrated major ultrastructural components, including tight junctions and microvilli, transporters, and functional brush-border and digestive enzymes. This study demonstrates that tissue-engineered small intestine possesses a well-differentiated epithelium with intact ion transporters/channels, functional brush-border enzymes, and similar ultrastructural components to native tissue, including progenitor cells, whether derived from mouse or human cells.

  18. Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology.

    PubMed

    Zachos, Nicholas C; Kovbasnjuk, Olga; Foulke-Abel, Jennifer; In, Julie; Blutt, Sarah E; de Jonge, Hugo R; Estes, Mary K; Donowitz, Mark

    2016-02-19

    Identification of Lgr5 as the intestinal stem cell marker as well as the growth factors necessary to replicate adult intestinal stem cell division has led to the establishment of the methods to generate "indefinite" ex vivo primary intestinal epithelial cultures, termed "mini-intestines." Primary cultures developed from isolated intestinal crypts or stem cells (termed enteroids/colonoids) and from inducible pluripotent stem cells (termed intestinal organoids) are being applied to study human intestinal physiology and pathophysiology with great expectations for translational applications, including regenerative medicine. Here we discuss the physiologic properties of these cultures, their current use in understanding diarrhea-causing host-pathogen interactions, and potential future applications.

  19. Tipping elements in the human intestinal ecosystem

    PubMed Central

    Lahti, Leo; Salojärvi, Jarkko; Salonen, Anne; Scheffer, Marten; de Vos, Willem M.

    2014-01-01

    The microbial communities living in the human intestine can have profound impact on our well-being and health. However, we have limited understanding of the mechanisms that control this complex ecosystem. Here, based on a deep phylogenetic analysis of the intestinal microbiota in a thousand western adults, we identify groups of bacteria that exhibit robust bistable abundance distributions. These bacteria are either abundant or nearly absent in most individuals, and exhibit decreased temporal stability at the intermediate abundance range. The abundances of these bimodally distributed bacteria vary independently, and their abundance distributions are not affected by short-term dietary interventions. However, their contrasting alternative states are associated with host factors such as ageing and overweight. We propose that the bistable groups reflect tipping elements of the intestinal microbiota, whose critical transitions may have profound health implications and diagnostic potential. PMID:25003530

  20. Tipping elements in the human intestinal ecosystem.

    PubMed

    Lahti, Leo; Salojärvi, Jarkko; Salonen, Anne; Scheffer, Marten; de Vos, Willem M

    2014-07-08

    The microbial communities living in the human intestine can have profound impact on our well-being and health. However, we have limited understanding of the mechanisms that control this complex ecosystem. Here, based on a deep phylogenetic analysis of the intestinal microbiota in a thousand western adults, we identify groups of bacteria that exhibit robust bistable abundance distributions. These bacteria are either abundant or nearly absent in most individuals, and exhibit decreased temporal stability at the intermediate abundance range. The abundances of these bimodally distributed bacteria vary independently, and their abundance distributions are not affected by short-term dietary interventions. However, their contrasting alternative states are associated with host factors such as ageing and overweight. We propose that the bistable groups reflect tipping elements of the intestinal microbiota, whose critical transitions may have profound health implications and diagnostic potential.

  1. Transmigration route of Campylobacter jejuni across polarized intestinal epithelial cells: paracellular, transcellular or both?

    PubMed Central

    2013-01-01

    Intact intercellular junctions and cellular matrix contacts are crucial structural components for the formation and maintenance of epithelial barrier functions in humans to control the commensal flora and protect against intruding microbes. Campylobacter jejuni is one of the most important zoonotic pathogens causing food-borne gastroenteritis and potentially more severe diseases such as reactive arthritis or Guillain–Barré syndrome. Crossing the intestinal epithelial barrier and host cell invasion by C. jejuni are considered to represent the primary reasons of gut tissue damage in humans and various animal model systems including monkeys, piglets, rabbits, hamsters and ferrets. C. jejuni is also able to invade underlying tissues such as the lamina propria, can enter the bloodstream, and possibly reach distinct organs such as spleen, liver or mesenteric lymph nodes. However, the molecular mechanisms as well as major bacterial and host cell factors involved in these activities are poorly understood. Various models exist by which the pathogen can trigger its own transmigration across polarized intestinal epithelial cells in vitro, the paracellular and/or transcellular mechanism. Recent studies suggest that bacterial factors such as flagellum, serine protease HtrA and lipooligosaccharide LOS may play an active role in bacterial transmigration. Here we review our knowledge on transmigration of C. jejuni as well as some other Campylobacter species, and discuss the pros and cons for the route(s) taken to travel across polarized epithelial cell monolayers. These studies provide fresh insights into the infection strategies employed by this important pathogen. PMID:24079544

  2. A Three-Dimensional Cell Culture Model To Study Enterovirus Infection of Polarized Intestinal Epithelial Cells

    PubMed Central

    Drummond, Coyne G.

    2015-01-01

    ABSTRACT Despite serving as the primary entry portal for coxsackievirus B (CVB), little is known about CVB infection of the intestinal epithelium, owing at least in part to the lack of suitable in vivo models and the inability of cultured cells to recapitulate the complexity and structure associated with the gastrointestinal (GI) tract. Here, we report on the development of a three-dimensional (3-D) organotypic cell culture model of Caco-2 cells to model CVB infection of the gastrointestinal epithelium. We show that Caco-2 cells grown in 3-D using the rotating wall vessel (RWV) bioreactor recapitulate many of the properties of the intestinal epithelium, including the formation of well-developed tight junctions, apical-basolateral polarity, brush borders, and multicellular complexity. In addition, transcriptome analyses using transcriptome sequencing (RNA-Seq) revealed the induction of a number of genes associated with intestinal epithelial differentiation and/or intestinal processes in vivo when Caco-2 cells were cultured in 3-D. Applying this model to CVB infection, we found that although the levels of intracellular virus production were similar in two-dimensional (2-D) and 3-D Caco-2 cell cultures, the release of infectious CVB was enhanced in 3-D cultures at early stages of infection. Unlike CVB, the replication of poliovirus (PV) was significantly reduced in 3-D Caco-2 cell cultures. Collectively, our studies show that Caco-2 cells grown in 3-D using the RWV bioreactor provide a cell culture model that structurally and transcriptionally represents key aspects of cells in the human GI tract and can thus be used to expand our understanding of enterovirus-host interactions in intestinal epithelial cells. IMPORTANCE Coxsackievirus B (CVB), a member of the enterovirus family of RNA viruses, is associated with meningitis, pericarditis, diabetes, dilated cardiomyopathy, and myocarditis, among other pathologies. CVB is transmitted via the fecal-oral route and

  3. Expression of polarity genes in human cancer.

    PubMed

    Lin, Wan-Hsin; Asmann, Yan W; Anastasiadis, Panos Z

    2015-01-01

    Polarity protein complexes are crucial for epithelial apical-basal polarity and directed cell migration. Since alterations of these processes are common in cancer, polarity proteins have been proposed to function as tumor suppressors or oncogenic promoters. Here, we review the current understanding of polarity protein functions in epithelial homeostasis, as well as tumor formation and progression. As most previous studies focused on the function of single polarity proteins in simplified model systems, we used a genomics approach to systematically examine and identify the expression profiles of polarity genes in human cancer. The expression profiles of polarity genes were distinct in different human tissues and classified cancer types. Additionally, polarity expression profiles correlated with disease progression and aggressiveness, as well as with identified cancer types, where specific polarity genes were commonly altered. In the case of Scribble, gene expression analysis indicated its common amplification and upregulation in human cancer, suggesting a tumor promoting function.

  4. Robust bioengineered 3D functional human intestinal epithelium

    PubMed Central

    Chen, Ying; Lin, Yinan; Davis, Kimberly M.; Wang, Qianrui; Rnjak-Kovacina, Jelena; Li, Chunmei; Isberg, Ralph R.; Kumamoto, Carol A.; Mecsas, Joan; Kaplan, David L.

    2015-01-01

    Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments. PMID:26374193

  5. Robust bioengineered 3D functional human intestinal epithelium.

    PubMed

    Chen, Ying; Lin, Yinan; Davis, Kimberly M; Wang, Qianrui; Rnjak-Kovacina, Jelena; Li, Chunmei; Isberg, Ralph R; Kumamoto, Carol A; Mecsas, Joan; Kaplan, David L

    2015-09-16

    Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

  6. Establishment of novel prediction system of intestinal absorption in humans using human intestinal tissues.

    PubMed

    Miyake, Masateru; Toguchi, Hajime; Nishibayashi, Toru; Higaki, Kazutaka; Sugita, Akira; Koganei, Kazutaka; Kamada, Nobuhiko; Kitazume, Mina T; Hisamatsu, Tadakazu; Sato, Toshiro; Okamoto, Susumu; Kanai, Takanori; Hibi, Toshifumi

    2013-08-01

    The objective of this study was to establish a novel prediction system of drug absorption in humans by utilizing human intestinal tissues. Based on the transport index (TI), a newly defined parameter, calculated by taking account of the change in drug concentrations because of precipitation on the apical side and the amounts accumulated in the tissue and transported to the basal side, the absorbability of drugs in rank order as well as the fraction of dose absorbed (Fa) in humans were estimated. Human intestinal tissues taken from ulcerative colitis or Crohn's disease patients were mounted in a mini-Ussing chamber and transport studies were performed to evaluate the permeation of drugs, including FD-4, a very low permeable marker, atenolol, a low permeable marker, and metoprolol, a high permeable marker. Although apparent permeability coefficients calculated by the conventional equation did not reflect human Fa values for FD-4, atenolol, and metoprolol, TI values were well correlated with Fa values, which are described by 100 · [1 - e (- f · (TI - α)) ]. Based on this equation, Fa values in humans for other test drugs were predicted successfully, indicating that our new system utilizing human intestinal tissues would be valuable for predicting oral drug absorption in humans.

  7. Human milk oligosaccharides: the novel modulator of intestinal microbiota.

    PubMed

    Jeong, Kyunghun; Nguyen, Vi; Kim, Jaehan

    2012-08-01

    Human milk, which nourishes the early infants, is a source of bioactive components for the infant growth, development and commensal formulation as well. Human milk oligosaccharide is a group of complex and diverse glycans that is apparently not absorbed in human gastrointestinal tract. Although most mammalian milk contains oligosaccharides, oligosaccharides in human milk exhibit unique features in terms of their types, amounts, sizes, and functionalities. In addition to the prevention of infectious bacteria and the development of early immune system, human milk oligosaccharides are able to facilitate the healthy intestinal microbiota. Bifidobacterial intestinal microbiota appears to be established by the unilateral interaction between milk oligosaccharides, human intestinal activity and commensals. Digestibility, membrane transportation and catabolic activity by bacteria and intestinal epithelial cells, all of which are linked to the structural of human milk oligosaccharides, are crucial in determining intestinal microbiota.

  8. Immunohistochemical detection of human intestinal spirochetosis.

    PubMed

    Ogata, Sho; Shimizu, Ken; Oda, Tomohiro; Tominaga, Susumu; Nakanishi, Kuniaki

    2016-12-01

    Human intestinal spirochetosis (HIS) is a colorectal infection by Brachyspira species of spiral bacteria. Immunohistochemical cross-reaction to an antibody for Treponema pallidum aids its histologic diagnosis. This study's aim was to analyze the immunohistochemical characteristics of HIS. In this analysis, on 223 specimens from 83 HIS cases, we focused on so-called fringe formation (a histologic hallmark of HIS), spiral organisms within mucus or within crypts, and strong immunopositive materials in the mucosa, together with their location and the types of lesions. Fringe formation was found in 81.6% of all specimens and spiral organisms within mucus or within crypts in 97.3% and 57.0%, respectively. Strong immunopositive materials were observed in the surface epithelial layer in 87.9%, in the subepithelial layer in 94.6%, and in deeper mucosa in 2.2% of all specimens. The positive rates in conventional adenomas (24.0%, n = 146) and hyperplastic nodules (100%, n = 17) were each different from that found in inflammation (70.8%, n = 24), and spiral organisms were seen more frequently in the right-side large intestine than in the left (within mucus, 100%, n = 104 versus 95.0%, n = 119; within crypts, 65.4%, n = 104 versus 49.6%, n = 119). Thus, immunohistochemistry was effective not only in supporting the diagnosis of HIS but also in highlighting spiral organisms within mucus or crypts that were invisible in routine histology. Possibly, these spiral organisms may spread throughout the entire large intestine, although there is a potential problem with antibody specificity.

  9. Human intestinal lipoproteins. Studies in chyluric subjects.

    PubMed

    Green, P H; Glickman, R M; Saudek, C D; Blum, C B; Tall, A R

    1979-07-01

    To explore the role of the human intestine as a source of apolipoproteins, we have studied intestinal lipoproteins and apoprotein secretion in two subjects with chyluria (mesenteric lymphatic-urinary fistulae). After oral corn oil, apolipoprotein A-I (apoA-I) and apolipoprotein A-II (apoA-II) output in urine increased in parallel to urinary triglyceride. One subject, on two occasions, after 40 g of corn oil, excreted 8.4 and 8.6 g of triglyceride together with 196 and 199 mg apoA-I and on one occasion, 56 mg apoA-II. The other subject, after 40 g corn oil, excreted 0.3 g triglyceride and 17.5 mg apoA-I, and, after 100 g of corn oil, excreted 44.8 mg apoA-I and 5.8 mg apoA-II. 14.5+/-2.1% of apoA-I and 17.7+/-4.3% of apoA-II in chylous urine was in the d < 1.006 fraction (chylomicrons and very low density lipoprotein). Calculations based on the amount of apoA-I and apoA-II excreted on triglyceride-rich lipoproteins revealed that for these lipid loads, intestinal secretion could account for 50 and 33% of the calculated daily synthetic rate of apoA-I and apoA-II, respectively. Similarly, subject 2 excreted 48-70% and 14% of the calculated daily synthetic rate of apoA-I and apoA-II, respectively. Chylous urine contained chylomicrons, very low density lipoproteins and high density lipoproteins, all of which contained apoA-I. Chylomicrons and very low density lipoproteins contained a previously unreported human apoprotein of 46,000 mol wt. We have called this apoprotein apoA-IV because of the similarity of its molecular weight and amino acid composition to rat apoA-IV. In sodium dodecyl sulfate gels, chylomicron apoproteins consisted of apoB 3.4+/-0.7%, apoA-IV 10.0+/-3.3%, apoE 4.4+/-0.3%, apoA-I 15.0+/-1.8%, and apoC and apoA-II 43.3+/-11.3%. Very low density lipoprotein contained more apoB and apoA-IV and less apoC than chylomicrons. Ouchterlony immunodiffusion of chylomicron apoproteins revealed the presence of apoC-I, apoC-II, and apoC-III. In contrast, plasma

  10. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids

    PubMed Central

    Finkbeiner, Stacy R.; Freeman, Jennifer J.; Wieck, Minna M.; El-Nachef, Wael; Altheim, Christopher H.; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S.; Grikscheit, Tracy C.; Teitelbaum, Daniel H.; Spence, Jason R.

    2015-01-01

    ABSTRACT Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue. PMID:26459240

  11. Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.

    PubMed

    Finkbeiner, Stacy R; Freeman, Jennifer J; Wieck, Minna M; El-Nachef, Wael; Altheim, Christopher H; Tsai, Yu-Hwai; Huang, Sha; Dyal, Rachel; White, Eric S; Grikscheit, Tracy C; Teitelbaum, Daniel H; Spence, Jason R

    2015-10-12

    Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving, such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes, new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), called human intestinal organoids (HIOs), have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However, given that HIOs are small three-dimensional structures grown in vitro, methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds, and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro, the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast, HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine, which need to be explored further to develop them into fully functional tissue.

  12. A Revised Model for Dosimetry in the Human Small Intestine

    SciTech Connect

    John Poston; Nasir U. Bhuiyan; R. Alex Redd; Neil Parham; Jennifer Watson

    2005-02-28

    A new model for an adult human gastrointestinal tract (GIT) has been developed for use in internal dose estimations to the wall of the GIT and to the other organs and tissues of the body from radionuclides deposited in the lumenal contents of the five sections of the GIT. These sections were the esophasgus, stomach, small intestine, upper large intestine, and the lower large intestine. The wall of each section was separated from its lumenal contents.

  13. Exploring food effects on indinavir absorption with human intestinal fluids in the mouse intestine.

    PubMed

    Holmstock, Nico; De Bruyn, Tom; Bevernage, Jan; Annaert, Pieter; Mols, Raf; Tack, Jan; Augustijns, Patrick

    2013-04-11

    Food can have a significant impact on the pharmacokinetics of orally administered drugs, as it may affect drug solubility as well as permeability. Since fed state conditions cannot easily be implemented in the presently available permeability tools, including the frequently used Caco-2 system, exploring food effects during drug development can be quite challenging. In this study, we investigated the effect of fasted and fed state conditions on the intestinal absorption of the HIV protease inhibitor indinavir using simulated and human intestinal fluids in the in situ intestinal perfusion technique in mice. Although the solubility of indinavir was 6-fold higher in fed state human intestinal fluids (FeHIF) as compared to fasted state HIF (FaHIF), the intestinal permeation of indinavir was 22-fold lower in FeHIF as compared to FaHIF. Dialysis experiments showed that only a small fraction of indinavir is accessible for absorption in FeHIF due to micellar entrapment, possibly explaining its low intestinal permeation. The presence of ritonavir, a known P-gp inhibitor, increased the intestinal permeation of indinavir by 2-fold in FaHIF, while there was no increase when using FeHIF. These data confirm that drug-food interactions form a complex interplay between solubility and permeability effects. The use of HIF in in situ intestinal perfusions holds great promise for biorelevant absorption evaluation as it allows to directly explore this complex solubility/permeability interplay on drug absorption.

  14. Comparative analysis of fecal microbiota and intestinal microbial metabolic activity in captive polar bears.

    PubMed

    Schwab, Clarissa; Gänzle, Michael

    2011-03-01

    The composition of the intestinal microbiota depends on gut physiology and diet. Ursidae possess a simple gastrointestinal system composed of a stomach, small intestine, and indistinct hindgut. This study determined the composition and stability of fecal microbiota of 3 captive polar bears by group-specific quantitative PCR and PCR-DGGE (denaturing gradient gel electrophoresis) using the 16S rRNA gene as target. Intestinal metabolic activity was determined by analysis of short-chain fatty acids in feces. For comparison, other Carnivora and mammals were included in this study. Total bacterial abundance was approximately log 8.5 DNA gene copies·(g feces)-1 in all 3 polar bears. Fecal polar bear microbiota was dominated by the facultative anaerobes Enterobacteriaceae and enterococci, and the Clostridium cluster I. The detection of the Clostridium perfringens α-toxin gene verified the presence of C. perfringens. Composition of the fecal bacterial population was stable on a genus level; according to results obtained by PCR-DGGE, dominant bacterial species fluctuated. The total short-chain fatty acid content of Carnivora and other mammals analysed was comparable; lactate was detected in feces of all carnivora but present only in trace amounts in other mammals. In comparison, the fecal microbiota and metabolic activity of captive polar bears mostly resembled the closely related grizzly and black bears.

  15. Efficient genetic engineering of human intestinal organoids using electroporation.

    PubMed

    Fujii, Masayuki; Matano, Mami; Nanki, Kosaku; Sato, Toshiro

    2015-10-01

    Gene modification in untransformed human intestinal cells is an attractive approach for studying gene function in intestinal diseases. However, because of the lack of practical tools, such studies have largely depended upon surrogates, such as gene-engineered mice or immortalized human cell lines. By taking advantage of the recently developed intestinal organoid culture method, we developed a methodology for modulating genes of interest in untransformed human colonic organoids via electroporation of gene vectors. Here we describe a detailed protocol for the generation of intestinal organoids by culture with essential growth factors in a basement membrane matrix. We also describe how to stably integrate genes via the piggyBac transposon, as well as precise genome editing using the CRISPR-Cas9 system. Beginning with crypt isolation from a human colon sample, genetically modified organoids can be obtained in 3 weeks.

  16. Three-Dimensional Coculture Of Human Small-Intestine Cells

    NASA Technical Reports Server (NTRS)

    Wolf, David; Spaulding, Glen; Goodwin, Thomas J.; Prewett, Tracy

    1994-01-01

    Complex three-dimensional masses of normal human epithelial and mesenchymal small-intestine cells cocultured in process involving specially designed bioreactors. Useful as tissued models for studies of growth, regulatory, and differentiation processes in normal intestinal tissues; diseases of small intestine; and interactions between cells of small intestine and viruses causing disease both in small intestine and elsewhere in body. Process used to produce other tissue models, leading to advances in understanding of growth and differentiation in developing organisms, of renewal of tissue, and of treatment of myriad of clinical conditions. Prior articles describing design and use of rotating-wall culture vessels include "Growing And Assembling Cells Into Tissues" (MSC-21559), "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), and "In Vitro, Matrix-Free Formation Of Solid Tumor Spheroids" (MSC-21843).

  17. Characterization of human foetal intestinal alkaline phosphatase. Comparison with the isoenzymes from the adult intestine and human tumour cell lines.

    PubMed Central

    Behrens, C M; Enns, C A; Sussman, H H

    1983-01-01

    The molecular structure of human foetal intestinal alkaline phosphatase was defined by high-resolution two-dimensional polyacrylamide-gel electrophoresis and amino acid inhibition studies. Comparison was made with the adult form of intestinal alkaline phosphatase, as well as with alkaline phosphatases isolated from cultured foetal amnion cells (FL) and a human tumour cell line (KB). Two non-identical subunits were isolated from the foetal intestinal isoenzyme, one having same molecular weight and isoelectric point as placental alkaline phosphatase, and the other corresponding to a glycosylated subunit of the adult intestinal enzyme. The FL-cell and KB-cell alkaline phosphatases were also found to contain two subunits similar to those of the foetal intestinal isoenzyme. Characterization of neuraminidase digests of the non-placental subunit showed it to be indistinguishable from the subunits of the adult intestinal isoenzyme. This implies that no new phosphatase structural gene is involved in the transition from the expression of foetal to adult intestinal alkaline phosphatase, but that the molecular changes involve suppression of the placental subunit and loss of neuraminic acid from the non-placental subunit. Enzyme-inhibition studies demonstrated an intermediate response to the inhibitors tested for the foetal intestinal, FL-cell and KB-cell isoenzymes when compared with the placental, adult intestinal and liver forms. This result is consistent with the mixed-subunit structure observed for the former set of isoenzymes. In summary, this study has defined the molecular subunit structure of the foetal intestinal form of alkaline phosphatase and has demonstrated its expression in a human tumour cell line. Images Fig. 1. PMID:6882358

  18. Distinct human stem cell populations in small and large intestine.

    PubMed

    Cramer, Julie M; Thompson, Timothy; Geskin, Albert; LaFramboise, William; Lagasse, Eric

    2015-01-01

    The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

  19. Glucagon effects on the human small intestine.

    PubMed

    Patel, G K; Whalen, G E; Soergel, K H; Wu, W C; Meade, R C

    1979-07-01

    In healthy volunteers, the effects of intravenously administered glucagon on small intestinal function was investigated. Bolus doses resulting in plasma glucagon concentrations of greater than 800 pg/ml (5 min after injection) abolished jejunal contractions for 4.4 +/- 0.4 (SEM) min after a latency period of 49 +/- 4 sec. During continuous intravenous glucagon infusion, jejunal dilatation and increase in mean transit time (MTT) occurred at plasma levels greater than 720 pg/ml, while inhibition of water and electrolyte absorption was observed only with plasma glucagon concentrations of 1760 +/- 114 pg/ml. Under these conditions, the propulsion of fasting intestinal contents was slowed without change in flow rate. The observed effects cannot be attributed to the simultaneously occurring rise in plasma insulin and glucose concentrations. Short-term increases in circulating glucagon concentration inhibit intestinal tone, contractions, and propulsion with only a minor effect on water and electrolyte absorption limited to a narrow concentration range of plasma glucagon. Neither effect occurs at glucagon levels likely to occur under physiologic concentrations. The latency period preceding the abolition of jejunal contractions suggests that glucagon does not act directly on intestinal smooth muscle cells.

  20. Expression and membrane localization of MCT isoforms along the length of the human intestine.

    PubMed

    Gill, Ravinder K; Saksena, Seema; Alrefai, Waddah A; Sarwar, Zaheer; Goldstein, Jay L; Carroll, Robert E; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

    2005-10-01

    Recent studies from our laboratory and others have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the luminal uptake of short-chain fatty acids (SCFAs) in the human intestine. Functional studies from our laboratory previously demonstrated kinetically distinct SCFA transporters on the apical and basolateral membranes of human colonocytes. Although apical SCFA uptake is mediated by the MCT1 isoform, the molecular identity of the basolateral membrane SCFA transporter(s) and whether this transporter is encoded by another MCT isoform is not known. The present studies were designed to assess the expression and membrane localization of different MCT isoforms in human small intestine and colon. Immunoblotting was performed with the purified apical and basolateral membranes from human intestinal mucosa obtained from organ donor intestine. Immunohistochemistry studies were done on paraffin-embedded sections of human colonic biopsy samples. Immunoblotting studies detected a protein band of approximately 39 kDa for MCT1, predominantly in the apical membranes. The relative abundance of MCT1 mRNA and protein increased along the length of the human intestine. MCT4 (54 kDa) and MCT5 (54 kDa) isoforms showed basolateral localization and were highly expressed in the distal colon. Immunohistochemical studies confirmed that human MCT1 antibody labeling was confined to the apical membranes, whereas MCT5 antibody staining was restricted to the basolateral membranes of the colonocytes. We speculate that distinct MCT isoforms may be involved in SCFA transport across the apical or basolateral membranes in polarized colonic epithelial cells.

  1. Understanding drug resistance in human intestinal protozoa.

    PubMed

    El-Taweel, Hend Aly

    2015-05-01

    Infections with intestinal protozoa continue to be a major health problem in many areas of the world. The widespread use of a limited number of therapeutic agents for their management and control raises concerns about development of drug resistance. Generally, the use of any antimicrobial agent should be accompanied by meticulous monitoring of its efficacy and measures to minimize resistance formation. Evidence for the occurrence of drug resistance in different intestinal protozoa comes from case studies and clinical trials, sometimes with a limited number of patients. Large-scale field-based assessment of drug resistance and drug sensitivity testing of clinical isolates are needed. Furthermore, the association of drug resistance with certain geographic isolates or genotypes deserves consideration. Drug resistance has been triggered in vitro and has been linked to modification of pyruvate:ferredoxin oxidoreductase, nitroreductases, antioxidant defense, or cytoskeletal system. Further mechanistic studies will have important implications in the development of second generation therapeutic agents.

  2. Tim-3 promotes intestinal homeostasis in DSS colitis by inhibiting M1 polarization of macrophages.

    PubMed

    Jiang, Xingwei; Yu, Jiahui; Shi, Qingzhu; Xiao, Yan; Wang, Wei; Chen, Guojiang; Zhao, Zhi; Wang, Renxi; Xiao, He; Hou, Chunmei; Feng, Jiannan; Ma, Yuanfang; Shen, Beifen; Wang, Lili; Li, Yan; Han, Gencheng

    2015-10-01

    Tim-3 is involved in the physiopathology of inflammatory bowel disease (IBD), but the underlying mechanism is unknown. Here, we demonstrated that, in mouse with DSS colitis, Tim-3 inhibited the polarization of pathogenic pro-inflammatory M1 macrophages, while Tim-3 downregulation or blockade resulted in an increased M1 response. Adoptive transfer of Tim-3-silenced macrophages worsened DSS colitis and enhanced inflammation, while Tim-3 overexpression attenuated DSS colitis by decreasing the M1 macrophage response. Co-culture of Tim-3-overexpressing macrophages with intestinal lymphocytes decreased the pro-inflammatory response. Tim-3 shaped intestinal macrophage polarization may be TLR-4 dependent since Tim-3 blockade failed to exacerbate colitis or increase M1 macrophage response in the TLR-4 KO model. Finally, Tim-3 signaling inhibited phosphorylation of IRF3, a TLR-4 downstream transcriptional factor regulating macrophage polarization. A better understanding of this pathway may shed new light on colitis pathogenesis and result in a new therapeutic strategy.

  3. Curcuminoid Demethylation as an Alternative Metabolism by Human Intestinal Microbiota.

    PubMed

    Burapan, Supawadee; Kim, Mihyang; Han, Jaehong

    2017-04-14

    Curcumin and other curcuminoids from Curcuma longa are important bioactive compounds exhibiting various pharmacological activities. In addition to the known reductive metabolism of curcuminoids, an alternative biotransformation of curcuminoids by human gut microbiota is reported herein. A curcuminoid mixture, composed of curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3), was metabolized by the human intestinal bacterium Blautia sp. MRG-PMF1. 1 and 2 were converted to new metabolites by the methyl aryl ether cleavage reaction. Two metabolites, demethylcurcumin (4) and bisdemethylcurcumin (5), were sequentially produced from 1, and demethyldemethoxycurcumin (6) was produced from 2. Until now, sequential reduction of the heptadienone backbone of curcuminoids was the only known metabolism to occur in the human intestine. In this study, a new intestinal metabolism of curcuminoids was discovered. Demethylation of curcuminoids produced three new colonic metabolites that were already known as promising synthetic curcumin analogues. The results could explain the observed beneficial effects of turmeric.

  4. Intestine.

    PubMed

    Smith, J M; Skeans, M A; Horslen, S P; Edwards, E B; Harper, A M; Snyder, J J; Israni, A K; Kasiske, B L

    2016-01-01

    Intestine and intestine-liver transplant plays an important role in the treatment of intestinal failure, despite decreased morbidity associated with parenteral nutrition. In 2014, 210 new patients were added to the intestine transplant waiting list. Among prevalent patients on the list at the end of 2014, 65% were waiting for an intestine transplant and 35% were waiting for an intestine-liver transplant. The pretransplant mortality rate decreased dramatically over time for all age groups. Pretransplant mortality was highest for adult candidates, at 22.1 per 100 waitlist years compared with less than 3 per 100 waitlist years for pediatric candidates, and notably higher for candidates for intestine-liver transplant than for candidates for intestine transplant without a liver. Numbers of intestine transplants without a liver increased from a low of 51 in 2013 to 67 in 2014. Intestine-liver transplants increased from a low of 44 in 2012 to 72 in 2014. Short-gut syndrome (congenital and other) was the main cause of disease leading to both intestine and intestine-liver transplant. Graft survival improved over the past decade. Patient survival was lowest for adult intestine-liver recipients and highest for pediatric intestine recipients.

  5. Transepithelial Transport of PAMAM Dendrimers Across Isolated Human Intestinal Tissue.

    PubMed

    Hubbard, Dallin; Enda, Michael; Bond, Tanner; Moghaddam, Seyyed Pouya Hadipour; Conarton, Josh; Scaife, Courtney; Volckmann, Eric; Ghandehari, Hamidreza

    2015-11-02

    Poly(amido amine) (PAMAM) dendrimers have shown transepithelial transport across intestinal epithelial barrier in rats and across Caco-2 cell monolayers. Caco-2 models innately lack mucous barriers, and rat isolated intestinal tissue has been shown to overestimate human permeability. This study is the first report of transport of PAMAM dendrimers across isolated human intestinal epithelium. It was observed that FITC labeled G4-NH2 and G3.5-COOH PAMAM dendrimers at 1 mM concentration do not have a statistically higher permeability compared to free FITC controls in isolated human jejunum and colonic tissues. Mannitol permeability was increased at 10 mM concentrations of G3.5-COOH and G4-NH2 dendrimers. Significant histological changes in human colonic and jejunal tissues were observed at G3.5-COOH and G4-NH2 concentrations of 10 mM implying that dose limiting toxicity may occur at similar concentrations in vivo. The permeability through human isolated intestinal tissue in this study was compared to previous rat and Caco-2 permeability data. This study implicates that PAMAM dendrimer oral drug delivery may be feasible, but it may be limited to highly potent drugs.

  6. Berberine Inhibits Intestinal Polyps Growth in Apc (min/+) Mice via Regulation of Macrophage Polarization

    PubMed Central

    Piao, Meiyu; Cao, Hailong; He, NaNa; Yang, Boli; Dong, Wenxiao; Xu, Mengque; Yan, Fang; Zhou, Bing

    2016-01-01

    Antitumor effect of berberine has been reported in a wide spectrum of cancer, however, the mechanisms of which are not fully understood. The aim of this study was to investigate the hypothesis that berberine suppresses tumorigenesis in the familial adenomatous polyposis (FAP) by regulating the macrophage polarization in Apc (min/+) mouse model. Berberine was given to Apc (min/+) mice for 12 weeks. Primary macrophages were isolated; after berberine treatment, the change in signaling cascade was determined. The total number and size of polyps were reduced remarkably in berberine group, compared with control group. A significant decrease in protein levels of F4/80, mannose receptor (MR), and COX-2 in stroma of intestinal polyps and an increase in the level of iNOS were observed after berberine treatment. The mRNA level of MR and Arg-1 in berberine group was significantly lower than those in IL-10 or IL-4 group, while no significant difference in mRNA levels of iNOS and CXCL10 was observed. The migration and invasiveness assays in vitro showed that berberine could reduce the capability of migration and invasiveness. These findings suggest that berberine attenuates intestinal tumorigenesis by inhibiting the migration and invasion of colorectal tumor cells via regulation of macrophage polarization. PMID:27493671

  7. Molecular Epidemiology of Human Intestinal Amoebas in Iran

    PubMed Central

    Hooshyar, H; Rostamkhani, P; Rezaian, M

    2012-01-01

    Many microscopic-based epidemiological surveys on the prevalence of human intestinal pathogenic and non-pathogenic protozoa including intestinal amoeba performed in Iran show a high prevalence of human intestinal amoeba in different parts of Iran. Such epidemiological studies on amoebiasis are confusing, mainly due to recently appreciated distinction between the Entamoeba histolytica, E. dispar and E. moshkovskii. Differential diagnosis can be done by some methods such as PCR-based methods, monoclonal antibodies and the analysis of isoenzyme typing, however the molecular study of these protozoa in Iran is low. Based on molecular studies, it seems that E. dispar is predominant species especially in the central and northern areas of Iran and amoebiasis due to E. histolytica is a rare infection in the country. It is suggested that infection with E. moshkovskii may be common among Iranians. Considering the importance of molecular epidemiology of amoeba in Iran and also the current data, the present study reviews the data currently available on the molecular distribution of intestinal human amoeba in Iran. PMID:23193500

  8. A model for Vibrio cholerae colonization of the human intestine.

    PubMed

    Spagnuolo, Anna Maria; Dirita, Victor; Kirschner, Denise

    2011-11-21

    Vibrio cholerae is a strict human pathogen that causes the disease cholera. It is an old-world pathogen that has re-emerged as a new threat since the early 1990s. V. cholerae colonizes the upper, small intestine where it produces a toxin that leads to watery diarrhea, characterizing the disease (Kahn et al., 1988). The dynamics of colonization by the bacteria of the intestines are largely unknown. Although a large initial infectious dose is required for infection, data suggests that only a smaller sub-population colonizes a portion of the small bowel leading to disease. There are many barriers to colonization in the intestines including peristalsis, fluid wash-out, viscosity of the mucus layer, and pH. We are interested in identifying the mechanisms that allow this sub-population of bacteria to survive and colonize the intestines when faced with these barriers. To elaborate the dynamics of V. cholerae infection, we have developed a mathematical model based on a convection-diffusion-reaction-swimming equation capturing bacterial dynamics coupled with Stokes equations governing fluid velocity where we developed a novel non-local boundary condition. Our results indicate that both host and bacterial factors contribute to bacterial density in the gut. Host factors include intestinal diffusion and convection rates while bacterial factors include adherence, motility and growth rates. This model can ultimately be used to test therapeutic strategies against V. cholerae.

  9. Cell and tissue polarity in the intestinal tract during tumourigenesis: cells still know the right way up, but tissue organization is lost.

    PubMed

    Fatehullah, Aliya; Appleton, Paul L; Näthke, Inke S

    2013-01-01

    Cell and tissue polarity are tightly coupled and are vital for normal tissue homeostasis. Changes in cellular and tissue organization are common to even early stages of disease, particularly cancer. The digestive tract is the site of the second most common cause of cancer deaths in the developed world. Tumours in this tissue arise in an epithelium that has a number of axes of cell and tissue polarity. Changes in cell and tissue polarity in response to genetic changes that are known to underpin disease progression provide clues about the link between molecular-, cellular- and tissue-based mechanisms that accompany cancer. Mutations in adenomatous polyposis coli (APC) are common to most colorectal cancers in humans and are sufficient to cause tumours in mouse intestine. Tissue organoids mimic many features of whole tissue and permit identifying changes at different times after inactivation of APC. Using gut organoids, we show that tissue polarity is lost very early during cancer progression, whereas cell polarity, at least apical-basal polarity, is maintained and changes only at later stages. These observations reflect the situation in tumours and validate tissue organoids as a useful system to investigate the relationship between cell polarity and tissue organization.

  10. Polarized distribution of inducible nitric oxide synthase regulates activity in intestinal epithelial cells

    PubMed Central

    Rumbo, Martin; Courjault-Gautier, Françoise; Sierro, Frédéric; Sirard, Jean-Claude; Felley-Bosco, Emanuela

    2005-01-01

    Summary Inducible nitric oxide synthase (iNOS) functions as a homodimer. In cell extracts, iNOS molecules partition both in cytosolic and particulate fractions, indicating that iNOS exist as soluble and membrane associated forms. In this study, iNOS features were investigated in human intestinal epithelial cells stimulated with cytokines and in duodenum from mice exposed to flagellin. Our experiments indicate that iNOS is mainly associated to the particulate fraction of cell extracts. Confocal microscopy showed a preferential localization of iNOS at the apical pole of intestinal epithelial cells. In particulate fractions, iNOS dimers were more abundant than in the cytosolic fraction. Similar observations were done in mouse duodenum samples. These results suggest that, in epithelial cells, iNOS activity is regulated by localization-dependent processes. PMID:15654882

  11. Human small intestinal epithelial cells differentiated from adult intestinal stem cells as a novel system for predicting oral drug absorption in humans.

    PubMed

    Takenaka, Toru; Harada, Naomoto; Kuze, Jiro; Chiba, Masato; Iwao, Takahiro; Matsunaga, Tamihide

    2014-11-01

    Adult intestinal stem cells (ISCs) possess both a long-term proliferation ability and differentiation capability into enterocytes. As a novel in vitro system for the evaluation of drug absorption, we characterized a human small intestinal epithelial cell (HIEC) monolayer that differentiated from adult ISCs. Continuous proliferation/differentiation from ISCs consistently conferred the capability of maturation of enterocytes to HIECs over 25 passages. The morphologically matured HIEC monolayer consisted of polarized columnar epithelia with dense microvilli, tight junctions, and desmosomes 8 days after seeding onto culture inserts. Transepithelial electrical resistance across the monolayer was 9-fold lower in HIECs (98.9 Ω × cm(2)) than in Caco-2 cells (900 Ω × cm(2)), which indicated that the looseness of the tight junctions in the HIEC monolayer was similar to that in the human small intestine (approximately 40 Ω × cm(2)). No significant differences were observed in the overall gene expression patterns of the major drug-metabolizing enzymes and transporters between the HIEC and Caco-2 cell monolayers. Furthermore, the functions of P-glycoprotein and breast cancer resistance protein in the HIEC monolayer were confirmed by the vectorial transport of marker substrates and their disappearance in the presence of specific inhibitors. The apparent drug permeability values of paracellularly transported compounds (fluorescein isothiocyanate-dextran 4000, atenolol, and terbutaline) and nucleoside transporter substrates (didanosine, ribavirin, and doxifluridine) in the HIEC monolayer were markedly higher than those of Caco-2 cells, whereas transcellularly transported drugs (pindolol and midazolam) were equally well permeated. In conclusion, the HIEC monolayer can serve as a novel and superior alternative to the conventional Caco-2 cell monolayer for predicting oral absorption in humans.

  12. Dual system of intestinal thiamine transport in humans

    SciTech Connect

    Hoyumpa, A.M. Jr.; Strickland, R.; Sheehan, J.J.; Yarborough, G.; Nichols, S.

    1982-05-01

    The transport of thiamine across the intestine has been characterized in rats but has not been adequately studied in humans. To determine the kinetics of thiamine intestinal transport directly in humans, mucosal tissues were obtained during routine endoscopy from normal-appearing sites at the second portion of the duodenum. With 3H-dextran as the marker of adherent volume, the uptake of 14C-thiamine hydrochloride by the excised mucosa was measured in vitro. By this method thiamine uptake was linear with tissue weight and with incubation time up to 5 min. Results showed that at low thiamine concentrations (0.2 to 2.0 microM), uptake was saturable whereas at high concentrations (5 to 50 microM), uptake was linear with thiamine concentrations. Pyrithiamine, anoxia, N-ethylmaleimide, and replacement of sodium chloride by mannitol reduced the uptake of 0.5 microM thiamine by 42%, 37%, 32% and 35%, respectively (p less than 0.05) but had no effect on the uptake of 20 microM thiamine. These data suggest that, as in the rat, the intestinal transport of thiamine in humans proceeds by a coexistent dual system. At physiologic concentrations, thiamine is transported primarily by an energy-requiring, sodium-dependent active process, whereas at higher pharmacologic concentrations thiamine uptake is predominantly a passive process.

  13. Evolution of Symbiotic Bacteria in the Distal Human Intestine

    PubMed Central

    Ley, Ruth E; Lozupone, Catherine A; Hamady, Micah; Martens, Eric C; Henrissat, Bernard; Coutinho, Pedro M; Minx, Patrick; Latreille, Philippe; Cordum, Holland; Van Brunt, Andrew; Kim, Kyung; Fulton, Robert S; Fulton, Lucinda A; Clifton, Sandra W; Wilson, Richard K; Knight, Robin D; Gordon, Jeffrey I

    2007-01-01

    The adult human intestine contains trillions of bacteria, representing hundreds of species and thousands of subspecies. Little is known about the selective pressures that have shaped and are shaping this community's component species, which are dominated by members of the Bacteroidetes and Firmicutes divisions. To examine how the intestinal environment affects microbial genome evolution, we have sequenced the genomes of two members of the normal distal human gut microbiota, Bacteroides vulgatus and Bacteroides distasonis, and by comparison with the few other sequenced gut and non-gut Bacteroidetes, analyzed their niche and habitat adaptations. The results show that lateral gene transfer, mobile elements, and gene amplification have played important roles in affecting the ability of gut-dwelling Bacteroidetes to vary their cell surface, sense their environment, and harvest nutrient resources present in the distal intestine. Our findings show that these processes have been a driving force in the adaptation of Bacteroidetes to the distal gut environment, and emphasize the importance of considering the evolution of humans from an additional perspective, namely the evolution of our microbiomes. PMID:17579514

  14. Sequential cancer mutations in cultured human intestinal stem cells.

    PubMed

    Drost, Jarno; van Jaarsveld, Richard H; Ponsioen, Bas; Zimberlin, Cheryl; van Boxtel, Ruben; Buijs, Arjan; Sachs, Norman; Overmeer, René M; Offerhaus, G Johan; Begthel, Harry; Korving, Jeroen; van de Wetering, Marc; Schwank, Gerald; Logtenberg, Meike; Cuppen, Edwin; Snippert, Hugo J; Medema, Jan Paul; Kops, Geert J P L; Clevers, Hans

    2015-05-07

    Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

  15. Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro

    PubMed Central

    Papafragkou, Efstathia; Weaver, James C.; Ferrante, Thomas C.; Bahinski, Anthony; Elkins, Christopher A.; Kulka, Michael; Ingber, Donald E.

    2017-01-01

    Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower ‘vascular’ channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis. PMID:28146569

  16. Human milk hyaluronan enhances innate defense of the intestinal epithelium.

    PubMed

    Hill, David R; Rho, Hyunjin K; Kessler, Sean P; Amin, Ripal; Homer, Craig R; McDonald, Christine; Cowman, Mary K; de la Motte, Carol A

    2013-10-04

    Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn.

  17. Nonsteroidal antiinflammatory drug-induced intestinal inflammation in humans

    SciTech Connect

    Bjarnason, I.; Zanelli, G.; Smith, T.; Prouse, P.; Williams, P.; Smethurst, P.; Delacey, G.; Gumpel, M.J.; Levi, A.J.

    1987-09-01

    This study examines the effects of nonsteroidal antiinflammatory drugs on the small intestine in humans. Using an /sup 111/In-leukocyte technique in patients with rheumatoid arthritis (n = 90) and osteoarthritis (n = 7), it appears that nonsteroidal antiinflammatory drugs cause small intestinal inflammation in two-thirds of patients on long-term treatment and on discontinuation, the inflammation may persist for up to 16 mo. The prevalence and magnitude of the intestinal inflammation was unrelated to the type and dose of nonsteroidal drugs and previous or concomitant second-line drug treatment. There was a significant inverse correlation (r = -0.29, p less than 0.05) between fecal /sup 111/In excretion and hemoglobin levels in patients treated with nonsteroidal antiinflammatory drugs. The kinetics of fecal indium 111 excretion in patients treated with nonsteroidal antiinflammatory drugs was almost identical to that of patients with small bowel Crohn's disease. Eighteen patients on nonsteroidal antiinflammatory drugs underwent a radiologic examination of the small bowel and 3 were found to have asymptomatic ileal disease with ulceration and strictures. Nineteen patients on nonsteroidal antiinflammatory drugs, 20 healthy controls, and 13 patients with Crohn's ileitis underwent a dual radioisotopic ileal function test with tauro 23 (/sup 75/Se) selena-25-homocholic acid and cobalt 58-labeled cyanocobalamine. On day 4, more than half of the patients with rheumatoid arthritis had evidence of bile acid malabsorption, but the ileal dysfunction was much milder than seen in patients with Crohn's ileitis.

  18. Intestinal microbiota modulates gluten-induced immunopathology in humanized mice.

    PubMed

    Galipeau, Heather J; McCarville, Justin L; Huebener, Sina; Litwin, Owen; Meisel, Marlies; Jabri, Bana; Sanz, Yolanda; Murray, Joseph A; Jordana, Manel; Alaedini, Armin; Chirdo, Fernando G; Verdu, Elena F

    2015-11-01

    Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk.

  19. Diversity of human small intestinal Streptococcus and Veillonella populations.

    PubMed

    van den Bogert, Bartholomeus; Erkus, Oylum; Boekhorst, Jos; de Goffau, Marcus; Smid, Eddy J; Zoetendal, Erwin G; Kleerebezem, Michiel

    2013-08-01

    Molecular and cultivation approaches were employed to study the phylogenetic richness and temporal dynamics of Streptococcus and Veillonella populations in the small intestine. Microbial profiling of human small intestinal samples collected from four ileostomy subjects at four time points displayed abundant populations of Streptococcus spp. most affiliated with S. salivarius, S. thermophilus, and S. parasanguinis, as well as Veillonella spp. affiliated with V. atypica, V. parvula, V. dispar, and V. rogosae. Relative abundances varied per subject and time of sampling. Streptococcus and Veillonella isolates were cultured using selective media from ileostoma effluent samples collected at two time points from a single subject. The richness of the Streptococcus and Veillonella isolates was assessed at species and strain level by 16S rRNA gene sequencing and genetic fingerprinting, respectively. A total of 160 Streptococcus and 37 Veillonella isolates were obtained. Genetic fingerprinting differentiated seven Streptococcus lineages from ileostoma effluent, illustrating the strain richness within this ecosystem. The Veillonella isolates were represented by a single phylotype. Our study demonstrated that the small intestinal Streptococcus populations displayed considerable changes over time at the genetic lineage level because only representative strains of a single Streptococcus lineage could be cultivated from ileostoma effluent at both time points.

  20. Intestinal Microbiota Modulates Gluten-Induced Immunopathology in Humanized Mice

    PubMed Central

    Galipeau, Heather J.; McCarville, Justin L.; Huebener, Sina; Litwin, Owen; Meisel, Marlies; Jabri, Bana; Sanz, Yolanda; Murray, Joseph A.; Jordana, Manel; Alaedini, Armin; Chirdo, Fernando G.; Verdu, Elena F.

    2016-01-01

    Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. The recent increase in CD incidence suggests that additional environmental factors, such as intestinal microbiota alterations, are involved in its pathogenesis. However, there is no direct evidence of modulation of gluten-induced immunopathology by the microbiota. We investigated whether specific microbiota compositions influence immune responses to gluten in mice expressing the human DQ8 gene, which confers moderate CD genetic susceptibility. Germ-free mice, clean specific-pathogen-free (SPF) mice colonized with a microbiota devoid of opportunistic pathogens and Proteobacteria, and conventional SPF mice that harbor a complex microbiota that includes opportunistic pathogens were used. Clean SPF mice had attenuated responses to gluten compared to germ-free and conventional SPF mice. Germ-free mice developed increased intraepithelial lymphocytes, markers of intraepithelial lymphocyte cytotoxicity, gliadin-specific antibodies, and a proinflammatory gliadin-specific T-cell response. Antibiotic treatment, leading to Proteobacteria expansion, further enhanced gluten-induced immunopathology in conventional SPF mice. Protection against gluten-induced immunopathology in clean SPF mice was reversed after supplementation with a member of the Proteobacteria phylum, an enteroadherent Escherichia coli isolated from a CD patient. The intestinal microbiota can both positively and negatively modulate gluten-induced immunopathology in mice. In subjects with moderate genetic susceptibility, intestinal microbiota changes may be a factor that increases CD risk. PMID:26456581

  1. [Influencing factors on infections of human intestinal helminthes in suburb of Shangyu City].

    PubMed

    Song-Lin, Hu

    2011-06-01

    The infections of human intestinal helminthes and socioeconomic status were investigated in suburb of Shangyu City in 1990 and 2005, respectively. The results showed that the economic status, the save drinking water and latrines, working environment, and health habits and consciousness of the residents improved obviously. The infection rate of intestinal helminthes decreased significantly and the prevalence of intestinal helminthosis was controlled effectively.

  2. Human milk and infant intestinal mucosal glycans guide succession of the neonatal intestinal microbiota.

    PubMed

    Newburg, David S; Morelli, Lorenzo

    2015-01-01

    Infants begin acquiring intestinal microbiota at parturition. Initial colonization by pioneer bacteria is followed by active succession toward a dynamic ecosystem. Keystone microbes engage in reciprocal transkingdom communication with the host, which is essential for human homeostasis and health; therefore, these bacteria should be considered mutualists rather than commensals. This review discusses the maternal role in providing infants with functional and stable microbiota. The initial fecal inoculum of microbiota results from the proximity of the birth canal and anus; the biological significance of this anatomic proximity could underlie observed differences in microbiota between vaginal and cesarean birth. Secondary sources of inocula include mouths and skin of kin, animals and objects, and the human milk microbiome, but guiding microbial succession may be a primary role of human milk. The unique glycans of human milk cannot be digested by the infant, but are utilized by mutualist bacteria. These prebiotic glycans support expansion of mutualist microbiota, which manifests as differences in microbiota among breastfed and artificially fed infants. Human milk glycans vary by maternal genotype. Milks of genetically distinct mothers and variations in infant mucosal glycan expression support discrete microbiota. Early colonization may permanently influence microbiota composition and function, with ramifications for health.

  3. Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

    PubMed Central

    Roy, Soumitra; Calcedo, Roberto; Medina-Jaszek, Angelica; Keough, Martin; Peng, Hui; Wilson, James M.

    2011-01-01

    Objective Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects. Methods The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA. Results Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested. Conclusion Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora. PMID:21980361

  4. Functional Characterization of Cholera Toxin Inhibitors Using Human Intestinal Organoids.

    PubMed

    Zomer-van Ommen, Domenique D; Pukin, Aliaksei V; Fu, Ou; Quarles van Ufford, Linda H C; Janssens, Hettie M; Beekman, Jeffrey M; Pieters, Roland J

    2016-07-28

    Preclinical drug testing in primary human cell models that recapitulate disease can significantly reduce animal experimentation and time-to-the-clinic. We used intestinal organoids to quantitatively study the potency of multivalent cholera toxin inhibitors. The method enabled the determination of IC50 values over a wide range of potencies (15 pM to 9 mM). The results indicate for the first time that an organoid-based swelling assay is a useful preclinical method to evaluate inhibitor potencies of drugs that target pathogen-derived toxins.

  5. [Progress in the knowledge of the intestinal human microbiota].

    PubMed

    Robles-Alonso, Virginia; Guarner, Francisco

    2013-01-01

    New sequencing technologies together with the development of bio-informatics allow a description of the full spectrum of the microbial communities that inhabit the human intestinal tract, as well as their functional contributions to host health. Most community members belong to the domain Bacteria, but Archaea, Eukaryotes (yeasts and protists), and Viruses are also present. Only 7 to 9 of the 55 known divisions or phyla of the domain Bacteria are detected in faecal or mucosal samples from the human gut. Most taxa belong to just two divisions: Bacteroidetes and Firmicutes, and the other divisions that have been consistently found are Proteobacteria, Actinobacteria, Fusobacteria, and Verrucomicrobia. Bacteroides, Faecalibacterium and Bifidobacterium are the most abundant genera but their relative proportion is highly variable across individuals. Full metagenomic analysis has identified more than 5 million non-redundant microbial genes encoding up to 20,000 biological functions related with life in the intestinal habitat. The overall structure of predominant genera in the human gut can be assigned into three robust clusters, which are known as "enterotypes". Each of the three enterotypes is identifiable by the levels of one of three genera: Bacteroides (enterotype 1), Prevotella (enterotype 2) and Ruminococcus (enterotype 3). This suggests that microbiota variations across individuals are stratified, not continuous. Next steps include the identification of changes that may play a role in certain disease states. A better knowledge of the contributions of microbial symbionts to host health will help in the design of interventions to improve symbiosis and combat disease.

  6. Derivation of Intestinal Organoids from Human Induced Pluripotent Stem Cells for Use as an Infection System.

    PubMed

    Forbester, Jessica L; Hannan, Nicholas; Vallier, Ludovic; Dougan, Gordon

    2016-08-31

    Intestinal human organoids (iHOs) provide an effective system for studying the intestinal epithelium and its interaction with various stimuli. By using combinations of different signaling factors, human induced pluripotent stem cells (hIPSCs) can be driven to differentiate down the intestinal lineage. Here, we describe the process for this differentiation, including the derivation of hindgut from hIPSCs, embedding hindgut into a pro-intestinal culture system and passaging the resulting iHOs. We then describe how to carry out microinjections to introduce bacteria to the apical side of the intestinal epithelial cells (IECs).

  7. Human intestinal epithelial cells produce proinflammatory cytokines in response to infection in a SCID mouse-human intestinal xenograft model of amebiasis.

    PubMed Central

    Seydel, K B; Li, E; Swanson, P E; Stanley, S L

    1997-01-01

    The protozoan parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with significant morbidity and mortality worldwide. E. histolytica infection appears to involve the initial attachment of amebic trophozoites to intestinal epithelial cells, followed by lysis of these cells and subsequent invasion into the submucosa. A recent in vitro study (L. Eckmann, S. L. Reed, J. R. Smith, and M. F. Kagnoff, J. Clin. Invest. 96:1269-1279, 1995) demonstrated that incubation of E. histolytica trophozoites with epithelial cell lines results in epithelial cell production of inflammatory cytokines, including interleukin-1 (IL-1) and IL-8, suggesting that intestinal epithelial cell production of cytokines might play a role in the inflammatory response and tissue damage seen in intestinal amebiasis. To determine whether intestinal epithelial cell production of IL-1 and IL-8 occurs in response to E. histolytica infection in vivo and as an approach to studying the specific interactions between amebic trophozoites and human intestine, we used a SCID mouse-human intestinal xenograft (SCID-HU-INT) model of disease, where human intestinal xenografts were infected with virulent E. histolytica trophozoites. Infection of xenografts with E. histolytica trophozoites resulted in extensive tissue damage, which was associated with the development of an early inflammatory response composed primarily of neutrophils. Using oligonucleotide primers that specifically amplify human IL-1beta and IL-8, we could demonstrate by reverse transcription PCR that mRNA for both IL-1beta and IL-8 is produced by human intestinal xenografts in response to amebic infection. The increase in human intestinal IL-1beta and IL-8 in response to invasive amebiasis was confirmed by enzyme-linked immunosorbent assays specific for human IL-1beta and IL-8. Using immunohistochemistry, we confirmed that human intestinal epithelial cells were the source of IL-8 in infected xenografts

  8. Ovine intestinal adenocarcinomas: histologic and phenotypic comparison with human colon cancer.

    PubMed

    Munday, John S; Brennan, Moira M; Jaber, Azhar M; Kiupel, Matti

    2006-04-01

    Approximately 7% of old, unthrifty sheep (Ovis aries) in New Zealand have intestinal adenocarcinomas. To investigate whether these sheep might be used as a model of human colonic neoplasia, the biologic behavior and histologic appearance of ovine intestinal adenocarcinomas were compared with those reported for human colonic adenocarcinomas. We collected 50 intestinal tracts with grossly visible intestinal neoplasia from slaughtered sheep. Neoplasms were assessed using World Health Organization guidelines for assessment of human colonic adenocarcinomas. All ovine adenocarcinomas developed in the small intestine. In contrast, only 4% of human intestinal tumors develop at this location, whereas the majority develop in the colon. A visible polyp is present within 89% of human colonic adenocarcinomas, whereas polyps were present in only 46% of the ovine neoplasms. Intestinal wall infiltration by the neoplastic cells and rates of lymph node (84% in sheep; 61% in humans) and distant (52% in sheep; 17% in humans) metastases were comparable between ovine and human adenocarcinomas. However, ovine adenocarcinomas developed more peritoneal and fewer hepatic metastases than human adenocarcinomas. Histologic grading of ovine tumors revealed cell differentiation similar to that reported within human colonic adenocarcinomas. In conclusion, ovine intestinal adenocarcinomas, like human colonic adenocarcinomas, typically arise spontaneously and consistently develop widespread metastases. In addition, tumors appear histologically similar between these species. Therefore, sheep may provide a model of advanced human colonic cancer, possibly allowing evaluation of novel therapeutics and surgical procedures.

  9. Alpha2 adrenoceptors regulate proliferation of human intestinal epithelial cells

    PubMed Central

    Schaak, S; Cussac, D; Cayla, C; Devedjian, J; Guyot, R; Paris, H; Denis, C

    2000-01-01

    BACKGROUND AND AIMS—Previous studies on rodents have suggested that catecholamines stimulate proliferation of the intestinal epithelium through activation of α2 adrenoceptors located on crypt cells. The occurrence of this effect awaits demonstration in humans and the molecular mechanisms involved have not yet been elucidated. Here, we examined the effect of α2 agonists on a clone of Caco2 cells expressing the human α2A adrenoceptor.
METHODS—Cells were transfected with a bicistronic plasmid containing the α2C10 and neomycin phosphotransferase genes. G418 resistant clones were assayed for receptor expression using radioligand binding. Receptor functionality was assessed by testing its ability to couple Gi proteins and to inhibit cAMP production. Mitogen activated protein kinase (MAPK) phosphorylation was followed by western blot, and cell proliferation was estimated by measuring protein and DNA content.
RESULTS—Permanent transfection of Caco2 cells allowed us to obtain a clone (Caco2-3B) expressing α2A adrenoceptors at a density similar to that found in normal human intestinal epithelium. Caco2-3B retained morphological features and brush border enzyme expression characteristic of enterocytic differentiation. The receptor was coupled to Gi2/Gi3 proteins and its stimulation caused marked diminution of forskolin induced cAMP production. Treatment of Caco2-3B with UK14304 (α2 agonist) induced a rapid increase in the phosphorylation state of MAPK, extracellular regulated protein kinase 1 (Erk1), and 2 (Erk2). This event was totally abolished in pertussis toxin treated cells and in the presence of kinase inhibitors (genistein or PD98059). It was unaffected by protein kinase C downregulation but correlated with a transient increase in Shc tyrosine phosphorylation. Finally, sustained exposure of Caco2-3B to UK14304 resulted in modest but significant acceleration of cell proliferation. None of these effects was observed in the parental cell line Caco2.

  10. Multiscale analysis of the murine intestine for modeling human diseases

    PubMed Central

    Lyons, Jesse; Herring, Charles A.; Banerjee, Amrita; Simmons, Alan J.

    2015-01-01

    When functioning properly, the intestine is one of the key interfaces between the human body and its environment. It is responsible for extracting nutrients from our food and excreting our waste products. It provides an environment for a host of healthful microbes and serves as a first defense against pathogenic ones. These processes require tight homeostatic controls, which are provided by the interactions of a complex mix of epithelial, stromal, neural and immune cells, as well as the resident microflora. This homeostasis can be disrupted by invasive microbes, genetic lesions, and carcinogens, resulting in diseases such Clostridium difficile infection, inflammatory bowel disease (IBD) and cancer. Enormous strides have been made in understanding how this important organ functions in health and disease using everything from cell culture systems to animal models to human tissue samples. This has resulted in better therapies for all of these diseases, but there is still significant room for improvement. In the United States alone, 14000 people per year die of C. difficile, up to 1.6 million people suffer from IBD, and more than 50000 people die every year from colon cancer. Because these and other intestinal diseases arise from complex interactions between the different components of the gut ecosystem, we propose that systems approaches that address this complexity in an integrative manner may eventually lead to improved therapeutics that deliver lasting cures. This review will discuss the use of systems biology for studying intestinal diseases in vivo with particular emphasis on mouse models. Additionally, it will focus on established experimental techniques that have been used to drive this systems-level analysis, and emerging techniques that will push this field forward in the future. PMID:26040649

  11. Electromagnetic radiation from ingested sources in the human intestine between 150 MHz and 1.2 GHz.

    PubMed

    Chirwa, Lawrence C; Hammond, Paul A; Roy, Scott; Cumming, David R S

    2003-04-01

    The conventional method of diagnosing disorders of the human gastro-intestinal (GI) tract is by sensors embedded in cannulae that are inserted through the anus, mouth, or nose. However, these cannulae cause significant patient discomfort and cannot be used in the small intestine. As a result, there is considerable ongoing work in developing wireless sensors that can be used in the small intestine. The radiation characteristics of sources in the GI tract cannot be readily calculated due to the complexity of the human body and its composite tissues, each with different electrical characteristics. In addition, the compact antennas used are electrically small, making them inefficient radiators. This paper presents radiation characteristics for sources in the GI tract that should allow for the optimum design of more efficient telemetry systems. The characteristics are determined using the finite-difference time-domain method with a realistic antenna model on an established fully segmented human body model. Radiation intensity outside the body was found to have a Gaussian-form relationship with frequency. Maximum radiation occurs between 450 and 900 MHz. The gut region was found generally to inhibit vertically polarized electric fields more than horizontally polarized fields.

  12. Perceiving polarization with the naked eye: characterization of human polarization sensitivity

    PubMed Central

    Temple, Shelby E.; McGregor, Juliette E.; Miles, Camilla; Graham, Laura; Miller, Josie; Buck, Jordan; Scott-Samuel, Nicholas E.; Roberts, Nicholas W.

    2015-01-01

    Like many animals, humans are sensitive to the polarization of light. We can detect the angle of polarization using an entoptic phenomenon called Haidinger's brushes, which is mediated by dichroic carotenoids in the macula lutea. While previous studies have characterized the spectral sensitivity of Haidinger's brushes, other aspects remain unexplored. We developed a novel methodology for presenting gratings in polarization-only contrast at varying degrees of polarization in order to measure the lower limits of human polarized light detection. Participants were, on average, able to perform the task down to a threshold of 56%, with some able to go as low as 23%. This makes humans the most sensitive vertebrate tested to date. Additionally, we quantified a nonlinear relationship between presented and perceived polarization angle when an observer is presented with a rotatable polarized light field. This result confirms a previous theoretical prediction of how uniaxial corneal birefringence impacts the perception of Haidinger's brushes. The rotational dynamics of Haidinger's brushes were then used to calculate corneal retardance. We suggest that psychophysical experiments, based upon the perception of polarized light, are amenable to the production of affordable technologies for self-assessment and longitudinal monitoring of visual dysfunctions such as age-related macular degeneration. PMID:26136441

  13. Perceiving polarization with the naked eye: characterization of human polarization sensitivity.

    PubMed

    Temple, Shelby E; McGregor, Juliette E; Miles, Camilla; Graham, Laura; Miller, Josie; Buck, Jordan; Scott-Samuel, Nicholas E; Roberts, Nicholas W

    2015-07-22

    Like many animals, humans are sensitive to the polarization of light. We can detect the angle of polarization using an entoptic phenomenon called Haidinger's brushes, which is mediated by dichroic carotenoids in the macula lutea. While previous studies have characterized the spectral sensitivity of Haidinger's brushes, other aspects remain unexplored. We developed a novel methodology for presenting gratings in polarization-only contrast at varying degrees of polarization in order to measure the lower limits of human polarized light detection. Participants were, on average, able to perform the task down to a threshold of 56%, with some able to go as low as 23%. This makes humans the most sensitive vertebrate tested to date. Additionally, we quantified a nonlinear relationship between presented and perceived polarization angle when an observer is presented with a rotatable polarized light field. This result confirms a previous theoretical prediction of how uniaxial corneal birefringence impacts the perception of Haidinger's brushes. The rotational dynamics of Haidinger's brushes were then used to calculate corneal retardance.We suggest that psychophysical experiments, based upon the perception of polarized light, are amenable to the production of affordable technologies for self-assessment and longitudinal monitoring of visual dysfunctions such as age-related macular degeneration.

  14. An in vivo model of human small intestine using pluripotent stem cells.

    PubMed

    Watson, Carey L; Mahe, Maxime M; Múnera, Jorge; Howell, Jonathan C; Sundaram, Nambirajan; Poling, Holly M; Schweitzer, Jamie I; Vallance, Jefferson E; Mayhew, Christopher N; Sun, Ying; Grabowski, Gregory; Finkbeiner, Stacy R; Spence, Jason R; Shroyer, Noah F; Wells, James M; Helmrath, Michael A

    2014-11-01

    Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the crypt-villus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies.

  15. Distribution of the IgG Fc Receptor, FcRn, in the Human Fetal Intestine

    PubMed Central

    Shah, Uzma; Dickinson, Bonny L.; Blumberg, Richard S.; Simister, Neil E.; Lencer, Wayne I.; Walker, W. Allan

    2010-01-01

    The intestinal Fc receptor, FcRn, functions in the maternofetal transfer of gamma globulin (IgG) in the neonatal rodent. In humans, most of this transfer is presumed to occur in utero via the placenta. Although the fetus swallows amniotic fluid that contains immunoglobulin, it is unknown whether this transfer also occurs via the fetal intestine. A human FcRn has been identified in the syncytiotrophoblast that mediates the maternofetal transfer of antibody. It has also been identified in human fetal intestine and is postulated to function in IgG transport. We hypothesize that the human fetal intestinal FcRn may play a role in IgG transport from the amniotic fluid into the fetal circulation. The aim of this study was to characterize the distribution of the FcRn along the human fetal intestine. Lysates prepared from human fetal intestine and from a nonmalignant human fetal intestinal epithelial cell line (H4) were subjected to Western blot analysis and probed using anti-FcRn antibodies. A 42-kD band, consistent with the known molecular weight of the FcRn, was detected along the human fetal intestine and in H4 cells. Expression of the human FcRn was confirmed with immunohistochemistry. Our study demonstrates the expression of FcRn along the human fetal intestine and in a human nonmalignant fetal intestinal epithelial cell line (H4), which by location indicates that FcRn could play a role in the uptake and transport of IgG in the human fetus. PMID:12538789

  16. A Novel Model of P-Glycoprotein Inhibitor Screening Using Human Small Intestinal Organoids.

    PubMed

    Zhao, Junfang; Zeng, Zhiyang; Sun, Jialiang; Zhang, Yuanjin; Li, Dali; Zhang, Xueli; Liu, Mingyao; Wang, Xin

    2017-03-01

    P-glycoprotein (P-gp), an important efflux transporter in intestine, regulates the bioavailability of orally taken drugs. To develop an in vitro model that preferably mimics the physiological microenvironment of human intestine, we employed the three-dimensionally (3D) cultured organoids from human normal small intestinal epithelium. It was observed that the intestinal crypts could efficiently form cystic organoid structure with the extension of culture time. Furthermore, the physiological expression of ABCB1 was detected at both mRNA and protein levels in cultured organoids. Rhodamine 123 (Rh123), a typical substrate of P-gp, was actively transported across 3D organoids and accumulated in the luminal space. This transport process was also inhibited by verapamil and mitotane. In summary, the above-mentioned model based on human small intestinal 3D organoids is suitable to imitate the small intestinal epithelium and could be used as a novel in vitro model especially for P-gp inhibitor screening.

  17. Dynamic Change of Polarity in Primary Cultured Spheroids of Human Colorectal Adenocarcinoma and Its Role in Metastasis.

    PubMed

    Okuyama, Hiroaki; Kondo, Jumpei; Sato, Yumi; Endo, Hiroko; Nakajima, Aya; Piulats, Jose M; Tomita, Yasuhiko; Fujiwara, Takeshi; Itoh, Yu; Mizoguchi, Akira; Ohue, Masayuki; Inoue, Masahiro

    2016-04-01

    Intestinal epithelial cells possess apical-basal polarity, which governs the exchange of nutrients and waste. Perturbation of cell polarity appears to be a general feature of cancers, although most colorectal cancers are differentiated adenocarcinomas, in which polarity is maintained to some extent. Little is known about the role of dysregulated polarity in cancer. The cancer tissue-originated spheroid method was applied to the preparation and culture of spheroids. Spheroids were cultured in suspension or in type I collagen gel. Polarity was assessed by IHC of apical markers and electron microscopy. Two types of polarity status in spheroids were observed: apical-in, with apical membrane located at cavities inside the spheroids in type I collagen gel; and apical-out, with apical membrane located at the outermost layer of spheroids in suspension. These polarities were highly interchangeable. Inhibitors of Src and dynamin attenuated the polarity switch. In patients, clusters of cancer cells that invaded vessels had both apical-in and apical-out morphologic features, whereas primary and metastatic tumors had apical-in features. In a mouse liver metastasis model, apical-out spheroids injected into the portal vein became apical-in spheroids in the liver within a few days. Inhibitors of Src and dynamin significantly decreased liver metastasis. Polarity switching was observed in spheroids and human cancer. The polarity switch was critical in an experimental liver metastasis model.

  18. Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms.

    PubMed

    Beatty, Jennifer K; Akierman, Sarah V; Motta, Jean-Paul; Muise, Stacy; Workentine, Matthew L; Harrison, Joe J; Bhargava, Amol; Beck, Paul L; Rioux, Kevin P; McKnight, Gordon Webb; Wallace, John L; Buret, Andre G

    2017-02-22

    Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.

  19. R1: Immunohistochemical study of mucins in human intestinal spirochetosis.

    PubMed

    Ogata, Sho; Shimizu, Ken; Tominaga, Susumu; Nakanishi, Kuniaki

    2017-02-08

    Most patients with human intestinal spirochetosis (HIS; a colorectal bacterial infection caused by Brachyspira species) seem asymptomatic, and its pathogenicity remains unclear. Recently, alterations in mucin expression were reported in animal Brachyspira infection. The present question was "Is mucin expression altered in HIS?". Using antibodies for MUCs 1, 2, 4, 5 AC, and 6, we immunohistochemically compared 215 specimens from 83 histology-confirmed HIS cases with 106 specimens from 26 non-HIS cases. Positive staining (which included even focal positive staining) was rated "high (+)" or "low (+)". Results were analysed for four categories of lesions, and associations between MUC expression and spirochetal presence were also analysed. In the "specimens without polyps or adenocarcinoma" category: high (+) MUC2-positivity was more frequent in HIS than in control. In the hyperplasia/serrated polyp category: in HIS (vs. control), the MUC5AC-positivity rate was lower, while high (+) MUC4-positivity was more frequent. In the conventional adenoma category: in HIS (vs. control), the MUC1-positivity rate was lower, while both high (+) MUC2-positivity and high (+) MUC5AC-positivity were less frequent. In the adenocarcinoma category: high (+) MUC2-positivity was more frequent in HIS than in control. Among the above mucins, only MUC1-positivity was significantly associated with an absence of the so-called fringe formation, an absence of spiral organisms within mucus, and an absence of strong immunopositive materials within the epithelial layer and within the subepithelial layer. The results suggest that Brachyspira infection or a related change in the microbiome may alter the large intestine mucin-expression profile in humans.

  20. Cell polarity proteins: common targets for tumorigenic human viruses

    PubMed Central

    Javier, RT

    2012-01-01

    Loss of polarity and disruption of cell junctions are common features of epithelial-derived cancer cells, and mounting evidence indicates that such defects have a direct function in the pathology of cancer. Supporting this idea, results with several different human tumor viruses indicate that their oncogenic potential depends in part on a common ability to inactivate key cell polarity proteins. For example, adenovirus (Ad) type 9 is unique among human Ads by causing exclusively estrogen-dependent mammary tumors in experimental animals and in having E4 region-encoded open reading frame 1 (E4-ORF1) as its primary oncogenic determinant. The 125-residue E4-ORF1 protein consists of two separate protein-interaction elements, one of which defines a PDZ domain-binding motif (PBM) required for E4-ORF1 to induce both cellular transformation in vitro and tumorigenesis in vivo. Most notably, the E4-ORF1 PBM mediates interactions with a selected group of cellular PDZ proteins, three of which include the cell polarity proteins Dlg1, PATJ and ZO-2. Data further indicate that these interactions promote disruption of cell junctions and a loss of cell polarity. In addition, one or more of the E4-ORF1-interacting cell polarity proteins, as well as the cell polarity protein Scribble, are common targets for the high-risk human papillomavirus (HPV) E6 or human T-cell leukemia virus type 1 (HTLV-1) Tax oncoproteins. Underscoring the significance of these observations, in humans, high-risk HPV and HTLV-1 are causative agents for cervical cancer and adult T-cell leukemia, respectively. Consequently, human tumor viruses should serve as powerful tools for deciphering mechanisms whereby disruption of cell junctions and loss of cell polarity contribute to the development of many human cancers. This review article discusses evidence supporting this hypothesis, with an emphasis on the human Ad E4-ORF1 oncoprotein. PMID:19029943

  1. Human Intestinal Enteroids: a New Model To Study Human Rotavirus Infection, Host Restriction, and Pathophysiology

    PubMed Central

    Saxena, Kapil; Blutt, Sarah E.; Ettayebi, Khalil; Zeng, Xi-Lei; Broughman, James R.; Crawford, Sue E.; Karandikar, Umesh C.; Sastri, Narayan P.; Conner, Margaret E.; Opekun, Antone R.; Graham, David Y.; Qureshi, Waqar; Sherman, Vadim; Foulke-Abel, Jennifer; In, Julie; Kovbasnjuk, Olga; Zachos, Nicholas C.; Donowitz, Mark

    2015-01-01

    ABSTRACT Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion. IMPORTANCE Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of

  2. Quantitative prediction of intestinal metabolism in humans from a simplified intestinal availability model and empirical scaling factor.

    PubMed

    Kadono, Keitaro; Akabane, Takafumi; Tabata, Kenji; Gato, Katsuhiko; Terashita, Shigeyuki; Teramura, Toshio

    2010-07-01

    This study aimed to establish a practical and convenient method of predicting intestinal availability (F(g)) in humans for highly permeable compounds at the drug discovery stage, with a focus on CYP3A4-mediated metabolism. We constructed a "simplified F(g) model," described using only metabolic parameters, assuming that passive diffusion is dominant when permeability is high and that the effect of transporters in epithelial cells is negligible. Five substrates for CYP3A4 (alprazolam, amlodipine, clonazepam, midazolam, and nifedipine) and four for both CYP3A4 and P-glycoprotein (P-gp) (nicardipine, quinidine, tacrolimus, and verapamil) were used as model compounds. Observed fraction of drug absorbed (F(a)F(g)) values for these compounds were calculated from in vivo pharmacokinetic (PK) parameters, whereas in vitro intestinal intrinsic clearance (CL(int,intestine)) was determined using human intestinal microsomes. The CL(int,intestine) for the model compounds corrected with that of midazolam was defined as CL(m,index) and incorporated into a simplified F(g) model with empirical scaling factor. Regardless of whether the compound was a P-gp substrate, the F(a)F(g) could be reasonably fitted by the simplified F(g) model, and the value of the empirical scaling factor was well estimated. These results suggest that the effects of P-gp on F(a) and F(g) are substantially minor, at least in the case of highly permeable compounds. Furthermore, liver intrinsic clearance (CL(int,liver)) can be used as a surrogate index of intestinal metabolism based on the relationship between CL(int,liver) and CL(m,index). F(g) can be easily predicted using a simplified F(g) model with the empirical scaling factor, enabling more confident selection of drug candidates with desirable PK profiles in humans.

  3. Planar Cell Polarity Signaling: From Fly Development to Human Disease

    PubMed Central

    Simons, Matias; Mlodzik, Marek

    2009-01-01

    Most, if not all, cell types and tissues display several aspects of polarization. In addition to the ubiquitous epithelial cell polarity along the apical-basolateral axis, many epithelial tissues and organs are also polarized within the plane of the epithelium. This is generally referred to as planar cell polarity (PCP; or historically, tissue polarity). Genetic screens in Drosophila pioneered the discovery of core PCP factors, and subsequent work in vertebrates has established that the respective pathways are evolutionarily conserved. PCP is not restricted only to epithelial tissues but is also found in mesenchymal cells, where it can regulate cell migration and cell intercalation. Moreover, particularly in vertebrates, the conserved core PCP signaling factors have recently been found to be associated with the orientation or formation of cilia. This review discusses new developments in the molecular understanding of PCP establishment in Drosophila and vertebrates; these developments are integrated with new evidence that links PCP signaling to human disease. PMID:18710302

  4. Metabolism of green tea catechins by the human small intestine.

    PubMed

    Schantz, Markus; Erk, Thomas; Richling, Elke

    2010-10-01

    Numerous studies have shown that green tea polyphenols can be degraded in the colon, and there is abundant knowledge about the metabolites of these substances that appear in urine and plasma after green tea ingestion. However, there is very little information on the extent and nature of intestinal degradation of green tea catechins in humans. Therefore, the aim of this study was to examine in detail the microbial metabolism and chemical stability of these polyphenols in the small intestine using a well-established ex vivo model. For this purpose, fresh ileostomy fluids from two probands were incubated for 24 h under anaerobic conditions with (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin 3-O-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatchin 3-O-gallate (EGCG) and gallic acid (GA). After lyophilisation and extraction, metabolites were separated, identified and quantified by high performance liquid chromatography-photodiode array detection (HPLC-DAD) and HPLC-ESI-tandem mass spectrometry. Two metabolites of EC and C (3', 4', 5'-trihydroxyphenyl-γ-valerolactone and 3', 4'-dihydroxyphenyl-γ-valerolactone) were identified. In addition, 3', 4', 5'-trihydroxyphenyl-γ-valerolactone was detected as a metabolite of EGC, and (after 24-h incubation) pyrogallol as a degradation product of GA. Cleavage of the GA esters of EGCG and ECG was also observed, with variations dependent on the sources (probands) of the ileal fluids, which differed substantially microbiotically. The results provide new information about the degradation of green tea catechins in the gastrointestinal tract, notably that microbiota-dependent liberation of GA esters may occur before these compounds reach the colon.

  5. Effect of Ceftaroline on Normal Human Intestinal Microflora▿

    PubMed Central

    Panagiotidis, Georgios; Bäckström, Tobias; Asker-Hagelberg, Charlotte; Jandourek, Alena; Weintraub, Andrej; Nord, Carl Erik

    2010-01-01

    Ceftaroline is a new broad-spectrum cephalosporin being developed for the treatment of serious bacterial infections, including those caused by aerobic Gram-positive and Gram-negative bacteria. The purpose of the present study was to investigate the effect of administration of ceftaroline on the intestinal flora of healthy subjects. Twelve healthy subjects (6 males and 6 females), 20 to 41 years of age, received ceftaroline (600 mg) by intravenous infusion every 12 h (q12h) for 7 days. Plasma and feces were collected for determination of ceftaroline concentration and analysis of fecal flora. Fecal specimens were cultured on nonselective and selective media. Different colony types were counted, isolated in pure culture, and identified to the genus level. All new strains of colonizing bacteria were tested for susceptibility to ceftaroline. The concentrations of ceftaroline in plasma were as follows: on day 2, 17.5 to 34.8 mg/liter; on day 5, 19.7 to 33.2 mg/liter; and on day 7, 18.0 to 29.8 mg/liter. No ceftaroline concentrations were found on day −1, 9, 14, or 21. No measurable concentrations in feces were found on day −1, 2, 5, 7, 9, 14, or 21. There was a minor impact on the numbers of Escherichia coli strains, while the numbers of enterococci and Candida albicans strains were not affected. There were moderate decreases in the numbers of bifidobacteria and lactobacilli during the first 7 days, while the numbers of clostridia increased during the same period. No impact on the numbers of Bacteroides bacteria was noticed. No new colonizing aerobic or anaerobic bacteria resistant to ceftaroline (MIC ≥ 4 mg/liter) were found. Ceftaroline had no significant ecological impact on the human intestinal microflora. PMID:20231399

  6. Cloning and expression of the human vasoactive intestinal peptide receptor.

    PubMed Central

    Sreedharan, S P; Robichon, A; Peterson, K E; Goetzl, E J

    1991-01-01

    Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator found in the central and peripheral nervous system. Distinct subsets of neural, respiratory, gastrointestinal, and immune cells bear specific high-affinity receptors for VIP, which are associated with a guanine nucleotide-binding (G) protein capable of activating adenylate cyclase. A cDNA clone (GPRN1) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line of leukemic pre-B lymphoblasts and the HT-29 line of colon carcinoma cells. The deduced 362-amino acid polypeptide sequence encoded by GPRN1 shares a seven-transmembrane-segment hydropathicity profile with other G protein-coupled receptors. Northern blot analyses identified a 2.7-kilobase transcript of the VIP receptor in Nalm 6 and HT-29 cells as well as in tissues from rat brain, colon, heart, lung, kidney, spleen, and small intestine. COS-6 cells transfected with GPRN1 bound 125I-labeled VIP specifically with a dissociation constant (Kd) of 2.5 nM. VIP--and less effectively secretin, peptide histidine isoleucine (PHI), and glucagon competitively displaced bound 125I-VIP from transfected COS-6 cells, with potencies in the order VIP greater than secretin = PHI much greater than glucagon. VIP stimulated adenylate cyclase activity in stably transfected Chinese hamster ovary K1 cells, inducing a 3-fold increase in the intracellular level of cAMP. When the antisense orientation of the VIP receptor clone was introduced into HT-29 cells, there was a 50% suppression of the specific binding of 125I-VIP and of the VIP-induced increase in cAMP level, relative to untransfected cells. The VIP receptor cloned exhibits less than or equal to 24% homology with other receptors in the same superfamily and thus represents a subset of G protein-coupled receptors for peptide ligands. Images PMID:1675791

  7. Human colostrum oligosaccharides modulate major immunologic pathways of immature human intestine

    PubMed Central

    He, YingYing; Liu, ShuBai; Leone, Serena; Newburg, David S.

    2014-01-01

    The immature neonatal intestinal immune system hyperreacts to newly colonizing unfamiliar bacteria. The hypothesis that human milk oligosaccharides from colostrum (cHMOS) can directly modulate the signaling pathways of the immature mucosa was tested. Modulation of cytokine immune signaling by HMOS was measured ex vivo in intact immature (fetal) human intestinal mucosa. From the genes whose transcription was modulated by colostrum HMOS (cHMOS), Ingenuity Pathway Analysis identified networks controlling immune cell communication, intestinal mucosal immune system differentiation, and homeostasis. cHMOS attenuate pathogen-associated molecular pattern (PAMP)-stimulated acute phase inflammatory cytokine protein levels (IL-8, IL-6, MCP-1/2, IL-1β), while elevating cytokines involved in tissue repair and homeostasis. 3’-, 4-, and 6’-galactosyllactoses of cHMOS account for specific immunomodulation of PIC-induced IL-8 levels. cHMOS attenuate mucosal responses to surface inflammatory stimuli during early development, while enhancing signals that support maturation of the intestinal mucosal immune system. PMID:24691111

  8. Human mini-guts: new insights into intestinal physiology and host–pathogen interactions

    PubMed Central

    In, Julie G.; Foulke-Abel, Jennifer; Estes, Mary K.; Zachos, Nicholas C.; Kovbasnjuk, Olga; Donowitz, Mark

    2016-01-01

    The development of indefinitely propagating human ‘mini-guts’ has led to a rapid advance in gastrointestinal research related to transport physiology, developmental biology, pharmacology, and pathophysiology. These mini-guts, also called enteroids or colonoids, are derived from LGR5+ intestinal stem cells isolated from the small intestine or colon. Addition of WNT3A and other growth factors promotes stemness and results in viable, physiologically functional human intestinal or colonic cultures that develop a crypt–villus axis and can be differentiated into all intestinal epithelial cell types. The success of research using human enteroids has highlighted the limitations of using animals or in vitro, cancer-derived cell lines to model transport physiology and pathophysiology. For example, curative or preventive therapies for acute enteric infections have been limited, mostly due to the lack of a physiological human intestinal model. However, the human enteroid model enables specific functional studies of secretion and absorption in each intestinal segment as well as observations of the earliest molecular events that occur during enteric infections. This Review describes studies characterizing these human mini-guts as a physiological model to investigate intestinal transport and host pathogen interactions. PMID:27677718

  9. Human mini-guts: new insights into intestinal physiology and host-pathogen interactions.

    PubMed

    In, Julie G; Foulke-Abel, Jennifer; Estes, Mary K; Zachos, Nicholas C; Kovbasnjuk, Olga; Donowitz, Mark

    2016-11-01

    The development of indefinitely propagating human 'mini-guts' has led to a rapid advance in gastrointestinal research related to transport physiology, developmental biology, pharmacology, and pathophysiology. These mini-guts, also called enteroids or colonoids, are derived from LGR5(+) intestinal stem cells isolated from the small intestine or colon. Addition of WNT3A and other growth factors promotes stemness and results in viable, physiologically functional human intestinal or colonic cultures that develop a crypt-villus axis and can be differentiated into all intestinal epithelial cell types. The success of research using human enteroids has highlighted the limitations of using animals or in vitro, cancer-derived cell lines to model transport physiology and pathophysiology. For example, curative or preventive therapies for acute enteric infections have been limited, mostly due to the lack of a physiological human intestinal model. However, the human enteroid model enables specific functional studies of secretion and absorption in each intestinal segment as well as observations of the earliest molecular events that occur during enteric infections. This Review describes studies characterizing these human mini-guts as a physiological model to investigate intestinal transport and host-pathogen interactions.

  10. Involvement of Concentrative Nucleoside Transporter 1 in Intestinal Absorption of Trifluridine Using Human Small Intestinal Epithelial Cells.

    PubMed

    Takahashi, Koichi; Yoshisue, Kunihiro; Chiba, Masato; Nakanishi, Takeo; Tamai, Ikumi

    2015-09-01

    TAS-102, which is effective for refractory metastatic colorectal cancer, is a combination drug of anticancer trifluridine (FTD; which is derived from pyrimidine nucleoside) and FTD-metabolizing enzyme inhibitor tipiracil hydrochloride (TPI) at a molecular ratio of 1:0.5. To evaluate the intestinal absorption mechanism of FTD, the uptake and transcellular transport of FTD by human small intestinal epithelial cell (HIEC) monolayer as a model of human intestinal epithelial cells was investigated. The uptake and membrane permeability of FTD by HIEC monolayers were saturable, Na(+) -dependent, and inhibited by nucleosides. These transport characteristics are mostly comparable with those of concentrative nucleoside transporters (CNTs). Moreover, the uptake of FTD by CNT1-expressing Xenopus oocytes was the highest among human CNT transporters. The obtained Km and Vmax values of FTD by CNT1 were 69.0 μM and 516 pmol/oocyte/30 min, respectively. The transcellular transport of FTD by Caco-2 cells, where CNT1 is heterologously expressed, from apical to basolateral side was greater than that by Mock cells. In conclusion, these results demonstrated that FTD exhibits high oral absorption by the contribution of human CNT1.

  11. An in vivo model of human small intestine using pluripotent stem cells

    PubMed Central

    Watson, Carey L; Mahe, Maxime M; Múnera, Jorge; Howell, Jonathan C; Sundaram, Nambirajan; Poling, Holly M; Schweitzer, Jamie I; Vallance, Jefferson E; Mayhew, Christopher N; Sun, Ying; Grabowski, Gregory; Finkbeiner, Stacy R; Spence, Jason R; Shroyer, Noah F; Wells, James M; Helmrath, Michael A

    2015-01-01

    Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant1,2. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds3,4. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)5,6 that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the cryptvillus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response7–9. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies. PMID:25326803

  12. Human intestinal absorption--neutral molecules and ionic species.

    PubMed

    Abraham, Michael H

    2014-07-01

    Analysis of percentage human intestinal absorption (%HIA) for 280 drugs shows that an excellent fit can be obtained using only three descriptors for neutral molecules with a SD of 13.9%. Use of descriptors for individual cations and anions does not lead to any better goodness-of-fit. It is noted that diffusion coefficients in water for ionized molecules are almost identical to those for the corresponding neutral molecules. Comparison of equation coefficients for HIA with those for other processes shows that HIA resembles diffusion in water but does not resemble permeation through biological bilayers. It is shown that compound substituent effects on HIA are near those for diffusion but are far away from substituent effects on permeation through a typical bilayer. Calculations indicate that rates of permeation through an unstirred mucosal layer are of the same order as experimental rates of permeation in HIA. It is concluded that for the 280 compound set, diffusion through the unstirred mucosal layer is the rate determining step. The effect on pK(a) in transfer of acids and bases from water to another solvent, and of diffusion past a negative charge in a phase/bilayer is also considered.

  13. Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis.

    PubMed

    Malyukova, Irina; Murray, Karen F; Zhu, Chengru; Boedeker, Edgar; Kane, Anne; Patterson, Kathleen; Peterson, Jeffrey R; Donowitz, Mark; Kovbasnjuk, Olga

    2009-01-01

    Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.

  14. Characterization of intracellular pteroylpolyglutamate hydrolase (PPH) from human intestinal mucosa

    SciTech Connect

    Wang, T.T.Y.; Chandler, C.J.; Halsted, C.H.

    1986-03-01

    There are two forms of pteroylpolyglutamate hydrolase (PPH) in the human intestinal mucosa, one in the brush border membrane and the other intracellular; brush border PPH is an exopeptidase with optimal activity at pH 6.5 and a requirement for zinc. The presence study characterized human intracellular PPH and compared its properties to those of brush border PPH. Intracellular PPH was purified 30-fold. The enzyme had a MW of 75,000 by gel filtration, was optimally active at pH 4.5, and had an isoelectric point at pH 8.0. In contrast to brush border PPH, intracellular PPH was unstable at increasing temperatures, was unaffected by dialysis against chelating agents and showed no requirement for Zn/sup 2 +/. Using PteGlu/sub 2/(/sup 14/C)Glu as substrate, they demonstrated a K/sub m/ of 1.2 ..mu..M and increasing affinity for folates with longer glutamate chains. Intracellular PPH required the complete folic acid (PteGlu) moiety and a ..gamma..-glutamyl linkage for activity. Using ion exchange chromatography and an HPLC method to determine the hydrolytic products of the reaction, they found intracellular PPH could cleave both internal and terminal ..gamma..-glutamyl linkages, with PteGlu as an end product. After subcellular fractionation of the mucosa, PPH was found in the lysosomes. In summary, the distinct characteristics of brush border and intracellular PPH suggest that the two hydrolases serve different roles in folate metabolism.

  15. Identification of Human Intestinal Bacteria that Promote or Inhibit Inflammation

    DTIC Science & Technology

    2012-11-01

    microbiota  composition  is  critical  in  intestinal  inflammation  and  their  perturbations  are...Evidence   suggests   that   intestinal   microbiota  is  a  very  important  factor   in   IBD   (Kaser   et   al.,   2009...treatment   of   intestinal   inflammation.   Pertinent   adjustments   in   the   microbiota   of   patients  

  16. Human intestinal parasites in primary school children in Kampala, Uganda.

    PubMed

    Kabatereine, N B; Kemijumbi, J; Kazibwe, F; Onapa, A W

    1997-05-01

    A cross sectional survey on intestinal parasite infections was carried out in 5,313 pupils between the ages of ten and fifteen years in 98 primary schools in Kampala. The aim was to identify the types and distribution of intestinal parasites and to estimate the prevalence in school children. Trichuris trichiura (28%), Ascaris lumbricoides (17%) and hookworms (12.9%) were common infections among the children. Other less commonly found parasites were S.mansoni, Strongyloides stercolaris, Taenia sp, Enterobius vermicularis, Giardia lamblia, Entamoeba coli and E. histolytica. Refuse dumps are probably a significant source of transmission of intestinal helminthic infections in Kampala.

  17. The metabolic profile of acteoside produced by human or rat intestinal bacteria or intestinal enzyme in vitro employed UPLC-Q-TOF-MS.

    PubMed

    Cui, Qingling; Pan, Yingni; Xu, Xiaotong; Zhang, Wenjie; Wu, Xiao; Qu, Shouhe; Liu, Xiaoqiu

    2016-03-01

    Acteoside, the main and representative phenylethanoid glycosides of Herba Cistanches, possesses wide bioactivities but low oral bioavailability. It may serve as the prodrug and be converted into the active forms in gastrointestinal tract, which mainly occurred in intestinal tract composed of intestinal bacteria and intestinal enzyme. Intestinal bacteria, a new drug target, take a significant role on exerting pharmacological effects of drugs by oral administration. In this paper, acteoside was incubated with human or rat intestinal bacteria or rat intestinal enzyme for 36 h to seek metabolites responsible for pharmacodynamics. The samples were analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Besides the parent compound, 14 metabolites were detected and identified based on their retention times and fragmentation patterns in their MS spectra including 8 degradation metabolites, 2 isomers in intestinal bacteria and intestinal enzyme samples and 4 parent metabolites only found in intestinal enzymes. The metabolic pathway of acteoside was thus proposed. Identification of these metabolites of acteoside by the intestinal bacteria or intestinal enzyme gave an insight to clarify pharmacological mechanism of traditional Chinese medicines and identify the real active molecules.

  18. In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development

    PubMed Central

    Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H.; Nagy, Melinda S.; Chin, Alana M.; Thomson, Matthew; Klein, Ophir D.

    2017-01-01

    ABSTRACT The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research. PMID:27927684

  19. Type 1 diabetes: role of intestinal microbiome in humans and mice.

    PubMed

    Boerner, Brian P; Sarvetnick, Nora E

    2011-12-01

    Type 1 diabetes is a disease involving autoimmune destruction of pancreatic beta cells in genetically predisposed individuals. Identifying factors that trigger initiation and progression of autoimmunity may provide opportunities for directed prophylactic and therapeutic measures to prevent and/or treat type 1 diabetes. The human intestinal microbiome is a complex, symbiotic ecological community that influences human health and development, including the development and maintenance of the human immune system. The role of the intestinal microbiome in autoimmunity has garnered significant attention, and evidence suggests a particular role for intestinal microbiome alterations in autoimmune disease development, including type 1 diabetes. This review will examine the role of the intestinal microbiome in the development and function of the immune system and how this relates to the development of autoimmunity. Data from animal and human studies linking alterations in the intestinal microbiome and intestinal integrity with type 1 diabetes will be closely examined. Finally, we will examine the interactions between the intestinal microbiome and dietary exposures and how these interactions may further influence autoimmunity and type 1 diabetes development.

  20. In vitro patterning of pluripotent stem cell-derived intestine recapitulates in vivo human development.

    PubMed

    Tsai, Yu-Hwai; Nattiv, Roy; Dedhia, Priya H; Nagy, Melinda S; Chin, Alana M; Thomson, Matthew; Klein, Ophir D; Spence, Jason R

    2017-03-15

    The intestine plays a central role in digestion, nutrient absorption and metabolism, with individual regions of the intestine having distinct functional roles. Many examples of region-specific gene expression in the adult intestine are known, but how intestinal regional identity is established during development is a largely unresolved issue. Here, we have identified several genes that are expressed in a region-specific manner in the developing human intestine. Using human embryonic stem cell-derived intestinal organoids, we demonstrate that the duration of exposure to active FGF and WNT signaling controls regional identity. Short-term exposure to FGF4 and CHIR99021 (a GSK3β inhibitor that stabilizes β-catenin) resulted in organoids with gene expression patterns similar to developing human duodenum, whereas longer exposure resulted in organoids similar to ileum. When region-specific organoids were transplanted into immunocompromised mice, duodenum-like organoids and ileum-like organoids retained their regional identity, demonstrating that regional identity of organoids is stable after initial patterning occurs. This work provides insights into the mechanisms that control regional specification of the developing human intestine and provides new tools for basic and translational research.

  1. Metabolism of puerarin and daidzin by human intestinal bacteria and their relation to in vitro cytotoxicity.

    PubMed

    Kim, D H; Yu, K U; Bae, E A; Han, M J

    1998-06-01

    When puerarin or daidzin were incubated for 24 h with human intestinal bacteria, two metabolites, daidzein and calycosin, were produced from them, respectively. The metabolic time course of puerarin was as follows: at an early time, puerarin was converted to daidzin, and then calycosin. The metabolic time course of daidzin by human intestinal bacteria was also similar to that of puerarin. The in vitro cytotoxicities of these metabolites, calycosin and daidzein, were superior to those of puerarin and daidzein.

  2. Human Intestinal Barrier Function in Health and Disease

    PubMed Central

    König, Julia; Wells, Jerry; Cani, Patrice D; García-Ródenas, Clara L; MacDonald, Tom; Mercenier, Annick; Whyte, Jacqueline; Troost, Freddy; Brummer, Robert-Jan

    2016-01-01

    The gastrointestinal tract consists of an enormous surface area that is optimized to efficiently absorb nutrients, water, and electrolytes from food. At the same time, it needs to provide a tight barrier against the ingress of harmful substances, and protect against a reaction to omnipresent harmless compounds. A dysfunctional intestinal barrier is associated with various diseases and disorders. In this review, the role of intestinal permeability in common disorders such as infections with intestinal pathogens, inflammatory bowel disease, irritable bowel syndrome, obesity, celiac disease, non-celiac gluten sensitivity, and food allergies will be discussed. In addition, the effect of the frequently prescribed drugs proton pump inhibitors and non-steroidal anti-inflammatory drugs on intestinal permeability, as well as commonly used methods to assess barrier function will be reviewed. PMID:27763627

  3. Diet and the development of the human intestinal microbiome

    PubMed Central

    Voreades, Noah; Kozil, Anne; Weir, Tiffany L.

    2014-01-01

    The important role of the gut microbiome in maintaining human health has necessitated a better understanding of the temporal dynamics of intestinal microbial communities as well as the host and environmental factors driving these dynamics. Genetics, mode of birth, infant feeding patterns, antibiotic usage, sanitary living conditions and long term dietary habits contribute to shaping the composition of the gut microbiome. This review focuses primarily on diet, as it is one of the most pivotal factors in the development of the human gut microbiome from infancy to the elderly. The infant gut microbiota is characterized by a high degree of instability, only reaching a state similar to that of adults by 2–3 years of age; consistent with the establishment of a varied solid food diet. The diet-related factors influencing the development of the infant gut microbiome include whether the child is breast or formula-fed as well as how and when solid foods are introduced. In contrast to the infant gut, the adult gut microbiome is resilient to large shifts in community structure. Several studies have shown that dietary changes induce transient fluctuations in the adult microbiome, sometimes in as little as 24 h; however, the microbial community rapidly returns to its stable state. Current knowledge of how long-term dietary habits shape the gut microbiome is limited by the lack of long-term feeding studies coupled with temporal gut microbiota characterization. However, long-term weight loss studies have been shown to alter the ratio of the Bacteroidetes and Firmicutes, the two major bacterial phyla residing in the human gastrointestinal tract. With aging, diet-related factors such as malnutrition are associated with microbiome shifts, although the cause and effect relationship between these factors has not been established. Increased pharmaceutical usage is also more prevalent in the elderly and can contribute to reduced gut microbiota stability and diversity. Foods containing

  4. Transformation of trollioside and isoquercetin by human intestinal flora in vitro.

    PubMed

    Yuan, Ming; Shi, Duo-Zhi; Wang, Teng-Yu; Zheng, Shi-Qi; Liu, Li-Jia; Sun, Zhen-Xiao; Wang, Ru-Feng; Ding, Yi

    2016-03-01

    The present study was designed to determine the intestinal bacterial metabolites of trollioside and isoquercetin and their antibacterial activities. A systematic in vitro biotransformation investigation on trollioside and isoquercetin, including metabolite identification, metabolic pathway deduction, and time course, was accomplished using a human intestinal bacterial model. The metabolites were analyzed and identified by HPLC and HPLC-MS. The antibacterial activities of trollioside, isoquercetin, and their metabolites were evaluated using the broth microdilution method with berberine as a positive control, and their potency was measured as minimal inhibitory concentration (MIC). Our results indicated that trollioside and isoquercetin were metabolized by human intestinal flora through O-deglycosylation, yielding aglycones proglobeflowery acid and quercetin, respectively The antibacterial activities of both metabolites were more potent than that of their parent compounds. In conclusion, trollioside and isoquercetin are totally and rapidly transformed by human intestinal bacteria in vitro and the transformation favors the improvement of the antibacterial activities of the parent compounds.

  5. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

    PubMed

    Vuille-dit-Bille, Raphael N; Camargo, Simone M; Emmenegger, Luca; Sasse, Tom; Kummer, Eva; Jando, Julia; Hamie, Qeumars M; Meier, Chantal F; Hunziker, Schirin; Forras-Kaufmann, Zsofia; Kuyumcu, Sena; Fox, Mark; Schwizer, Werner; Fried, Michael; Lindenmeyer, Maja; Götze, Oliver; Verrey, François

    2015-04-01

    Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression.

  6. Characteristic Analysis of Intestinal Transport in Enterocyte-Like Cells Differentiated from Human Induced Pluripotent Stem Cells.

    PubMed

    Kodama, Nao; Iwao, Takahiro; Katano, Takahiro; Ohta, Kinya; Yuasa, Hiroaki; Matsunaga, Tamihide

    2016-10-01

    We previously demonstrated that differentiated enterocytes from human induced pluripotent stem (iPS) cells exhibited drug-metabolizing activities and cytochrome P450 CYP3A4 inducibility. The aim of this study was to apply human iPS cell-derived enterocytes in pharmacokinetic studies by investigating the characteristics of drug transport into enterocyte-like cells. Human iPS cells cultured on feeder cells were differentiated into endodermal cells using activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, epidermal growth factor and small-molecule compounds induced the maturation of the intestinal stem cell-like cells. After differentiation, we performed transepithelial electrical resistance (TEER) measurements, immunofluorescence staining, and transport studies. TEER values increased in a time-dependent manner and reached approximately 100 Ω × cm(2) Efflux transport of Hoechst 33342, a substrate of breast cancer resistance protein (BCRP), was observed and inhibited by the BCRP inhibitor Ko143. The uptake of peptide transporter 1 substrate glycylsarcosine was also confirmed and suppressed when the temperature was lowered to 4°C. Using immunofluorescence staining, villin and Na(+)-K(+) ATPase were expressed. These results suggest that human iPS cell-derived enterocytes had loose tight junctions, polarity, as well as uptake and efflux transport functions. In addition, the rank order of apparent membrane permeability coefficient (Papp) values of these test compounds across the enterocyte-like cell membrane corresponded to the fraction absorbance (Fa) values. Therefore, differentiated enterocytes from human iPS cells may provide a useful comprehensive evaluation model of drug transport and metabolism in the small intestine.

  7. Practical techniques for detection of Toll-like receptor-4 in the human intestine.

    PubMed

    Ungaro, Ryan; Abreu, Maria T; Fukata, Masayuki

    2009-01-01

    The human intestine has evolved in the presence of a diverse array of luminal microorganisms. In order to maintain intestinal homeostasis, mucosal immune responses to theses microorganisms must be tightly regulated. The intestine needs to be able to respond to pathogenic organisms while at the same time maintain tolerance to normal commensal flora. Toll-like receptors (TLRs) play an important role in this delicate balance. TLRs are transmembrane noncatalytic receptor proteins that induce activation of innate and adaptive immune responses to microorganisms by recognizing structurally conserved molecular patterns of microbes. Expression of TLRs by intestinal epithelial cell is normally down-regulated to maintain immune tolerance to the luminal microorganisms.One of the challenges of TLR research in the human intestine is that it is difficult for many experimental methods to detect very low expression of TLRs within the intestinal mucosa. Quantitative methods such as PCR are limited in their ability to detect TLR expression by specific cell types within a tissue sample, which can be important when studying the contribution of TLR signaling to pathological conditions. In this regard, immunohistochemistry (IHC) is advantageous in that one can visualize the distribution and localization of target proteins within both normal and pathologic parts of a given tissue sample. We found that a subset of human colorectal cancers over-express TLR4 by means of immunofluorescence (IF) and IHC methods. Localization of TLR4 within cancer tissue often appears to be patchy, making IHC an appropriate way to examine these changes. We will describe our current techniques to detect TLR4 in paraffin-embedded human large intestine sections. Establishing a practical IHC technique that may provide consistent results between laboratories will significantly enhance understanding of the role of TLRs in human intestinal health and disease.

  8. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine.

    PubMed

    Chang, C C; Sakashita, N; Ornvold, K; Lee, O; Chang, E T; Dong, R; Lin, S; Lee, C Y; Strom, S C; Kashyap, R; Fung, J J; Farese, R V; Patoiseau, J F; Delhon, A; Chang, T Y

    2000-09-08

    By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

  9. In Vitro Polarization of Colonoids to Create an Intestinal Stem Cell Compartment

    PubMed Central

    Attayek, Peter J.; Ahmad, Asad A.; Wang, Yuli; Williamson, Ian; Sims, Christopher E.; Magness, Scott T.; Allbritton, Nancy L.

    2016-01-01

    The polarity of proliferative and differentiated cellular compartments of colonic crypts is believed to be specified by gradients of key mitogens and morphogens. Indirect evidence demonstrates a tight correlation between Wnt- pathway activity and the basal-luminal patterning; however, to date there has been no direct experimental manipulation demonstrating that a chemical gradient of signaling factors can produce similar patterning under controlled conditions. In the current work, colonic organoids (colonoids) derived from cultured, multicellular organoid fragments or single stem cells were exposed in culture to steep linear gradients of two Wnt-signaling ligands, Wnt-3a and R-spondin1. The use of a genetically engineered Sox9-Sox9EGFP:CAGDsRED reporter gene mouse model and EdU-based labeling enabled crypt patterning to be quantified in the developing colonoids. Colonoids derived from multicellular fragments cultured for 5 days under a Wnt-3a or a combined Wnt-3a and R-spondin1 gradient were highly polarized with proliferative cells localizing to the region of the higher morphogen concentration. In a Wnt-3a gradient, Sox9EGFP polarization was 7.3 times greater than that of colonoids cultured in the absence of a gradient; and the extent of EdU polarization was 2.2 times greater than that in the absence of a gradient. Under a Wnt-3a/R-spondin1 gradient, Sox9EGFP polarization was 8.2 times greater than that of colonoids cultured in the absence of a gradient while the extent of EdU polarization was 10 times greater than that in the absence of a gradient. Colonoids derived from single stem cells cultured in Wnt-3a/R-spondin1 gradients were most highly polarized demonstrated by a Sox9EGFP polarization 20 times that of colonoids grown in the absence of a gradient. This data provides direct evidence that a linear gradient of Wnt signaling factors applied to colonic stem cells is sufficient to direct patterning of the colonoid unit in culture. PMID:27100890

  10. Deoxycholic acid formation in gnotobiotic mice associated with human intestinal bacteria.

    PubMed

    Narushima, Seiko; Itoha, Kikuji; Miyamoto, Yukiko; Park, Sang-Hee; Nagata, Keiko; Kuruma, Kazuo; Uchida, Kiyohisa

    2006-09-01

    In humans and animals, intestinal flora is indispensable for bile acid transformation. The goal of our study was to establish gnotobiotic mice with intestinal bacteria of human origin in order to examine the role of intestinal bacteria in the transformation of bile acids in vivo using the technique of gnotobiology. Eight strains of bile acid-deconjugating bacteria were isolated from ex-germ-free mice inoculated with a human fecal dilution of 10(-6), and five strains of 7alpha-dehydroxylating bacteria were isolated from the intestine of limited human flora mice inoculated only with clostridia. The results of biochemical tests and 16S rDNA sequence analysis showed that seven out of eight bile acid-deconjugating strains belong to a bacteroides cluster (Bacteroides vulgatus, B. distasonis, and B. uniformis), and one strain had high similarity with Bilophila wadsworthia. All five strains that converted cholic acid to deoxycholic acid had greatest similarity with Clostridium hylemonae. A combination of 10 isolated strains converted taurocholic acid into deoxycholic acid both in vitro and in the mouse intestine. These results indicate that the predominant bacteria, mainly Bacteroides, in human feces comprise one of the main bacterial groups for the deconjugation of bile acids, and clostridia may play an important role in 7aplha-dehydroxylation of free-form primary bile acids in the intestine although these strains are not predominant. The gnotobiotic mouse with bacteria of human origin could be a useful model in studies of bile acid metabolism by human intestinal bacteria in vivo.

  11. A General O-Glycosylation System Important to the Physiology of a Major Human Intestinal Symbiont

    PubMed Central

    Fletcher, C. Mark; Coyne, Michael J.; Villa, Otto F.; Chatzidaki-Livanis, Maria; Comstock, Laurie E.

    2009-01-01

    SUMMARY The Bacteroides are a numerically dominant genus of the human intestinal microbiota. These organisms harbor a rare bacterial pathway for incorporation of exogenous fucose into capsular polysaccharides and glycoproteins. The infrequency of glycoprotein synthesis by bacteria prompted a more detailed analysis of this process. Here, we demonstrate that Bacteroides fragilis has a general O-glycosylation system. The proteins targeted for glycosylation include those predicted to be involved in protein folding, protein-protein interactions, peptide degradation, as well as surface lipoproteins. Protein glycosylation is central to the physiology of B. fragilis and is necessary for the organism to competitively colonize the mammalian intestine. We provide evidence that general O-glycosylation systems are conserved among intestinal Bacteroides species and likely contribute to the predominance of Bacteroides in the human intestine. PMID:19379697

  12. Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite.

    PubMed

    Wawrzyniak, Ivan; Texier, Catherine; Poirier, Philippe; Viscogliosi, Eric; Tan, Kevin S W; Delbac, Frédéric; El Alaoui, Hicham

    2012-09-01

    Blastocystis spp. are unicellular anaerobic intestinal parasites of both humans and animals and the most prevalent ones found in human stool samples. Their association with various gastrointestinal disorders raises the questions of its pathogenicity and of the molecular mechanisms involved. Since secreted proteases are well-known to be implicated in intestinal parasite virulence, we intended to determine whether Blastocystis spp. possess such pathogenic factors. In silico analysis of the Blastocystis subtype 7 (ST7) genome sequence highlighted 22 genes coding proteases which were predicted to be secreted. We characterized the proteolytic activities in the secretory products of Blastocystis ST7 using specific protease inhibitors. Two cysteine proteases, a cathepsin B and a legumain, were identified in the parasite culture supernatant by gelatin zymographic SDS-PAGE gel and MS/MS analysis. These proteases might act on intestinal cells and disturb gut function. This work provides serious molecular candidates to link Blastocystis spp. and intestinal disorders.

  13. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes.

    PubMed

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N; Brantley, Scott J; Paine, Mary F; Oberlies, Nicholas H

    2011-02-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC(50)) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and < 10 µM, respectively, using HIM as the enzyme source and 2.8, 4.3, and < 10 µM, respectively, using recombinant CYP3A4 as the enzyme source. These in vitro inhibitory potencies, which are within the range of those reported for two CYP3A inhibitory components in grapefruit juice, suggest that these triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study.

  14. Dietary fat composition, food matrix and relative polarity modulate the micellarization and intestinal uptake of carotenoids from vegetables and fruits.

    PubMed

    Mashurabad, Purna Chandra; Palika, Ravindranadh; Jyrwa, Yvette Wilda; Bhaskarachary, K; Pullakhandam, Raghu

    2017-02-01

    Dietary fat increases carotenoid bioavailability by facilitating their transfer to the aqueous micellar fraction during digestion. However, the specific effect of both quantity and type of dietary fat required for optimal carotenoid absorption remained unexplored. In the present study, the effect of amount and type of vegetable oils on carotenoid micellarization from carrot, spinach, drumstick leaves and papaya using in vitro digestion/Caco-2 cell model have been assessed. Although, dietary fat (0.5-10% w/w) significantly increased the micellarization of carotenoids from all the test foods, the extent of increase was determined by the food matrix (papaya > drumstick = spinach > carrot) and polarity of carotenoids (lutein > β-carotene = α-carotene > lycopene). Among the dietary fats tested the carotenoid micellarization was twofold to threefold higher with dietary fat rich in unsaturated fatty acids (olive oil = soybean oil = sunflower oil) compared to saturated fatty acids (peanut oil = palm oil > coconut oil). Intestinal cell uptake of lutein exceeded that of β-carotene from micellar fraction of spinach leaves digested with various oils. However, cellular uptake of β-carotene is depended on the carotenoid content in micellar fraction rather than the type of fat used. Together these results suggest that food matrix, polarity of carotenoids and type of dietary fat determines the extent of carotenoid micellarization from vegetables and fruits.

  15. Antigen-Presenting Human γδ T Cells Promote Intestinal CD4(+) T Cell Expression of IL-22 and Mucosal Release of Calprotectin.

    PubMed

    Tyler, Christopher J; McCarthy, Neil E; Lindsay, James O; Stagg, Andrew J; Moser, Bernhard; Eberl, Matthias

    2017-03-22

    The cytokine IL-22 plays a critical role in mucosal barrier defense, but the mechanisms that promote IL-22 expression in the human intestine remain poorly understood. As human microbe-responsive Vγ9/Vδ2 T cells are abundant in the gut and recognize microbiota-associated metabolites, we assessed their potential to induce IL-22 expression by intestinal CD4(+) T cells. Vγ9/Vδ2 T cells with characteristics of APCs were generated from human blood and intestinal organ cultures, then cocultured with naive and memory CD4(+) T cells obtained from human blood or the colon. The potency of blood and intestinal γδ T-APCs was compared with that of monocytes and dendritic cells, by assessing CD4(+) T cell phenotypes and proliferation as well as cytokine and transcription factor profiles. Vγ9/Vδ2 T cells in human blood, colon, and terminal ileum acquired APC functions upon microbial activation in the presence of microenvironmental signals including IL-15, and were capable of polarizing both blood and colonic CD4(+) T cells toward distinct effector fates. Unlike monocytes or dendritic cells, gut-homing γδ T-APCs employed an IL-6 independent mechanism to stimulate CD4(+) T cell expression of IL-22 without upregulating IL-17. In human intestinal organ cultures, microbial activation of Vγ9/Vδ2 T cells promoted mucosal secretion of IL-22 and ICOSL/TNF-α-dependent release of the IL-22 inducible antimicrobial protein calprotectin without modulating IL-17 expression. In conclusion, human γδ T-APCs stimulate CD4(+) T cell responses distinct from those induced by myeloid APCs to promote local barrier defense via mucosal release of IL-22 and calprotectin. Targeting of γδ T-APC functions may lead to the development of novel gut-directed immunotherapies and vaccines.

  16. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    PubMed

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-08-29

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  17. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    PubMed Central

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-01-01

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

  18. Transgenic milk containing recombinant human lactoferrin modulates the intestinal flora in piglets.

    PubMed

    Hu, Wenping; Zhao, Jie; Wang, Jianwu; Yu, Tian; Wang, Jing; Li, Ning

    2012-06-01

    Lactoferrin (LF) is a beneficial multifunctional protein in milk. The objective of this study was to determine whether bovine transgenic milk containing recombinant human lactoferrin (rhLF) can modulate intestinal flora in the neonatal pig as an animal model for the human infant. We fed 7-day-old piglets (i) ordinary whole milk (OM), (ii) a 1:1 mixture of OM and rhLF milk (MM), or (iii) rhLF milk (LFM). LFM provided better average daily mass gain than OM (P = 0.007). PCR-denaturing gradient gel electrophoresis and 16S rDNA sequencing analysis revealed that the LFM piglets exhibited more diversity of the intestinal flora than the OM group. Except for the colon in the LFM group, an increasing trend in microbial diversity occurred from the duodenum to the colon. Fecal flora was not different across different ages or different treatment groups, but a cluster analysis showed that the fecal flora of OM- and MM-fed piglets had a higher degree of similarity than that of LFM-fed piglets. Based on culture-based bacterial counts of intestinal content samples, concentrations of Salmonella spp. in the colon and of Escherichia coli throughout the intestine were reduced with LFM (P < 0.01). Concentrations of Bifidobacterium spp. in the ileum and of Lactobacillus spp. throughout the intestine were also increased with LFM (P ≤ 0.01). We suggest that rhLF can modulate the intestinal flora in piglets.

  19. The human milk oligosaccharide 2′-fucosyllactose augments the adaptive response to extensive intestinal

    PubMed Central

    Hawkins, Jennifer A.; Ollberding, Nicholas J.; Karns, Rebekah; Morrow, Ardythe L.; Helmrath, Michael A.

    2015-01-01

    Intestinal resection resulting in short bowel syndrome (SBS) carries a heavy burden of long-term morbidity, mortality, and cost of care, which can be attenuated with strategies that improve intestinal adaptation. SBS infants fed human milk, compared with formula, have more rapid intestinal adaptation. We tested the hypothesis that the major noncaloric human milk oligosaccharide 2′-fucosyllactose (2′-FL) contributes to the adaptive response after intestinal resection. Using a previously described murine model of intestinal adaptation, we demonstrated increased weight gain from 21 to 56 days (P < 0.001) and crypt depth at 56 days (P < 0.0095) with 2′-FL supplementation after ileocecal resection. Furthermore, 2′-FL increased small bowel luminal content microbial alpha diversity following resection (P < 0.005) and stimulated a bloom in organisms of the genus Parabacteroides (log2-fold = 4.1, P = 0.035). Finally, transcriptional analysis of the intestine revealed enriched ontologies and pathways related to antimicrobial peptides, metabolism, and energy processing. We conclude that 2′-FL supplementation following ileocecal resection increases weight gain, energy availability through microbial community modulation, and histological changes consistent with improved adaptation. PMID:26702137

  20. Circulating intestine-derived exosomal miR-328 in plasma, a possible biomarker for estimating BCRP function in the human intestines.

    PubMed

    Gotanda, Keisuke; Hirota, Takeshi; Saito, Jumpei; Fukae, Masato; Egashira, Yu; Izumi, Noritomo; Deguchi, Mariko; Kimura, Miyuki; Matsuki, Shunji; Irie, Shin; Ieiri, Ichiro

    2016-08-30

    A variant in the breast cancer resistance protein (BCRP) gene, 421C> A is a useful biomarker for describing large inter-individual differences in the pharmacokinetics of sulfasalazine (SASP), a BCRP substrate. However, large intra-genotypic variability still exists in spite of the incorporation of this variant into the pharmacokinetics of SASP. Since miR-328 negatively regulates BCRP expression in human tissues, we hypothesized that exosomal miR-328 in plasma, which leaks from the intestines, is a possible biomarker for estimating BCRP activity in the human intestines. We established an immunoprecipitation-based quantitative method for circulating intestine-derived miR-328 in plasma using an anti-glycoprotein A33 antibody. A clinical study was conducted with an open-label, non-randomized, and single-arm design involving 33 healthy participants. Intestine-derived exosomal miR-328 levels positively correlated (P < 0.05) with SASP AUC0-48, suggesting that subjects with high miR-328 levels have low intestinal BCRP activity, resulting in the high AUC of SASP. Circulating intestine-derived exosomal miR-328 in plasma has potential as a possible biomarker for estimating BCRP function in the human intestines.

  1. Study of the Biotransformation of Tongmai Formula by Human Intestinal Flora and Its Intestinal Permeability across the Caco-2 Cell Monolayer.

    PubMed

    Wu, Shuai; Xu, Wei; Wang, Fu-Rong; Yang, Xiu-Wei

    2015-10-15

    Tongmai formula (TMF) is a well-known Chinese medicinal preparation that contains isoflavones as its major bioactive constituents. As traditional Chinese medicines (TCMs) are usually used by oral administration, their fate inside the intestinal lumen, including their biotransformation by human intestinal flora (HIF) and intestinal absorption deserves study. In this work TMF extract was incubated with human intestinal bacteria under anaerobic conditions and the changes in the twelve main constituents of TMF were then investigated. Their intestinal permeabilities, i.e., the transport capability across the intestinal brush border were investigated with a human colon carcinoma cell line (Caco-2) cell monolayer model to predict the absorption mechanism. Meanwhile, rapid HPLC-DAD methods were established for the assay. According to the biotransformation curves of the twelve constituents and the permeability coefficients, the intestinal absorption capacity of the typical compounds was elevated from the levels of 10(-7) cm/s to 10(-5) cm/s from those of the original compounds in TMF. Among them the main isoflavone glycosides puerarin (4), mirificin (6) and daidzin (7) were transformed into the same aglycone, daidzein (10). Therefore it was predicted that the aglycone compounds might be the real active ingredients in TMF. The models used can represent a novel path for the TCM studies.

  2. Lipid profiling of polarized human monocyte-derived macrophages.

    PubMed

    Montenegro-Burke, J Rafael; Sutton, Jessica A; Rogers, Lisa M; Milne, Ginger L; McLean, John A; Aronoff, David M

    2016-12-01

    The highly orchestrated transcriptional and metabolic reprogramming during activation drastically transforms the main functions and physiology of human macrophages across the polarization spectrum. Lipids, for example, can modify protein function by acting remotely as signaling molecules but also locally by altering the physical properties of cellular membranes. These changes play key roles in the functions of highly plastic immune cells due to their involvement in inflammation, immune responses, phagocytosis and wound healing processes. We report an analysis of major membrane lipids of distinct phenotypes of resting (M0), classically activated (M1), alternatively activated (M2a) and deactivated (M2c) human monocyte derived macrophages from different donors. Samples were subjected to supercritical fluid chromatography-ion mobility-mass spectrometry analysis, which allowed separations based on lipid class, facilitating the profiling of their fatty acid composition. Different levels of arachidonic acid mobilization as well as other fatty acid changes were observed for different lipid classes in the distinct polarization phenotypes, suggesting the activation of highly orchestrated and specific enzymatic processes in the biosynthesis of lipid signaling molecules and cell membrane remodeling. Thromboxane A2 production appeared to be a specific marker of M1 polarization. These alterations to the global composition of lipid bi-layer membranes in the cell provide a potential methodology for the definition and determination of cellular and tissue activation states.

  3. Polarization-sensitive optical coherence tomography for imaging human atherosclerosis

    NASA Astrophysics Data System (ADS)

    Kuo, Wen-Chuan; Chou, Nai-Kuan; Chou, Chien; Lai, Chih-Ming; Huang, Huan-Jang; Wang, Shoei-Shen; Shyu, Jeou-Jong

    2007-05-01

    Polarization-sensitive optical coherence tomography (PS-OCT) combines the advantages of OCT with image contrast enhancement, which is based on its ability to detect phase retardation and the fast-axis angle. Both PS-OCT images and histopathology have demonstrated similar features that allowed differentiation of atherosclerotic structures (i.e., plaques) from normal tissue. Moreover, the picrosirius polarization method was used to confirm PS-OCT assessment of collagen in the fibrous cap of atherosclerotic plaques, and high-frequency (40 MHz) ultrasound images were used to identify calcium in the vessel wall. Our preliminary ex vivo investigation of human aortic specimens indicated that PS-OCT might help to identify atherosclerotic lesions.

  4. P-gp activity and inhibition in the different regions of human intestine ex vivo.

    PubMed

    Li, Ming; de Graaf, Inge A M; de Jager, Marina H; Groothuis, Geny M M

    2017-03-01

    Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Association of germ-free mice with a simplified human intestinal microbiota results in a shortened intestine.

    PubMed

    Slezak, Kathleen; Krupova, Zuzana; Rabot, Sylvie; Loh, Gunnar; Levenez, Florence; Descamps, Amandine; Lepage, Patricia; Doré, Joël; Bellier, Sylvain; Blaut, Michael

    2014-01-01

    Genetic, nutritional, and gut microbiota-derived factors have been proposed to play a role in the development of the whole intestine that is around 40% longer in PRM/Alf mice compared with other mouse strains. The PRM/Alf genotype explains 60% of this length difference. The remaining 40% are due to a maternal effect that could depend on the gut microbiota transmitted by the mother to their pups. Germ-free PRM/Alf mice and C3H/He mice were associated with a simplified human microbiota (SIHUMI) to study its impact on gut length. The small intestines of the SIHUMI-associated mice were 16.4% (PRM/Alf) and 9.7% (C3H/He) shorter than those of the corresponding germ-free counterparts. Temporal temperature gradient gel electrophoresis and quantitative real-time PCR revealed differences in microbiota composition between both SIHUMI-associated mouse strains. Anaerostipes caccae was one log lower in PRM/Alf mice than in C3H/He mice. Since polyamines and short-chain fatty acids (SCFAs) are important intestinal growth factors, their concentrations were explored. Cecal concentrations of putrescine, spermine, spermidine, and N-acetylspermine were 1.5-fold, 3.7-fold, 2.2-fold, and 1.4-fold higher, respectively, in the SIHUMI-C3H/He mice compared with the SIHUMI-PRM/Alf mice. In addition, cecal acetate, propionate, and butyrate concentrations in SIHUMI-C3H/He mice were 1.4-fold, 1.1-fold, and 2.1-fold higher, respectively, than in SIHUMI-PRM/Alf mice. These results indicate that polyamines and SCFAs did not promote gut lengthening in any of the two mouse strains. This suggests that as yet unknown factors provided by the SIHUMI prevented gut lengthening in the SIHUMI-associated mice compared with the germfree mice.

  6. Generating human intestinal tissues from pluripotent stem cells to study development and disease

    PubMed Central

    Sinagoga, Katie L; Wells, James M

    2015-01-01

    As one of the largest and most functionally complex organs of the human body, the intestines are primarily responsible for the breakdown and uptake of macromolecules from the lumen and the subsequent excretion of waste from the body. However, the intestine is also an endocrine organ, regulating digestion, metabolism, and feeding behavior. Intricate neuronal, lymphatic, immune, and vascular systems are integrated into the intestine and are required for its digestive and endocrine functions. In addition, the gut houses an extensive population of microbes that play roles in digestion, global metabolism, barrier function, and host–parasite interactions. With such an extensive array of cell types working and performing in one essential organ, derivation of functional intestinal tissues from human pluripotent stem cells (PSCs) represents a significant challenge. Here we will discuss the intricate developmental processes and cell types that are required for assembly of this highly complex organ and how embryonic processes, particularly morphogenesis, have been harnessed to direct differentiation of PSCs into 3-dimensional human intestinal organoids (HIOs) in vitro. We will further describe current uses of HIOs in development and disease research and how additional tissue complexity might be engineered into HIOs for better functionality and disease modeling. PMID:25792515

  7. Generating human intestinal tissues from pluripotent stem cells to study development and disease.

    PubMed

    Sinagoga, Katie L; Wells, James M

    2015-05-05

    As one of the largest and most functionally complex organs of the human body, the intestines are primarily responsible for the breakdown and uptake of macromolecules from the lumen and the subsequent excretion of waste from the body. However, the intestine is also an endocrine organ, regulating digestion, metabolism, and feeding behavior. Intricate neuronal, lymphatic, immune, and vascular systems are integrated into the intestine and are required for its digestive and endocrine functions. In addition, the gut houses an extensive population of microbes that play roles in digestion, global metabolism, barrier function, and host-parasite interactions. With such an extensive array of cell types working and performing in one essential organ, derivation of functional intestinal tissues from human pluripotent stem cells (PSCs) represents a significant challenge. Here we will discuss the intricate developmental processes and cell types that are required for assembly of this highly complex organ and how embryonic processes, particularly morphogenesis, have been harnessed to direct differentiation of PSCs into 3-dimensional human intestinal organoids (HIOs) in vitro. We will further describe current uses of HIOs in development and disease research and how additional tissue complexity might be engineered into HIOs for better functionality and disease modeling.

  8. A review of drug solubility in human intestinal fluids: implications for the prediction of oral absorption.

    PubMed

    Augustijns, Patrick; Wuyts, Benjamin; Hens, Bart; Annaert, Pieter; Butler, James; Brouwers, Joachim

    2014-06-16

    The purpose of this paper is to collate all recently published solubility data of orally administered drugs in human intestinal fluids (HIF) that were aspirated from the upper small intestine (duodenum and jejunum). The data set comprises in total 102 solubility values in fasted state HIF and 37 solubility values in fed state HIF, covering 59 different drugs. Despite differences in the protocol for HIF sampling and subsequent handling, this summary of HIF solubilities provides a critical reference data set to judge the value of simulated media for intestinal solubility estimation. In this regard, the review includes correlations between the reported solubilizing capacity of HIF and fasted or fed state simulated intestinal fluid (FaSSIF/FeSSIF). Correlating with HIF solubilities enables the optimal use of solubility measurements in simulated biorelevant media to obtain accurate estimates of intestinal solubility during drug development. Considering the fraction of poorly soluble new molecular entities in contemporary drug discovery, adequate prediction of intestinal solubility is critical for efficient lead optimization, early candidate profiling, and further development.

  9. Heparan sulfate and syndecan-1 are essential in maintaining murine and human intestinal epithelial barrier function

    PubMed Central

    Bode, Lars; Salvestrini, Camilla; Park, Pyong Woo; Li, Jin-Ping; Esko, Jeffrey D.; Yamaguchi, Yu; Murch, Simon; Freeze, Hudson H.

    2007-01-01

    Patients with protein-losing enteropathy (PLE) fail to maintain intestinal epithelial barrier function and develop an excessive and potentially fatal efflux of plasma proteins. PLE occurs in ostensibly unrelated diseases, but emerging commonalities in clinical observations recently led us to identify key players in PLE pathogenesis. These include elevated IFN-γ, TNF-α, venous hypertension, and the specific loss of heparan sulfate proteoglycans from the basolateral surface of intestinal epithelial cells during PLE episodes. Here we show that heparan sulfate and syndecan-1, the predominant intestinal epithelial heparan sulfate proteoglycan, are essential in maintaining intestinal epithelial barrier function. Heparan sulfate– or syndecan-1–deficient mice and mice with intestinal-specific loss of heparan sulfate had increased basal protein leakage and were far more susceptible to protein loss induced by combinations of IFN-γ, TNF-α, and increased venous pressure. Similarly, knockdown of syndecan-1 in human epithelial cells resulted in increased basal and cytokine-induced protein leakage. Clinical application of heparin has been known to alleviate PLE in some patients but its unknown mechanism and severe side effects due to its anticoagulant activity limit its usefulness. We demonstrate here that non-anticoagulant 2,3-de-O-sulfated heparin could prevent intestinal protein leakage in syndecan-deficient mice, suggesting that this may be a safe and effective therapy for PLE patients. PMID:18064305

  10. Insights from human congenital disorders of intestinal lipid metabolism

    PubMed Central

    Levy, Emile

    2015-01-01

    The intestine must challenge the profuse daily flux of dietary fat that serves as a vital source of energy and as an essential component of cell membranes. The fat absorption process takes place in a series of orderly and interrelated steps, including the uptake and translocation of lipolytic products from the brush border membrane to the endoplasmic reticulum, lipid esterification, Apo synthesis, and ultimately the packaging of lipid and Apo components into chylomicrons (CMs). Deciphering inherited disorders of intracellular CM elaboration afforded new insight into the key functions of crucial intracellular proteins, such as Apo B, microsomal TG transfer protein, and Sar1b GTPase, the defects of which lead to hypobetalipoproteinemia, abetalipoproteinemia, and CM retention disease, respectively. These “experiments of nature” are characterized by fat malabsorption, steatorrhea, failure to thrive, low plasma levels of TGs and cholesterol, and deficiency of liposoluble vitamins and essential FAs. After summarizing and discussing the functions and regulation of these proteins for reader’s comprehension, the current review focuses on their specific roles in malabsorptions and dyslipidemia-related intestinal fat hyperabsorption while dissecting the spectrum of clinical manifestations and managements. The influence of newly discovered proteins (proprotein convertase subtilisin/kexin type 9 and angiopoietin-like 3 protein) on fat absorption has also been provided. Finally, it is stressed how the overexpression or polymorphism status of the critical intracellular proteins promotes dyslipidemia and cardiometabolic disorders. PMID:25387865

  11. Intestinal Microbiota Distinguish Gout Patients from Healthy Humans

    PubMed Central

    Guo, Zhuang; Zhang, Jiachao; Wang, Zhanli; Ang, Kay Ying; Huang, Shi; Hou, Qiangchuan; Su, Xiaoquan; Qiao, Jianmin; Zheng, Yi; Wang, Lifeng; Koh, Eileen; Danliang, Ho; Xu, Jian; Lee, Yuan Kun; Zhang, Heping

    2016-01-01

    Current blood-based approach for gout diagnosis can be of low sensitivity and hysteretic. Here via a 68-member cohort of 33 healthy and 35 diseased individuals, we reported that the intestinal microbiota of gout patients are highly distinct from healthy individuals in both organismal and functional structures. In gout, Bacteroides caccae and Bacteroides xylanisolvens are enriched yet Faecalibacterium prausnitzii and Bifidobacterium pseudocatenulatum depleted. The established reference microbial gene catalogue for gout revealed disorder in purine degradation and butyric acid biosynthesis in gout patients. In an additional 15-member validation-group, a diagnosis model via 17 gout-associated bacteria reached 88.9% accuracy, higher than the blood-uric-acid based approach. Intestinal microbiota of gout are more similar to those of type-2 diabetes than to liver cirrhosis, whereas depletion of Faecalibacterium prausnitzii and reduced butyrate biosynthesis are shared in each of the metabolic syndromes. Thus the Microbial Index of Gout was proposed as a novel, sensitive and non-invasive strategy for diagnosing gout via fecal microbiota. PMID:26852926

  12. Intestinal Microbiota Distinguish Gout Patients from Healthy Humans.

    PubMed

    Guo, Zhuang; Zhang, Jiachao; Wang, Zhanli; Ang, Kay Ying; Huang, Shi; Hou, Qiangchuan; Su, Xiaoquan; Qiao, Jianmin; Zheng, Yi; Wang, Lifeng; Koh, Eileen; Danliang, Ho; Xu, Jian; Lee, Yuan Kun; Zhang, Heping

    2016-02-08

    Current blood-based approach for gout diagnosis can be of low sensitivity and hysteretic. Here via a 68-member cohort of 33 healthy and 35 diseased individuals, we reported that the intestinal microbiota of gout patients are highly distinct from healthy individuals in both organismal and functional structures. In gout, Bacteroides caccae and Bacteroides xylanisolvens are enriched yet Faecalibacterium prausnitzii and Bifidobacterium pseudocatenulatum depleted. The established reference microbial gene catalogue for gout revealed disorder in purine degradation and butyric acid biosynthesis in gout patients. In an additional 15-member validation-group, a diagnosis model via 17 gout-associated bacteria reached 88.9% accuracy, higher than the blood-uric-acid based approach. Intestinal microbiota of gout are more similar to those of type-2 diabetes than to liver cirrhosis, whereas depletion of Faecalibacterium prausnitzii and reduced butyrate biosynthesis are shared in each of the metabolic syndromes. Thus the Microbial Index of Gout was proposed as a novel, sensitive and non-invasive strategy for diagnosing gout via fecal microbiota.

  13. Construction vs. development: polarizing models of human gestation.

    PubMed

    Stith, Richard

    2014-12-01

    This essay argues that the polarization of our public debate over embryo-destructive research may be due, to a large extent, not to different valuations of individual human life but to different conceptions of the process of gestation, with one group treating the process as a making or construction and the other treating it as a development. These two incompatible models of reproduction are shown to explain the various positions commonly encountered in this debate over the treatment of embryos, and to a significant degree those encountered in the debate over abortion as well. Finally, the historical, theoretical, and intuitive strengths of each model are examined.

  14. Chemical form of selenium affects its uptake, transport and glutathione peroxidase activity in the human intestinal Caco-2 cell model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

  15. Identification of the transcriptional response of human intestinal mucosa to Lactobacillus plantarum WCFS1 in vivo

    PubMed Central

    Troost, Freddy J; van Baarlen, Peter; Lindsey, Patrick; Kodde, Andrea; de Vos, Willem M; Kleerebezem, Michiel; Brummer, Robert-Jan M

    2008-01-01

    Background There is limited knowledge on the extent and dynamics of the mucosal response to commensal and probiotic species in the human intestinal lumen. This study aimed to identify the acute, time-dependent responses of intestinal mucosa to commensal Lactobacillus plantarum WCFS1 in vivo in two placebo-controlled human intervention studies in healthy volunteers. Transcriptional changes in duodenal mucosa upon continuous intraduodenal infusion of L. plantarum WCFS1 for one- and six h, respectively, were studied using oro- and nasogastric intubations with dedicated orogastric catheters and tissue sampling by standard flexible gastroduodenoscopy. Results One- and six-h exposure of small intestinal mucosa to L. plantarum WCFS1 induced differential expression of 669 and 424 gene reporters, respectively. While short-term exposure to L. plantarum WCFS1 inhibited fatty acid metabolism and cell cycle progression, cells switched to a more proliferative phase after prolonged exposure with an overall expression profile characterized by upregulation of genes involved in lipid metabolism, cellular growth and development. Cell death and immune responses were triggered, but cell death-executing genes or inflammatory signals were not expressed. Proteome analysis showed differential expression of several proteins. Only the microsomal protein 'microsomal triglyceride transfer protein' was regulated on both the transcriptional and the protein level in all subjects. Conclusion Overall, this study showed that intestinal exposure to L. plantarum WCFS1 induced consistent, time-dependent transcriptional responses in healthy intestinal mucosa. This extensive exploration of the human response to L. plantarum WCFS1 could eventually provide molecular support for specific or probiotic activity of this strain or species, and exemplifies the strength of the applied technology to identify the potential bio-activity of microbes in the human intestine. PMID:18681965

  16. Hierarchical radial and polar organisation of chromosomes in human sperm.

    PubMed

    Millan, N M; Lau, P; Hann, M; Ioannou, D; Hoffman, D; Barrionuevo, M; Maxson, W; Ory, S; Tempest, H G

    2012-10-01

    It is well established that chromosomes occupy distinct positions within the interphase nuclei, conferring a potential functional implication to the genome. In addition, alterations in the nuclear organisation patterns have been associated with disease phenotypes (e.g. cancer or laminopathies). The human sperm is the smallest cell in the body with specific DNA packaging and the mission of delivering the paternal genome to the oocyte during fertilisation. Studies of nuclear organisation in the sperm have postulated nonrandom chromosome position and have proposed a chromocentre model with the centromeres facing toward the interior and the telomeres toward the periphery of the nucleus. Most studies have assessed the nuclear address in the sperm longitudinally predominantly using centromeric or telomeric probes and to a lesser extent with whole chromosome paints. To date, studies investigating the radial organisation of human sperm have been limited. The purpose of this study was to utilise whole chromosome paints for six clinically important chromosomes (18, 19, 21, 22, X, and Y) to investigate nuclear address by assessing their radial and longitudinal nuclear organisation. A total of 10,800 sperm were analysed in nine normozoospermic individuals. The results have shown nonrandom chromosome position for all chromosomes using both methods of analysis. We present novel radial and polar analysis of chromosome territory localization within the human sperm nucleus. Specifically, a hierarchical organisation was observed radially with chromosomes organised from the interior to the periphery (chromosomes 22, 21, Y, X, 19, and 18 respectively) and polar organisation from the sperm head to tail (chromosomes X, 19, Y, 22, 21, and 18, respectively). We provide evidence of defined nuclear organisation in the human sperm and discuss the function of organisation and potential possible clinical ramifications of these results in regards to male infertility and early human development.

  17. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells

    PubMed Central

    1975-01-01

    The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented. PMID:1202021

  18. Enhancing the intestinal absorption of molecules containing the polar guanidino functionality: a double-targeted prodrug approach

    PubMed Central

    Sun, Jing; Dahan, Arik; Amidon, Gordon L.

    2011-01-01

    A prodrug strategy was applied to guanidino-containing analogs to increase oral absorption via hPEPT1 and hVACVase. L-Valine, L-isoleucine and L-phenylalanine esters of [3-(hydroxymethyl)phenyl]guanidine (3-HPG) were synthesized and evaluated for transport and activation. In HeLa/hPEPT1 cells, Val-3-HPG and Ile-3-HPG exhibited high affinity to hPEPT1 (IC50: 0.65 and 0.63 mM, respectively), and all three L-amino acid esters showed higher uptake (2.6- to 9-fold) than the parent compound 3-HPG. Val-3-HPG and Ile-3-HPG demonstrated remarkable Caco-2 permeability enhancement, and Val-3-HPG exhibited comparable permeability to valacyclovir. In rat perfusion studies, Val-3-HPG and Ile-3-HPG permeabilities were significantly higher than 3-HPG, and exceeded/matched the high-permeability standard metoprolol, respectively. All the L-amino acid 3-HPG esters were effectively activated in HeLa and Caco-2 cell homogenates, and were found to be good substrates of hVACVase (kcat/Km in mM−1·s−1: Val-3-HPG, 3370; Ile-3-HPG, 1580; Phe-3-HPG, 1660). In conclusion, a prodrug strategy is effective at increasing the intestinal permeability of polar guanidino analogs via targeting hPEPT1 for transport and hVACVase for activation. PMID:19957998

  19. hPSC-derived lung and intestinal organoids as models of human fetal tissue.

    PubMed

    Aurora, Megan; Spence, Jason R

    2016-12-15

    In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC).

  20. Human Intestinal Raf Kinase Inhibitor Protein (RKIP) Catalyzes Prasugrel as a Bioactivation Hydrolase.

    PubMed

    Kazui, Miho; Ogura, Yuji; Hagihara, Katsunobu; Kubota, Kazuishi; Kurihara, Atsushi

    2016-01-01

    Prasugrel is a thienopyridine antiplatelet prodrug that undergoes rapid hydrolysis in vivo to a thiolactone metabolite by human carboxylesterase-2 (hCE2) during gastrointestinal absorption. The thiolactone metabolite is further converted to a pharmacologically active metabolite by cytochrome P450 isoforms. The aim of the current study was to elucidate hydrolases other than hCE2 involved in the bioactivation step of prasugrel in human intestine. Using size-exclusion column chromatography of a human small intestinal S9 fraction, another peak besides the hCE2 peak was observed to have prasugrel hydrolyzing activity, and this protein was found to have a molecular weight of about 20 kDa. This prasugrel hydrolyzing protein was successfully purified from a monkey small intestinal cytosolic fraction by successive four-step column chromatography and identified as Raf-1 kinase inhibitor protein (RKIP) by liquid chromatography-tandem mass spectrometry. Second, we evaluated the enzymatic kinetic parameters for prasugrel hydrolysis using recombinant human RKIP and hCE2 and estimated the contributions of these two hydrolyzing enzymes to the prasugrel hydrolysis reaction in human intestine, which were approximately 40% for hRKIP and 60% for hCE2. Moreover, prasugrel hydrolysis was inhibited by anti-hRKIP antibody and carboxylesterase-specific chemical inhibitor (bis p-nitrophenyl phosphate) by 30% and 60%, respectively. In conclusion, another protein capable of hydrolyzing prasugrel to its thiolactone metabolite was identified as RKIP, and this protein may play a significant role with hCE2 in prasugrel bioactivation in human intestine. RKIP is known to have diverse functions in many intracellular signaling cascades, but this is the first report describing RKIP as a hydrolase involved in drug metabolism.

  1. Study on the uptake and deglycosylation of the masked forms of zearalenone in human intestinal Caco-2 cells.

    PubMed

    Cirlini, Martina; Barilli, Amelia; Galaverna, Gianni; Michlmayr, Herbert; Adam, Gerhard; Berthiller, Franz; Dall'Asta, Chiara

    2016-12-01

    This study investigated for the first time the intestinal transfer of two modified forms of zearalenone (ZEN), zearalenone-14-glucoside (ZEN14Glc) and zearalenone-16-glucoside (ZEN16Glc), using polarized monolayers of Caco-2 cells. The cells were apically exposed to 40 μM of ZEN14Glc, ZEN16Glc and ZEN, separately. Results showed that, after apical administration, ZEN14Glc and ZEN16Glc can be detected in cellular extracts indicating uptake by intestinal cells. Moreover, the glucosylated forms were cleaved to release ZEN, with a different cleavage ability for the two conjugated isomers. In particular, ZEN16Glc seems to be less prone to deglycosylation compared to ZEN14Glc, probably on account of an increased steric hindrance. We could show that human cytosolic β-glucosidase is able to cleave ZEN14Glc liberating ZEN, but is unable to cleave ZEN16Glc.All metabolites could cross the cell membrane and be detected in the apical compartment, while ZEN was also found in the basolateral compartment together with the modified mycotoxin form.

  2. Attaching and effacing activities of rabbit and human enteropathogenic Escherichia coli in pig and rabbit intestines.

    PubMed Central

    Moon, H W; Whipp, S C; Argenzio, R A; Levine, M M; Giannella, R A

    1983-01-01

    Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine. The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy. It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections. The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC. The EPEC strains also varied in the frequency and extent of lesion production. For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system. Images PMID:6350186

  3. Consensus hologram QSAR modeling for the prediction of human intestinal absorption.

    PubMed

    Moda, Tiago L; Andricopulo, Adriano D

    2012-04-15

    Consistent in silico models for ADME properties are useful tools in early drug discovery. Here, we report the hologram QSAR modeling of human intestinal absorption using a dataset of 638 compounds with experimental data associated. The final validated models are consistent and robust for the consensus prediction of this important pharmacokinetic property and are suitable for virtual screening applications.

  4. Investigation of the interactions between Chrysanthemum morifolium flowers extract and intestinal bacteria from human and rat.

    PubMed

    Tao, Jin-Hua; Duan, Jin-Ao; Qian, Yi-Yun; Qian, Da-Wei; Guo, Jian-Ming

    2016-11-01

    Flos Chrysanthemi, dried flower of Chrysanthemum morifolium Ramat, has drawn much attention recently owing to its potential beneficial health effects for human. Flos Chrysanthemi products are usually taken orally and metabolized by intestinal microflora. However, there has been no investigation of the comprehensive metabolic profile of the Flos Chrysanthemi extract by intestinal flora owing to its chemical complexity and the limitations of analytical methods. In this paper, a rapid, sensitive and automated analysis method, ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry including MS(E) technology and automated data processing Metabolynx™ software, was developed and successfully applied for the biotransformation and metabolic profile of flavonoids in the Flos Chrysanthemi extract by intestinal flora from human and rat. A total of 32 metabolites were detected and tentatively identified in human and rat intestinal bacterial samples. These metabolites indicated that hydrolysis, hydroxylation, acetylation, methylation, hydrogenation and deoxygenation were the major conversion pathways of flavonoids in the Flos Chrysanthemi extract in vitro. Furthermore, the effects of the Flos Chrysanthemi extract on the growth of different intestinal bacteria were detected using an Emax precision microplate reader. Certain pathogenic bacteria such as Enterobacter, Enterococcus, Clostridium and Bacteroides were significantly inhibited by Flos Chrysanthemi, while commensal probiotics such as Lactobacillus and Bifidobacterium were moderately promoted. Our observation provided further evidence for the importance of intestinal bacteria in the metabolism and potential activity of the Flos Chrysanthemi extract. The results will also be helpful for the further pharmacokinetic study of Flos Chrysanthemi and to unravel how it works in vivo.

  5. Human-derived probiotic Lactobacillus reuteri strains differentially reduce intestinal inflammation.

    PubMed

    Liu, Yuying; Fatheree, Nicole Y; Mangalat, Nisha; Rhoads, Jon Marc

    2010-11-01

    Lactobacillus reuteri (L. reuteri) is a probiotic that inhibits the severity of enteric infections and modulates the immune system. Human-derived L. reuteri strains DSM17938, ATCC PTA4659, ATCC PTA 5289, and ATCC PTA 6475 have demonstrated strain-specific immunomodulation in cultured monocytoid cells, but information about how these strains affect inflammation in intestinal epithelium is limited. We determined the effects of the four different L. reuteri strains on lipopolysaccharide (LPS)-induced inflammation in small intestinal epithelial cells and in the ileum of newborn rats. IPEC-J2 cells (derived from the jejunal epithelium of a neonatal piglet) and IEC-6 cells (derived from the rat crypt) were treated with L. reuteri. Newborn rat pups were gavaged cow milk formula supplemented with L. reuteri strains in the presence or absence of LPS. Protein and mRNA levels of cytokines and histological changes were measured. We demonstrate that even though one L. reuteri strain (DSM 17938) did not inhibit LPS-induced IL-8 production in cultured intestinal cells, all strains significantly reduced intestinal mucosal levels of KC/GRO (∼IL-8) and IFN-γ when newborn rat pups were fed formula containing LPS ± L. reuteri. Intestinal histological damage produced by LPS plus cow milk formula was also significantly reduced by all four strains. Cow milk formula feeding (without LPS) produced mild gut inflammation, evidenced by elevated mucosal IFN-γ and IL-13 levels, a process that could be suppressed by strain 17938. Other cytokines and chemokines were variably affected by the different strains, and there was no toxic effect of L. reuteri on intestinal cells or mucosa. In conclusion, L. reuteri strains differentially modulate LPS-induced inflammation. Probiotic interactions with both epithelial and nonepithelial cells in vivo must be instrumental in modulating intrinsic anti-inflammatory effects in the intestine. We suggest that the terms anti- and proinflammatory be used only

  6. CFTR is a tumor suppressor gene in murine and human intestinal cancer

    PubMed Central

    Than, BLN; Linnekamp, JF; Starr, TK; Largaespada, DA; Rod, A; Zhang, Y; Bruner, V; Abrahante, J; Schumann, A; Luczak, T; Niemczyk, A; O’Sullivan, MG; Medema, JP; Fijneman, RJA; Meijer, GA; Van den Broek, E; Hodges, CA; Scott, PM; Vermeulen, L; Cormier, RT

    2016-01-01

    CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid–base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated ApcMin mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc+/+ mice aged to ~ 1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc+/+ Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease

  7. CFTR is a tumor suppressor gene in murine and human intestinal cancer.

    PubMed

    Than, B L N; Linnekamp, J F; Starr, T K; Largaespada, D A; Rod, A; Zhang, Y; Bruner, V; Abrahante, J; Schumann, A; Luczak, T; Niemczyk, A; O'Sullivan, M G; Medema, J P; Fijneman, R J A; Meijer, G A; Van den Broek, E; Hodges, C A; Scott, P M; Vermeulen, L; Cormier, R T

    2016-08-11

    CFTR, the cystic fibrosis (CF) gene, encodes for the CFTR protein that plays an essential role in anion regulation and tissue homeostasis of various epithelia. In the gastrointestinal (GI) tract CFTR promotes chloride and bicarbonate secretion, playing an essential role in ion and acid-base homeostasis. Cftr has been identified as a candidate driver gene for colorectal cancer (CRC) in several Sleeping Beauty DNA transposon-based forward genetic screens in mice. Further, recent epidemiological and clinical studies indicate that CF patients are at high risk for developing tumors in the colon. To investigate the effects of CFTR dysregulation on GI cancer, we generated Apc(Min) mice that carried an intestinal-specific knockout of Cftr. Our results indicate that Cftr is a tumor suppressor gene in the intestinal tract as Cftr mutant mice developed significantly more tumors in the colon and the entire small intestine. In Apc(+/+) mice aged to ~1 year, Cftr deficiency alone caused the development of intestinal tumors in >60% of mice. Colon organoid formation was significantly increased in organoids created from Cftr mutant mice compared with wild-type controls, suggesting a potential role of Cftr in regulating the intestinal stem cell compartment. Microarray data from the Cftr-deficient colon and the small intestine identified dysregulated genes that belong to groups of immune response, ion channel, intestinal stem cell and other growth signaling regulators. These associated clusters of genes were confirmed by pathway analysis using Ingenuity Pathway Analysis and gene set enrichment analysis (GSEA). We also conducted RNA Seq analysis of tumors from Apc(+/+) Cftr knockout mice and identified sets of genes dysregulated in tumors including altered Wnt β-catenin target genes. Finally we analyzed expression of CFTR in early stage human CRC patients stratified by risk of recurrence and found that loss of expression of CFTR was significantly associated with poor disease

  8. Identification of a human intestinal myeloid cell subset that regulates gut homeostasis.

    PubMed

    Barman, Soumik; Kayama, Hisako; Okuzaki, Daisuke; Ogino, Takayuki; Osawa, Hideki; Matsuno, Hiroshi; Mizushima, Tsunekazu; Mori, Masaki; Nishimura, Junichi; Takeda, Kiyoshi

    2016-11-01

    Inappropriate activation of T helper (Th) cells, such as Th1 and Th17 cells, is implicated in the pathogenesis of chronic inflammatory disorders including ulcerative colitis (UC). CX3CR1(high) macrophages contribute to intestinal homeostasis through various mechanisms in mice. However, whether mononuclear phagocytes with regulatory functions are present in the human colon is not clearly defined. We investigated whether innate myeloid cells that suppress activation of effector T cells exist in the human intestinal mucosa. Among intestinal lamina propria cells, Lin(-) HLA-DR(high) CD14(+) CD163(high) cells were subdivided into CD160(low) and CD160(high) cells. Both subsets produced high levels of IL-10. CD163(high) CD160(high) cells suppressed effector T cell proliferation, whereas CD163(high) CD160(low) cells induced Th17 differentiation. Patients with UC exhibited increased numbers of CD163(high) CD160(low) cells, while showing profoundly decreased numbers of CD163(high) CD160(high) cells. In this context, CD163(high) CD160(high) cells had higher CD80/CD86 expression and lower IL10RB expression, and these cells did not suppress effector T cell proliferation. The CD163(high) CD160(high) subset in normal intestinal mucosa inhibits inappropriate Th1/Th17 responses through suppression of their proliferation, and its number and suppressive activity are impaired in patients with UC. These findings indicate how human innate immune cells might prevent UC development.

  9. Reductive dechlorination of methoxychlor and DDT by human intestinal bacterium Eubacterium limosum under anaerobic conditions.

    PubMed

    Yim, You-Jin; Seo, Jiyoung; Kang, Su-Il; Ahn, Joong-Hoon; Hur, Hor-Gil

    2008-04-01

    Methoxychlor [1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane], a substitute for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), is a compound of environmental concern because of potential long-term health risks related to its endocrine-disrupting and carcinogenic potency. In order to determine the metabolic fate of methoxychlor and DDT in the human intestinal gut, Eubacterium limosum (ATCC 8486), a strict anaerobe isolated from the human intestine that is capable of O-demethylation toward O-methylated isoflavones, was used as a model intestinal microbial organism. Under anaerobic incubation conditions, E. limosum completely transformed methoxychlor and DDT in 16 days. Based on gas chromatography-mass chromatography analyses, the metabolites produced from methoxychlor and DDT by E. limosum were confirmed to be 1,1-dichloro-2,2-bis(p-methoxyphenyl)ethane (methoxydichlor) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), respectively. This study suggests that E. limosum in the human intestinal gut might be a participant in the reductive dechlorination of methoxychlor to the more antiandrogenic active methoxydichlor.

  10. Metabolism of Isoflavones Found in the Pueraria thomsonii Flower by Human Intestinal Microbiota.

    PubMed

    Hirayama, Kazuhiro; Matsuzuka, Yuki; Kamiya, Tomoyasu; Ikeguchi, Motoya; Takagaki, Kinya; Itoh, Kikuji

    2011-01-01

    Isoflavones contained in the root and flower of Kudzu (Pueraria lobata and related species) are suggested to be the critical component for its effects. Although metabolism of soy isoflavones has been well studied, the composition of isoflavones found in Kudzu is completely different from that of soy isoflavones. In the present study, we investigated whether isoflavones found in the flower of Pueraria thomsonii, a species of Kudzu, were metabolized by human fecal microbiota and murine small intestinal enzymes. Among 5 glycosidic isoflavones of the Pueraria thomsonii flower, tectorigenin 7-O-xylosylglucoside, tectoridin, genistin and glycitin were completely hydrolyzed by a homogenate of germfree mouse small intestine without contribution of bacteria. Released aglycones were not further metabolized, except that up to half of glycitein disappeared. Mouse small intestinal enzymes did not metabolize 6-hydroxygenistein 6,7-di-O-glucoside. Isoflavone aglycones as well as 6-hydroxygenistein 6,7-di-O-glucoside were highly metabolized by most of the human fecal suspensions. Metabolites were not detected with the present analytical methods in most cases. Although further investigations of the pharmacokinetics of Pueraria thomsonii flower isoflavones are needed, the results of the present study indicate active metabolism of Pueraria thomsonii flower isoflavones in the human intestine.

  11. Metabolism of heme and bilirubin in rat and human small intestinal mucosa.

    PubMed Central

    Hartmann, F; Bissell, D M

    1982-01-01

    Formation of heme, bilirubin, and bilirubin conjugates has been examined in mucosal cells isolated from the rat upper small intestine. Intact, viable cells were prepared by enzymatic dissociation using a combined vascular and luminal perfusion and incubated with an isotopically labeled precursor, delta-amino-[2,3-3H]levulinic acid. Labeled heme and bile pigment were formed with kinetics similar to those exhibited by hepatocytes. Moreover, the newly formed bilirubin was converted rapidly to both mono- and diglucuronide conjugates. In addition, cell-free extracts of small intestinal mucosa from rats or humans exhibited a bilirubin-UDP-glucuronyl transferase activity that was qualitatively similar to that present in liver. The data suggest that the small intestinal mucosa normally contributes to bilirubin metabolism. PMID:6806320

  12. Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells.

    PubMed

    Forbester, Jessica L; Goulding, David; Vallier, Ludovic; Hannan, Nicholas; Hale, Christine; Pickard, Derek; Mukhopadhyay, Subhankar; Dougan, Gordon

    2015-07-01

    The intestinal mucosa forms the first line of defense against infections mediated by enteric pathogens such as salmonellae. Here we exploited intestinal "organoids" (iHOs) generated from human induced pluripotent stem cells (hIPSCs) to explore the interaction of Salmonella enterica serovar Typhimurium with iHOs. Imaging and RNA sequencing were used to analyze these interactions, and clear changes in transcriptional signatures were detected, including altered patterns of cytokine expression after the exposure of iHOs to bacteria. S. Typhimurium microinjected into the lumen of iHOs was able to invade the epithelial barrier, with many bacteria residing within Salmonella-containing vacuoles. An S. Typhimurium invA mutant defective in the Salmonella pathogenicity island 1 invasion apparatus was less capable of invading the iHO epithelium. Hence, we provide evidence that hIPSC-derived organoids are a promising model of the intestinal epithelium for assessing interactions with enteric pathogens.

  13. Detection of fastidious mycobacteria in human intestines by the polymerase chain reaction.

    PubMed

    Dumonceau, J M; Van Gossum, A; Adler, M; Van Vooren, J P; Fonteyne, P A; De Beenhouwer, H; Portaels, F

    1997-05-01

    The aim of this study was to determine whether difficult-to-grow mycobacteria are present in human intestines. Intestinal tissue samples were subjected to both mycobacterial culture and a polymerase chain reaction (PCR) assay. After detection by PCR, species identity was determined by hybridizing the amplified 16S rRNA gene fragments with species-specific oligonucleotides. Intestinal biopsies from 63 patients with noninflammatory bowel diseases (n = 22), Crohn's disease (n = 31), or ulcerative colitis (n = 10) were analyzed. Culture and PCR revealed mycobacteria in four (6%) and 25 (40%) samples, respectively. Samples positive by PCR were negative with all probes specific to nine common cultivable species but were positive with Mycobacterium genavense-specific probe in 68% of cases. Mycobacterial isolates were identified as Mycobacterium gordonae and Mycobacterium chelonae. Findings were similar in Crohn's disease samples compared to non-Chron's disease samples. This study shows that difficult-to-grow mycobacteria can be detected by PCR in large and similar proportions of inflamed intestinal tissue from patients with inflammatory bowel disease and intestinal tissue that appears normal from patients with noninflammatory bowel disease.

  14. Recombinant Human Epidermal Growth Factor Accelerates Recovery of Mouse Small Intestinal Mucosa After Radiation Damage

    SciTech Connect

    Lee, Kang Kyoo; Jo, Hyang Jeong; Hong, Joon Pio; Lee, Sang-wook Sohn, Jung Sook; Moon, Soo Young; Yang, Sei Hoon; Shim, Hyeok; Lee, Sang Ho; Ryu, Seung-Hee; Moon, Sun Rock

    2008-07-15

    Purpose: To determine whether systemically administered recombinant human epidermal growth factor (rhEGF) accelerates the recovery of mouse small intestinal mucosa after irradiation. Methods and Materials: A mouse mucosal damage model was established by administering radiation to male BALB/c mice with a single dose of 15 Gy applied to the abdomen. After irradiation, rhEGF was administered subcutaneously at various doses (0.04, 0.2, 1.0, and 5.0 mg/kg/day) eight times at 2- to 3-day intervals. The evaluation methods included histologic changes of small intestinal mucosa, change in body weight, frequency of diarrhea, and survival rate. Results: The recovery of small intestinal mucosa after irradiation was significantly improved in the mice treated with a high dose of rhEGF. In the mice that underwent irradiation without rhEGF treatment, intestinal mucosal ulceration, mucosal layer damage, and severe inflammation occurred. The regeneration of villi was noticeable in mice treated with more than 0.2 mg/kg rhEGF, and the villi recovered fully in mice given more than 1 mg/kg rhEGF. The frequency of diarrhea persisting for more than 3 days was significantly greater in the radiation control group than in the rhEGF-treated groups. Conclusions: Systemic administration of rhEGF accelerates recovery from mucosal damage induced by irradiation. We suggest that rhEGF treatment shows promise for the reduction of small intestinal damage after irradiation.

  15. Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine.

    PubMed

    Drozdzik, Marek; Gröer, Christian; Penski, Jette; Lapczuk, Joanna; Ostrowski, Marek; Lai, Yurong; Prasad, Bhagwat; Unadkat, Jashvant D; Siegmund, Werner; Oswald, Stefan

    2014-10-06

    Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

  16. Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

    PubMed Central

    Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

    2002-01-01

    In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

  17. Human milk mucin 1 and mucin 4 inhibit Salmonella enterica serovar Typhimurium invasion of human intestinal epithelial cells in vitro.

    PubMed

    Liu, Bo; Yu, Zhuoteng; Chen, Ceng; Kling, David E; Newburg, David S

    2012-08-01

    Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate-labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

  18. Diversity of halophilic archaea in fermented foods and human intestines and their application.

    PubMed

    Lee, Han-Seung

    2013-12-01

    Archaea are prokaryotic organisms distinct from bacteria in the structural and molecular biological sense, and these microorganisms are known to thrive mostly at extreme environments. In particular, most studies on halophilic archaea have been focused on environmental and ecological researches. However, new species of halophilic archaea are being isolated and identified from high salt-fermented foods consumed by humans, and it has been found that various types of halophilic archaea exist in food products by culture-independent molecular biological methods. In addition, even if the numbers are not quite high, DNAs of various halophilic archaea are being detected in human intestines and much interest is given to their possible roles. This review aims to summarize the types and characteristics of halophilic archaea reported to be present in foods and human intestines and to discuss their application as well.

  19. A comparison of polarized and non-polarized human endometrial monolayer culture systems on murine embryo development

    PubMed Central

    Baghaban Eslami Nejad, Mohamad Reza; Rezazadeh Valojerdi, Mojtaba; Kazemi Ashtiani, Saeed

    2005-01-01

    Background Co-culture of embryos with various somatic cells has been suggested as a promising approach to improve embryo development. Despite numerous reports regarding the beneficial effects of epithelial cells from the female genital tract on embryo development in a co-culture system, little is known about the effect of these cells when being cultured under a polarized condition on embryo growth. Our study evaluated the effects of in vitro polarized cells on pre-embryo development. Methods Human endometrial tissue was obtained from uterine specimens excised at total hysterectomy performed for benign indications. Epithelial cells were promptly isolated and cultured either on extra-cellular matrix gel (ECM-Gel) coated millipore filter inserts (polarized) or plastic surfaces (non-polarized). The epithelial nature of the cells cultured on plastic was confirmed through immunohistochemistry, and polarization of cells cultured on ECM-Gel was evaluated by transmission electron microscopy (TEM). One or two-cell stage embryos of a superovulated NMRI mouse were then flushed and placed in culture with either polarized or non-polarized cells and medium alone. Development rates were determined for all embryos daily and statistically compared. At the end of the cultivation period, trophectoderm (TE) and inner cell mass (ICM) of expanded blastocysts from each group were examined microscopically. Results Endometrial epithelial cells cultured on ECM-Gel had a highly polarized columnar shape as opposed to the flattened shape of the cells cultured on a plastic surface. The two-cell embryos cultured on a polarized monolayer had a higher developmental rate than those from the non-polarized cells. There was no statistically significant difference; still, the blastocysts from the polarized monolayer, in comparison with the non-polarized group, had a significantly higher mean cell number. The development of one-cell embryos in the polarized and non-polarized groups showed no

  20. Subtractive transcriptomics : establishing polarity drives human endothelial morphogenesis

    SciTech Connect

    Glesne, D. A.; Zhang, W.; Mandava, S.; Ursos, L.; Buell, M. E.; Makowski, L.; Rodi, D. J.; Biosciences Division

    2006-04-15

    Although investigations of mature normal and tumor-derived capillaries have resulted in characterization of these structures at the phenotypic level, less is known regarding the initial molecular cues for cellular assembly of endothelial cells into human capillaries. Here, we employ a novel combination of microenvironmental manipulation and microarray data filtration over narrowly delineated temporal data series to identify the morphogenesis component apart from the proliferation component, as pooled human microvascular-derived endothelial cells are induced to form capillary-like structures in vitro in a murine tumor-derived matrix. The 217 morphogenesis-specific genes identified using this subtractive transcriptomics approach are mostly independent of the angiogenic proteins currently used as therapeutic targets for aberrant angiogenesis. Quantitative real-time PCR was used to validate 20% of these transcripts. Immunofluorescent analysis of proliferating and tube-forming cells validates at the protein level the morphogenesis-specific expression pattern of 16 of the 217 gene products identified. The transcripts that are selectively up-regulated in tube-forming endothelial cells reveal a temporal expression pattern of genes primarily associated with intracellular trafficking, guided migration, cytoskeletal reorganization, cellular adhesion, and proliferation inhibition. These data show that a sequential upregulation of genes that establish and maintain polarity occurs during migration and morphogenesis of in vitro human endothelial cells undergoing tubulogenesis; some of which may well be effective as novel antiangiogenic drug targets.

  1. Doublecortin-like kinase 1-positive enterocyte - a new cell type in human intestine.

    PubMed

    Leppänen, Joni; Helminen, Olli; Huhta, Heikki; Kauppila, Joonas H; Miinalainen, Ilkka; Ronkainen, Veli-Pekka; Saarnio, Juha; Lehenkari, Petri P; Karttunen, Tuomo J

    2016-11-01

    Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.

  2. Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium

    PubMed Central

    Walsham, Alistair D. S.; MacKenzie, Donald A.; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L.; Juge, Nathalie; Schüller, Stephanie

    2016-01-01

    Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains. PMID:26973622

  3. Short- and long-term effects of oral vancomycin on the human intestinal microbiota

    PubMed Central

    Isaac, Sandrine; Scher, Jose U.; Djukovic, Ana; Jiménez, Nuria; Littman, Dan R.; Abramson, Steven B.; Pamer, Eric G.; Ubeda, Carles

    2017-01-01

    Background Oral vancomycin remains the mainstay of therapy for severe infections produced by Clostridium difficile, the most prevalent cause of healthcare-associated infectious diarrhoea in developed countries. However, its short- and long-term effects on the human intestinal microbiota remain largely unknown. Methods We utilized high-throughput sequencing to analyse the effects of vancomycin on the faecal human microbiota up to 22 weeks post-antibiotic cessation. The clinical relevance of the observed microbiota perturbations was studied in mice. Results During vancomycin therapy, most intestinal microbiota genera and operational taxonomic units (OTUs) were depleted in all analysed subjects, including all baseline OTUs from the phylum Bacteroidetes. This was accompanied by a vast expansion of genera associated with infections, including Klebsiella and Escherichia/Shigella. Following antibiotic cessation, marked differences in microbiota resilience were observed among subjects. While some individuals recovered a microbiota close to baseline composition, in others, up to 89% of abundant OTUs could no longer be detected. The clinical relevance of the observed microbiota changes was further demonstrated in mice, which developed analogous microbiota alterations. During vancomycin treatment, mice were highly susceptible to intestinal colonization by an antibiotic-resistant pathogen and, upon antibiotic cessation, a less-resilient microbiota allowed higher levels of pathogen colonization. Conclusions Oral vancomycin induces drastic and consistent changes in the human intestinal microbiota. Upon vancomycin cessation, the microbiota recovery rate varied considerably among subjects, which could influence, as validated in mice, the level of susceptibility to pathogen intestinal colonization. Our results demonstrate the negative long-term effects of vancomycin, which should be considered as a fundamental aspect of the cost–benefit equation for antibiotic prescription. PMID

  4. Megaselia scalaris causing human intestinal myiasis in Egypt.

    PubMed

    Mazayad, Said A M; Rifaat, Manal M A

    2005-04-01

    Megaselia scalaris is a worldwide distributed insect of medical importance. In a laboratory-based study, stool samples with undefined maggot infestation were examined and the presence of M. scalaris maggots was confirmed. Binocular stereo-microscopy was used for identification of the maggots. Larvae were allowed to develop into adults onto a human stool culture. The larvae and the emerged flies were identified using standard keys. This may be the first report of M. scalaris as a causative agent of human myiasis in Egypt. Details of the third instar larva, pupa and adults were given.

  5. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids.

    PubMed

    Matano, Mami; Date, Shoichi; Shimokawa, Mariko; Takano, Ai; Fujii, Masayuki; Ohta, Yuki; Watanabe, Toshiaki; Kanai, Takanori; Sato, Toshiro

    2015-03-01

    Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

  6. Hydrolysis of pyrethroids by human and rat tissues: Examination of intestinal, liver and serum carboxylesterases

    SciTech Connect

    Crow, J. Allen; Borazjani, Abdolsamad; Potter, Philip M.; Ross, Matthew K. . E-mail: mross@cvm.msstate.edu

    2007-05-15

    Hydrolytic metabolism of pyrethroid insecticides in humans is one of the major catabolic pathways that clear these compounds from the body. Rodent models are often used to determine the disposition and clearance rates of these esterified compounds. In this study the distribution and activities of esterases that catalyze pyrethroid metabolism have been investigated in vitro using several human and rat tissues, including small intestine, liver and serum. The major esterase in human intestine is carboxylesterase 2 (hCE2). We found that the pyrethroid trans-permethrin is effectively hydrolyzed by a sample of pooled human intestinal microsomes (5 individuals), while deltamethrin and bioresmethrin are not. This result correlates well with the substrate specificity of recombinant hCE2 enzyme. In contrast, a sample of pooled rat intestinal microsomes (5 animals) hydrolyze trans-permethrin 4.5-fold slower than the sample of human intestinal microsomes. Furthermore, it is demonstrated that pooled samples of cytosol from human or rat liver are {approx} 2-fold less hydrolytically active (normalized per mg protein) than the corresponding microsomal fraction toward pyrethroid substrates; however, the cytosolic fractions do have significant amounts ({approx} 40%) of the total esteratic activity. Moreover, a 6-fold interindividual variation in carboxylesterase 1 protein expression in human hepatic cytosols was observed. Human serum was shown to lack pyrethroid hydrolytic activity, but rat serum has hydrolytic activity that is attributed to a single CE isozyme. We purified the serum CE enzyme to homogeneity to determine its contribution to pyrethroid metabolism in the rat. Both trans-permethrin and bioresmethrin were effectively cleaved by this serum CE, but deltamethrin, esfenvalerate, alpha-cypermethrin and cis-permethrin were slowly hydrolyzed. Lastly, two model lipase enzymes were examined for their ability to hydrolyze pyrethroids. However, no hydrolysis products could be

  7. Comparative genomics analysis of Streptococcus isolates from the human small intestine reveals their adaptation to a highly dynamic ecosystem.

    PubMed

    Van den Bogert, Bartholomeus; Boekhorst, Jos; Herrmann, Ruth; Smid, Eddy J; Zoetendal, Erwin G; Kleerebezem, Michiel

    2013-01-01

    The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.

  8. Metabolomics analysis of Cistus monspeliensis leaf extract on energy metabolism activation in human intestinal cells.

    PubMed

    Shimoda, Yoichi; Han, Junkyu; Kawada, Kiyokazu; Smaoui, Abderrazak; Isoda, Hiroko

    2012-01-01

    Energy metabolism is a very important process to improve and maintain health from the point of view of physiology. It is well known that the intracellular ATP production is contributed to energy metabolism in cells. Cistus monspeliensis is widely used as tea, spices, and medical herb; however, it has not been focusing on the activation of energy metabolism. In this study, C. monspeliensis was investigated as the food resources by activation of energy metabolism in human intestinal epithelial cells. C. monspeliensis extract showed high antioxidant ability. In addition, the promotion of metabolites of glycolysis and TCA cycle was induced by C. monspeliensis treatment. These results suggest that C. monspeliensis extract has an ability to enhance the energy metabolism in human intestinal cells.

  9. Diagnosis of edema and inflammation in human intestines using ultrawideband radar

    NASA Astrophysics Data System (ADS)

    Smith, Sonny; Narayanan, Ram M.; Messaris, Evangelos

    2015-05-01

    Human intestines are vital organs, which are often subjected to chronic issues. In particular, Crohn's disease is a bowel aliment resulting in inflammation along the lining of one's digestive tract. Moreover, such an inflammatory condition causes changes in the thickness of the intestines; and we posit induce changes in the dielectric properties detectable by radar. This detection hinges on the increase in fluid content in the afflicted area, which is described by effective medium approximations (EMA). In this paper, we consider one of the constitutive parameters (i.e. relative permittivity) of different human tissues and introduce a simple numerical, electromagnetic multilayer model. We observe how the increase in water content in one layer can be approximated to predict the effective permittivity of that layer. Moreover, we note trends in how such an accumulation can influence the total effective reflection coefficient of the multiple layers.

  10. Metabolomics Analysis of Cistus monspeliensis Leaf Extract on Energy Metabolism Activation in Human Intestinal Cells

    PubMed Central

    Shimoda, Yoichi; Han, Junkyu; Kawada, Kiyokazu; Smaoui, Abderrazak; Isoda, Hiroko

    2012-01-01

    Energy metabolism is a very important process to improve and maintain health from the point of view of physiology. It is well known that the intracellular ATP production is contributed to energy metabolism in cells. Cistus monspeliensis is widely used as tea, spices, and medical herb; however, it has not been focusing on the activation of energy metabolism. In this study, C. monspeliensis was investigated as the food resources by activation of energy metabolism in human intestinal epithelial cells. C. monspeliensis extract showed high antioxidant ability. In addition, the promotion of metabolites of glycolysis and TCA cycle was induced by C. monspeliensis treatment. These results suggest that C. monspeliensis extract has an ability to enhance the energy metabolism in human intestinal cells. PMID:22523469

  11. Human intestinal gas measurement systems: in vitro fermentation and gas capsules.

    PubMed

    Ou, Jian Zhen; Yao, C K; Rotbart, Asaf; Muir, Jane G; Gibson, Peter R; Kalantar-zadeh, Kourosh

    2015-04-01

    The biological and clinical significance of the human gut microbiome is currently attracting worldwide attention. While rRNA and DNA technologies led to a quantum leap in our understanding of the numbers and types of gut microorganisms, much less is known about these microorganisms' activity in situ and in real time. Accurately measuring their byproducts, including intestinal gases, may offer unique biomarkers for specific gut microbiota, accelerating our understanding of the relationships among intestinal gases, the metabolic activity of the gut microbiome, and human health states. Here we present two novel techniques, namely in vitro fermentation and gas capsule systems, for measuring and assessing selected gas species. We discuss new developments with these technologies and the methods of their implementation and provide an overall review of their operation.

  12. Generation of L cells in mouse and human small intestine organoids.

    PubMed

    Petersen, Natalia; Reimann, Frank; Bartfeld, Sina; Farin, Henner F; Ringnalda, Femke C; Vries, Robert G J; van den Brink, Stieneke; Clevers, Hans; Gribble, Fiona M; de Koning, Eelco J P

    2014-02-01

    Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts. We show that short-chain fatty acids selectively increase the number of L cells, resulting in an elevation of GLP-1 release. This is accompanied by the upregulation of transcription factors associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L cells in mouse and human crypts as a potential basis for novel therapeutic strategies in patients with type 2 diabetes.

  13. Metabolism of liriodendrin and syringin by human intestinal bacteria and their relation to in vitro cytotoxicity.

    PubMed

    Kim, D H; Lee, K T; Bae, E A; Han, M J; Park, H J

    1999-02-01

    When liriodendrin or syringin was incubated for 24 h with human intestinal bacteria, two metabolites, (+)-syringaresinol-beta-D-glucopyranoside and (+)-syringaresinol, from liriodendrin and one metabolite, synapyl alcohol, from syringin were produced. The metabolic time course of liriodendrin was as follows: at early time, liriodendrin was converted to (+)-syringaresinol-beta-D-glucopyranoside, and then (+)-syringaresinol. The in vitro cytotoxicities of these metabolites, (+)-syringaresinol and synapyl alcohol, were superior to those of liriodendrin and syringin.

  14. A bioassay using intestinal organoids to measure CFTR modulators in human plasma.

    PubMed

    Dekkers, R; Vijftigschild, L A W; Vonk, A M; Kruisselbrink, E; de Winter-de Groot, K M; Janssens, H M; van der Ent, C K; Beekman, J M

    2015-03-01

    Treatment efficacies of drugs depend on patient-specific pharmacokinetic and pharmacodynamic properties. Here, we developed an assay to measure functional levels of the CFTR potentiator VX-770 in human plasma and observed that VX-770 in plasma from different donors induced variable CFTR function in intestinal organoids. This assay can help to understand variability in treatment response to CFTR potentiators by functionally modeling individual pharmacokinetics.

  15. Prebiotic effects of almonds and almond skins on intestinal microbiota in healthy adult humans.

    PubMed

    Liu, Zhibin; Lin, Xiuchun; Huang, Guangwei; Zhang, Wen; Rao, Pingfan; Ni, Li

    2014-04-01

    Almonds and almond skins are rich in fiber and other components that have potential prebiotic properties. In this study we investigated the prebiotic effects of almond and almond skin intake in healthy humans. A total of 48 healthy adult volunteers consumed a daily dose of roasted almonds (56 g), almond skins (10 g), or commercial fructooligosaccharides (8 g) (as positive control) for 6 weeks. Fecal samples were collected at defined time points and analyzed for microbiota composition and selected indicators of microbial activity. Different strains of intestinal bacteria had varying degrees of growth sensitivity to almonds or almond skins. Significant increases in the populations of Bifidobacterium spp. and Lactobacillus spp. were observed in fecal samples as a consequence of almond or almond skin supplementation. However, the populations of Escherichia coli did not change significantly, while the growth of the pathogen Clostridum perfringens was significantly repressed. Modification of the intestinal microbiota composition induced changes in bacterial enzyme activities, specifically a significant increase in fecal β-galactosidase activity and decreases in fecal β-glucuronidase, nitroreductase and azoreductase activities. Our observations suggest that almond and almond skin ingestion may lead to an improvement in the intestinal microbiota profile and a modification of the intestinal bacterial activities, which would induce the promotion of health beneficial factors and the inhibition of harmful factors. Thus we believe that almonds and almond skins possess potential prebiotic properties.

  16. Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization▿

    PubMed Central

    Schmiedel, Dinah; Epple, Hans-Jörg; Loddenkemper, Christoph; Ignatius, Ralf; Wagner, Jutta; Hammer, Bettina; Petrich, Annett; Stein, Harald; Göbel, Ulf B.; Schneider, Thomas; Moter, Annette

    2009-01-01

    Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS. PMID:19279178

  17. Identification of astilbin metabolites produced by human intestinal bacteria using UPLC-Q-TOF/MS.

    PubMed

    Zhao, Min; Xu, Jun; Qian, Dawei; Guo, Jianming; Jiang, Shu; Shang, Er-xin; Duan, Jin-ao

    2014-07-01

    Astilbin, mainly isolated from a commonly used herbal medicine, Smilax glabra Roxb (SGR), exhibits a variety of pharmacological activities and biological effects. It is metabolized by intestinal bacteria after oral administration which leads to the variation of ethnopharmacological profile of this traditional medicine. However, little is known on the interactions of this active compound with intestinal bacteria, which would be very helpful in unravelling how SGR works. In this study, different pure bacteria from human feces were isolated and were used to investigate their conversion capability of astilbin. Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx(TM) software was used to analyze astilbin and its metabolites. The parent compound and two metabolites (quercetin and eriodictyol) were detected in the isolated bacterial samples compared with blank samples. Quercetin was present in Enterococcus sp. 8B, 8-2 and 9-2 samples. Eriodictyol was only identified in Enterococcus sp. 8B sample. The metabolic routes and metabolites of astilbin produced by the different intestinal bacteria are reported for the first time. This will be useful for the investigation of the pharmacokinetic study of astilbin in vivo and the role of different intestinal bacteria in the metabolism of natural compounds.

  18. Human cytokine responses induced by Gram-positive cell walls of normal intestinal microbiota

    PubMed Central

    Chen, T; Isomäki, P; Rimpiläinen, M; Toivanen, P

    1999-01-01

    The normal microbiota plays an important role in the health of the host, but little is known of how the human immune system recognizes and responds to Gram-positive indigenous bacteria. We have investigated cytokine responses of peripheral blood mononuclear cells (PBMC) to Gram-positive cell walls (CW) derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (Eu.a.), Eubacterium limosum(Eu.l.), Lactobacillus casei(L.c.), and Lactobacillus fermentum (L.f.). Our results indicate that Gram-positive CW of the normal intestinal microbiota can induce cytokine responses of the human PBMC. The profile, level and kinetics of these responses are similar to those induced by lipopolysaccharide (LPS) or CW derived from a pathogen, Streptococcus pyogenes (S.p.). Bacterial CW are capable of inducing production of a proinflammatory cytokine, tumour necrosis factor-alpha (TNF-α), and an anti-inflammatory cytokine, IL-10, but not that of IL-4 or interferon-gamma (IFN-γ). Monocytes are the main cell population in PBMC to produce TNF-α and IL-10. Induction of cytokine secretion is serum-dependent; both CD14-dependent and -independent pathways are involved. These findings suggest that the human cytokine responses induced by Gram-positive CW of the normal intestinal microbiota are similar to those induced by LPS or Gram-positive CW of the pathogens. PMID:10540188

  19. Evidence of native starch degradation with human small intestinal maltase-glucoamylase (recombinant).

    PubMed

    Ao, Zihua; Quezada-Calvillo, Roberto; Sim, Lyann; Nichols, Buford L; Rose, David R; Sterchi, Erwin E; Hamaker, Bruce R

    2007-05-29

    Action of human small intestinal brush border carbohydrate digesting enzymes is thought to involve only final hydrolysis reactions of oligosaccharides to monosaccharides. In vitro starch digestibility assays use fungal amyloglucosidase to provide this function. In this study, recombinant N-terminal subunit enzyme of human small intestinal maltase-glucoamylase (rhMGAM-N) was used to explore digestion of native starches from different botanical sources. The susceptibilities to enzyme hydrolysis varied among the starches. The rate and extent of hydrolysis of amylomaize-5 and amylomaize-7 into glucose were greater than for other starches. Such was not observed with fungal amyloglucosidase or pancreatic alpha-amylase. The degradation of native starch granules showed a surface furrowed pattern in random, radial, or tree-like arrangements that differed substantially from the erosion patterns of amyloglucosidase or alpha-amylase. The evidence of raw starch granule degradation with rhMGAM-N indicates that pancreatic alpha-amylase hydrolysis is not a requirement for native starch digestion in the human small intestine.

  20. Geomagnetic Variations of Near-polar Regions and Human Health

    NASA Astrophysics Data System (ADS)

    Tchistova, Z. B.; Kutinov, Y. G.

    to conclude, that geomagnetic and geoelectric fields produced the biological effect, that is correlated with solar activity variations. The result of many years investigation of geomagnetic variations on near-polar territory, the authors found the facts of sharp increasing of intensity of the short wave' variations near the points of fault crossing. Analysis of character of geomagnetic variations can give information on the alleged effects of electromagnetic fields on human health. Spectral analysis shows rhythms with periods of 98, 105, 125, 130, 145, 160, 170, 180, 200, 235, 250, 270, 330, 395 sec. The rhythms with period of 98 -180 sec. coincide with rhythms of human cardiovascular activity. This concurrence entail occurrence of a magnetic resonance.

  1. The action of berry phenolics against human intestinal pathogens.

    PubMed

    Puupponen-Pimiä, Riitta; Nohynek, Liisa; Alakomi, Hanna-Leena; Oksman-Caldentey, Kirsi-Marja

    2005-01-01

    Phenolic compounds present in berries selectively inhibit the growth of human gastrointestinal pathogens. Especially cranberry, cloudberry, raspberry, strawberry and bilberry possess clear antimicrobial effects against e.g. salmonella and staphylococcus. Complex phenolic polymers, such as ellagitannins, are strong antibacterial agents present in cloudberry, raspberry and strawberry. Berry phenolics seem to affect the growth of different bacterial species with different mechanisms. Adherence of bacteria to epithelial surfaces is a prerequisite for colonization and infection of many pathogens. Antimicrobial activity of berries may also be related to anti-adherence activity of the berries. Utilization of enzymes in berry processing increases the amount of phenolics and antimicrobial activity of the berry products. Antimicrobial berry compounds are likely to have many important applications in the future as natural antimicrobial agents for food industry as well as for medicine.

  2. Effects of human fecal flora on intestinal morphology and mucosal immunity in human flora-associated piglet.

    PubMed

    Che, C; Pang, X; Hua, X; Zhang, B; Shen, J; Zhu, J; Wei, H; Sun, L; Chen, P; Cui, L; Zhao, L; Yang, Q

    2009-03-01

    Human flora-associated (HFA) piglet model was established to examine the effects of gut microbes from a different donor species on the intestinal morphology and mucosal immunity. Newborn germ-free piglets, obtained by caesarean section, were orally inoculated with a human and a porcine faecal suspension, and artificially fed to establish a HFA group (n = 7) and pig flora-associated (PFA) group (n = 7), respectively. All pigs were killed 6 weeks later. Tissue samples from duodenum, jejunum, ileum and colon were collected and studied by histochemistry and immunohistochemistry methods for intestinal morphological analyses and detection of immunocompetent cells. In summary, both groups of pigs performed well but HFA pigs had a somewhat better daily weight gain, and their jejunal villus height and crypt depth were significantly higher. In comparison with PFA pigs, the number of intraepithelial lymphocytes in jejunum was lower but the number of goblet cells containing neutral mucins was significantly increased in HFA pigs. No difference was observed in the number of mast cells. The areas of IgA producing cells and CD4(+) T cells in the jejunum and IgG producing cells in the small intestine were significantly higher in HFA pigs. However, the areas of MHC class II expressing cells were significantly increased in the duodenum and colon. Additionally, the amount of Bifidobacteria spp. was significantly higher in HFA pigs. This study confirms that the composition of gut microbes differentially affects the host intestinal mucosal immunity and suggests that commensal bacteria have great effects on intestinal health and development.

  3. Prediction of human drug clearance by multiple metabolic pathways: integration of hepatic and intestinal microsomal and cytosolic data.

    PubMed

    Cubitt, Helen E; Houston, J Brian; Galetin, Aleksandra

    2011-05-01

    The current study assesses hepatic and intestinal glucuronidation, sulfation, and cytochrome P450 (P450) metabolism of raloxifene, quercetin, salbutamol, and troglitazone using different in vitro systems. The fraction metabolized by conjugation and P450 metabolism was estimated in liver and intestine, and the importance of multiple metabolic pathways on accuracy of clearance prediction was assessed. In vitro intrinsic sulfation clearance (CL(int, SULT)) was determined in human intestinal and hepatic cytosol and compared with hepatic and intestinal microsomal glucuronidation (CL(int, UGT)) and P450 clearance (CL(int, CYP)) expressed per gram of tissue. Hepatic and intestinal cytosolic scaling factors of 80.7 mg/g liver and 18 mg/g intestine were estimated from published data. Scaled CL(int, SULT) ranged between 0.7 and 11.4 ml · min(-1) · g(-1) liver and 0.1 and 3.3 ml · min(-1) · g(-1) intestine (salbutamol and quercetin were the extremes). Salbutamol was the only compound with a high extent of sulfation (51 and 28% of total CL(int) for liver and intestine, respectively) and also significant renal clearance (26-57% of observed plasma clearance). In contrast, the clearance of quercetin was largely accounted for by glucuronidation. Drugs metabolized by multiple pathways (raloxifene and troglitazone) demonstrated improved prediction of intravenous clearance using data from all hepatic pathways (44-86% of observed clearance) compared with predictions based only on the primary pathway (22-36%). The assumption of no intestinal first pass resulted in underprediction of oral clearance for raloxifene, troglitazone, and quercetin (3-22% of observed, respectively). Accounting for the intestinal contribution to oral clearance via estimated intestinal availability improved prediction accuracy for raloxifene and troglitazone (within 2.5-fold of observed). Current findings emphasize the importance of both hepatic and intestinal conjugation for in vitro-in vivo extrapolation

  4. Host-microbe interactions in the neonatal intestine: role of human milk oligosaccharides.

    PubMed

    Donovan, Sharon M; Wang, Mei; Li, Min; Friedberg, Iddo; Schwartz, Scott L; Chapkin, Robert S

    2012-05-01

    The infant intestinal microbiota is shaped by genetics and environment, including the route of delivery and early dietary intake. Data from germ-free rodents and piglets support a critical role for the microbiota in regulating gastrointestinal and immune development. Human milk oligosaccharides (HMO) both directly and indirectly influence intestinal development by regulating cell proliferation, acting as prebiotics for beneficial bacteria and modulating immune development. We have shown that the gut microbiota, the microbial metatranscriptome, and metabolome differ between porcine milk-fed and formula-fed (FF) piglets. Our goal is to define how early nutrition, specifically HMO, shapes host-microbe interactions in breast-fed (BF) and FF human infants. We an established noninvasive method that uses stool samples containing intact sloughed epithelial cells to quantify intestinal gene expression profiles in human infants. We hypothesized that a systems biology approach, combining i) HMO composition of the mother's milk with the infant's gut gene expression and fecal bacterial composition, ii) gene expression, and iii short-chain fatty acid profiles would identify important mechanistic pathways affecting intestinal development of BF and FF infants in the first few months of life. HMO composition was analyzed by HLPC Chip/time-of-flight MS and 3 HMO clusters were identified using principle component analysis. Initial findings indicated that both host epithelial cell mRNA expression and the microbial phylogenetic profiles provided strong feature sets that distinctly classified the BF and FF infants. Ongoing analyses are designed to integrate the host transcriptome, bacterial phylogenetic profiles, and functional metagenomic data using multivariate statistical analyses.

  5. Ex vivo permeability experiments in excised rat intestinal tissue and in vitro solubility measurements in aspirated human intestinal fluids support age-dependent oral drug absorption.

    PubMed

    Annaert, Pieter; Brouwers, Joachim; Bijnens, Ann; Lammert, Frank; Tack, Jan; Augustijns, Patrick

    2010-01-31

    The possible influence of advanced age on intestinal drug absorption was investigated by determining the effects of aging on (i) solubility of model drugs in human intestinal fluids (HIF) obtained from two age groups (18-25 years; 62-72 years); and (ii) transepithelial permeation of model drugs across intestinal tissue excised from young, adult and old rats. Average equilibrium solubility values for 10 poorly soluble compounds in HIF aspirated from both age groups showed high interindividual variability, but did not reveal significant differences. Characterization of the HIF from both age groups demonstrated comparable pH profiles, while concentrations of individual bile salts showed pronounced variability between individuals, however without statistical differences between age groups. Transepithelial permeation of the transcellular probe metoprolol was significantly increased in old rats (38 weeks) compared to the younger age groups, while the modulatory role of P-glycoprotein in transepithelial talinolol transport was observed in adult and old rats but not in young rats. In conclusion, age-dependent permeability of intestinal tissue (rather than age-dependent luminal drug solubility) may contribute to altered intestinal drug absorption in older patients compared to young adults.

  6. Crohn disease--associated adherent-invasive E. coli bacteria target mouse and human Peyer's patches via long polar fimbriae.

    PubMed

    Chassaing, Benoit; Rolhion, Nathalie; de Vallée, Amélie; Salim, Sa'ad Y; Prorok-Hamon, Maelle; Neut, Christel; Campbell, Barry J; Söderholm, Johan D; Hugot, Jean-Pierre; Colombel, Jean-Frédéric; Darfeuille-Michaud, Arlette

    2011-03-01

    Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC)--a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyer's patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2(-/-) mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients.

  7. Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs.

    PubMed

    Nishimuta, Haruka; Nakagawa, Tetsuya; Nomura, Naruaki; Yabuki, Masashi

    2013-11-01

    1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds. 2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A. 3. Hepatic intrinsic clearance (CLint) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CLint values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CLint values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans. 4. In conclusion, dogs showed the most similar metabolic activity to humans.

  8. Fiber-based polarization-sensitive OCT of the human retina with correction of system polarization distortions

    PubMed Central

    Braaf, Boy; Vermeer, Koenraad A.; de Groot, Mattijs; Vienola, Kari V.; de Boer, Johannes F.

    2014-01-01

    In polarization-sensitive optical coherence tomography (PS-OCT) the use of single-mode fibers causes unpredictable polarization distortions which can result in increased noise levels and erroneous changes in calculated polarization parameters. In the current paper this problem is addressed by a new Jones matrix analysis method that measures and corrects system polarization distortions as a function of wavenumber by spectral analysis of the sample surface polarization state and deeper located birefringent tissue structures. This method was implemented on a passive-component depth-multiplexed swept-source PS-OCT system at 1040 nm which was theoretically modeled using Jones matrix calculus. High-resolution B-scan images are presented of the double-pass phase retardation, diattenuation, and relative optic axis orientation to show the benefits of the new analysis method for in vivo imaging of the human retina. The correction of system polarization distortions yielded reduced phase retardation noise, and better estimates of the diattenuation and the relative optic axis orientation in weakly birefringent tissues. The clinical potential of the system is shown by en face visualization of the phase retardation and optic axis orientation of the retinal nerve fiber layer in a healthy volunteer and a glaucoma patient with nerve fiber loss. PMID:25136498

  9. Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells--A Review.

    PubMed

    Kamiloglu, Senem; Capanoglu, Esra; Grootaert, Charlotte; Van Camp, John

    2015-09-08

    Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells.

  10. Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice.

    PubMed

    von Klitzing, Eliane; Öz, Fulya; Ekmekciu, Ira; Escher, Ulrike; Bereswill, Stefan; Heimesaat, Markus M

    2017-03-01

    Secondary abiotic mice generated by broad-spectrum antibiotic treatment provide a valuable tool for association studies with microbiota derived from different vertebrate hosts. We here generated human microbiota-associated (hma) mice by human fecal microbiota transplantation of secondary abiotic mice and performed a comprehensive survey of the intestinal microbiota dynamics in offspring of hma mice over 18 weeks following weaning as compared to their mothers applying both cultural and molecular methods. Mice were maintained under standard hygienic conditions with open cages, handled under aseptic conditions, and fed autoclaved chow and water. Within 1 week post weaning, fecal loads of commensal enterobacteria and enterococci had decreased, whereas obligate anaerobic bacteria such as Bacteroides/Prevotella species and clostridia were stably colonizing the intestines of hma offspring at high loads. Lactobacilli numbers were successively increasing until 18 weeks post weaning in both hma offspring and mothers, whereas by then, bifidobacteria were virtually undetectable in the former only. Interestingly, fecal lactobacilli and bifidobacteria were higher in mothers as compared to their offspring at 5 and 18 weeks post weaning. We conclude that the intestinal microbiota composition changes in offspring of hma mice, but also their mothers over time particularly affecting aerobic and microaerobic species.

  11. Comprehensive Survey of Intestinal Microbiota Changes in Offspring of Human Microbiota-Associated Mice

    PubMed Central

    von Klitzing, Eliane; Öz, Fulya; Ekmekciu, Ira; Escher, Ulrike; Bereswill, Stefan; Heimesaat, Markus M.

    2017-01-01

    Secondary abiotic mice generated by broad-spectrum antibiotic treatment provide a valuable tool for association studies with microbiota derived from different vertebrate hosts. We here generated human microbiota-associated (hma) mice by human fecal microbiota transplantation of secondary abiotic mice and performed a comprehensive survey of the intestinal microbiota dynamics in offspring of hma mice over 18 weeks following weaning as compared to their mothers applying both cultural and molecular methods. Mice were maintained under standard hygienic conditions with open cages, handled under aseptic conditions, and fed autoclaved chow and water. Within 1 week post weaning, fecal loads of commensal enterobacteria and enterococci had decreased, whereas obligate anaerobic bacteria such as Bacteroides/Prevotella species and clostridia were stably colonizing the intestines of hma offspring at high loads. Lactobacilli numbers were successively increasing until 18 weeks post weaning in both hma offspring and mothers, whereas by then, bifidobacteria were virtually undetectable in the former only. Interestingly, fecal lactobacilli and bifidobacteria were higher in mothers as compared to their offspring at 5 and 18 weeks post weaning. We conclude that the intestinal microbiota composition changes in offspring of hma mice, but also their mothers over time particularly affecting aerobic and microaerobic species. PMID:28386472

  12. Anthocyanin Absorption and Metabolism by Human Intestinal Caco-2 Cells—A Review

    PubMed Central

    Kamiloglu, Senem; Capanoglu, Esra; Grootaert, Charlotte; Van Camp, John

    2015-01-01

    Anthocyanins from different plant sources have been shown to possess health beneficial effects against a number of chronic diseases. To obtain any influence in a specific tissue or organ, these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity. One of the key factors affecting the bioavailability of anthocyanins is their transport through the gut epithelium. The Caco-2 cell line, a human intestinal epithelial cell model derived from a colon carcinoma, has been proven to be a good alternative to animal studies for predicting intestinal absorption of anthocyanins. Studies investigating anthocyanin absorption by Caco-2 cells report very low absorption of these compounds. However, the bioavailability of anthocyanins may be underestimated since the metabolites formed in the course of digestion could be responsible for the health benefits associated with anthocyanins. In this review, we critically discuss recent findings reported on the anthocyanin absorption and metabolism by human intestinal Caco-2 cells. PMID:26370977

  13. Subversion of human intestinal mucosa innate immunity by a Crohn's disease-associated E. coli.

    PubMed

    Jarry, A; Crémet, L; Caroff, N; Bou-Hanna, C; Mussini, J M; Reynaud, A; Servin, A L; Mosnier, J F; Liévin-Le Moal, V; Laboisse, C L

    2015-05-01

    Adherent-invasive Escherichia coli (AIEC), associated with Crohn's disease, are likely candidate contributory factors in the disease. However, signaling pathways involved in human intestinal mucosa innate host response to AIEC remain unknown. Here we use a 3D model of human intestinal mucosa explant culture to explore the effects of the AIEC strain LF82 on two innate immunity platforms, i.e., the inflammasome through evaluation of caspase-1 status, and NFκB signaling. We showed that LF82 bacteria enter and survive within a few intestinal epithelial cells and macrophages, without altering the mucosa overall architecture. Although 4-h infection with a Salmonella strain caused crypt disorganization, caspase-1 activation, and mature IL-18 production, LF82 bacteria were unable to activate caspase-1 and induce IL-18 production. In parallel, LF82 bacteria activated NFκB signaling in epithelial cells through IκBα phosphorylation, NFκBp65 nuclear translocation, and TNFα secretion. In addition, NFκB activation was crucial for the maintenance of epithelial homeostasis upon LF82 infection. In conclusion, here we decipher at the whole-mucosa level the mechanisms of the LF82-induced subversion of innate immunity that, by maintaining host cell integrity, ensure intracellular bacteria survival.

  14. Vasoactive Intestinal Peptide Inhibits Human Small-Cell Lung Cancer Proliferation in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Maruno, Kaname; Absood, Afaf; Said, Sami I.

    1998-11-01

    Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.

  15. hPSC-derived lung and intestinal organoids as models of human fetal tissue

    PubMed Central

    Aurora, Megan; Spence, Jason R.

    2016-01-01

    In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC). PMID:27287882

  16. Intestinal epithelial culture under an air-liquid interface: a tool for studying human and mouse esophagi.

    PubMed

    Yokobori, T; Suzuki, S; Miyazaki, T; Sohda, M; Sakai, M; Tanaka, N; Ozawa, D; Hara, K; Honjo, H; Altan, B; Fukuchi, M; Ishii, H; Iwatsuki, M; Sugimachi, K; Sudo, T; Iwaya, T; Nishida, N; Mimori, K; Kuwano, H; Mori, M

    2016-10-01

    This study investigated whether an intestinal epithelial culture method can be applied to mouse and human esophageal cultures. The esophagi harvested from 1-day-old mice and adult humans were maintained in collagen gels. A commercially available culture medium for human embryonic stem cells was used for the human esophageal culture. We discovered that the intestinal epithelial culture method can be successfully applied to both mouse and human esophageal cultures. The long-term cultured esophageal organoids were rod-like luminal structures lined with myofibroblasts. We discovered that regeneration of the esophageal mucosal surface can be almost completely achieved in vitro, and the advantage of this method is that organoid cultures may be generated using host-derived fibroblasts as a niche. This method is a promising tool for mouse and human research in intestinal biology, carcinogenesis, and regenerative medicine.

  17. Interleukin 2 modulates ion secretion and cell proliferation in cultured human small intestinal enterocytes

    PubMed Central

    O'Loughlin, E; Pang, G; Noltorp, R; Koina, C; Batey, R; Clancy, R

    2001-01-01

    AIMS—To determine if interleukin 2 (IL-2) alters epithelial transport and barrier function in cultured human small intestinal enterocytes.
METHODS—Confluent monolayers of small intestinal cells derived from duodenal biopsies were treated with IL-2 0.2-50 U/ml for 24 hours prior to study. Transport measurements were performed under short circuited conditions in Ussing chambers, with and without the secretagogues forskolin and 3-isobutyl-1-methyl xanthine (IBMX). Serosal to mucosal flux of 3[H] mannitol (permeability) and 3[H] thymidine uptake (proliferation) were measured. IL-2 receptor and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA were identified using reverse transcription-polymerase chain reaction (RT-PCR).
RESULTS—IL-2 did not alter baseline electrical parameters but caused a significant increase in cAMP dependent chloride secretion. The effect was mediated by the IL-2 receptor and paralleled a rapid increase in tyrosine phosphorylation, janus kinase 1, and signal transducers and activators of transcription (STATs) 1, 3, and 5. IL-2 significantly increased proliferation but at a lower dose than observed for enhanced secretion but did not alter permeability. IL-2 receptor β and γc chains and CFTR mRNA were identified by RT-PCR.
CONCLUSIONS—IL-2 treatment enhances cAMP stimulated chloride secretion and cellular proliferation in a human small intestinal cell line expressing a functional IL-2 receptor.


Keywords: interleukin 2; ion secretion; cell proliferation; enterocytes; small intestine PMID:11600465

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

    PubMed Central

    Couvigny, Benoît; de Wouters, Tomas; Kaci, Ghalia; Jacouton, Elsa; Delorme, Christine; Doré, Joël; Renault, Pierre; Blottière, Hervé M.

    2015-01-01

    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. PMID:25946041

  19. Lysophosphatidylcholine enhances carotenoid uptake from mixed micelles by Caco-2 human intestinal cells.

    PubMed

    Sugawara, T; Kushiro, M; Zhang, H; Nara, E; Ono, H; Nagao, A

    2001-11-01

    Despite the interest in the beneficial roles of dietary carotenoids in human health, little is known about their solubilization from foods to mixed bile micelles during digestion and the intestinal uptake from the micelles. We investigated the absorption of carotenoids solubilized in mixed micelles by differentiated Caco-2 human intestinal cells, which is a useful model for studying the absorption of dietary compounds by intestinal cells. The micelles were composed of 1 micromol/L carotenoids, 2 mmol/L sodium taurocholate, 100 micromol/L monoacylglycerol, 33.3 micromol/L fatty acid and phospholipid (0-200 micromol/L). The phospholipid content of micelles had profound effects on the cellular uptake of carotenoids. Uptake of micellar beta-carotene and lutein was greatly suppressed by phosphatidylcholine (PC) in a dose-dependent manner, whereas lysophosphatidylcholine (lysoPC), the lipolysis product of PC by phospholipase A2 (PLA2), markedly enhanced both beta-carotene and lutein uptake. The addition of PLA2 from porcine pancreas to the medium also enhanced the uptake of carotenoids from micelles containing PC. Caco-2 cells could take up 15 dietary carotenoids, including epoxy carotenoids, such as violaxanthin, neoxanthin and fucoxanthin, from micellar carotenoids, and the uptakes showed a linear correlation with their lipophilicity, defined as the distribution coefficient in 1-octanol/water (log P(ow)). These results suggest that pancreatic PLA2 and lysoPC are important in regulating the absorption of carotenoids in the digestive tract and support a simple diffusion mechanism for carotenoid absorption by the intestinal epithelium.

  20. Tea Catechin Auto-oxidation Dimers are Accumulated and Retained by Caco-2 Human Intestinal Cells

    PubMed Central

    Neilson, Andrew P.; Song, Brian J.; Sapper, Teryn N.; Bomser, Joshua A.; Ferruzzi, Mario G.

    2010-01-01

    Despite the presence of bioactive catechin B-ring auto-oxidation dimers in tea, little is known regarding their absorption in humans. Our hypothesis for this research is that catechin auto-oxidation dimers are present in teas and are absorbable by human intestinal epithelial cells. Dimers [theasinensins (THSNs) and P-2 analogs) were quantified in commercial teas by HPLC-MS. (−)-Epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCG) homodimers were present at 10–43 and 0–62 µmol/g leaf, respectively. EGC-EGCG heterodimers were present at 0–79 µmol/g. The potential intestinal absorption of these dimers was assessed using Caco-2 intestinal cells. Catechin monomers and dimers were detected in cells exposed to media containing monomers and preformed dimers. Accumulation of dimers was significantly greater than monomers from test media. Three h accumulation of EGC and EGCG was 0.19– 0.55% and 1.24–1.35% respectively. Comparatively, 3h accumulation of the EGC P-2 analog, and THSNs C/E was 0.89 ± 0.28% and 1.53 ± 0.36%. Accumulation of P-2, and THSNs A/D was 6.93 ± 2.1%, and 10.1 ± 3.6%. EGCG-EGC heterodimer P-2 analog, and THSN B 3h accumulation was 4.87 ± 2.2%, and 4.65 ± 2.8% respectively. One h retention of P-2, and THSNs A/D was 171 ± 22%, and 29.6 ± 9.3% of accumulated amount suggesting intracellular oxidative conversion of THSNs to P-2. These data suggest that catechin dimers present in the gut lumen may be readily absorbed by intestinal epithelium. PMID:20579525

  1. Multispectral tissue characterization for intestinal anastomosis optimization

    NASA Astrophysics Data System (ADS)

    Cha, Jaepyeong; Shademan, Azad; Le, Hanh N. D.; Decker, Ryan; Kim, Peter C. W.; Kang, Jin U.; Krieger, Axel

    2015-10-01

    Intestinal anastomosis is a surgical procedure that restores bowel continuity after surgical resection to treat intestinal malignancy, inflammation, or obstruction. Despite the routine nature of intestinal anastomosis procedures, the rate of complications is high. Standard visual inspection cannot distinguish the tissue subsurface and small changes in spectral characteristics of the tissue, so existing tissue anastomosis techniques that rely on human vision to guide suturing could lead to problems such as bleeding and leakage from suturing sites. We present a proof-of-concept study using a portable multispectral imaging (MSI) platform for tissue characterization and preoperative surgical planning in intestinal anastomosis. The platform is composed of a fiber ring light-guided MSI system coupled with polarizers and image analysis software. The system is tested on ex vivo porcine intestine tissue, and we demonstrate the feasibility of identifying optimal regions for suture placement.

  2. Intestinal Commitment and Maturation of Human Pluripotent Stem Cells Is Independent of Exogenous FGF4 and R-spondin1

    PubMed Central

    Tamminen, Kaisa; Balboa, Diego; Toivonen, Sanna; Pakarinen, Mikko P.; Wiener, Zoltan; Alitalo, Kari; Otonkoski, Timo

    2015-01-01

    Wnt/beta-catenin signaling plays a central role in guiding the differentiation of the posterior parts of the primitive gut tube into intestinal structures in vivo and some studies suggest that FGF4 is another crucial factor for intestinal development. The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids. The most prominent induction of the well-established intestinal marker gene CDX2 was achieved when hPSC-derived definitive endoderm cells were treated with Wnt agonist molecule CHIR99021 during differentiation to hindgut. FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage. When hindgut stage cells were further cultured in 3D, they formed self-renewing organoid structures containing all major intestinal cell types even without exogenous R-spondin1 (RSPO1), a crucial factor for the culture of epithelial organoids derived from adult intestine. This may be explained by the presence of a mesenchymal compartment in the hPSC-derived organoids. Addition of WNT3A increased the expression of the Paneth cell marker Lysozyme in hPSC-derived organoid cultures, whereas FGF4 inhibited both the formation and maturation of intestinal-like organoids. Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro. PMID:26230325

  3. Intestinal Commitment and Maturation of Human Pluripotent Stem Cells Is Independent of Exogenous FGF4 and R-spondin1.

    PubMed

    Tamminen, Kaisa; Balboa, Diego; Toivonen, Sanna; Pakarinen, Mikko P; Wiener, Zoltan; Alitalo, Kari; Otonkoski, Timo

    2015-01-01

    Wnt/beta-catenin signaling plays a central role in guiding the differentiation of the posterior parts of the primitive gut tube into intestinal structures in vivo and some studies suggest that FGF4 is another crucial factor for intestinal development. The aim of this study was to define the effects of Wnt and FGF4 on intestinal commitment in vitro by establishing conditions for differentiation of human pluripotent stem cells (hPSC) into posterior endoderm (hindgut) and further to self-renewing intestinal-like organoids. The most prominent induction of the well-established intestinal marker gene CDX2 was achieved when hPSC-derived definitive endoderm cells were treated with Wnt agonist molecule CHIR99021 during differentiation to hindgut. FGF4 was found to be dispensable during intestinal commitment, but it had an early role in repressing development towards the hepatic lineage. When hindgut stage cells were further cultured in 3D, they formed self-renewing organoid structures containing all major intestinal cell types even without exogenous R-spondin1 (RSPO1), a crucial factor for the culture of epithelial organoids derived from adult intestine. This may be explained by the presence of a mesenchymal compartment in the hPSC-derived organoids. Addition of WNT3A increased the expression of the Paneth cell marker Lysozyme in hPSC-derived organoid cultures, whereas FGF4 inhibited both the formation and maturation of intestinal-like organoids. Similar hindgut and organoid cultures were established from human induced pluripotent stem cells, implying that this approach can be used to create patient-specific intestinal tissue models for disease modeling in vitro.

  4. Immune phenotypes of microglia in human neurodegenerative disease: challenges to detecting microglial polarization in human brains.

    PubMed

    Walker, Douglas G; Lue, Lih-Fen

    2015-08-19

    Inflammatory responses in the brain, which can be demonstrated by changes in properties of microglia, the brain-resident macrophages, are a common feature of human neurodegenerative diseases. Different monocyte/macrophage phenotypes have been defined by changes in expression of cytokines, receptors and other markers as a response to different classes of stimuli. Monocytes, macrophages and microglia can have a range of phenotypes with associated properties depending on their microenvironment. Macrophage/microglia polarization states have been defined as classical activation (M1), alternative activation (M2a), type II alternative activation (M2b) or acquired deactivation (M2c). Available markers for identifying microglial phenotypes in human brains are still limited; those available provide incomplete information on the functions or polarization states of microglia observed in tissues from diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis. The most widely used marker to describe activated microglia in human brains, particularly diseased brains, has been HLA-DR, the major histocompatibility complex II protein. HLA-DR-positive microglia can have a wide range of activation morphologies that are affected not only by disease pathology, but also by their differentiation states and brain regions. Two other widely used markers to identify microglia in human brains are ionized calcium binding adaptor molecule-1 and CD68. Although their expression changes in diseased brains, these markers do not show specificity for different phenotypes. Over the years there have been studies with additional markers that attempt to further define microglial properties, particularly in Alzheimer's disease brains. Most studies have employed immunohistochemical techniques to identify microglia in tissue sections, but recent advances in this field have allowed gene expression profiling of microglia upon immediate isolation from brains. We will review which markers

  5. Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2.

    PubMed

    Stremmel, Wolfgang; Staffer, Simone; Gan-Schreier, Hongying; Wannhoff, Andreas; Bach, Margund; Gauss, Annika

    2016-09-01

    Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.

  6. Intestinal Capillariasis

    DTIC Science & Technology

    1987-12-01

    bhIll inenais, the tiny nematode causing Intestinal capillariasis In humans, Is a Iunique parasite. It is one of the newest parasites that has been...Capillariaphilippinensis, the tiny nematode causing intestinal capillariasis in humans, is a unique parasite. It is one of the newest parasites that has been shown to...stichocytes surrounding the oesophagus. The posterior half of the nematode is wider than the anterior half and contains the digestive tract and the

  7. Mast cell expression of the serotonin1A receptor in guinea pig and human intestine.

    PubMed

    Wang, Guo-Du; Wang, Xi-Yu; Zou, Fei; Qu, Meihua; Liu, Sumei; Fei, Guijun; Xia, Yun; Needleman, Bradley J; Mikami, Dean J; Wood, Jackie D

    2013-05-15

    Serotonin [5-hydroxytryptamine (5-HT)] is released from enterochromaffin cells in the mucosa of the small intestine. We tested a hypothesis that elevation of 5-HT in the environment of enteric mast cells might degranulate the mast cells and release mediators that become paracrine signals to the enteric nervous system, spinal afferents, and secretory glands. Western blotting, immunofluorescence, ELISA, and pharmacological analysis were used to study expression of 5-HT receptors by mast cells in the small intestine and action of 5-HT to degranulate the mast cells and release histamine in guinea pig small intestine and segments of human jejunum discarded during Roux-en-Y gastric bypass surgeries. Mast cells in human and guinea pig preparations expressed the 5-HT1A receptor. ELISA detected spontaneous release of histamine in guinea pig and human preparations. The selective 5-HT1A receptor agonist 8-hydroxy-PIPAT evoked release of histamine. A selective 5-HT1A receptor antagonist, WAY-100135, suppressed stimulation of histamine release by 5-HT or 8-hydroxy-PIPAT. Mast cell-stabilizing drugs, doxantrazole and cromolyn sodium, suppressed the release of histamine evoked by 5-HT or 8-hydroxy-PIPAT in guinea pig and human preparations. Our results support the hypothesis that serotonergic degranulation of enteric mast cells and release of preformed mediators, including histamine, are mediated by the 5-HT1A serotonergic receptor. Association of 5-HT with the pathophysiology of functional gastrointestinal disorders (e.g., irritable bowel syndrome) underlies a question of whether selective 5-HT1A receptor antagonists might have therapeutic application in disorders of this nature.

  8. Human intestinal parasites in non-biting synanthropic flies in Ogun State, Nigeria.

    PubMed

    Adenusi, Adedotun Adesegun; Adewoga, Thomas O Sunday

    2013-01-01

    Filth-feeding and breeding, non-biting synanthropic flies have been incriminated in the dissemination of human enteropathogens in the environment. This study determined the species of non-biting synanthropic flies associated with four filthy sites in Ilishan, Ogun State, southwest Nigeria, and assessed their potentials for mechanical transmission of human intestinal parasites. 7190 flies identified as Musca domestica (33.94%), Chrysomya megacephala (26.01%), Musca sorbens (23.23%), Lucilia cuprina (8.76%), Calliphora vicina (4.59%), Sarcophaga sp. (2.78%) and Fannia scalaris (0.70%) were examined for human intestinal parasites by the formol-ether concentration and modified Ziehl-Neelsen techniques. Eggs of the following parasites: Ascaris lumbricoides (34.08%), Trichuris trichiura (25.87%), hookworms (20.45%), Taenia sp. (2.36%), Hymenolepis nana (1.11%), Enterobius vermicularis (0.56%), Strongyloides stercoralis (larvae; 3.89%) and cysts of Entamoeba histolytica/dispar (27.26%), Entamoeba coli (22.67%), Giardia lamblia (3.34%) and Cryptosporidium sp. (1.81%) were isolated from the body surfaces and or gut contents of 75.24% of 719 pooled fly batches. The helminths A. lumbricoides and T. trichiura and the protozoans, E. histolytica/dispar and E. coli were the dominant parasites detected, both on body surfaces and in the gut contents of flies. C. megacephala was the highest carrier of parasites (diversity and number). More parasites were isolated from the gut than from body surfaces (P < 0.05). Flies from soiled ground often carried more parasites than those from abattoir, garbage or open-air market. Synanthropic fly species identified in this study can be of potential epidemiological importance as mechanical transmitters of human intestinal parasites acquired naturally from filth and carried on their body surfaces and or in the gut, because of their vagility and feeding mechanisms.

  9. Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites

    PubMed Central

    Yamamoto, Denise; Hernandes, Rodrigo T.; Liberatore, Ana Maria A.; Abe, Cecilia M.; de Souza, Rodrigo B.; Romão, Fabiano T.; Sperandio, Vanessa; Koh, Ivan H.

    2017-01-01

    Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo. PMID:28178312

  10. Human milk oligosaccharide effects on intestinal function and inflammation after preterm birth in pigs.

    PubMed

    Rasmussen, Stine O; Martin, Lena; Østergaard, Mette V; Rudloff, Silvia; Roggenbuck, Michael; Nguyen, Duc Ninh; Sangild, Per T; Bering, Stine B

    2017-02-01

    Human milk oligosaccharides (HMOs) may mediate prebiotic and anti-inflammatory effects in newborns. This is particularly important for preterm infants who are highly susceptible to intestinal dysfunction and necrotizing enterocolitis (NEC). We hypothesized that HMO supplementation of infant formula (IF) improves intestinal function, bacterial colonization and NEC resistance immediately after preterm birth, as tested in a preterm pig model. Mixtures of HMOs were investigated in intestinal epithelial cells and in preterm pigs (n=112) fed IF supplemented without (CON) or with a mixture of four HMOs (4-HMO) or >25 HMOs (25-HMO, 5-10 g/L given for 5 or 11 days). The 25-HMO blend decreased cell proliferation and both HMO blends decreased lipopolysaccharide-induced interleukin-8 secretion in IPEC-J2 cells, relative to control (P<.05). All HMOs were found in urine and feces of HMO-treated pigs, and short-chain fatty acids in the colon were higher in HMO vs. CON pigs (P<.05). After 5 days, NEC lesions were similar between HMO and CON pigs and 25-HMO increased colon weights (P<.01). After 11 days, the 4-HMO diet did not affect NEC (56 vs. 79%, P=.2) but increased dehydration and diarrhea (P<.05) and expression of immune-related genes (IL10, IL12, TGFβ, TLR4; P<.05). Bacterial adherence and diversity was unchanged after HMO supplementation.

  11. Escherichia albertii, a novel human enteropathogen, colonizes rat enterocytes and translocates to extra-intestinal sites.

    PubMed

    Yamamoto, Denise; Hernandes, Rodrigo T; Liberatore, Ana Maria A; Abe, Cecilia M; Souza, Rodrigo B de; Romão, Fabiano T; Sperandio, Vanessa; Koh, Ivan H; Gomes, Tânia A T

    2017-01-01

    Diarrhea is the second leading cause of death of children up to five years old in the developing countries. Among the etiological diarrheal agents are atypical enteropathogenic Escherichia coli (aEPEC), one of the diarrheagenic E. coli pathotypes that affects children and adults, even in developed countries. Currently, genotypic and biochemical approaches have helped to demonstrate that some strains classified as aEPEC are actually E. albertii, a recently recognized human enteropathogen. Studies on particular strains are necessary to explore their virulence potential in order to further understand the underlying mechanisms of E. albertii infections. Here we demonstrated for the first time that infection of fragments of rat intestinal mucosa is a useful tool to study the initial steps of E. albertii colonization. We also observed that an E. albertii strain can translocate from the intestinal lumen to Mesenteric Lymph Nodes and liver in a rat model. Based on our finding of bacterial translocation, we investigated how E. albertii might cross the intestinal epithelium by performing infections of M-like cells in vitro to identify the potential in vivo translocation route. Altogether, our approaches allowed us to draft a general E. albertii infection route from the colonization till the bacterial spreading in vivo.

  12. Human Milk Oligosaccharides in Premature Infants: Absorption, Excretion and Influence on the Intestinal Microbiota

    PubMed Central

    Underwood, Mark A.; Gaerlan, Stephanie; De Leoz, M. Lorna A.; Dimapasoc, Lauren; Kalanetra, Karen M.; Lemay, Danielle G.; German, J. Bruce; Mills, David A.; Lebrilla, Carlito B.

    2015-01-01

    Background Human milk oligosaccharides (HMOs) shape the intestinal microbiota in term infants. In premature infants, alterations in the intestinal microbiota (dysbiosis) are associated with risk of necrotizing enterocolitis and sepsis and the influence of HMOs on the microbiota is unclear. Methods Milk, urine, and stool specimens from 14 mother-premature infant dyads were investigated by mass spectrometry for HMO composition. The stools were analyzed by next-generation sequencing (NGS) to complement a previous analysis. Results Percentages of fucosylated and sialylated HMOs were highly variable between individuals but similar in urine, feces and milk within dyads. Differences in urine and fecal HMO composition suggest variability in absorption. Secretor status of the mother correlated with the urine and fecal content of specific HMO structures. Trends toward higher levels of Proteobacteria and lower levels of Firmicutes, were noted in premature infants of non-secretor mothers. Specific HMO structures in the milk, urine and feces were associated with alterations in fecal Proteobacteria and Firmicutes. Conclusion HMOs may influence the intestinal microbiota in premature infants. Specific HMOs, for example those associated with secretor mothers, may have a protective effect by decreasing pathogens associated with sepsis and necrotizing enterocolitis while other HMOs may increase dysbiosis in this population. PMID:26322410

  13. Infection with fully mature Corynosoma cf. validum causes ulcers in the human small intestine.

    PubMed

    Takahashi, Keitaro; Ito, Takahiro; Sato, Tomonobu; Goto, Mitsuru; Kawamoto, Toru; Fujinaga, Akihiro; Yanagawa, Nobuyuki; Saito, Yoshinori; Nakao, Minoru; Hasegawa, Hideo; Fujiya, Mikihiro

    2016-06-01

    Corynosoma is a parasite that can normally be found in the intestinal tract of fish-eating mammals, particularly in seals and birds. The present case proposed that Corynosoma could attain full maturity in the human intestine. A 70-year-old female complained of abdominal pain. A computed tomography (CT) scan revealed a swelling of the intraperitoneal lymph nodes with no responsible lesion. Video capsule endoscopy and double-balloon endoscopy detected several ulcerations and one parasite in the ileum, which was tightly attached at the bottom of the ulcerations. The parasite was cylindrical and measured approximately 10 mm (long) x 3 mm (wide). Pathologically, the worm had a four-layered body wall and contained embryonated eggs. The sequences of the parasite-derived nuclear ribosomal DNA fragment and mitochondrial DNA fragment of cox1 were almost identical to those of Corynosoma validum. The patient's abdominal pain immediately improved after the administration of pyrantel pamoate (1,500 mg). Corynosoma was possibly the responsible disease in a patient who complained of abdominal pain and in whom no responsible lesion was detected by CT, gastroduodenoscopy or colonoscopy. Examinations of the small intestines should be aggressively performed in such cases.

  14. Functional modulation of human intestinal epithelial cell responses by Bifidobacterium infantis and Lactobacillus salivarius

    PubMed Central

    O'Hara, Ann M; O'Regan, Padraig; Fanning, Áine; O'Mahony, Caitlin; MacSharry, John; Lyons, Anne; Bienenstock, John; O'Mahony, Liam; Shanahan, Fergus

    2006-01-01

    Intestinal epithelial cells (IECs) and dendritic cells (DCs) play a pivotal role in antigen sampling and the maintenance of gut homeostasis. However, the interaction of commensal bacteria with the intestinal surface remains incompletely understood. Here we investigated immune cell responses to commensal and pathogenic bacteria. HT-29 human IECs were incubated with Bifidobacterium infantis 35624, Lactobacillus salivarius UCC118 or Salmonella typhimurium UK1 for varying times, or were pretreated with a probiotic for 2 hr prior to stimulation with S. typhimurium or flagellin. Gene arrays were used to examine inflammatory gene expression. Nuclear factor (NF)-κB activation, interleukin (IL)-8 secretion, pathogen adherence to IECs, and mucin-3 (MUC3) and E-cadherin gene expression were assayed by TransAM assay, enzyme-linked immunosorbent assay (ELISA), fluorescence, and real-time reverse transcriptase–polymerase chain reaction (RT-PCR), respectively. IL-10 and tumour necrosis factor (TNF)-α secretion by bacteria-treated peripheral blood-derived DCs were measured using ELISA. S. typhimurium increased expression of 36 of the 847 immune-related genes assayed, including NF-κB and IL-8. The commensal bacteria did not alter expression levels of any of the 847 genes. However, B. infantis and L. salivarius attenuated both IL-8 secretion at baseline and S. typhimurium-induced pro-inflammatory responses. B. infantis also limited flagellin-induced IL-8 protein secretion. The commensal bacteria did not increase MUC3 or E-cadherin expression, or interfere with pathogen binding to HT-29 cells, but they did stimulate IL-10 and TNF-α secretion by DCs. The data demonstrate that, although the intestinal epithelium is immunologically quiescent when it encounters B. infantis or L. salivarius, these commensal bacteria exert immunomodulatory effects on intestinal immune cells that mediate host responses to flagellin and enteric pathogens. PMID:16771855

  15. Human milk oligosaccharides: evolution, structures and bioselectivity as substrates for intestinal bacteria.

    PubMed

    German, J Bruce; Freeman, Samara L; Lebrilla, Carlito B; Mills, David A

    2008-01-01

    Human milk contains a high concentration of diverse soluble oligosaccharides, carbohydrate polymers formed from a small number of monosaccharides. Novel methods combining liquid chromatography with high resolution mass spectrometry have identified approximately 200 unique oligosaccharides structures varying from 3 to 22 sugars. The increasing complexity of oligosaccharides follows the general pattern of mammalian evolution though the concentration and diversity of these structures in homo sapiens are strikingly. There is also diversity among human mothers in oligosaccharides. Milks from randomly selected mothers contain as few as 23 and as many as 130 different oligosaccharides. The functional implications of this diversity are not known. Despite the role of milk to serve as a sole nutrient source for mammalian infants, the oligosaccharides in milk are not digestible by human infants. This apparent paradox raises questions about the functions of these oligosaccharides and how their diverse molecular structures affect their functions. The nutritional function most attributed to milk oligosaccharides is to serve as prebiotics - a form of indigestible carbohydrate that is selectively fermented by desirable gut microflora. This function was tested by purifying human milk oligosaccharides and providing these as the sole carbon source to various intestinal bacteria. Indeed, the selectively of providing the complex mixture of oligosaccharides pooled from human milk samples is remarkable. Among a variety of Bifidobacteria tested only Bifidobacteria longum biovar infantis was able to grow extensively on human milk oligosaccharides as sole carbon source. The genomic sequence of this strain revealed approximately 700 genes that are unique to infantis, including a variety of co-regulated glycosidases, relative to other Bifidobacteria, implying a co-evolution of human milk oligosaccharides and the genetic capability of select intestinal bacteria to utilize them. The goal of

  16. Metabolism of Kaempferia parviflora polymethoxyflavones by human intestinal bacterium Bautia sp. MRG-PMF1.

    PubMed

    Kim, Mihyang; Kim, Nayoung; Han, Jaehong

    2014-12-24

    Poylmethoxyflavones (PMFs) are major bioactive flavonoids, which exhibit various biological activities, such as anticancer effects. The biotransformation of PMFs and characterization of a PMF-metabolizing human intestinal bacterium were studied herein for the first time. Hydrolysis of aryl methyl ether functional groups by human fecal samples was observed from the bioconversion of various PMFs. Activity-guided screening for PMF-metabolizing intestinal bacteria under anaerobic conditions resulted in the isolation of a strict anaerobic bacterium, which was identified as Blautia sp. MRG-PMF1. The isolated MRG-PMF1 was able to metabolize various PMFs to the corresponding demethylated flavones. The microbial conversion of bioactive 5,7-dimethoxyflavone (5,7-DMF) and 5,7,4'-trimethoxyflavone (5,7,4'-TMF) was studied in detail. 5,7-DMF and 5,7,4'-TMF were completely metabolized to 5,7-dihydroxyflavone (chrysin) and 5,7,4'-trihydroxyflavone (apigenin), respectively. From a kinetics study, the methoxy group on the flavone C-7 position was found to be preferentially hydrolyzed. 5-Methoxychrysin, the intermediate of 5,7-DMF metabolism by Blautia sp. MRG-PMF1, was isolated and characterized by nuclear magnetic resonance spectroscopy. Apigenin was produced from the sequential demethylation of 5,7,4'-TMF, via 5,4'-dimethoxy-7-hydroxyflavone and 7,4'-dihydroxy-5-methoxyflavone (thevetiaflavone). Not only demethylation activity but also deglycosylation activity was exhibited by Blautia sp. MRG-PMF1, and various flavonoids, including isoflavones, flavones, and flavanones, were found to be metabolized to the corresponding aglycones. The unprecedented PMF demethylation activity of Blautia sp. MRG-PMF1 will expand our understanding of flavonoid metabolism in the human intestine and lead to novel bioactive compounds.

  17. Combined Effects of Lipophilic Phycotoxins (Okadaic Acid, Azapsiracid-1 and Yessotoxin) on Human Intestinal Cells Models

    PubMed Central

    Ferron, Pierre-Jean; Dumazeau, Kevin; Beaulieu, Jean-François; Le Hégarat, Ludovic; Fessard, Valérie

    2016-01-01

    Phycotoxins are monitored in seafood because they can cause food poisonings in humans. Phycotoxins do not only occur singly but also as mixtures in shellfish. The aim of this study was to evaluate the in vitro toxic interactions of binary combinations of three lipophilic phycotoxins commonly found in Europe (okadaic acid (OA), yessotoxin (YTX) and azaspiracid-1 (AZA-1)) using the neutral red uptake assay on two human intestinal cell models, Caco-2 and the human intestinal epithelial crypt-like cells (HIEC). Based on the cytotoxicity of individual toxins, we studied the interactions between toxins in binary mixtures using the combination index-isobologram equation, a method widely used in pharmacology to study drug interactions. This method quantitatively classifies interactions between toxins in mixtures as synergistic, additive or antagonistic. AZA-1/OA, and YTX/OA mixtures showed increasing antagonism with increasing toxin concentrations. In contrast, the AZA-1/YTX mixture showed increasing synergism with increasing concentrations, especially for mixtures with high YTX concentrations. These results highlight the hazard potency of AZA-1/YTX mixtures with regard to seafood intoxication. PMID:26907345

  18. Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers.

    PubMed

    Stetinová, Vera; Smetanová, Libuse; Kholová, Dagmar; Svoboda, Zbynek; Kvetina, Jaroslav

    2009-09-01

    This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

  19. Smoking Cessation Induces Profound Changes in the Composition of the Intestinal Microbiota in Humans

    PubMed Central

    Biedermann, Luc; Zeitz, Jonas; Mwinyi, Jessica; Sutter-Minder, Eveline; Rehman, Ateequr; Ott, Stephan J.; Steurer-Stey, Claudia; Frei, Anja; Frei, Pascal; Scharl, Michael; Loessner, Martin J.; Vavricka, Stephan R.; Fried, Michael; Schreiber, Stefan; Schuppler, Markus; Rogler, Gerhard

    2013-01-01

    Background The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation. Methods During the observational period of 9 weeks repetitive stool samples were collected. Based on abundance of 16S rRNA genes bacterial composition was analysed and compared to 10 control subjects (5 continuing smokers and 5 non-smokers) by means of Terminal Restriction Fragment Length Polymorphism analysis and high-throughput sequencing. Results Profound shifts in the microbial composition after smoking cessation were observed with an increase of Firmicutes and Actinobacteria and a lower proportion of Bacteroidetes and Proteobacteria on the phylum level. In addition, after smoking cessation there was an increase in microbial diversity. Conclusions These results indicate that smoking is an environmental factor modulating the composition of human gut microbiota. The observed changes after smoking cessation revealed to be similar to the previously reported differences in obese compared to lean humans and mice respectively, suggesting a potential pathogenetic link between weight gain and smoking cessation. In addition they give rise to a potential association of smoking status and the course of IBD. PMID:23516617

  20. Initiation of an inflammatory response in resident intestinal lamina propria cells -use of a human organ culture model.

    PubMed

    Schröder-Braunstein, Jutta; Gras, Judith; Brors, Benedikt; Schwarz, Sonja; Szikszai, Timea; Lasitschka, Felix; Wabnitz, Guido; Heidtmann, Antje; Lee, Young-Seon; Schiessling, Serin; Leowardi, Christine; Al-Saeedi, Mohammed; Ulrich, Alexis; Engelke, Antonia; Winter, Johannes; Samstag, Yvonne; Giese, Thomas; Meuer, Stefan

    2014-01-01

    Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL1B, IL6, IL8, IL23A, TNFA, CXCL2, and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. Furthermore, genes never been previously associated with intestinal inflammation were identified using this model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease.

  1. Transesterification of a series of 12 parabens by liver and small-intestinal microsomes of rats and humans.

    PubMed

    Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Kitamura, Shigeyuki

    2014-02-01

    Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1-C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1-C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively.

  2. Intestinal parasite co-infection among pulmonary tuberculosis cases without human immunodeficiency virus infection in a rural county in China.

    PubMed

    Li, Xin-Xu; Chen, Jia-Xu; Wang, Li-Xia; Tian, Li-Guang; Zhang, Yu-Ping; Dong, Shuang-Pin; Hu, Xue-Guang; Liu, Jian; Wang, Feng-Feng; Wang, Yue; Yin, Xiao-Mei; He, Li-Jun; Yan, Qiu-Ye; Zhang, Hong-Wei; Xu, Bian-Li; Zhou, Xiao-Nong

    2014-01-01

    Epidemiologic studies of co-infection with tuberculosis (TB) and intestinal parasites in humans have not been extensively investigated in China. A cross-section study was conducted in a rural county of Henan Province, China. Pulmonary TB (PTB) case-patients receiving treatment for infection with Mycobacterium tuberculosis and healthy controls matched for geographic area, age, and sex were surveyed by using questionnaires. Fecal and blood specimens were collected for detection of intestinal parasites, routine blood examination, and infection with human immunodeficiency virus. The chi-square test was used for univariate analysis and multivariate logistic regression models were used to adjust for potential confounding factors. A total of 369 persons with PTB and 366 healthy controls were included; all participants were negative for human immunodeficiency virus. The overall prevalence of intestinal parasites in persons with PTB was 14.9%, including intestinal protozoa (7.9%) and helminthes (7.6%). The infection spectrum of intestinal parasites was Entamoeba spp. (1.4%), Blastocystis hominis (6.2%), Trichomonas hominis (0.3%), Clonorchis sinensis (0.3%), Ascaris lumbricoides (0.5%), Trichuris trichiura (2.2%), and hookworm (4.6%). The prevalence of intestinal parasites showed no significant difference between persons with PTB and healthy controls after adjusting for potential confounding factors. There was no factor that affected infection rates for intestinal parasites between the two groups. Infection with intestinal parasites of persons with PTB was associated with female sex (adjusted odds ratio [AOR] = 2.05, 95% confidence interval [CI] = 1.01-4.17), body mass index ≤ 19 (AOR = 3.02, 95% CI = 1.47-6.20), and anemia (AOR = 2.43, 95% CI = 1.17-5.03). Infection of healthy controls was only associated with an annual labor time in farmlands > 2 months (AOR = 4.50, 95% CI = 2.03-10.00). In addition, there was no significant trend between rates of infection with

  3. In vitro and in vivo small intestinal metabolism of CYP3A and UGT substrates in preclinical animals species and humans: species differences.

    PubMed

    Komura, Hiroshi; Iwaki, Masahiro

    2011-11-01

    Intestinal first-pass metabolism has a great impact on the bioavailability of cytochrome P450 3A4 (CYP3A) and/or uridine 5'-diphosphate (UDP)-glucoronosyltranferase (UGT) substrates in humans. In vitro and in vivo intestinal metabolism studies are essential for clarifying pharmacokinetics in animal species and for predicting the effects of human intestinal metabolism. We review species differences in intestinal metabolism both in vitro and in vivo. Based on mRNA expression levels, the major intestinal CYP3A isoform is CYP3A4 for humans, CYP3A4 (3A8) for monkeys, CYP3A9 for rats, cyp3a13 for mice, and CYP3A12 for dogs. Additionally, the intestinal-specific UGT would be UGT1A10 for humans, UGT1A8 for monkeys, and UGT1A7 for rats. In vitro and in vivo intestinal metabolism of CYP3A substrates were larger in monkeys than in humans, although a correlation in intestinal availability between monkeys and humans has been reported. Little information is available regarding species differences in in vitro and in vivo UGT activities; however, UGT-mediated in vivo intestinal metabolism has been demonstrated for raloxifene in humans and for baicalein in rats. Further assessment of intestinal metabolism, particularly for UGT substrates, is required to clarify the entire picture of species differences.

  4. High-speed fiber based polarization-sensitive optical coherence tomography of in vivo human skin.

    PubMed

    Saxer, C E; de Boer, J F; Park, B H; Zhao, Y; Chen, Z; Nelson, J S

    2000-09-15

    A high-speed single-mode fiber-based polarization-sensitive optical coherence tomography (PS OCT) system was developed. With a polarization modulator, Stokes parameters of reflected flight for four input polarization states are measured as a function of depth. A phase modulator in the reference arm of a Michelson interferometer permits independent control of the axial scan rate and carrier frequency. In vivo PS OCT images of human skin are presented, showing subsurface structures that are not discernible in conventional OCT images. A phase retardation image in tissue is calculated based on the reflected Stokes parameters of the four input polarization states.

  5. Intestinal Epithelial Cell Regulation of Adaptive Immune Dysfunction in Human Type 1 Diabetes

    PubMed Central

    Graves, Christina L.; Li, Jian; LaPato, Melissa; Shapiro, Melanie R.; Glover, Sarah C.; Wallet, Mark A.; Wallet, Shannon M.

    2017-01-01

    Environmental factors contribute to the initiation, progression, and maintenance of type 1 diabetes (T1D), although a single environmental trigger for disease has not been identified. Studies have documented the contribution of immunity within the gastrointestinal tract (GI) to the expression of autoimmunity at distal sites. Intestinal epithelial cells (IECs) regulate local and systemic immunologic homeostasis through physical and biochemical interactions with innate and adaptive immune populations. We hypothesize that a loss in the tolerance-inducing nature of the GI tract occurs within T1D and is due to altered IECs’ innate immune function. As a first step in addressing this hypothesis, we contrasted the global immune microenvironment within the GI tract of individuals with T1D as well as evaluated the IEC-specific effects on adaptive immune cell phenotypes. The soluble and cellular immune microenvironment within the duodenum, the soluble mediator profile of primary IECs derived from the same duodenal tissues, and the effect of the primary IECs’ soluble mediator profile on T-cell expansion and polarization were evaluated. Higher levels of IL-17C and beta-defensin 2 (BD-2) mRNA in the T1D-duodenum were observed. Higher frequencies of type 1 innate lymphoid cells (ILC1) and CD8+CXCR3+ T-cells (Tc1) were also observed in T1D-duodenal tissues, concomitant with lower frequencies of type 3 ILC (ILC3) and CD8+CCR6+ T-cells (Tc17). Higher levels of proinflammatory mediators (IL-17C and BD-2) in the absence of similar changes in mediators associated with homeostasis (interleukin 10 and thymic stromal lymphopoietin) were also observed in T1D-derived primary IEC cultures. T1D-derived IEC culture supernatants induced more robust CD8+ T-cell proliferation along with enhanced polarization of Tc1 populations, at the expense of Tc17 polarization, as well as the expansion of CXCR3+CCR6+/− Tregs, indicative of a Th1-like and less regulatory phenotype. These data demonstrate

  6. Failure of d-psicose absorbed in the small intestine to metabolize into energy and its low large intestinal fermentability in humans.

    PubMed

    Iida, Tetsuo; Hayashi, Noriko; Yamada, Takako; Yoshikawa, Yuko; Miyazato, Shoko; Kishimoto, Yuka; Okuma, Kazuhiro; Tokuda, Masaaki; Izumori, Ken

    2010-02-01

    Experiments with rats have produced data on the metabolism and energy value of d-psicose; however, no such data have been obtained in humans. The authors assessed the availability of d-psicose absorbed in the small intestine by measuring carbohydrate energy expenditure (CEE) by indirect calorimetry. They measured the urinary excretion rate by quantifying d-psicose in urine for 48 hours. To examine d-psicose fermentation in the large intestine, the authors measured breath hydrogen gas and fermentability using 35 strains of intestinal bacteria. Six healthy subjects participated in the CEE test, and 14 participated in breath hydrogen gas and urine tests. d-Psicose fermentation subsequent to an 8-week adaptation period was also assessed by measuring hydrogen gas in 8 subjects. d-Psicose absorbed in the small intestine was not metabolized into energy, unlike glucose, because CEE did not increase within 3 hours of d-psicose ingestion (0.35 g/kg body weight [BW]). The accumulated d-psicose urinary excretion rates were around 70% for 0.34, 0.17, and 0.08 g/kg BW of ingested d-psicose. Low d-psicose fermentability was observed in intestinal bacteria and breath hydrogen gas tests, in which fructooligosaccharide (0.34, 0.17, and 0.08 g/kg BW) was used as a positive control because its available energy is known to be 8.4 kJ/g. Based on the results of the plot of breath hydrogen concentration vs calories ingested, the energy value of d-psicose was expected to be less than 1.6 kJ/g. Incremental d-psicose fermentability subsequent to an adaptation period was not observed.

  7. Effects of transgalactosylated disaccharides on the human intestinal microflora and their metabolism.

    PubMed

    Ito, M; Kimura, M; Deguchi, Y; Miyamori-Watabe, A; Yajima, T; Kan, T

    1993-06-01

    The effects of transgalactosylated disaccharide (TD) intake on human fecal microflora and their metabolism were investigated in 12 Japanese males. TD is a mixture of sugars, galactosyl galactose, and galactosyl glucose, synthesized from lactose through the transgalactosylation reaction of Streptococcus thermophilus beta-galactosidase. Volunteers took 15 g of the test sugar daily for 6 days. The TD ingestion increased the number of bifidobacteria and lactobacilli, but decreased the number of Bacteroidaceae and Candida spp. in the feces. The ratio of bifidobacteria to total bacteria increased from 0.28 to 0.51. TD decreased the fecal concentrations of propionic acid, isobutyric acid, isovaleric acid, and valeric acid. This sugar also lowered the fecal pH, and the concentrations of fecal ammonia, p-cresol, and indole. Moreover, a positive correlation was found between the concentration of ammonia, and that of branched-chain fatty acids (isobutyric acid and isovaleric acid), p-cresol, and indole. All of these compounds are produced from amino acids through deamination by the intestinal bacteria. The depression of amino acid fermentation by intestinal bacteria may be involved in the reduction of fecal ammonia. These results suggest that a part of the transgalactosylated disaccharides passes into the colon, inducing changes in the colonic microflora composition, hastening carbohydrate fermentation, and depressing amino acid fermentation in the human gut.

  8. Transport and function of syntaxin 3 in human epithelial intestinal cells.

    PubMed

    Breuza, L; Fransen, J; Le Bivic, A

    2000-10-01

    To follow the transport of human syntaxin (Syn) 3 to the apical surface of intestinal cells, we produced and expressed in Caco-2 cells a chimera made of the entire Syn3 coding sequence and the extracellular domain of the human transferrin receptor (TfR). This chimera (Syn3TfR) was localized to the apical membrane and was transported along the direct apical pathway, suggesting that this is also the case for endogenous Syn3. To test the potential role of Syn3 in apical transport, we overexpressed it in Caco-2 cells and measured the efficiency of apical and basolateral delivery of several endogenous markers. We observed a strong inhibition of apical delivery of sucrase-isomaltase (SI), an apical transmembrane protein, and of alpha-glucosidase, an apically secreted protein. No effect was observed on the basolateral delivery of Ag525, a basolateral antigen, strongly suggesting that Syn3 is necessary for efficient delivery of proteins to the apical surface of intestinal cells.

  9. Cockroaches as carriers of human intestinal parasites in two localities in Ethiopia.

    PubMed

    Kinfu, Addisu; Erko, Berhanu

    2008-11-01

    A study was undertaken to assess the role of cockroaches as potential carriers of human intestinal parasites in Addis Ababa and Ziway, Ethiopia. A total of 6480 cockroaches were trapped from the two localities from October 2006 to March 2007. All the cockroaches trapped in Addis Ababa (n=2240) and almost 50% (2100/4240) of those trapped in Ziway were identified as Blattella germanica. The rest of the cockroaches trapped in Ziway were identified as Periplaneta brunnea (24.52%), Pycnoscelus surinamensis (16.03%) and Supella longipalpa (9.90%). Microscopic examination of the external body washes of pooled cockroaches and individual gut contents revealed that cockroaches are carriers of Entamoeba coli and Entamoeba histolytica/dispar cysts as well as Enterobius vermicularis, Trichuris trichiura, Taenia spp. and Ascaris lumbricoides ova. Besides their role as a nuisance, the present study further confirms that cockroaches serve as carriers of human intestinal parasites. The possible association of cockroaches with allergic conditions such as asthma is also discussed. Hence, appropriate control measures should be taken particularly to make hotels and residential areas free of cockroaches as they represent a health risk.

  10. Polarization sensitive optical coherence tomography in the human eye

    PubMed Central

    Pircher, Michael; Hitzenberger, Christoph K.; Schmidt-Erfurth, Ursula

    2011-01-01

    Optical coherence tomography (OCT) has become a well established imaging tool in ophthalmology. The unprecedented depth resolution that is provided by this technique yields valuable information on different ocular tissues ranging from the anterior to the posterior eye segment. Polarization sensitive OCT (PS-OCT) extends the concept of OCT and utilizes the information that is carried by polarized light to obtain additional information on the tissue. Several structures in the eye (e.g. cornea, retinal nerve fiber layer, retinal pigment epithelium) alter the polarization state of the light and show therefore a tissue specific contrast in PS-OCT images. First this review outlines the basic concepts of polarization changing light–tissue interactions and gives a short introduction in PS-OCT instruments for ophthalmic imaging. In a second part a variety of different applications of this technique are presented in ocular imaging that are ranging from the anterior to the posterior eye segment. Finally the benefits of the method for imaging different diseases as, e.g., age related macula degeneration (AMD) or glaucoma is demonstrated. PMID:21729763

  11. The Intestinal Transport of Bovine Milk Exosomes Is Mediated by Endocytosis in Human Colon Carcinoma Caco-2 Cells and Rat Small Intestinal IEC-6 Cells123

    PubMed Central

    Wolf, Tovah; Baier, Scott R; Zempleni, Janos

    2015-01-01

    Background: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs in tissues and body fluids is encapsulated in exosomes, thereby conferring protection against degradation and a pathway for intestinal transport. MicroRNAs in cow milk are bioavailable in humans. Objective: This research assessed the transport mechanism of bovine milk exosomes, and therefore microRNAs, in human and rodent intestinal cells. Methods: The intestinal transport of bovine milk exosomes and microRNAs was assessed using fluorophore-labeled bovine milk exosomes in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. Transport kinetics and mechanisms were characterized using dose-response studies, inhibitors of vesicle transport, carbohydrate competitors, proteolysis of surface proteins on cells and exosomes, and transepithelial transport in transwell plates. Results: Exosome transport exhibited saturation kinetics at 37°C [Michaelis constant (Km) = 55.5 ± 48.6 μg exosomal protein/200 μL of media; maximal transport rate = 0.083 ± 0.057 ng of exosomal protein · 81,750 cells−1 · h−1] and decreased by 64% when transport was measured at 4°C, consistent with carrier-mediated transport in Caco-2 cells. Exosome uptake decreased by 61–85% under the following conditions compared with controls in Caco-2 cells: removal of exosome and cell surface proteins by proteinase K, inhibition of endocytosis and vesicle trafficking by synthetic inhibitors, and inhibition of glycoprotein binding by carbohydrate competitors. When milk exosomes, at a concentration of 5 times the Km, were added to the upper chamber in transwell plates, Caco-2 cells accumulated miR-29b and miR-200c in the lower chamber, and reverse transport was minor. Transport characteristics were similar in IEC-6 cells and Caco-2 cells, except that substrate affinity and transporter capacity were lower and higher, respectively. Conclusion: The uptake of bovine milk exosomes is

  12. Binding of diarrheagenic Escherichia coli to 32- to 33-kilodalton human intestinal brush border proteins.

    PubMed Central

    Manjarrez-Hernandez, A; Gavilanes-Parra, S; Chavez-Berrocal, M E; Molina-Lopez, J; Cravioto, A

    1997-01-01

    We have detected human intestinal brush border proteins to which Escherichia coli strains adhere by means of a blotting-nitrocellulose method in which the binding of radiolabeled bacteria to sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated intestinal cell membranes was evaluated. The brush border fraction contained several polypeptides that bound only adherent E. coli strains. The most prominent and consistent of these proteins had apparent molecular masses of 32 to 33 kDa. Additional polypeptides ranging from 50 to 70, from 105 to 130, and from 180 to 200 kDa were also recognized by adherent E. coli strains, although with less intensity (in accordance with the number of bound bacteria to these polypeptides). Independently of the pattern of adherence (localized [LA], diffuse [DA], or aggregative [AggA]) all HEp-2-adhering strains recognized, with different intensities, the 32- to 33-kDa brush border proteins, whereas nonadhesive strains did not. The relative avidity of an LA strain to bind to the 32- to 33-kDa proteins was approximately seven- and sixfold higher than the binding of strains with aggregative and diffuse adherence, respectively. Thus, it is reasonable to think that LA, DA, and AggA strains have a common adhesin that mediates binding to the 32- to 33-kDa bands. Inhibition experiments using HEp-2 cells demonstrated that isolated 32- to 33-kDa proteins or specific antiserum blocked preferentially bacterial adherence of the LA pattern. Delipidization and protein digestion of the human brush borders confirmed that E. coli bound to structures of a proteinaceous nature. Deglycosylation studies and sodium meta-periodate oxidation of the intestinal cell membranes decreased bacterial binding activity significantly, indicating that E. coli bound to carbohydrate moieties in the glycoproteins. These results suggest that binding of E. coli strains, mainly of the LA phenotype, to the 32- to 33-kDa proteins could play a role in colonization through

  13. Doxycycline protects human intestinal cells from hypoxia/reoxygenation injury: Implications from an in-vitro hypoxia model.

    PubMed

    Hummitzsch, Lars; Zitta, Karina; Berndt, Rouven; Kott, Matthias; Schildhauer, Christin; Parczany, Kerstin; Steinfath, Markus; Albrecht, Martin

    2017-04-15

    Intestinal ischemia/reperfusion (I/R) injury is a grave clinical emergency and associated with high morbidity and mortality rates. Based on the complex underlying mechanisms, a multimodal pharmacological approach seems necessary to prevent intestinal I/R injury. The antibiotic drug doxycycline, which exhibits a wide range of pleiotropic therapeutic properties, might be a promising candidate for also reducing I/R injury in the intestine. To investigate possible protective effects of doxycycline on intestinal I/R injury, human intestinal CaCo-2 cells were exposed to doxycycline at clinically relevant concentrations. In order to mimic I/R injury, CaCo-2 were thereafter subjected to hypoxia/reoxygenation by using our recently described two-enzyme in-vitro hypoxia model. Investigations of cell morphology, cell damage, apoptosis and hydrogen peroxide formation were performed 24h after the hypoxic insult. Hypoxia/reoxygenation injury resulted in morphological signs of cell damage, elevated LDH concentrations in the respective culture media (P<0.001) and increased protein expression of proapoptotic caspase-3 (P<0.05) in the intestinal cultures. These events were associated with increased levels hydrogen peroxide (P<0.001). Preincubation of CaCo-2 cells with different concentrations of doxycycline (5µM, 10µM, 50µM) reduced the hypoxia induced signs of cell damage and LDH release (P<0.001 for all concentrations). The reduction of cellular damage was associated with a reduced expression of caspase-3 (5µM, P<0.01; 10µM, P<0.01; 50µM, P<0.05), while hydrogen peroxide levels remained unchanged. In summary, doxycycline protects human intestinal cells from hypoxia/reoxygenation injury in-vitro. Further animal and clinical studies are required to prove the protective potential of doxycycline on intestinal I/R injury under in-vivo conditions.

  14. Dysfunctions at human intestinal barrier by water-borne protozoan parasites: lessons from cultured human fully differentiated colon cancer cell lines.

    PubMed

    Liévin-Le Moal, Vanessa

    2013-06-01

    Some water-borne protozoan parasites induce diseases through their membrane-associated functional structures and virulence factors that hijack the host cellular molecules and signalling pathways leading to structural and functional lesions in the intestinal barrier. In this Microreview we analyse the insights on the mechanisms of pathogenesis of Entamoeba intestinalis, Giardia and Cryptosporidium observed in the human colon carcinoma fully differentiated colon cancer cell lines, cell subpopulations and clones expressing the structural and functional characteristics of highly specialized fully differentiated epithelial cells lining the intestinal epithelium and mimicking structurally and functionally an intestinal barrier.

  15. Free fucose is a danger signal to human intestinal epithelial cells.

    PubMed

    Chow, Wai Ling; Lee, Yuan Kun

    2008-03-01

    Fucose is present in foods, and it is a major component of human mucin glycoproteins and glycolipids. l-Fucose can also be found at the terminal position of many cell-surface oligosaccharide ligands that mediate cell-recognition and adhesion-signalling pathways. Mucin fucose can be released through the hydrolytic activity of pathogens and indigenous bacteria, leading to the release of free fucose into the intestinal lumen. The immunomodulating effects of free fucose on intestinal epithelial cells (enterocyte-like Caco-2) were investigated. It was found that the presence of l-fucose up regulated genes and secretion of their encoded proteins that are involved in both the innate and adaptive immune responses, possibly via the toll-like receptor-2 signalling pathway. These include TNFSF5, TNFSF7, TNF-alpha, IL12, IL17 and IL18. Besides modulating immune reactions in differentiated Caco-2 cells, fucose induced a set of cytokine genes that are involved in the development and proliferation of immune cells. These include the bone morphogenetic proteins (BMP) BMP2, BMP4, IL5, thrombopoietin and erythropoietin. In addition, the up regulated gene expression of fibroblast growth factor-2 may help to promote epithelial cell restitution in conjunction with the enhanced expression of transforming growth factor-beta mRNA. Since the exogenous fucose was not metabolised by the differentiated Caco-2 cells as a carbon source, the reactions elicited were suggested to be a result of the direct interaction of fucose and differentiated Caco-2 cells. The presence of free fucose may signal the invasion of mucin-hydrolysing microbial cells and breakage of the mucosal barrier. The intestinal epithelial cells respond by up regulation and secretion of cytokines, pre-empting the actual invasion of pathogens.

  16. Associations between common intestinal parasites and bacteria in humans as revealed by qPCR.

    PubMed

    O'Brien Andersen, L; Karim, A B; Roager, H M; Vigsnæs, L K; Krogfelt, K A; Licht, T R; Stensvold, C R

    2016-09-01

    Several studies have shown associations between groups of intestinal bacterial or specific ratios between bacterial groups and various disease traits. Meanwhile, little is known about interactions and associations between eukaryotic and prokaryotic microorganisms in the human gut. In this work, we set out to investigate potential associations between common single-celled parasites such as Blastocystis spp. and Dientamoeba fragilis and intestinal bacteria. Stool DNA from patients with intestinal symptoms were selected based on being Blastocystis spp.-positive (B+)/negative (B-) and D. fragilis-positive (D+)/negative (D-), and split into four groups of 21 samples (B+ D+, B+ D-, B- D+, and B- D-). Quantitative PCR targeting the six bacterial taxa Bacteroides, Prevotella, the butyrate-producing clostridial clusters IV and XIVa, the mucin-degrading Akkermansia muciniphila, and the indigenous group of Bifidobacterium was subsequently performed, and the relative abundance of these bacteria across the four groups was compared. The relative abundance of Bacteroides in B- D- samples was significantly higher compared with B+ D- and B+ D+ samples (P < 0.05 and P < 0.01, respectively), and this association was even more significant when comparing all parasite-positive samples with parasite-negative samples (P < 0.001). Additionally, our data revealed that a low abundance of Prevotella and a higher abundance of Clostridial cluster XIVa was associated with parasite-negative samples (P < 0.05 and P < 0.01, respectively). Our data support the theory that Blastocystis alone or combined with D. fragilis is associated with gut microbiota characterized by low relative abundances of Bacteroides and Clostridial cluster XIVa and high levels of Prevotella.

  17. PGE2 is a direct and robust mediator of anion/fluid secretion by human intestinal epithelial cells

    PubMed Central

    Fujii, Satoru; Suzuki, Kohei; Kawamoto, Ami; Ishibashi, Fumiaki; Nakata, Toru; Murano, Tatsuro; Ito, Go; Shimizu, Hiromichi; Mizutani, Tomohiro; Oshima, Shigeru; Tsuchiya, Kiichiro; Nakamura, Tetsuya; Araki, Akihiro; Ohtsuka, Kazuo; Okamoto, Ryuichi; Watanabe, Mamoru

    2016-01-01

    Intestinal epithelial cells (IECs) play an indispensable role in maintaining body fluid balance partly through their ability to regulate anion/fluid secretion. Yet in various inflammatory gastrointestinal diseases, over-secretion of anions results in symptoms such as severe diarrhoea. Endogenous mediators, such as vasoactive intestinal peptide or prostaglandin E2 (PGE2), regulate intestinal anion/fluid secretion, but their direct effect on purified human IECs has never been described in detail. Based on a previously described intestinal organoid swelling model, we established a 3D-scanner-assisted quantification method to evaluate the anion/fluid secretory response of cultured human IECs. Among various endogenous secretagogues, we found that PGE2 had the lowest EC50 value with regard to the induction of swelling of the jejunal and colonic organoids. This PGE2-mediated swelling response was dependent on environmental Cl− concentrations as well as on several channels and transporters as shown by a series of chemical inhibitor studies. The concomitant presence of various inflammatory cytokines with PGE2 failed to modulate the PGE2-mediated organoid swelling response. Therefore, the present study features PGE2 as a direct and robust mediator of anion/fluid secretion by IECs in the human intestine. PMID:27827428

  18. Intestinal microbiology in early life: specific prebiotics can have similar functionalities as human-milk oligosaccharides.

    PubMed

    Oozeer, Raish; van Limpt, Kees; Ludwig, Thomas; Ben Amor, Kaouther; Martin, Rocio; Wind, Richèle D; Boehm, Günther; Knol, Jan

    2013-08-01

    Human milk is generally accepted as the best nutrition for newborns and has been shown to support the optimal growth and development of infants. On the basis of scientific insights from human-milk research, a specific mixture of nondigestible oligosaccharides has been developed, with the aim to improve the intestinal microbiota in early life. The mixture has been extensively studied and has been shown to be safe and to have potential health benefits that are similar to those of human milk. The specific mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides has been found to affect the development of early microbiota and to increase the Bifidobacterium amounts as observed in human-milk-fed infants. The resulting gut ecophysiology is characterized by high concentrations of lactate, a slightly acidic pH, and specific short-chain fatty acid profiles, which are high in acetate and low in butyrate and propionate. Here, we have summarized the main findings of dietary interventions with these specific oligosaccharides on the gut microbiota in early life. The gut ecophysiology in early life may have consequences for the metabolic, immunologic, and even neurologic development of the child because reports increasingly substantiate the important function of gut microbes in human health. This review highlights major findings in the field of early gut colonization and the potential impact of early nutrition in healthy growth and development.

  19. SREBP-2 negatively regulates FXR-dependent transcription of FGF19 in human intestinal cells.

    PubMed

    Miyata, Masaaki; Hata, Tatsuya; Yamazoe, Yasushi; Yoshinari, Kouichi

    2014-01-10

    Sterol regulatory element-binding protein-2 (SREBP-2) is a basic helix-loop-helix-leucine zipper transcription factor that positively regulates transcription of target genes involved in cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in human intestinal expression of fibroblast growth factor (FGF)19, which is an endocrine hormone involved in the regulation of lipid and glucose metabolism. Overexpression of constitutively active SREBP-2 decreased FGF19 mRNA levels in human colon-derived LS174T cells. In reporter assays, active SREBP-2 overexpression suppressed GW4064/FXR-mediated increase in reporter activities in regions containing the IR-1 motif (+848 to +5200) in the FGF19 gene. The suppressive effect disappeared in reporter activities in the region containing the IR-1 motif when the mutation was introduced into the IR-1 motif. In electrophoretic mobility shift assays, binding of the FXR/retinoid X receptor α heterodimer to the IR-1 motif was attenuated by adding active SREBP-2, but SREBP-2 binding to the IR-1 motif was not observed. In chromatin immunoprecipitation assays, specific binding of FXR to the IR-1-containing region of the FGF19 gene (+3214 to +3404) was increased in LS174T cells by treatment with cholesterol and 25-hydroxycholesterol. Specific binding of SREBP-2 to FXR was observed in glutathione-S-transferase (GST) pull-down assays. These results suggest that SREBP-2 negatively regulates the FXR-mediated transcriptional activation of the FGF19 gene in human intestinal cells.

  20. Intestinal Short Chain Fatty Acids and their Link with Diet and Human Health

    PubMed Central

    Ríos-Covián, David; Ruas-Madiedo, Patricia; Margolles, Abelardo; Gueimonde, Miguel; de los Reyes-Gavilán, Clara G.; Salazar, Nuria

    2016-01-01

    The colon is inhabited by a dense population of microorganisms, the so-called “gut microbiota,” able to ferment carbohydrates and proteins that escape absorption in the small intestine during digestion. This microbiota produces a wide range of metabolites, including short chain fatty acids (SCFA). These compounds are absorbed in the large bowel and are defined as 1-6 carbon volatile fatty acids which can present straight or branched-chain conformation. Their production is influenced by the pattern of food intake and diet-mediated changes in the gut microbiota. SCFA have distinct physiological effects: they contribute to shaping the gut environment, influence the physiology of the colon, they can be used as energy sources by host cells and the intestinal microbiota and they also participate in different host-signaling mechanisms. We summarize the current knowledge about the production of SCFA, including bacterial cross-feedings interactions, and the biological properties of these metabolites with impact on the human health. PMID:26925050

  1. Supersaturation and Precipitation of Posaconazole Upon Entry in the Upper Small Intestine in Humans.

    PubMed

    Hens, Bart; Brouwers, Joachim; Corsetti, Maura; Augustijns, Patrick

    2016-09-01

    The purpose of this study was to explore gastrointestinal dissolution, supersaturation and precipitation of the weakly basic drug posaconazole in humans, and to assess the impact of formulation pH and type on these processes. In a cross-over study, two posaconazole suspensions (40 mg dispersed in 240 mL water at pH 1.6 and pH 7.1, respectively) were intragastrically administered; subsequently, gastric and duodenal fluids were aspirated. In parallel, blood samples were collected. Additionally, posaconazole was intragastrically administered as a solution (20 mg in 240 mL water, pH 1.6). When posaconazole was administered as an acidified suspension, supersaturated duodenal concentrations of posaconazole were observed for approximately 45 min. However, extensive intestinal precipitation was observed. Administration of the neutral suspension resulted in subsaturated concentrations with a mean duodenal AUC0-120 min and Cmax being approximately twofold lower than for the acidified suspension. The mean plasma AUC0-8 h of posaconazole was also twofold higher following administration of the acidified suspension. Similar to the acidified suspension, significant intestinal precipitation (up to 92%) was observed following intragastric administration of the posaconazole solution. This study demonstrated for the first time the gastrointestinal behavior of a weakly basic drug administered in different conditions, and its impact on systemic exposure.

  2. Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

    PubMed Central

    Bluett, M K; Abumrad, N N; Arab, N; Ghishan, F K

    1986-01-01

    D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose. PMID:3800877

  3. Stereomicroscopic 3D-pattern profiling of murine and human intestinal inflammation reveals unique structural phenotypes

    PubMed Central

    Rodriguez-Palacios, Alex; Kodani, Tomohiro; Kaydo, Lindsey; Pietropaoli, Davide; Corridoni, Daniele; Howell, Scott; Katz, Jeffry; Xin, Wei; Pizarro, Theresa T.; Cominelli, Fabio

    2015-01-01

    Histology is fundamental to assess two-dimensional intestinal inflammation; however, inflammatory bowel diseases (IBDs) are often indistinguishable microscopically on the basis of mucosal biopsies. Here, we use stereomicroscopy (SM) to rapidly profile the entire intestinal topography and assess inflammation. We examine the mucosal surface of >700 mice (encompassing >16 strains and various IBD-models), create a profiling catalogue of 3D-stereomicroscopic abnormalities and demonstrate that mice with comparable histological scores display unique sub-clusters of 3D-structure-patterns of IBD pathology, which we call 3D-stereoenterotypes, and which are otherwise indiscernible histologically. We show that two ileal IBD-stereoenterotypes (‘cobblestones' versus ‘villous mini-aggregation') cluster separately within two distinct mouse lines of spontaneous ileitis, suggesting that host genetics drive unique and divergent inflammatory 3D-structural patterns in the gut. In humans, stereomicroscopy reveals ‘liquefaction' lesions and hierarchical fistulous complexes, enriched with clostridia/segmented filamentous bacteria, running under healthy mucosa in Crohn's disease. We suggest that stereomicroscopic (3D-SMAPgut) profiling can be easily implemented and enable the comprehensive study of inflammatory 3D structures, genetics and flora in IBD. PMID:26154811

  4. Human intestinal Vdelta1+ lymphocytes recognize tumor cells of epithelial origin

    PubMed Central

    1996-01-01

    gammadelta T cells can be grouped into discrete subsets based upon their expression of T cell receptor (TCR) variable (V) region families, their tissue distribution, and their specificity. Vdelta2+ T cells constitute the majority of gammadelta T cells in peripheral blood whereas Vdelta1+T cells reside preferentially in skin epithelium and in the intestine. gammadelta T cells are envisioned as first line host defense mechanisms capable of providing a source of immune effector T cells and immunomodulating cytokines such as interleukin (IL) 4 or interferon (IFN) gamma. We describe here the fine specificity of three distinct gammadelta+ tumor-infiltrating lymphocytes (TIL) obtained from patients with primary or metastatic colorectal cancer, that could be readily expanded in vitro in the presence of IL-1beta and IL-7. Irrespective of donor, these individual gammadelta T cells exhibited a similar pattern of reactivity defined by recognition of autologous and allogeneic colorectal cancer cells, renal cell cancer, pancreatic cancer, and a freshly isolated explant from human intestine as measured by cytolytic T cell responses and by IFN-gamma release. In contrast, tumors of alternate histologies were not lysed, including lung cancer, squamous cell cancer, as well as the natural/lymphocyte-activated killer cell-sensitive hematopoietic cell lines T2, C1R, or Daudi. The cell line K562 was only poorly lysed when compared with colorectal cancer targets. Target cell reactivity mediated by Vdelta1+ T cells was partially blocked with Abs directed against the TCR, the beta2 or beta7 integrin chains, or fibronectin receptor. Marker analysis using flow cytometry revealed that all three gammadelta T cell lines exhibit a similar phenotype. Analysis of the gammadelta TCR junctional suggested exclusive usage of the Vdelta1/Ddelta3/Jdelta1 TCR segments with extensive (< or = 29 bp) N/P region diversity. T cell recognition of target cells did not appear to be a major histocompatibility

  5. Transcobalamin derived from bovine milk stimulates apical uptake of vitamin B12 into human intestinal epithelial cells.

    PubMed

    Hine, Brad; Boggs, Irina; Green, Ralph; Miller, Joshua W; Hovey, Russell C; Humphrey, Rex; Wheeler, Thomas T

    2014-11-01

    Intestinal uptake of vitamin B12 (hereafter B12) is impaired in a significant proportion of the human population. This impairment is due to inherited or acquired defects in the expression or function of proteins involved in the binding of diet-derived B12 and its uptake into intestinal cells. Bovine milk is an abundant source of bioavailable B12 wherein it is complexed with transcobalamin. In humans, transcobalamin functions primarily as a circulatory protein, which binds B12 following its absorption and delivers it to peripheral tissues via its cognate receptor, CD320. In the current study, the transcobalamin-B12 complex was purified from cows' milk and its ability to stimulate uptake of B12 into cultured bovine, mouse and human cell lines was assessed. Bovine milk-derived transcobalamin-B12 complex was absorbed by all cell types tested, suggesting that the uptake mechanism is conserved across species. Furthermore, the complex stimulated the uptake of B12 via the apical surface of differentiated Caco-2 human intestinal epithelial cells. These findings suggest the presence of an alternative transcobalamin-mediated uptake pathway for B12 in the human intestine other than that mediated by the gastric glycoprotein, intrinsic factor. Our findings highlight the potential for transcobalamin-B12 complex derived from bovine milk to be used as a natural bioavailable alternative to orally administered free B12 to overcome B12 malabsorption.

  6. Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression

    PubMed Central

    Gong, Xiaoming; Tsai, Shu-Whei; Yan, Bingfang; Rubin, Lewis P

    2006-01-01

    Background Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1) catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression. Results We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARγ bind to their respective DNA elements and synergistically transactivate BCMO1 expression. Conclusion We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARγ and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene. PMID:16504037

  7. An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.

    PubMed

    Li, Hong; Jin, Hyo-Eon; Shim, Won-Sik; Shim, Chang-Koo

    2013-10-01

    The Biopharmaceutics Classification System (BCS) was developed to facilitate estimation of the in vivo pharmacokinetic performance of drugs from human intestinal permeability and solubility. However, the measurement of human in vivo intestinal permeability, unlike that of solubility, is problematic and inefficient. Thus, rat in vitro intestinal permeability results obtained via the Ussing chamber technique are often used instead. However, these data could be unreliable due to difficulty in maintaining the viability of the dissected intestinal membrane in the Ussing chamber. Therefore, a more efficient method to obtain a reliable in vitro permeability is mandatory. Here, we propose a new approach by introducing a novel factor called the permeability ratio (PR). Basically, PR is a rat in vitro intestinal permeability obtained from the Ussing chamber, which is then corrected by the permeability of lucifer yellow, a paracellular permeability marker. To prove the validity of the method, 12 model drugs representing different BCS classes were tested, and the correlation with human in vivo intestinal permeability was high. More importantly, the new method perfectly classified all 12 model drugs. The results indicate that PR is a reliable factor with high correlation to human in vivo intestinal permeability, which can further be used to accurately predict the BCS classification.

  8. Delphinidin Reduces Glucose Uptake in Mice Jejunal Tissue and Human Intestinal Cells Lines through FFA1/GPR40.

    PubMed

    Hidalgo, Jorge; Teuber, Stefanie; Morera, Francisco J; Ojeda, Camila; Flores, Carlos A; Hidalgo, María A; Núñez, Lucía; Villalobos, Carlos; Burgos, Rafael A

    2017-04-05

    Anthocyanins are pigments with antihyperglycemic properties, and they are potential candidates for developing functional foods for the therapy or prevention of Diabetes mellitus type 2 (DM2). The mechanism of these beneficial effects of anthocyanins are, however, hard to explain, given their very low bioavailability due to poor intestinal absorption. We propose that free fatty acid receptor 1 (FFA1, also named GPR40), is involved in an inhibitory effect of the anthocyanidin delphinidin over intestinal glucose absorption. We show the direct effects of delphinidin on the intestine using jejunum samples from RF/J mice, and the human intestinal cell lines HT-29, Caco-2, and NCM460. By the use of specific pharmacological antagonists, we determined that delphinidin inhibits glucose absorption in both mouse jejunum and a human enterocytic cell line in a FFA1-dependent manner. Delphinidin also affects the function of sodium-glucose cotransporter 1 (SGLT1). Intracellular signaling after FFA1 activation involved cAMP increase and cytosolic Ca(2+) oscillations originated from intracellular Ca(2+) stores and were followed by store-operated Ca(2+) entry. Taken together, our results suggest a new GPR-40 mediated local mechanism of action for delphinidin over intestinal cells that may in part explain its antidiabetic effect. These findings are promising for the search for new prevention and pharmacological treatment strategies for DM2 management.

  9. Bifidobacteria Prevent Tunicamycin-Induced Endoplasmic Reticulum Stress and Subsequent Barrier Disruption in Human Intestinal Epithelial Caco-2 Monolayers

    PubMed Central

    Akiyama, Takuya; Oishi, Kenji

    2016-01-01

    Endoplasmic reticulum (ER) stress is caused by accumulation of unfolded and misfolded proteins in the ER, thereby compromising its vital cellular functions in protein production and secretion. Genome wide association studies in humans as well as experimental animal models linked ER stress in intestinal epithelial cells (IECs) with intestinal disorders including inflammatory bowel diseases. However, the mechanisms linking the outcomes of ER stress in IECs to intestinal disease have not been clarified. In this study, we investigated the impact of ER stress on intestinal epithelial barrier function using human colon carcinoma-derived Caco-2 monolayers. Tunicamycin-induced ER stress decreased the trans-epithelial electrical resistance of Caco-2 monolayers, concomitant with loss of cellular plasma membrane integrity. Epithelial barrier disruption in Caco-2 cells after ER stress was not caused by caspase- or RIPK1-dependent cell death but was accompanied by lysosomal rupture and up-regulation of the ER stress markers Grp78, sXBP1 and Chop. Interestingly, several bifidobacteria species inhibited tunicamycin-induced ER stress and thereby diminished barrier disruption in Caco-2 monolayers. Together, these results showed that ER stress compromises the epithelial barrier function of Caco-2 monolayers and demonstrate beneficial impacts of bifidobacteria on ER stress in IECs. Our results identify epithelial barrier loss as a potential link between ER stress and intestinal disease development, and suggest that bifidobacteria could exert beneficial effects on this phenomenon. PMID:27611782

  10. Effect of variations in the amounts of P-glycoprotein (ABCB1), BCRP (ABCG2) and CYP3A4 along the human small intestine on PBPK models for predicting intestinal first pass.

    PubMed

    Bruyère, Arnaud; Declèves, Xavier; Bouzom, Francois; Ball, Kathryn; Marques, Catie; Treton, Xavier; Pocard, Marc; Valleur, Patrice; Bouhnik, Yoram; Panis, Yves; Scherrmann, Jean-Michel; Mouly, Stephane

    2010-10-04

    It is difficult to predict the first-pass effect in the human intestine due to a lack of scaling factors for correlating in vitro and in vivo data. We have quantified cytochrome P450/3A4 (CYP3A4) and two ABC transporters, P-glycoprotein (P-gp, ABCB1) and the breast cancer resistant protein BCRP (ABCG2), throughout the human small intestine to determine the scaling factors for predicting clearance from intestinal microsomes and develop a physiologically based pharmacokinetic (PBPK) model. CYP3A4, P-gp and BCRP proteins were quantified by Western blotting and/or enzyme activities in small intestine samples from 19 donors, and mathematical trends of these expressions with intestinal localization were established. Microsome fractions were prepared and used to calculate the amount of microsomal protein per gram of intestine (MPPGI). Our results showed a trend in CYP3A4 expression decrease from the upper to the lower small intestine while P-gp expression is increasing. In contrast, BCRP expression did not vary significantly with position, but varied greatly between individuals. The MPPGI (mg microsomal protein per centimeter intestine) remained constant along the length of the small intestine, at about 1.55 mg/cm. Moreover, intrinsic clearance measured with specific CYP3A4 substrates (midazolam and an in-house Servier drug) and intestinal microsomes was well correlated with the amount of CYP3A4 (R(2) > 0.91, p < 0.01). In vivo data were more accurately predicted using PBPK models of blood concentrations of these two substrates based on the segmental distributions of these enzymes and MPPGI determined in this study. Thus, these mathematical trends can be used to predict drug absorption at different intestinal sites and their metabolism can be predicted with the MPPGI.

  11. Vasoactive intestinal peptide (VIP) induces malignant transformation of the human prostate epithelial cell line RWPE-1.

    PubMed

    Fernández-Martínez, Ana B; Bajo, Ana M; Isabel Arenas, M; Sánchez-Chapado, Manuel; Prieto, Juan C; Carmena, María J

    2010-12-18

    The carcinogenic potential of vasoactive intestinal peptide (VIP) was analyzed in non-tumor human prostate epithelial cells (RWPE-1) and in vivo xenografts. VIP induced morphological changes and a migratory phenotype consistent with stimulation of expression/activity of metalloproteinases MMP-2 and MMP-9, decreased E-cadherin-mediated cell-cell adhesion, and increased cell motility. VIP increased cyclin D1 expression and cell proliferation that was blocked after VPAC(1)-receptor siRNA transfection. Similar effects were seen in RWPE-1 tumors developed by subcutaneous injection of VIP-treated cells in athymic nude mice. VIP acts as a cytokine in RWPE-1 cell transformation conceivably through epithelial-mesenchymal transition (EMT), reinforcing VIP role in prostate tumorigenesis.

  12. Automatic segmentation and classification of human intestinal parasites from microscopy images.

    PubMed

    Suzuki, Celso T N; Gomes, Jancarlo F; Falcão, Alexandre X; Papa, João P; Hoshino-Shimizu, Sumie

    2013-03-01

    Human intestinal parasites constitute a problem in most tropical countries, causing death or physical and mental disorders. Their diagnosis usually relies on the visual analysis of microscopy images, with error rates that may range from moderate to high. The problem has been addressed via computational image analysis, but only for a few species and images free of fecal impurities. In routine, fecal impurities are a real challenge for automatic image analysis. We have circumvented this problem by a method that can segment and classify, from bright field microscopy images with fecal impurities, the 15 most common species of protozoan cysts, helminth eggs, and larvae in Brazil. Our approach exploits ellipse matching and image foresting transform for image segmentation, multiple object descriptors and their optimum combination by genetic programming for object representation, and the optimum-path forest classifier for object recognition. The results indicate that our method is a promising approach toward the fully automation of the enteroparasitosis diagnosis.

  13. Human chorionic gonadotropin promotes expression of protein absorption factors in the intestine of goldfish (Carassius auratus).

    PubMed

    Zhou, Y; Hao, G; Zhong, H; Wu, Q; Lu, S Q; Zhao, Q; Liu, Z

    2015-07-27

    Protein use is crucial for the ovulation and spawning of fish. Currently, limited information is available regarding the expression of protein absorption factors during the breeding seasons of teleosts and thus how various proteins involved in this process is not well-understood. The expression of CDX2, CREB, gluatamate dehydrogenase, LAT2, aminopeptidase N, PepT1, and SP1 were significantly elevated from the non-breeding season to the breeding season in female goldfish, and all proteins except PepT1 and SP1 were elevated in male goldfish. Injection of human chorionic gonadotropin upregulated the expression of all proteins except for aminopeptidase N in female goldfish and SP1 in male goldfish, suggesting a luteinizing hormone-inductive effect on protein absorption factors. Protein use in the intestine is increased during the breeding seasons as a result of increased luteinizing hormone.

  14. Substrate specificity and some properties of phenol sulfotransferase from human intestinal Caco-2 cells

    SciTech Connect

    Baranczyk-Kuzma, A.; Garren, J.A.; Hidalgo, I.J.; Borchardt, R.T. )

    1991-01-01

    The phase 2 metabolic reactions, sulfation and glucuronidation, were studied in a human colon carcinoma cell line (Caco-2), which has been developed as a model of intestinal enterocytes. Phenol sulfotransferase was isolated from Caco-2 cells cultured for 7, 14 and 21 days. The enzyme catalyzed the sulfation of both p-nitrophenol and catecholamines as well as most catecholamine metabolites. The affinity (K{sub m}) of PST for dopamine was much higher than for p-nitrophenol, and the specific activity of PST with both substrates increased with the age of the cells. The thermal stability of Caco-2 PST increased with cell age and was not dependent on the acceptor substrate used. The thermolabile PST from 7-day old cells was more sensitive to NEM than was the thermostable enzyme from 21-day old cells. No UDP-glucuronyltransferase activity was detected in 7-, 14- and 21-day old Caco-2 cells with any of the methods used.

  15. Activation of Intestinal Human Pregnane X Receptor Protects against Azoxymethane/Dextran Sulfate Sodium–Induced Colon Cancer

    PubMed Central

    Cheng, Jie; Fang, Zhong-Ze; Nagaoka, Kenjiro; Okamoto, Minoru; Qu, Aijuan; Tanaka, Naoki; Kimura, Shioko

    2014-01-01

    The role of intestinal human pregnane X receptor (PXR) in colon cancer was determined through investigation of the chemopreventive role of rifaximin, a specific agonist of intestinal human PXR, toward azoxymethane (AOM)/dextran sulfate sodium (DSS)–induced colon cancer. Rifaximin treatment significantly decreased the number of colon tumors induced by AOM/DSS treatment in PXR-humanized mice, but not wild-type or Pxr-null mice. Additionally, rifaximin treatment markedly increased the survival rate of PXR-humanized mice, but not wild-type or Pxr-null mice. These data indicated a human PXR–dependent therapeutic chemoprevention of rifaximin toward AOM/DSS-induced colon cancer. Nuclear factor κ-light-chain-enhancer of activated B cells–mediated inflammatory signaling was upregulated in AOM/DSS-treated mice, and inhibited by rifaximin in PXR-humanized mice. Cell proliferation and apoptosis were also modulated by rifaximin treatment in the AOM/DSS model. In vitro cell-based assays further revealed that rifaximin regulated cell apoptosis and cell cycle in a human PXR-dependent manner. These results suggested that specific activation of intestinal human PXR exhibited a chemopreventive role toward AOM/DSS-induced colon cancer by mediating anti-inflammation, antiproliferation, and proapoptotic events. PMID:25277138

  16. Innervation of enteric mast cells by primary spinal afferents in guinea pig and human small intestine.

    PubMed

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Qu, Meihua; Xia, Yun; Needleman, Bradley J; Mikami, Dean J; Wood, Jackie D

    2014-10-01

    Mast cells express the substance P (SP) neurokinin 1 receptor and the calcitonin gene-related peptide (CGRP) receptor in guinea pig and human small intestine. Enzyme-linked immunoassay showed that activation of intramural afferents by antidromic electrical stimulation or by capsaicin released SP and CGRP from human and guinea pig intestinal segments. Electrical stimulation of the afferents evoked slow excitatory postsynaptic potentials (EPSPs) in the enteric nervous system. The slow EPSPs were mediated by tachykinin neurokinin 1 and CGRP receptors. Capsaicin evoked slow EPSP-like responses that were suppressed by antagonists for protease-activated receptor 2. Afferent stimulation evoked slow EPSP-like excitation that was suppressed by mast cell-stabilizing drugs. Histamine and mast cell protease II were released by 1) exposure to SP or CGRP, 2) capsaicin, 3) compound 48/80, 4) elevation of mast cell Ca²⁺ by ionophore A23187, and 5) antidromic electrical stimulation of afferents. The mast cell stabilizers cromolyn and doxantrazole suppressed release of protease II and histamine when evoked by SP, CGRP, capsaicin, A23187, electrical stimulation of afferents, or compound 48/80. Neural blockade by tetrodotoxin prevented mast cell protease II release in response to antidromic electrical stimulation of mesenteric afferents. The results support a hypothesis that afferent innervation of enteric mast cells releases histamine and mast cell protease II, both of which are known to act in a diffuse paracrine manner to influence the behavior of enteric nervous system neurons and to elevate the sensitivity of spinal afferent terminals.

  17. Innervation of enteric mast cells by primary spinal afferents in guinea pig and human small intestine

    PubMed Central

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Qu, Meihua; Xia, Yun; Needleman, Bradley J.; Mikami, Dean J.

    2014-01-01

    Mast cells express the substance P (SP) neurokinin 1 receptor and the calcitonin gene-related peptide (CGRP) receptor in guinea pig and human small intestine. Enzyme-linked immunoassay showed that activation of intramural afferents by antidromic electrical stimulation or by capsaicin released SP and CGRP from human and guinea pig intestinal segments. Electrical stimulation of the afferents evoked slow excitatory postsynaptic potentials (EPSPs) in the enteric nervous system. The slow EPSPs were mediated by tachykinin neurokinin 1 and CGRP receptors. Capsaicin evoked slow EPSP-like responses that were suppressed by antagonists for protease-activated receptor 2. Afferent stimulation evoked slow EPSP-like excitation that was suppressed by mast cell-stabilizing drugs. Histamine and mast cell protease II were released by 1) exposure to SP or CGRP, 2) capsaicin, 3) compound 48/80, 4) elevation of mast cell Ca2+ by ionophore A23187, and 5) antidromic electrical stimulation of afferents. The mast cell stabilizers cromolyn and doxantrazole suppressed release of protease II and histamine when evoked by SP, CGRP, capsaicin, A23187, electrical stimulation of afferents, or compound 48/80. Neural blockade by tetrodotoxin prevented mast cell protease II release in response to antidromic electrical stimulation of mesenteric afferents. The results support a hypothesis that afferent innervation of enteric mast cells releases histamine and mast cell protease II, both of which are known to act in a diffuse paracrine manner to influence the behavior of enteric nervous system neurons and to elevate the sensitivity of spinal afferent terminals. PMID:25147231

  18. Vertebrate records in polar sediments: Biological responses to past climate change and human activities

    NASA Astrophysics Data System (ADS)

    Sun, L. G.; Emslie, S. D.; Huang, T.; Blais, J. M.; Xie, Z. Q.; Liu, X. D.; Yin, X. B.; Wang, Y. H.; Huang, W.; Hodgson, D. A.; Smol, J. P.

    2013-11-01

    Biological responses to climate and environmental changes in remote polar regions are of increasing interest in global change research. Terrestrial and marine polar ecosystems have suffered from impacts of both rapid climate change and intense human activities, and large fluctuations in the population sizes of seabirds, seals, and Antarctic krill have been observed in the past decades. To understand the mechanisms driving these regime shifts in polar ecosystems, it is important to first distinguish the influences of natural forcing from anthropogenic activities. Therefore, investigations of past changes of polar ecosystems prior to human contact are relevant for placing recent human-induced changes within a long-term historical context. Here we focus our review on the fossil, sub-fossil, archaeological, and biogeochemical remains of marine vertebrates in polar sediments. These remains include well-preserved tissues such as bones, hairs and feathers, and biogeochemical markers and other proxy indicators, including deposits of guano and excrement, which can accumulate in lake and terrestrial sediments over thousands of years. Analyses of these remains have provided insight into both natural and anthropogenic impacts on marine vertebrates over millennia and have helped identify the causal agents for these impacts. Furthermore, land-based seabirds and marine mammals have been shown to play an important role as bio-vectors in polar environments as they transport significant amounts of nutrients and anthropogenic contaminants between ocean and terrestrial ecosystems.

  19. Listeria monocytogenes Inhibits Serotonin Transporter in Human Intestinal Caco-2 Cells.

    PubMed

    Latorre, E; Pradilla, A; Chueca, B; Pagán, R; Layunta, E; Alcalde, A I; Mesonero, J E

    2016-10-01

    Listeria monocytogenes is a Gram-positive bacterium that can cause a serious infection. Intestinal microorganisms have been demonstrated to contribute to intestinal physiology not only through immunological responses but also by modulating the intestinal serotonergic system. Serotonin (5-HT) is a neuromodulator that is synthesized in the intestinal epithelium and regulates the whole intestinal physiology. The serotonin transporter (SERT), located in enterocytes, controls intestinal 5-HT availability and therefore serotonin's effects. Infections caused by L. monocytogenes are well described as being due to the invasion of intestinal epithelial cells; however, the effect of L. monocytogenes on the intestinal epithelium remains unknown. The main aim of this work, therefore, was to study the effect of L. monocytogenes on SERT. Caco2/TC7 cell line was used as an enterocyte-like in vitro model, and SERT functional and molecular expression assays were performed. Our results demonstrate that living L. monocytogenes inhibits serotonin uptake by reducing SERT expression at the brush border membrane. However, neither inactivated L. monocytogenes nor soluble metabolites were able to affect SERT. The results also demonstrate that L. monocytogenes yields TLR2 and TLR10 transcriptional changes in intestinal epithelial cells and suggest that TLR10 is potentially involved in the inhibitory effect observed on SERT. Therefore, L. monocytogenes, through TLR10-mediated SERT inhibition, may induce increased intestinal serotonin availability and potentially contributing to intestinal physiological changes and the initiation of the inflammatory response.

  20. Ultrasound Elasticity Imaging for Detecting Intestinal Fibrosis and Inflammation in Rats and Humans With Crohn’s Disease

    PubMed Central

    Stidham, Ryan W.; Xu, Jingping; Johnson, Laura A.; Kim, Kang; Moons, David S.; Mckenna, Barbara J.; Rubin, Jonathan M.; Higgins, Peter D. R.

    2016-01-01

    BACKGROUND Intestinal fibrosis causes many complications of Crohn’s disease (CD). Available biomarkers and imaging modalities lack sufficient accuracy to distinguish intestinal inflammation from fibrosis. Transcutaneous ultrasound elasticity imaging (UEI) is a promising, noninvasive approach for measuring tissue mechanical properties. We hypothesized that UEI could differentiate inflammatory from fibrotic bowel wall changes in both animal models of colitis and humans with CD. METHODS Female Lewis rats underwent weekly trinitrobenzene sulfonic acid enemas yielding models of acute inflammatory colitis (n = 5) and chronic intestinal fibrosis (n = 6). UEI scanning used a novel speckle-tracking algorithm to estimate tissue strain. Resected bowel segments were evaluated for evidence of inflammation and fibrosis. Seven consecutive patients with stenotic CD were studied with UEI and their resected stenotic and normal bowel segments were evaluated by ex vivo elastometry and histopathology. RESULTS Transcutaneous UEI normalized strain was able to differentiate acutely inflamed (−2.07) versus chronic fibrotic (−1.10) colon in rat models of inflammatory bowel disease (IBD; P = .037). Transcutaneous UEI normalized strain also differentiated stenotic (−0.87) versus adjacent normal small bowel (−1.99) in human CD (P = .0008), and this measurement also correlated well with ex vivo elastometry (r = −0.81). CONCLUSIONS UEI can differentiate inflammatory from fibrotic intestine in rat models of IBD and can differentiate between fibrotic and unaffected intestine in a pilot study in humans with CD. UEI represents a novel technology with potential to become a new objective measure of progression of intestinal fibrosis. Prospective clinical studies in CD are needed. PMID:21784048

  1. Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey.

    PubMed

    Cataloluk, Osman; Gogebakan, Bulent

    2004-07-01

    The prevalence of different resistance genes was investigated in lactobacilli of human and dairy origin by PCR. The presence of erm, van, tet, and cat-TC genes were determined in 16 raw milk, 15 cream, 10 yogurt, 50 hand-made cheese, and 20 industrially produced white-cheese samples of dairy origin and 16 mouth, 32 fecal, and 36 vaginal samples from different subjects of human origin. Lactobacilli of dairy and human origin were found to carry only erm(B) and tet(M) genes. The majority of the isolates, Lactobacillus crispatus (61), Lactobacillus gasseri (49), Lactobacillus plantarum (80) studied were found to harbor either erm(B) or tet(M) gene or both. No resistant lactobacilli was found in raw-milk and cream samples. All the human fecal samples and the majority of vaginal (29 of 36) and mouth (10 of 14) samples were found to carry the resistance genes. While a third of the hand-made cheeses carried resistant lactobacilli only one industrially produced cheese was found to carry resistant lactobacilli. Furthermore, the genes were found in the non-starter species, Lactobacillus acidophilus and Lb. plantarum, indicating that industrially produced cheeses in this respect could be considered more favorable. These results indicate that drug resistance seems to be very common in Turkey. Even though the number of dairy samples harboring the resistance genes (17 of 111) is smaller in regards to human samples, 10% of them were still found to carry the resistance genes as well. The presence of the resistance genes in majority of the samples of human origin and in minority of the samples of dairy origin indicates that drug resistance may be acquired in the intestinal tract during passage and spread to dairy products by the hands of workers during production.

  2. Improved biocompatibility of small intestinal submucosa (SIS) following conditioning by human endothelial cells.

    PubMed

    Woods, A M; Rodenberg, E J; Hiles, M C; Pavalko, F M

    2004-02-01

    Small intestinal submucosa (SIS) is a naturally occurring tissue matrix composed of extracellular matrix proteins and various growth factors. SIS is derived from the porcine jejunum and functions as a remodeling scaffold for tissue repair. While SIS has proven to be a useful biomaterial for implants in vivo, problems associated with endothelialization and thrombogenicity of SIS implants may limit its vascular utility. The goal of this study was to determine if the biological properties of SIS could be improved by growing human umbilical vein endothelial cells (HUVEC) on SIS and allowing these cells to deposit human basement membrane proteins on the porcine substrate to create what we have called "conditioned" SIS (c-SIS). Using an approach in which HUVEC were grown for 2 weeks on SIS and then removed via a technique that leaves behind an intact basement membrane, we hypothesized that the surface properties of SIS might be improved. We found that when re-seeded on c-SIS, HUVEC exhibited enhanced organization of cell junctions and had increased metabolic activity compared to cells on native SIS (n-SIS). Furthermore, HUVEC grown on c-SIS released lower amounts of the pro-inflammatory prostaglandin PGI2 into the media compared to cells grown on n-SIS. Additionally, we found that adhesion of resting or activated human platelets to c-SIS was significantly decreased compared to n-SIS suggesting that, in addition to improved cell growth characteristics, conditioning SIS with human basement membrane proteins might decrease its thrombogenic potential. In summary, conditioning of porcine SIS by human endothelial cells improves key biological properties of the material that may improve its usefulness as remodeling scaffold for tissue repair. Identification of critical modifications of SIS by human endothelial cells should help guide future efforts to develop more biocompatible vascular grafts.

  3. Differential modulation of human intestinal bifidobacterium populations after consumption of a wild blueberry (Vaccinium angustifolium) drink.

    PubMed

    Guglielmetti, Simone; Fracassetti, Daniela; Taverniti, Valentina; Del Bo', Cristian; Vendrame, Stefano; Klimis-Zacas, Dorothy; Arioli, Stefania; Riso, Patrizia; Porrini, Marisa

    2013-08-28

    Bifidobacteria are gaining increasing interest as health-promoting bacteria. Nonetheless, the genus comprises several species, which can exert different effects on human host. Previous studies showed that wild blueberry drink consumption could selectively increase intestinal bifidobacteria, suggesting an important role for the polyphenols and fiber present in wild blueberries. This study evaluated the modulation of the most common and abundant bifidobacterial taxonomic groups inhabiting the human gut in the same fecal samples. The analyses carried out showed that B. adolescentis, B. breve, B. catenulatum/pseudocatelulatum, and B. longum subsp. longum were always present in the group of subjects enrolled, whereas B. bifidum and B. longum subsp. infantis were not. Furthermore, it was found that the most predominant bifidobacterial species were B. longum subsp. longum and B. adolescentis. The results obtained revealed a high interindividual variability; however, a significant increase of B. longum subsp. infantis cell concentration was observed in the feces of volunteers after the wild blueberry drink treatment. This bifidobacterial group was shown to possess immunomodulatory abilities and to relieve symptoms and promote the regression of several gastrointestinal disorders. Thus, an increased cell concentration of B. longum subsp. infantis in the human gut could be considered of potential health benefit. In conclusion, wild blueberry consumption resulted in a specific bifidogenic effect that could positively affect certain populations of bifidobacteria with demonstrated health-promoting properties.

  4. Alteration of a human intestinal microbiota under extreme life environment in the Antarctica.

    PubMed

    Jin, Jong-Sik; Touyama, Mutsumi; Yamada, Shin; Yamazaki, Takashi; Benno, Yoshimi

    2014-01-01

    The human intestinal microbiota (HIM) settles from birth and continues to change phenotype by some factors (e.g. host's diet) throughout life. However, the effect of extreme life environment on human HIM composition is not well known. To understand HIM fluctuation under extreme life environment in humans, fecal samples were collected from six Japanese men on a long Antarctic expedition. They explored Antarctica for 3 months and collected their fecal samples at once-monthly intervals. Using terminal restriction fragment length polymorphism (T-RFLP) and real time polymerase chain reaction (PCR) analysis, the composition of HIM in six subjects was investigated. Three subjects presented restoration of HIM after the expedition compared versus before and during the expedition. Two thirds samples collected during the expedition belonged to the same cluster in dendrogram. However, all through the expedition, T-RFLP patterns showed interindividual variability. Especially, Bifidobacterium spp. showed a tendency to decrease during and restore after the expedition. A reduction of Bifidobacterium spp. was observed in five subjects the first 1 month of the expedition. Bacteroides thetaiotaomicron, which is thought to proliferate during emotional stress, significantly decreased in one subject, indicating that other factors in addition to emotional stress may affect the composition of HIM in this study. These findings could be helpful to understand the effect of extreme life environment on HIM.

  5. Carboxylated nanodiamonds are neither cytotoxic nor genotoxic on liver, kidney, intestine and lung human cell lines.

    PubMed

    Paget, V; Sergent, J A; Grall, R; Altmeyer-Morel, S; Girard, H A; Petit, T; Gesset, C; Mermoux, M; Bergonzo, P; Arnault, J C; Chevillard, S

    2014-08-01

    Although nanodiamonds (NDs) appear as one of the most promising nanocarbon materials available so far for biomedical applications, their risk for human health remains unknown. Our work was aimed at defining the cytotoxicity and genotoxicity of two sets of commercial carboxylated NDs with diameters below 20 and 100 nm, on six human cell lines chosen as representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung). Cytotoxicity of NDs was assessed by measuring cell impedance (xCELLigence® system) and cell survival/death by flow cytometry while genotoxicity was assessed by γ-H2Ax foci detection, which is considered the most sensitive technique for studying DNA double-strand breaks. To validate and check the sensitivity of the techniques, aminated polystyrene nanobeads were used as positive control in all assays. Cell incorporation of NDs was also studied by flow cytometry and luminescent N-V center photoluminescence (confirmed by Raman microscopy), to ensure that nanoparticles entered the cells. Overall, we show that NDs effectively entered the cells but NDs do not induce any significant cytotoxic or genotoxic effects on the six cell lines up to an exposure dose of 250 µg/mL. Taken together these results strongly support the huge potential of NDs for human nanomedicine but also their potential as negative control in nanotoxicology studies.

  6. Total Body Irradiation in the "Hematopoietic" Dose Range Induces Substantial Intestinal Injury in Non-Human Primates.

    PubMed

    Wang, Junru; Shao, Lijian; Hendrickson, Howard P; Liu, Liya; Chang, Jianhui; Luo, Yi; Seng, John; Pouliot, Mylene; Authier, Simon; Zhou, Daohong; Allaben, William; Hauer-Jensen, Martin

    2015-11-01

    The non-human primate has been a useful model for studies of human acute radiation syndrome (ARS). However, to date structural changes in various parts of the intestine after total body irradiation (TBI) have not been systematically studied in this model. Here we report on our current study of TBI-induced intestinal structural injury in the non-human primate after doses typically associated with hematopoietic ARS. Twenty-four non-human primates were divided into three groups: sham-irradiated control group; and total body cobalt-60 (60Co) 6.7 Gy gamma-irradiated group; and total body 60Co 7.4 Gy gamma-irradiated group. After animals were euthanized at day 4, 7 and 12 postirradiation, sections of small intestine (duodenum, proximal jejunum, distal jejunum and ileum) were collected and fixed in 10% formalin. The intestinal mucosal surface length, villus height and crypt depths were assessed by computer-assisted image analysis. Plasma citrulline levels were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Total bone marrow cells were counted and hematopoietic stem/progenitor cells in bone marrow were analyzed by flow cytometer. Histopathologically, all segments exhibited conspicuous disappearance of plicae circulares and prominent atrophy of crypts and villi. Intestinal mucosal surface length was significantly decreased in all intestinal segments on day 4, 7 and 12 after irradiation (P < 0.02-P < 0.001). Villus height was significantly reduced in all segments on day 4 and 7 (P = 0.02-0.005), whereas it had recovered by day 12 (P > 0.05). Crypt depth was also significantly reduced in all segments on day 4, 7 and 12 after irradiation (P < 0.04-P < 0.001). Plasma citrulline levels were dramatically reduced after irradiation, consistent with intestinal mucosal injury. Both 6.7 and 7.4 Gy TBI reduced total number of bone marrow cells. And further analysis showed that the number and function of CD45(+)CD34(+) hematopoietic stem/progenitors in bone

  7. Metformin Transport by a Newly Cloned Proton-Stimulated Organic Cation Transporter (Plasma Membrane Monoamine Transporter) Expressed in Human Intestine

    PubMed Central

    Zhou, Mingyan; Xia, Li; Wang, Joanne

    2009-01-01

    Metformin is a widely used oral antihyperglycemic drug for the treatment of type II diabetes mellitus. The intestinal absorption of metformin is dose-dependent and involves an active, saturable uptake process. Metformin has been shown to be transported by the human organic cation transporters 1 and 2 (hOCT1–2). We recently cloned and characterized a novel proton-activated organic cation transporter, plasma membrane monoamine transporter (PMAT). We previously showed that PMAT transports many classic organic cations (e.g., monoamine neurotransmitters, 1-methyl-4-phenylpyridinium) in a pH-dependent manner and its mRNA is expressed in multiple human tissues. The goal of this study is to investigate whether metformin is a substrate of PMAT and whether PMAT plays a role in the intestinal uptake of metformin. Using Madin-Darby canine kidney cells stably expressing human PMAT, we showed that metformin is avidly transported by PMAT, with an apparent affinity (Km = 1.32 mM) comparable to those reported for hOCT1–2. Interestingly, the concentration-velocity profile of PMAT-mediated metformin uptake is sigmoidal, with a Hill coefficient of 2.64. PMAT-mediated metformin transport is greatly stimulated by acidic pH, with the uptake rate being ~4-fold higher at pH 6.6 than at pH 7.4. Using a polyclonal antibody against PMAT, we showed that the PMAT protein (58 kDa) was expressed in human small intestine and concentrated on the tips of the mucosal epithelial layer. Taken together, our results suggest that PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorption of metformin and possibly other cationic drugs. PMID:17600084

  8. Epidemiology of infections with intestinal parasites and human immunodeficiency virus (HIV) among sugar-estate residents in Ethiopia.

    PubMed

    Fontanet, A L; Sahlu, T; Rinke de Wit, T; Messele, T; Masho, W; Woldemichael, T; Yeneneh, H; Coutinho, R A

    2000-04-01

    Intestinal parasitic infections could play an important role in the progression of infection with human immunodeficiency virus (HIV), by further disturbing the immune system whilst it is already engaged in the fight against HIV. HIV and intestinal parasitic infections were investigated in 1239, randomly selected individuals, aged 15-54 years, living on a sugar estate in central Ethiopia. Intestinal parasites were identified in faecal samples (one/subject) using direct, concentration, and (for Strongyloides stercoralis larvae) Baermann methods. HIV serological status was determined using ELISA, with ELISA-positive samples confirmed as positive by western blotting. Most (70.1%) of the subjects were infected with at least one intestinal parasite and 3.1% were seropositive (but asymptomatic) for HIV. The intestinal parasites identified in the study population were amoebic parasites (Entamoeba histolytica/Enta. dispar) (24.6%), hookworms (23.8%), Ascaris lumbricoides (22.2%), Trichuris trichiura (19.5%), S. stercoralis (13.0%), Taenia saginata (4.5%), Giardia lamblia (3.0%), and Enterobius vermicularis (1.3%). Overall, the HIV-positives were no more or less likely to carry intestinal parasites than the HIV-negatives (76.2% v. 69.9%; P > 0.05). However, when each parasite was considered separately, amoebic parasites were found to be more common in the HIV-positives than the HIV-negatives (43.7% v. 24.0%; P < 0.05). This difference remained significant in a multivariate analysis, after controlling for the socio-demographic characteristics of the study participants. In conclusion, there was moderate interaction between intestinal parasites and HIV at the asymptomatic stage of HIV infection. The observed association between amoebic and HIV infections requires confirmation in a prospective study, allowing for the analysis of biological mechanisms involved in the association.

  9. Advancing the use of Lactobacillus acidophilus surface layer protein A for the treatment of intestinal disorders in humans

    PubMed Central

    Sahay, Bikash; Ge, Yong; Colliou, Natacha; Zadeh, Mojgan; Weiner, Chelsea; Mila, Ashley; Owen, Jennifer L; Mohamadzadeh, Mansour

    2015-01-01

    Intestinal immunity is subject to complex and fine-tuned regulation dictated by interactions of the resident microbial community and their gene products with host innate cells. Deterioration of this delicate process may result in devastating autoinflammatory diseases, including inflammatory bowel disease (IBD), which primarily comprises Crohn's disease (CD) and ulcerative colitis (UC). Efficacious interventions to regulate proinflammatory signals, which play critical roles in IBD, require further scientific investigation. We recently demonstrated that rebalancing intestinal immunity via the surface layer protein A (SlpA) from Lactobacillus acidophilus NCFM potentially represents a feasible therapeutic approach to restore intestinal homeostasis. To expand on these findings, we established a new method of purifying bacterial SlpA, a new SlpA-specific monoclonal antibody, and found no SlpA-associated toxicity in mice. Thus, these data may assist in our efforts to determine the immune regulatory efficacy of SlpA in humans. PMID:26647142

  10. Partitioning of polar and non-polar neutral organic chemicals into human and cow milk.

    PubMed

    Geisler, Anett; Endo, Satoshi; Goss, Kai-Uwe

    2011-10-01

    The aim of this work was to develop a predictive model for milk/water partition coefficients of neutral organic compounds. Batch experiments were performed for 119 diverse organic chemicals in human milk and raw and processed cow milk at 37°C. No differences (<0.3 log units) in the partition coefficients of these types of milk were observed. The polyparameter linear free energy relationship model fit the calibration data well (SD=0.22 log units). An experimental validation data set including hormones and hormone active compounds was predicted satisfactorily by the model. An alternative modelling approach based on log K(ow) revealed a poorer performance. The model presented here provides a significant improvement in predicting enrichment of potentially hazardous chemicals in milk. In combination with physiologically based pharmacokinetic modelling this improvement in the estimation of milk/water partitioning coefficients may allow a better risk assessment for a wide range of neutral organic chemicals.

  11. Evaluation of physicochemical properties and intestinal permeability of six dietary polyphenols in human intestinal colon adenocarcinoma Caco-2 cells.

    PubMed

    Rastogi, Himanshu; Jana, Snehasis

    2016-02-01

    Phenolic compounds are common ingredients in many dietary supplements and functional foods. However, data concerning physicochemical properties and permeability of polyphenols on the intestinal epithelial cells are scarce. The aims of this study were to determine the experimental partition coefficient (Log P), and parallel artificial membrane permeability assay (PAMPA), to characterize the bi-directional transport of six phenolic compounds viz. caffeic acid, chrysin, gallic acid, quercetin, resveratrol and rutin in Caco-2 cells. The experimental Log P values of six polyphenols were correlated (R (2) = 0.92) well with the calculated Log P values. The apparent permeability (P app) range of all polyphenols in PAMPA for the apical (AP) to basolateral (BL) was 1.18 ± 0.05 × 10(-6) to 5.90 ± 0.16 × 10(-6) cm/s. The apparent Caco-2 permeability (P app) range for the AP-BL was 0.96 ± 0.03 × 10(-6) to 3.80 ± 0.45 × 10(-6) cm/s. The efflux ratio of P app (BL → AP) to P app (AP → BL) for all phenolics was <2, suggesting greater permeability in the absorptive direction. Six compounds exhibited strong correlations between Log P and PAMPA/Caco-2 cell monolayer permeation data. Dietary six polyphenols were poorly absorbed through PAMPA and Caco-2 cells, and their transepithelial transports were mainly by passive diffusion.

  12. Enabling the Intestinal Absorption of Highly Polar Anti-Viral Agents: Ion-Pair Facilitated Membrane Permeation of Zanamivir Heptyl Ester and Guanidino Oseltamivir

    PubMed Central

    Miller, Jonathan M.; Dahan, Arik; Gupta, Deepak; Varghese, Sheeba; Amidon, Gordon L.

    2012-01-01

    Anti-viral drugs often suffer from poor intestinal permeability, preventing their delivery via the oral route. The goal of this work was to enhance the intestinal absorption of the low-permeability anti-viral agents zanamivr heptyl ester (ZHE) and guanidino oseltamivir (GO) utilizing an ion-pairing approach, as a critical step toward making them oral drugs. The counterion 1-hydroxy-2-napthoic acid (HNAP) was utilized to enhance the lipophilicity and permeability of the highly polar drugs. HNAP substantially increased the log P of the drugs by up to 3.7 log units. Binding constants (K11aq) of 388 M−1 for ZHE-HNAP and 2.91 M−1 for GO.-HNAP were obtained by applying a quasi-equilibrium transport model to double-reciprocal plots of apparent octanol-buffer distribution coefficients versus HNAP concentration. HNAP enhanced the apparent permeability (Papp) of both compounds across Caco-2 cell monolayers in a concentration-dependent manner, as substantial Papp (0.8 – 3.0 × 10−6 cm/s) was observed in the presence of 6–24 mM HNAP, whereas no detectable transport was observed without counterion. Consistent with a quasi-equilibrium transport model, a linear relationship with slope near 1 was obtained from a log-log plot of Caco-2 Papp versus HNAP concentration, supporting the ion-pair mechanism behind the permeability enhancement. In the rat jejunal perfusion assay, the addition of HNAP failed to increase the effective permeability (Peff) of GO. However, the rat jejunal permeability of ZHE was significantly enhanced by the addition of HNAP in a concentration-dependent manner, from essentially zero without HNAP to 4.0 × 10−5 cm/s with 10 mM HNAP, matching the Peff of the high-permeability standard metoprolol. The success of ZHE-HNAP was explained by its >100-fold stronger K11aq versus GO-HNAP, making ZHE-HNAP less prone to dissociation and ion-exchange with competing endogenous anions and able to remain intact during membrane permeation. Overall, this work

  13. Studies on the bioavailability of zinc in humans: intestinal interaction of tin and zinc.

    PubMed

    Solomons, N W; Marchini, J S; Duarte-Favaro, R M; Vannuchi, H; Dutra de Oliveira, J E

    1983-04-01

    Mineral/mineral interactions at the intestinal level are important in animal nutrition and toxicology, but only limited understanding of their extent or importance in humans has been developed. An inhibitory interaction of dietary tin on zinc retention has been recently described from human metabolic studies. We have explored the tin/zinc interaction using the change-in-plasma-zinc-concentration method with a standard dosage of 12.5 mg of zinc as zinc sulfate in 100 ml of Coca-Cola. Sn/Zn ratios of 2:1, 4:1, and 8:1, constituted by addition of 25, 50, and 100 mg of tin as stannous chloride, had no significant overall effect on zinc uptake. The 100-mg dose of tin produced noxious gastrointestinal symptoms. Addition of iron as ferrous sulfate to form ratios of Sn/Fe/Zn of 1:1:1 and 2:2:1 with the standard zinc solution and the appropriate doses of tin produced a reduction of zinc absorption not dissimilar from that seen previously with zinc and iron alone, and addition of picolinic acid did not influence the uptake of zinc from the solution with the 2:2:1 Sn/Fe/Zn ratio.

  14. Study of the adhesion of Bifidobacterium bifidum MIMBb75 to human intestinal cell lines.

    PubMed

    Guglielmetti, Simone; Tamagnini, Isabella; Minuzzo, Mario; Arioli, Stefania; Parini, Carlo; Comelli, Elena; Mora, Diego

    2009-08-01

    The aim of this study was to investigate the adhesive phenotype of the human intestinal isolate Bifidobacterium bifidum MIMBb75 to human colon carcinoma cell lines. We have previously shown that the adhesion of this strain to Caco-2 cells is mediated by an abundant surface lipoprotein named BopA. In this study, we found that this strain adheres to Caco-2 and HT-29 cells, and that its adhesion strongly depends on the environmental conditions, including the presence of sugars and bile salts and the pH. Considerably more adhesion to a Caco-2 monolayer occurred in the presence of fucose and mannose and less when MIMBb75 grew in Oxgall bile salts compared to standard environmental conditions. In particular, growth in Oxgall bile salts reduced the adhesion ability of MIMBb75 and modified the SDS-PAGE profile of the cell wall associated proteins of the strain. The pH markedly affected both adhesion to Caco-2 and bacterial autoaggregation. Finally, experiments with sodium metaperiodate suggested that not only proteinaceous determinants are involved in the adhesion process of B. bifidum. In conclusion, it seems that the colonization strategy of this bacterium can be influenced by factors varying along the gastrointestinal tract, such as the presence of specific sugars and bile salts and the pH, possibly limiting the adhesion of B. bifidum to only restricted distal sites of the gut.

  15. Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome.

    PubMed

    Zuber, Julien; Shonts, Brittany; Lau, Sai-Ping; Obradovic, Aleksandar; Fu, Jianing; Yang, Suxiao; Lambert, Marion; Coley, Shana; Weiner, Joshua; Thome, Joseph; DeWolf, Susan; Farber, Donna L; Shen, Yufeng; Caillat-Zucman, Sophie; Bhagat, Govind; Griesemer, Adam; Martinez, Mercedes; Kato, Tomoaki; Sykes, Megan

    2016-10-01

    A paradigm in transplantation states that graft-infiltrating T cells are largely non-alloreactive "bystander" cells. However, the origin and specificity of allograft T cells over time has not been investigated in detail in animals or humans. Here, we use polychromatic flow cytometry and high throughput TCR sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intra-graft host-vs-graft (HvG) and graft-vs-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted long-term in the graft. Early expansion of GvH clones in the graft correlated with rapid replacement of donor APCs by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2D(Hi) CD8+ alpha beta T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady state tissue-resident phenotype, but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in non-rejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.

  16. Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome

    PubMed Central

    Zuber, Julien; Obradovic, Aleksandar; Fu, Jianing; Yang, Suxiao; Lambert, Marion; Coley, Shana; Weiner, Joshua; Thome, Joseph; DeWolf, Susan; Farber, Donna L.; Shen, Yufeng; Caillat-Zucman, Sophie; Bhagat, Govind; Griesemer, Adam; Martinez, Mercedes; Kato, Tomoaki; Sykes, Megan

    2016-01-01

    A paradigm in transplantation states that graft-infiltrating T cells are largely non-alloreactive “bystander” cells. However, the origin and specificity of allograft T cells over time has not been investigated in detail in animals or humans. Here, we use polychromatic flow cytometry and high throughput TCR sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intra-graft host-vs-graft (HvG) and graft-vs-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted long-term in the graft. Early expansion of GvH clones in the graft correlated with rapid replacement of donor APCs by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ alpha beta T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady state tissue-resident phenotype, but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in non-rejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population. PMID:28239678

  17. Molecular Paleoparasitological Hybridization Approach as Effective Tool for Diagnosing Human Intestinal Parasites from Scarce Archaeological Remains

    PubMed Central

    Jaeger, Lauren Hubert; Iñiguez, Alena Mayo

    2014-01-01

    Paleoparasitology is the science that uses parasitological techniques for diagnosing parasitic diseases in the past. Advances in molecular biology brought new insights into this field allowing the study of archaeological material. However, due to technical limitations a proper diagnosis and confirmation of the presence of parasites is not always possible, especially in scarce and degraded archaeological remains. In this study, we developed a Molecular Paleoparasitological Hybridization (MPH) approach using ancient DNA (aDNA) hybridization to confirm and complement paleoparasitological diagnosis. Eight molecular targets from four helminth parasites were included: Ascaris sp., Trichuris trichiura, Enterobius vermicularis, and Strongyloides stercoralis. The MPH analysis using 18th century human remains from Praça XV cemetery (CPXV), Rio de Janeiro, Brazil, revealed for the first time the presence E. vermicularis aDNA (50%) in archaeological sites of Brazil. Besides, the results confirmed T. trichiura and Ascaris sp. infections. The prevalence of infection by Ascaris sp. and E. vermicularis increased considerably when MPH was applied. However, a lower aDNA detection of T. trichiura (40%) was observed when compared to the diagnosis by paleoparasitological analysis (70%). Therefore, based on these data, we suggest a combination of Paleoparasitological and MPH approaches to verify the real panorama of intestinal parasite infection in human archeological samples. PMID:25162694

  18. Soluble Human Intestinal Lactoferrin Receptor: Ca(2+)-Dependent Binding to Sepharose-Based Matrices.

    PubMed

    Oshima, Yuta; Seki, Kohei; Shibuya, Masataka; Naka, Yuki; Yokoyama, Tatsuya; Sato, Atsushi

    2016-01-01

    A soluble form of human intestinal lactoferrin receptor (shLFR) is identical to human intelectin-1 (hITLN-1), a galactofuranose-binding protein that acts as a host defense against invading pathogenic microorganisms. We found that recombinant shLFR, expressed in mammalian cells (CHO DG44, COS-1, and RK13), binds tightly to Sepharose 4 Fast Flow (FF)-based matrices in a Ca(2+)-dependent manner. This binding of shLFR to Sepharose 4 FF-based matrices was inhibited by excess D-galactose, but not by D-glucose, suggesting that shLFR recognizes repeating units of α-1,6-linked D-galactose in Sepharose 4 FF. Furthermore, shLFR could bind to both Sepharose 4B- and Sepharose 6B-based matrices that were not crosslinked in a similar manner as to Sepharose 4 FF-based matrices. Therefore, shLFR (hITLN-1) binds to Sepharose-based matrices in a Ca(2+)-dependent manner. This binding property is most likely related to the ability, as host defense lectins, to recognize sepharose (agarobiose)-like structures present on the surface of invading pathogenic microorganisms.

  19. Effect of the artificial sweetener, sucralose, on small intestinal glucose absorption in healthy human subjects.

    PubMed

    Ma, Jing; Chang, Jessica; Checklin, Helen L; Young, Richard L; Jones, Karen L; Horowitz, Michael; Rayner, Christopher K

    2010-09-01

    It has been reported that the artificial sweetener, sucralose, stimulates glucose absorption in rodents by enhancing apical availability of the transporter GLUT2. We evaluated whether exposure of the proximal small intestine to sucralose affects glucose absorption and/or the glycaemic response to an intraduodenal (ID) glucose infusion in healthy human subjects. Ten healthy subjects were studied on two separate occasions in a single-blind, randomised order. Each subject received an ID infusion of sucralose (4 mM in 0.9% saline) or control (0.9% saline) at 4 ml/min for 150 min (T = - 30 to 120 min). After 30 min (T = 0), glucose (25 %) and its non-metabolised analogue, 3-O-methylglucose (3-OMG; 2.5 %), were co-infused intraduodenally (T = 0-120 min; 4.2 kJ/min (1 kcal/min)). Blood was sampled at frequent intervals. Blood glucose, plasma glucagon-like peptide-1 (GLP-1) and serum 3-OMG concentrations increased during ID glucose/3-OMG infusion (P < 0.005 for each). However, there were no differences in blood glucose, plasma GLP-1 or serum 3-OMG concentrations between sucralose and control infusions. In conclusion, sucralose does not appear to modify the rate of glucose absorption or the glycaemic or incretin response to ID glucose infusion when given acutely in healthy human subjects.

  20. Challenges of culturing human norovirus in three-dimensional organoid intestinal cell culture models.

    PubMed

    Papafragkou, Efstathia; Hewitt, Joanne; Park, Geun Woo; Greening, Gail; Vinjé, Jan

    2014-01-01

    Human noroviruses are the most common cause of acute gastroenteritis worldwide. Recently, cell culture systems have been described using either human embryonic intestinal epithelial cells (Int-407) or human epithelial colorectal adenocarcinoma cells (Caco-2) growing on collagen-I porous micro carrier beads in a rotating bioreactor under conditions of physiological fluid shear. Here, we describe the efforts from two independent laboratories to implement this three dimensional (3D) cell culture system for the replication of norovirus. Int-407 and Caco-2 were grown in a rotating bioreactor for up to 28 days. Prior to infection, cells were screened for the presence of microvilli by electron microscopy and stained for junction proteins (zonula occludens-1, claudin-1, and β-catenin). Differentiated 3D cells were transferred to 24-well plates and infected with bacteria-free filtrates of various norovirus genotypes (GI.1, GI.3, GI.8, GII.2, GII.4, GII.7, and GII.8). At 12 h, 24 h, and 48 h post inoculation, viral RNA from both cells and supernatants were collected and analyzed for norovirus RNA by real-time reverse transcription PCR. Despite observations of high expression of junction proteins and microvilli development in stained thin sections, our data suggest no significant increase in viral titer based on norovirus RNA copy number during the first 48 h after inoculation for the different samples and virus culture conditions tested. Our combined efforts demonstrate that 3D cell culture models using Int-407 or Caco-2 cells do not support norovirus replication and highlight the complexity and difficulty of developing a reproducible in vitro cell culture system for human norovirus.

  1. Systemic and mucosal immune responses following oral adenoviral delivery of influenza vaccine to the human intestine by radio controlled capsule

    PubMed Central

    Kim, Leesun; Martinez, C. Josefina; Hodgson, Katie A.; Trager, George R.; Brandl, Jennifer R.; Sandefer, Erik P.; Doll, Walter J.; Liebowitz, Dave; Tucker, Sean N.

    2016-01-01

    There are several benefits of oral immunization including the ability to elicit mucosal immune responses that may protect against pathogens that invade through a mucosal surface. Our understanding of human immune biology is hampered by the difficulty in isolating mucosal cells from humans, and the fact that animal models may or may not completely mirror human intestinal immunobiology. In this human pharmacodynamic study, a novel adenovirus vector-based platform expressing influenza hemagglutinin was explored. We used radio-controlled capsules to deliver the vaccine to either the jejunum or the ileum. The resulting immune responses induced by immunization at each of the intestinal sites were investigated. Both intestinal sites were capable of inducing mucosal and systemic immune responses to influenza hemagglutinin, but ileum delivery induced higher numbers of antibody secreting cells of IgG and IgA isotypes, increased mucosal homing B cells, and higher number of vaccine responders. Overall, these data provided substantial insights into human mucosal inductive sites, and aided in the design and selection of indications that could be used with this oral vaccine platform. PMID:27881837

  2. Microbiota/Host Crosstalk Biomarkers: Regulatory Response of Human Intestinal Dendritic Cells Exposed to Lactobacillus Extracellular Encrypted Peptide

    PubMed Central

    Al-Hassi, Hafid O.; Mann, Elizabeth R.; Urdaci, María C.; Knight, Stella C.; Margolles, Abelardo

    2012-01-01

    The human gastrointestinal tract is exposed to a huge variety of microorganisms, either commensal or pathogenic; at this site, a balance between immunity and immune tolerance is required. Intestinal dendritic cells (DCs) control the mechanisms of immune response/tolerance in the gut. In this paper we have identified a peptide (STp) secreted by Lactobacillus plantarum, characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains, and lacks cleavage sites for the major intestinal proteases. When studied in vitro, STp expanded the ongoing production of regulatory IL-10 in human intestinal DCs from healthy controls. STp-primed DC induced an immunoregulatory cytokine profile and skin-homing profile on stimulated T-cells. Our data suggest that some of the molecular dialogue between intestinal bacteria and DCs may be mediated by immunomodulatory peptides, encoded in larger extracellular proteins, secreted by commensal bacteria. These peptides may be used for the development of nutraceutical products for patients with IBD. In addition, this kind of peptides seem to be absent in the gut of inflammatory bowel disease patients, suggesting a potential role as biomarker of gut homeostasis. PMID:22606249

  3. 4-Nonylphenol reduces cell viability and induces apoptosis and ER-stress in a human epithelial intestinal cell line.

    PubMed

    Lepretti, M; Paolella, G; Giordano, D; Marabotti, A; Gay, F; Capaldo, A; Esposito, C; Caputo, I

    2015-10-01

    4-Nonylphenol is a widely diffused and stable environmental contaminant, originating from the degradation of alkyl phenol ethoxylates, common surfactants employed in several industrial applications. Due to its hydrophobic nature, 4-nonylphenol can easily accumulate in living organisms, including humans, where it displays a wide range of toxic effects. Since the gastrointestinal tract represents the main route by which 4-nonylphenol enters the body, the intestine may be one of the first organs to be damaged by chronic exposure to this pollutant through the diet. In the present study, we investigated the effects of 4-nonylphenol on a human intestinal epithelial cell line (Caco-2 cells). We demonstrated that 4-nonylphenol was cytotoxic to cells, as revealed by a decrease of the cell number and the decrement of mitochondrial functionality after 24 h of treatment. 4-Nonylphenol also reduced the number of cells entering into S-phase and interfered with epidermal growth factor signalling, with consequent negative effects on cell survival. In addition, 4-nonylphenol induced apoptosis, involving the activation of caspase-3, and triggered an endoplasmic reticulum-stress response, as revealed by over-expression of GRP78 (78 kDa glucose-regulated protein) and activation of XBP1 (X-box binding protein-1). Together, these findings support the hypothesis that prolonged exposure to 4-nonylphenol through the diet may lead to local damage at the level of intestinal mucosa, with potentially negative consequences for intestinal homeostasis and functionality.

  4. Microbiota/host crosstalk biomarkers: regulatory response of human intestinal dendritic cells exposed to Lactobacillus extracellular encrypted peptide.

    PubMed

    Bernardo, David; Sánchez, Borja; Al-Hassi, Hafid O; Mann, Elizabeth R; Urdaci, María C; Knight, Stella C; Margolles, Abelardo

    2012-01-01

    The human gastrointestinal tract is exposed to a huge variety of microorganisms, either commensal or pathogenic; at this site, a balance between immunity and immune tolerance is required. Intestinal dendritic cells (DCs) control the mechanisms of immune response/tolerance in the gut. In this paper we have identified a peptide (STp) secreted by Lactobacillus plantarum, characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains, and lacks cleavage sites for the major intestinal proteases. When studied in vitro, STp expanded the ongoing production of regulatory IL-10 in human intestinal DCs from healthy controls. STp-primed DC induced an immunoregulatory cytokine profile and skin-homing profile on stimulated T-cells. Our data suggest that some of the molecular dialogue between intestinal bacteria and DCs may be mediated by immunomodulatory peptides, encoded in larger extracellular proteins, secreted by commensal bacteria. These peptides may be used for the development of nutraceutical products for patients with IBD. In addition, this kind of peptides seem to be absent in the gut of inflammatory bowel disease patients, suggesting a potential role as biomarker of gut homeostasis.

  5. Glutamine pretreatment reduces IL-8 production in human intestinal epithelial cells by limiting IkappaBalpha ubiquitination.

    PubMed

    Hubert-Buron, Aurélie; Leblond, Jonathan; Jacquot, Arnaud; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2006-06-01

    Glutamine, the most abundant amino acid in the human body, plays several important roles in the intestine. Recent studies showed that glutamine regulates protein metabolism and intestinal inflammation among other mechanisms by reducing proinflammatory cytokine release. Because regulation of the inflammatory response was shown to be linked to proteolysis regulation, we hypothesized that glutamine pretreatment could act on IL-8 production in human intestinal epithelial cells through the regulation of inhibitor kappaB (IkappaB) ubiquitination. The HCT-8 cells were pretreated for 24 h with 0.6, 2, or 10 mmol/L glutamine. IL-8 concentration and IkappaB (free and ubiquitinated) expressions were assessed by ELISA and immunoblotting, respectively. A pretreatment with 10 mmol/L glutamine decreased IL-8 production under both basal and proinflammatory conditions (both P < 0.05). In the presence of a proteasome inhibitor (MG132), the ubiquitin-IkappaBalpha complex expression was not significantly modified by glutamine under basal conditions but decreased significantly under proinflammatory conditions (P < 0.05). After the addition of 10 mmol/L of glutamine, the free IkappaBalpha expression increased under basal and stimulated conditions (both P < 0.05). A glutamine pretreatment of 10 mmol/L did not affect ubiquitin expression or proteasome activity. This study indicates that glutamine pretreatment may reduce the intestinal inflammatory response by limiting the proteolysis of IkappaBalpha.

  6. Glucagon-like peptide-1 (GLP-1) induces M2 polarization of human macrophages via STAT3 activation.

    PubMed

    Shiraishi, Daisuke; Fujiwara, Yukio; Komohara, Yoshihiro; Mizuta, Hiroshi; Takeya, Motohiro

    2012-08-24

    It is known that glucagon-like peptide-1 (GLP-1) is a hormone secreted postprandially from the L-cells of the small intestine and regulates glucose homeostasis. GLP-1 is now used for the treatment of diabetes because of its beneficial role against insulin resistance. The GLP-1 receptor (GLP-1R) is expressed on many cell types, including macrophages, and GLP-1 suppresses the development of atherosclerosis by inhibiting macrophage function. However, there have so far been few studies that have investigated the significance of GLP-1/GLP-1R signaling in macrophage activation. In the present study, we examined the effect of GLP-1 and exenatide, a GLP-1R agonist, on human monocyte-derived macrophage (HMDM) activation. We found that GLP-1 induced signal transducer and activator of transcription 3 (STAT3) activation. Silencing of GLP-1R suppressed the GLP-1-induced STAT3 activation. In addition, alternatively activated (M2) macrophage-related molecules, such as IL-10, CD163, and CD204 in HMDM, were significantly upregulated by GLP-1. Furthermore, the co-culture of 3T3-L1 adipocytes with GLP-1-treated RAW 264.7 macrophages increased the secretion of adiponectin compared to co-culture of the 3T3-L1 adipocytes with untreated RAW 264.7 macrophages. Our results demonstrate that GLP-1 induces macrophage polarization toward the M2 phenotype, which may contribute to the protective effects of GLP-1 against diabetes and cardiovascular diseases.

  7. Assessment of the mode of action underlying development of rodent small intestinal tumors following oral exposure to hexavalent chromium and relevance to humans

    PubMed Central

    Proctor, Deborah M.; Suh, Mina; Haws, Laurie C.; Kirman, Christopher R.; Harris, Mark A.

    2013-01-01

    Chronic exposure to high concentrations of hexavalent chromium (Cr(VI)) in drinking water causes intestinal adenomas and carcinomas in mice, but not in rats. Cr(VI) causes damage to intestinal villi and crypt hyperplasia in mice after only one week of exposure. After two years of exposure, intestinal damage and crypt hyperplasia are evident in mice (but not rats), as are intestinal tumors. Although Cr(VI) has genotoxic properties, these findings suggest that intestinal tumors in mice arise as a result of chronic mucosal injury. To better understand the mode of action (MOA) of Cr(VI) in the intestine, a 90-day drinking water study was conducted to collect histological, biochemical, toxicogenomic and pharmacokinetic data in intestinal tissues. Using MOA analyses and human relevance frameworks proposed by national and international regulatory agencies, the weight of evidence supports a cytotoxic MOA with the following key events: (a) absorption of Cr(VI) from the intestinal lumen, (b) toxicity to intestinal villi, (c) crypt regenerative hyperplasia and (d) clonal expansion of mutations within the crypt stem cells, resulting in late onset tumorigenesis. This article summarizes the data supporting each key event in the MOA, as well as data that argue against a mutagenic MOA for Cr(VI)-induced intestinal tumors. PMID:23445218

  8. Glycosylation of the major polar tube protein of Encephalitozoon hellem, a microsporidian parasite that infects humans.

    PubMed

    Xu, Yanji; Takvorian, Peter M; Cali, Ann; Orr, George; Weiss, Louis M

    2004-11-01

    The microsporidia are ubiquitous, obligate intracellular eukaryotic spore-forming parasites infecting a wide range of invertebrates and vertebrates, including humans. The defining structure of microsporidia is the polar tube, which forms a hollow tube through which the sporoplasm is transferred to the host cell. Research on the molecular and cellular biology of the polar tube has resulted in the identification of three polar tube proteins: PTP1, PTP2, and PTP3. The major polar tube protein, PTP1, accounts for at least 70% of the mass of the polar tube. In the present study, PTP1 was found to be posttranslationally modified. Concanavalin A (ConA) bound to PTP1 and to the polar tube of several different microsporidia species. Analysis of the glycosylation of Encephalitozoon hellem PTP1 suggested that it is modified by O-linked mannosylation, and ConA binds to these O-linked mannose residues. Mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose-binding molecule. A CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to E. hellem infection compared to wild-type CHO cells. These data suggest that the O-mannosylation of PTP1 may have functional significance for the ability of microsporidia to invade their host cells.

  9. Vasoactive intestinal polypeptide immunoreactivity in the human cerebellum: qualitative and quantitative analyses

    PubMed Central

    Benagiano, Vincenzo; Flace, Paolo; Lorusso, Loredana; Rizzi, Anna; Bosco, Lorenzo; Cagiano, Raffaele; Ambrosi, Glauco

    2009-01-01

    Although autoradiographic, reverse transcription-polymerase chain reaction and immunohistochemical studies have demonstrated receptors for vasoactive intestinal polypeptide (VIP) in the cerebellum of various species, immunohistochemistry has never shown immunoreactivity for VIP within cerebellar neuronal bodies and processes. The present study aimed to ascertain whether VIP immunoreactivity really does exist in the human cerebellum by making a systematic analysis of samples removed post-mortem from all of the cerebellar lobes. The study was carried out using light microscopy immunohistochemical techniques based on a set of four different antibodies (three polyclonal and one monoclonal) against VIP, carefully selected on the basis of control tests performed on human colon. All of the antibodies used showed VIP-immunoreactive neuronal bodies and processes distributed in the cerebellar cortex and subjacent white matter of all of the cerebellum lobes, having similar qualitative patterns of distribution. Immunoreactive neurons included subpopulations of the main neuron types of the cortex. Statistical analysis of the quantitative data on the VIP immunoreactivity revealed by the different antibodies in the different cerebellar lobes did not demonstrate any significant differences. In conclusion, using four different anti-VIP antibodies, the first evidence of VIP immunoreactivity is herein supplied in the human post-mortem cerebellum, with similar qualitative/quantitative patterns of distribution among the different cerebellum lobes. Owing to the function performed by VIP as a neurotransmitter/neuromodulator, it is a candidate for a role in intrinsic and extrinsic (projective) circuits of the cerebellum, in agreement with previous demonstrations of receptors for VIP in the cerebellar cortex and nuclei. As VIP signalling pathways are implicated in the regulation of cognitive and psychic functions, cerebral blood flow and metabolism, processes of histomorphogenesis

  10. Structural and Biochemical Characterization of a Novel Aminopeptidase from Human Intestine*

    PubMed Central

    Tykvart, Jan; Bařinka, Cyril; Svoboda, Michal; Navrátil, Václav; Souček, Radko; Hubálek, Martin; Hradilek, Martin; Šácha, Pavel; Lubkowski, Jacek; Konvalinka, Jan

    2015-01-01

    N-acetylated α-linked acidic dipeptidase-like protein (NAALADase L), encoded by the NAALADL1 gene, is a close homolog of glutamate carboxypeptidase II, a metallopeptidase that has been intensively studied as a target for imaging and therapy of solid malignancies and neuropathologies. However, neither the physiological functions nor structural features of NAALADase L are known at present. Here, we report a thorough characterization of the protein product of the human NAALADL1 gene, including heterologous overexpression and purification, structural and biochemical characterization, and analysis of its expression profile. By solving the NAALADase L x-ray structure, we provide the first experimental evidence that it is a zinc-dependent metallopeptidase with a catalytic mechanism similar to that of glutamate carboxypeptidase II yet distinct substrate specificity. A proteome-based assay revealed that the NAALADL1 gene product possesses previously unrecognized aminopeptidase activity but no carboxy- or endopeptidase activity. These findings were corroborated by site-directed mutagenesis and identification of bestatin as a potent inhibitor of the enzyme. Analysis of NAALADL1 gene expression at both the mRNA and protein levels revealed the small intestine as the major site of protein expression and points toward extensive alternative splicing of the NAALADL1 gene transcript. Taken together, our data imply that the NAALADL1 gene product's primary physiological function is associated with the final stages of protein/peptide digestion and absorption in the human digestive system. Based on these results, we suggest a new name for this enzyme: human ileal aminopeptidase (HILAP). PMID:25752612

  11. Spectroscopic issues in optical polarization of 3He gas for Magnetic Resonance Imaging of human lungs

    NASA Astrophysics Data System (ADS)

    Dohnalik, T.; Głowacz, B.; Olejniczak, Z.; Pałasz, T.; Suchanek, M.; Wojna, A.

    2013-10-01

    The Magnetic Resonance Imaging (MRI) of human lungs for diagnostic purposes became possible by using nuclear spin hyperpolarized noble gases, such as 3He. One of the methods to polarize 3He is the Metastability Exchange Optical Pumping (MEOP), which up to now has been performed at low pressure of about 1 mbar and in low magnetic field below 0.1 T (standard conditions). The equilibrium nuclear polarization can reach up to 80%, but it is dramatically reduced during the subsequent gas compression to the atmospheric pressure that is necessary for the lungs examination. Further polarization losses occur during the transportation of the gas to the hospital scanner. It was shown recently that up to 50% polarization can be obtained at elevated pressure exceeding 20 mbar, by using magnetic field higher than 0.1 T (nonstandard conditions). Therefore, following the construction of the low-field MEOP polarizer located in the lab, a dedicated portable unit was developed, which uses the magnetic field of the 1.5 T MR medical scanner and works in the continuous-flow regime. The first in Poland MRI images of human lungs in vivo were obtained on the upgraded to 3He resonance frequency Siemens Sonata medical scanner. An evident improvement in the image quality was achieved when using the new technique. The paper shows how spectroscopic measurements of 3He carried out in various experimental conditions led both to useful practical results and to significant progress in understanding fundamental processes taking place during MEOP.

  12. Influence of rifampicin on the expression and function of human intestinal cytochrome P450 enzymes

    PubMed Central

    Glaeser, H; Drescher, S; Eichelbaum, M; Fromm, M F

    2005-01-01

    List of nonstandard abbreviations mlpc multilumen perfusion catheter TMPD: transmucosal potential difference a.u. arbitrary unit Aims To investigate the potential induction by rifampicin of intestinal CYP2C8, CYP2C9, CYP2D6 and CYP3A4 using preparations of human enterocytes. Methods Using a multilumen perfusion catheter shed human enterocytes were collected from 6 healthy subjects before and after 10 days of 600 mg day−1 oral rifampicin administration. The protein expression of CYP2C8, CYP2C9, CYP2D6 and CYP3A4 as well as that of CYP3A4 mRNA was determined using Western blotting and RT-PCR, respectively. Results CYP3A4 mRNA expression in shed enterocytes increased from 74.6 ± 44.2 to 143.2 ± 68.4 a.u. (P < 0.05, 95% CI: 21.8–115.3). Expression of CYP2C8 and CYP2C9 increased from 5.1 ± 0.9 to 10.4 ± 2.3 pmol mg−1 protein (P < 0.01, 95% CI: 2.8–7.7) and from 4.2 ± 1.4 to 5.7 ± 1.1 pmol mg−1 protein (P < 0.01, 95% CI: 0.6–2.4), respectively. No significant difference in CYP2D6 expression before and during rifampicin intake was observed. Rifampicin administration also resulted in a significant induction of CYP3A4 protein (34.1 ± 10.7 vs. 113.9 ± 31.1 pmol mg−1 protein (P < 0.001, 95% CI: 51.8–107.6)). Ex vivo incubation of enterocyte homogenates with verapamil resulted in a significantly increased production of the metabolites formed via CYP3A4 (D-617: 125.9 ± 118.8 vs. 277.2 ± 145.5 pmol min−1 mg−1 protein (P < 0.05, 95% CI: 30.1–272.5); norverapamil: 113.0 ± 57.9 vs. 398.4 ± 148.2 pmol min−1 mg−1 protein (P < 0.05, 95% CI: 47.2–523.6)). Conclusion Our findings indicate that shed enterocytes are a useful tool to study the expression, regulation and function of drug metabolizing enzymes. Induction of intestinal CYP2C8 and CYP2C9 might contribute in part to rifampicin – mediated drug interactions, in addition to their hepatic counterparts and intestinal and hepatic CYP3A4. PMID:15676042

  13. Determination of tolerable fatty acids and cholera toxin concentrations using human intestinal epithelial cells and BALB/c mouse macrophages.

    PubMed

    Tamari, Farshad; Tychowski, Joanna; Lorentzen, Laura

    2013-05-30

    The positive role of fatty acids in the prevention and alleviation of non-human and human diseases have been and continue to be extensively documented. These roles include influences on infectious and non-infectious diseases including prevention of inflammation as well as mucosal immunity to infectious diseases. Cholera is an acute intestinal illness caused by the bacterium Vibrio cholerae. It occurs in developing nations and if left untreated, can result in death. While vaccines for cholera exist, they are not always effective and other preventative methods are needed. We set out to determine tolerable concentrations of three fatty acids (oleic, linoleic and linolenic acids) and cholera toxin using mouse BALB/C macrophages and human intestinal epithelial cells, respectively. We solubilized the above fatty acids and used cell proliferation assays to determine the concentration ranges and specific concentrations of the fatty acids that are not detrimental to human intestinal epithelial cell viability. We solubilized cholera toxin and used it in an assay to determine the concentration ranges and specific concentrations of cholera toxin that do not statistically decrease cell viability in BALB/C macrophages. We found the optimum fatty acid concentrations to be between 1-5 ng/μl, and that for cholera toxin to be < 30 ng per treatment. This data may aid future studies that aim to find a protective mucosal role for fatty acids in prevention or alleviation of cholera infections.

  14. In vitro metabolism of the glycosidic sweeteners, stevia mixture and enzymatically modified stevia in human intestinal microflora.

    PubMed

    Koyama, E; Kitazawa, K; Ohori, Y; Izawa, O; Kakegawa, K; Fujino, A; Ui, M

    2003-03-01

    Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified stevia) by LC/MS/ESI analysis. Degradation was examined by incubating stevia mixture, enzymatically modified stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers) for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified stevia, stevioside and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation is consistent with previous rat metabolism studies. Similarly, enzymatically modified stevia appeared to be metabolized via stevia components and, finally, to steviol. This study suggests that there are apparently no species differences in intestinal metabolism of stevia mixture between rats and humans.

  15. Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo

    PubMed Central

    Finkbeiner, Stacy R.; Hill, David R.; Altheim, Christopher H.; Dedhia, Priya H.; Taylor, Matthew J.; Tsai, Yu-Hwai; Chin, Alana M.; Mahe, Maxime M.; Watson, Carey L.; Freeman, Jennifer J.; Nattiv, Roy; Thomson, Matthew; Klein, Ophir D.; Shroyer, Noah F.; Helmrath, Michael A.; Teitelbaum, Daniel H.; Dempsey, Peter J.; Spence, Jason R.

    2015-01-01

    Summary Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation. PMID:26050928

  16. Effect of linear alkylbenzene sulfonate (LAS) on human intestinal Caco-2 cells at non cytotoxic concentrations.

    PubMed

    Bradai, Mohamed; Han, Junkyu; Omri, Abdelfatteh El; Funamizu, Naoyuki; Sayadi, Sami; Isoda, Hiroko

    2016-08-01

    Linear alkylbenzene sulfonate (LAS) is a cytotoxic synthetic anionic surfactant widely present in the environment due to its large-scale production and intensive use in the detergency field. In this study, we investigated the effect of LAS (CAS No. 25155-30-0) at non cytotoxic concentrations on human intestinal Caco-2 cells using different in vitro bioassays. As results, LAS increased Caco-2 cell proliferation at concentrations ranging from 1 to 15 ppm, more significantly for shorter exposure time (24 h), confirmed using flow cytometry and trypan blue exclusion methods. Moreover, proteomics analysis revealed that this effect was associated with an over-expression of elongation factor 2 and dipeptidyl peptidase 3, and a down-regulation of 14-3-3 protein theta, confirmed at mRNA level using real-time PCR. These findings suggest that LAS at non cytotoxic concentrations, similar to those observed at wastewater treatment plants outlets, increases the growth rate of colon cancer cells, raising thereby its tumor promotion effect potential.

  17. Assessment of the prebiotic effect of quinoa and amaranth in the human intestinal ecosystem.

    PubMed

    Gullón, Beatriz; Gullón, Patricia; Tavaria, Freni K; Yáñez, Remedios

    2016-09-14

    Quinoa and amaranth belong to the group of the so called "superfoods" and have a nutritional composition that confers multiple benefits. In this work, we explored the possibility of these foods exhibiting a prebiotic effect. These pseudocereals were subjected to an in vitro digestion and used as carbon sources in batch cultures with faecal human inocula. The effects on the microbiota composition and their metabolic products were determined by assessment of variations in pH, short-chain fatty acid (SCFA) production and changes in the dynamic bacterial populations by fluorescence in situ hybridization (FISH). After 48 h of incubation, the total SCFAs were 106.5 mM for quinoa and 108.83 mM for amaranth, in line with the decrease in pH. Considerable differences (p < 0.05) were found in certain microbial groups, including Bifidobacterium spp., Lactobacillus-Enterococcus, Atopobium, Bacteroides-Prevotella, Clostridium coccoides-Eubacterium rectale, Faecalibacterium prausnitzii and Roseburia intestinalis. Our research suggests that these pseudocereals can have the prebiotic potential and that their intake may improve dysbiosis or maintain the gastrointestinal health through a balanced intestinal microbiota, although additional studies are necessary.

  18. Investigation of intestinal parasites in pig feces that are also human pathogens.

    PubMed

    Uysal, Hayriye Kirkoyun; Boral, Ozden; Metiner, Kemal; Ilgaz, Atilla

    2009-01-01

    A total of 238 pig fecal specimens were collected from pig farms in Corlu (Tekirdağ), Ayazma, and Arnavutköy (Istanbul) during the summer. Out of the 238 pig specimens, 105 were from pigs younger than 6 months and 133 from pigs older than 6 months. These were investigated for intestine parasites in particular the ones that are human pathogens. Cryptosporidium spp. was detected In 21 fecal specimens (8.8%), Giardia spp. in 9 (3.7%), Balantidium coli cysts in 4 (1.6%) and Ascaris suum eggs in 9 (4.1%). Giardia lamblia were found in 8 (7.6%) of 105 pigs younger than 6 months, Cryptosporidium spp. in 12 (11.4%), Balantidium coli cysts in 2 (1.5%). In the pigs older than 6 months Giardia lamblia were found in 1 (0.7%), Cryptosporidium spp. in 9 (6.7%), Balantidium coli cysts in 2 (1.5%). and Ascaris suum eggs in 9 (6.7%). The difference in the rate of G. lamblia (p=0.01) in pigs less than 6 months and of A. suum in those over 6 months was found to be statistically significant (p=0.005). Our results revealed that pigs are important sources of these parasites.

  19. Transepithelial transports of rare sugar D-psicose in human intestine.

    PubMed

    Hishiike, Takashi; Ogawa, Masahiro; Hayakawa, Shigeru; Nakajima, Daichi; O'Charoen, Siwaporn; Ooshima, Hisaka; Sun, Yuanxia

    2013-07-31

    D-Psicose (Psi), the C3-epimer of D-fructose (Fru), is a noncalorie sugar with a lower glycemic response. The trans-cellular pathway of Psi in human enterocytes was investigated using a Caco-2 cell monolayer. The permeation rate of Psi across the monolayer was not affected by the addition of phlorizin, an inhibitor of sugar transporter SGLT1, whereas it was accelerated by treatment with forskolin, a GLUT5-gene inducer, clearly showing that GLUT5 is involved in the transport of Psi. The permeability of Psi was suppressed in the presence of D-glucose (Glc) and Fru, suggesting that the three monosaccharides are transported via the same transporter. Since GLUT2, the predominant sugar transporter on the basolateral membrane of enterocytes, mediates the transport of Glc and Fru, Psi might be mediated by GLUT2. The present study shows that Psi is incorporated from the intestinal lumen into enterocytes via GLUT5 and is released to the lamina propria via GLUT2.

  20. Fermentation in the human large intestine: its physiologic consequences and the potential contribution of prebiotics.

    PubMed

    Macfarlane, George T; Macfarlane, Sandra

    2011-11-01

    The human large intestine harbors a complex microbiota containing many hundreds of different bacterial species. Although structure/function relationships between different components of the microbiota are unclear, this complex multicellular entity plays an important role in maintaining homeostasis in the body. Many of the physiologic properties of the microbiota can be attributed to fermentation and the production of short-chain fatty acids (SCFAs), particularly acetate, propionate, and butyrate. In healthy people, fermentation processes are largely controlled by the amounts and different types of substrate, particularly complex carbohydrates that are accessible to bacteria in the colonic ecosystem. However, other factors impact on bacterial metabolism in the large gut, including large bowel transit time, the availability of inorganic terminal electron acceptors, such as nitrate and sulfate, and gut pH. They all affect the types and levels of SCFA that can be formed by the microbiota. This is important because to a large extent, acetate, propionate, and butyrate have varying physiologic effects in different body tissues. Prebiotics such as galactooligosaccharides together with inulins and their fructooligosaccharide derivatives have been shown to modify the species composition of the colonic microbiota, and in various degrees, to manifest several health-promoting properties related to enhanced mineral absorption, laxation, potential anticancer properties, lipid metabolism, and anti-inflammatory and other immune effects, including atopic disease. Many of these phenomena can be linked to their digestion and SCFA production by bacteria in the large gut.

  1. Prediction of Human intestinal absorption of compounds using artificial intelligence techniques.

    PubMed

    Kumar, Rajnish; Sharma, Anju; Siddiqui, Mohammed Haris; Tiwari, Rajesh Kumar

    2017-04-04

    Information about Pharmacokinetics of compounds is an essential component of drug design and development. Modeling the pharmacokinetic properties require identification of the factors effecting absorption, distribution, metabolism and excretion of compounds. There have been continuous attempts in the prediction of absorption of compounds using various Artificial intelligence methods in the effort to reduce the attrition rate of drug candidates entering to preclinical and clinical trials. Currently, there are large numbers of individual predictive models available for absorption using machine learning approaches. In current work, we are presenting a comprehensive study of prediction of absorption. Six Artificial intelligence methods namely, Support vector machine, k- nearest neighbor, Probabilistic neural network, Artificial neural network, Partial least square and Linear discriminant analysis were used for prediction of absorption of compounds with prediction accuracy of 91.54%, 88.33%, 84.30%, 86.51%, 79.07% and 80.08% respectively. Comparative analysis of all the six prediction models suggested that Support vector machine with Radial basis function based kernel is comparatively better for binary classification of compounds using human intestinal absorption and may be useful at preliminary stages of drug design and development.

  2. In vitro extraction and fermentation of polyphenols from grape seeds (Vitis vinifera) by human intestinal microbiota.

    PubMed

    Zhou, Li; Wang, Wei; Huang, Jun; Ding, Yu; Pan, Zhouqiang; Zhao, Ya; Zhang, Renkang; Hu, Bing; Zeng, Xiaoxiong

    2016-04-01

    The effects of several parameters on the extraction yield of total polyphenols from grape seeds by pressurized liquid extraction were investigated. The highest recovery of total polyphenols occurred at 80 °C within 5 min, and a single extraction allowed a recovery of more than 97% of total polyphenols. Following the purification with macroporous resin, the effects of grape polyphenols (>94.8%) on human intestinal microbiota were monitored over 36 h incubation by fluorescence in situ hybridization, and short-chain fatty acids (SCFAs) were measured by HPLC. The result showed that the grape polyphenols promoted the changes in the relevant microbial populations and shifted the profiles of SCFAs. Fermentation of grape polyphenols resulted in a significant increase in the numbers of Bifidobacterium spp. and Lactobacillus-Enterococcus group and inhibition in the growth of the Clostridium histolyticum group and the Bacteroides-Prevotella group, with no significant effect on the population of total bacteria. The findings suggest that grape polyphenols have potential prebiotic effects on modulating the gut microbiota composition and generating SCFAs that contribute to the improvements of host health.

  3. Transport of Antihypertensive Peptide RVPSL, Ovotransferrin 328-332, in Human Intestinal Caco-2 Cell Monolayers.

    PubMed

    Ding, Long; Wang, Liying; Zhang, Yan; Liu, Jingbo

    2015-09-23

    The objective of this study was to investigate the transepithelial transport of RVPSL (Arg-Val-Pro-Ser-Leu), an egg-white-derived peptide with angiotensin I-converting enzyme (ACE) inhibitory and antihypertensive activity, in human intestinal Caco-2 cell monolayers. Results revealed that RVPSL could be passively transported across Caco-2 cell monolayers. However, during the process of transport, 36.31% ± 1.22% of the initial RVPSL added to the apical side was degraded, but this degradation decreased to 23.49% ± 0.68% when the Caco-2 cell monolayers were preincubated with diprotin A (P < 0.001), suggesting that RVPSL had a low resistance to various brush border membrane peptidases. When transport from the apical side to the basolateral side was investigated, the apparent permeability coefficient (Papp) was (6.97 ± 1.11) × 10(-6) cm/s. The transport route of RVPSL appears to be the paracellular pathway via tight junctions, as only cytochalasin D, a disruptor of tight junctions (TJs), significantly increased the transport rate (P < 0.001). In addition, the relationship between the structure of RVPSL and transport across Caco-2 cell monolayers was studied by mutation of RVPSL. It was found that N-terminal Pro residues were more beneficial for transport of pentapeptides across Caco-2 cell monolayers than Arg and Val. Furthermore, RVPSL could be more easily transported as smaller peptides, especially in the form of dipeptides and tripeptides.

  4. Influence of Phenol-Enriched Olive Oils on Human Intestinal Immune Function

    PubMed Central

    Martín-Peláez, Sandra; Castañer, Olga; Solà, Rosa; Motilva, María José; Castell, Margarida; Pérez-Cano, Francisco José; Fitó, Montserrat

    2016-01-01

    Olive oil (OO) phenolic compounds (PC) are able to influence gut microbial populations and metabolic output. Our aim was to investigate whether these compounds and changes affect the mucosal immune system. In a randomized, controlled, double blind cross-over human trial, for three weeks, preceded by two-week washout periods, 10 hypercholesterolemic participants ingested 25 mL/day of three raw virgin OO differing in their PC concentration and origin: (1) an OO containing 80 mg PC/kg (VOO); (2) a PC-enriched OO containing 500 mg PC/kg from OO (FVOO); and (3) a PC-enriched OO containing a mixture of 500 mg PC/kg from OO and thyme (1:1, FVOOT). Intestinal immunity (fecal immunoglobulin A (IgA) and IgA-coated bacteria) and inflammation markers (C-reactive protein (CRP) and fecal interleukin 6 (IL-6), tumor necrosis factor α (TNFα) and calprotectin) was analyzed. The ingestion of high amounts of OO PC, as contained in FVOO, tended to increase the proportions of IgA-coated bacteria and increased plasma levels of CRP. However, lower amounts of OO PC (VOO) and the combination of two PC sources (FVOOT) did not show significant effects on the variables investigated. Results indicate a potential stimulation of the immune system with very high doses of OO PC, which should be further investigated. PMID:27077879

  5. CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells

    PubMed Central

    Gracz, Adam D.; Fuller, Megan K.; Wang, Fengchao; Li, Linheng; Stelzner, Matthias; Dunn, James C.Y.; Martin, Martin G.; Magness, Scott T.

    2013-01-01

    Recent seminal studies have rapidly advanced the understanding of intestinal epithelial stem cell (IESC) biology in murine models. However, the lack of techniques suitable for isolation and subsequent downstream analysis of IESCs from human tissue has hindered the application of these findings toward the development of novel diagnostics and therapies with direct clinical relevance. This study demonstrates that the cluster of differentiation genes CD24 and CD44 are differentially expressed across LGR5 positive “active” stem cells as well as HOPX positive “facultative” stem cells. Fluorescence-activated cell sorting enables differential enrichment of LGR5 cells (CD24−/CD44+) and HOPX (CD24+/CD44+) cells for gene expression analysis and culture. These findings provide the fundamental methodology and basic cell surface signature necessary for isolating and studying intestinal stem cell populations in human physiology and disease. PMID:23553902

  6. Human Regional Pulmonary Gas Exchange with Xenon Polarization Transfer (XTC)

    NASA Astrophysics Data System (ADS)

    Muradian, Iga; Butler, James; Hrovat, Mirko; Topulos, George; Hersman, Elizabeth; Ruset, Iulian; Covrig, Silviu; Frederick, Eric; Ketel, Stephen; Hersman, F. W.; Patz, Samuel

    2007-03-01

    Xenon Transfer Contrast (XTC) is an existing imaging method (Ruppert et al, Magn Reson Med, 51:676-687, 2004) that measures the fraction F of ^129Xe magnetization that diffuses from alveolar gas spaces to septal parenchymal tissue in lungs in a specified exchange time. As previously implemented, XTC is a 2-breath method and has been demonstrated in anesthetized animals. To use XTC in humans and to avoid issues associated with obtaining identical gas volumes on subsequent breath-hold experiments as well as precise image registration in post-processing, a single breath XTC method was developed that acquires three consecutive gradient echo images in an 8s acquisition. We report here initial measurements of the mean and variance of F for 5 normal healthy subjects as well as 7 asymptomatic smokers. The experiments were performed at two lung volumes (˜45 and 65% of TLC). We found that both the mean and variance of F increased with smoking history. In comparison, standard pulmonary function tests such as DLCO FEV1 showed no correlation with smoking history.

  7. Improvement in Human Immune Function with Changes in Intestinal Microbiota by Salacia reticulata Extract Ingestion: A Randomized Placebo-Controlled Trial

    PubMed Central

    Oda, Yuriko; Ueda, Fumitaka; Utsuyama, Masanori; Kamei, Asuka; Kakinuma, Chihaya; Abe, Keiko; Hirokawa, Katsuiku

    2015-01-01

    Plants belonging to the genus Salacia in the Hippocrateaceae family are known to inhibit sugar absorption. In a previous study, administration of Salacia reticulata extract in rats altered the intestinal microbiota and increased expression of immune-relevant genes in small intestinal epithelial cells. This study aimed to investigate the effect of S. reticulata extract in human subjects by examining the gene expression profiles of blood cells, immunological indices, and intestinal microbiota. The results revealed an improvement in T-cell proliferation activity and some other immunological indices. In addition, the intestinal microbiota changed, with an increase in Bifidobacterium and a decrease in Clostridium bacteria. The expression levels of many immune-relevant genes were altered in blood cells. We concluded that S. reticulata extract ingestion in humans improved immune functions and changed the intestinal microbiota. Trial Registration: UMIN Clinical Trials Registry UMIN000011732 PMID:26630568

  8. Crohn disease–associated adherent-invasive E. coli bacteria target mouse and human Peyer’s patches via long polar fimbriae

    PubMed Central

    Chassaing, Benoit; Rolhion, Nathalie; de Vallée, Amélie; Salim, Sa’ad Y.; Prorok-Hamon, Maelle; Neut, Christel; Campbell, Barry J.; Söderholm, Johan D.; Hugot, Jean-Pierre; Colombel, Jean-Frédéric; Darfeuille-Michaud, Arlette

    2011-01-01

    Crohn disease (CD) is a multifactorial disease in which an abnormal immune response in the gastrointestinal (GI) tract leads to chronic inflammation. The small intestine, particularly the ileum, of patients with CD is colonized by adherent-invasive E. coli (AIEC) — a pathogenic group of E. coli able to adhere to and invade intestinal epithelial cells. As the earliest inflammatory lesions are microscopic erosions of the epithelium lining the Peyer’s patches (PPs), we investigated the ability of AIEC bacteria to interact with PPs and the virulence factors involved. We found that AIEC bacteria could interact with mouse and human PPs via long polar fimbriae (LPF). An LPF-negative AIEC mutant was highly impaired in its ability to interact with mouse and human PPs and to translocate across monolayers of M cells, specialized epithelial cells at the surface of PPs. The prevalence of AIEC strains harboring the lpf operon was markedly higher in CD patients compared with controls. In addition, increased numbers of AIEC, but not LPF-deficient AIEC, bacteria were found interacting with PPs from Nod2–/– mice compared with WT mice. In conclusion, we have identified LPF as a key factor for AIEC to target PPs. This could be the missing link between AIEC colonization and the presence of early lesions in the PPs of CD patients. PMID:21339647

  9. Survival of human parainfluenza viruses in the South Polar environment.

    PubMed Central

    Parkinson, A J; Muchmore, H G; Scott, E N; Scott, L V

    1983-01-01

    The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season. PMID:6314897

  10. Birefringence measurements in human skin using polarization-sensitive optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Pierce, Mark C.; Strasswimmer, John; Park, B. H.; Cense, Barry; de Boer, Johannes F.

    2004-07-01

    Optical coherence tomography enables cross-sectional imaging of tissue structure to depths of around 1.5 mm, at high-resolution and in real-time. Incorporation of polarization-sensitivity enables the birefringent properties of tissues to be visualized and quantified. We present polarization-sensitive optical coherence tomography images and quantitative birefringence analysis of in vivo human skin. From measurements on a sample of 5 human volunteers, mean double-pass phase retardation rates of 0.340 +/- 0.143, 0.250 +/- 0.076 and 0.592 +/- 0.142°/μm were obtained for normal skin at the dorsal hand, temple and lower back regions respectively. Compared to these values measured in normal skin, a reduction in birefringence was observed and quantified in human skin following thermal injury. Conversely, increased birefringence was consistently measured at skin sites following wound healing and repair.

  11. Animal models of intestinal fibrosis: new tools for the understanding of pathogenesis and therapy of human disease

    PubMed Central

    Rieder, Florian; Kessler, Sean; Sans, Miquel

    2012-01-01

    Fibrosis is a serious condition complicating chronic inflammatory processes affecting the intestinal tract. Advances in this field that rely on human studies have been slow and seriously restricted by practical and logistic reasons. As a consequence, well-characterized animal models of intestinal fibrosis have emerged as logical and essential systems to better define and understand the pathophysiology of fibrosis. In point of fact, animal models allow the execution of mechanistic studies as well as the implementation of clinical trials with novel, pathophysiology-based therapeutic approaches. This review provides an overview of the currently available animal models of intestinal fibrosis, taking into consideration the methods of induction, key characteristics of each model, and underlying mechanisms. Currently available models will be classified into seven categories: spontaneous, gene-targeted, chemical-, immune-, bacteria-, and radiation-induced as well as postoperative fibrosis. Each model will be discussed in regard to its potential to create research opportunities to gain insights into the mechanisms of intestinal fibrosis and stricture formation and assist in the development of effective and specific antifibrotic therapies. PMID:22878121

  12. Bacillus megaterium SF185 induces stress pathways and affects the cell cycle distribution of human intestinal epithelial cells.

    PubMed

    Di Luccia, B; D'Apuzzo, E; Varriale, F; Baccigalupi, L; Ricca, E; Pollice, A

    2016-09-01

    The interaction between the enteric microbiota and intestinal cells often involves signal molecules that affect both microbial behaviour and host responses. Examples of such signal molecules are the molecules secreted by bacteria that induce quorum sensing mechanisms in the producing microorganism and signal transduction pathways in the host cells. The pentapeptide competence and sporulation factor (CSF) of Bacillus subtilis is a well characterized quorum sensing factor that controls competence and spore formation in the producing bacterium and induces cytoprotective heat shock proteins in intestinal epithelial cells. We analysed several Bacillus strains isolated from human ileal biopsies of healthy volunteers and observed that some of them were unable to produce CSF but still able to act in a CSF-like fashion on model intestinal epithelial cells. One of those strains belonging to the Bacillus megaterium species secreted at least two factors with effects on intestinal HT29 cells: a peptide smaller than 3 kDa able to induce heat shock protein 27 (hsp27) and p38-MAPK, and a larger molecule able to induce protein kinase B (PKB/Akt) with a pro-proliferative effect.

  13. Impact of Different Fecal Processing Methods on Assessments of Bacterial Diversity in the Human Intestine

    PubMed Central

    Hsieh, Yu-Hsin; Peterson, Courtney M.; Raggio, Anne; Keenan, Michael J.; Martin, Roy J.; Ravussin, Eric; Marco, Maria L.

    2016-01-01

    The intestinal microbiota are integral to understanding the relationships between nutrition and health. Therefore, fecal sampling and processing protocols for metagenomic surveys should be sufficiently robust, accurate, and reliable to identify the microorganisms present. We investigated the use of different fecal preparation methods on the bacterial community structures identified in human stools. Complete stools were collected from six healthy individuals and processed according to the following methods: (i) randomly sampled fresh stool, (ii) fresh stool homogenized in a blender for 2 min, (iii) randomly sampled frozen stool, and (iv) frozen stool homogenized in a blender for 2 min, or (v) homogenized in a pneumatic mixer for either 10, 20, or 30 min. High-throughput DNA sequencing of the 16S rRNA V4 regions of bacterial community DNA extracted from the stools showed that the fecal microbiota remained distinct between individuals, independent of processing method. Moreover, the different stool preparation approaches did not alter intra-individual bacterial diversity. Distinctions were found at the level of individual taxa, however. Stools that were frozen and then homogenized tended to have higher proportions of Faecalibacterium, Streptococcus, and Bifidobacterium and decreased quantities of Oscillospira, Bacteroides, and Parabacteroides compared to stools that were collected in small quantities and not mixed prior to DNA extraction. These findings indicate that certain taxa are at particular risk for under or over sampling due to protocol differences. Importantly, homogenization by any method significantly reduced the intra-individual variation in bacteria detected per stool. Our results confirm the robustness of fecal homogenization for microbial analyses and underscore the value of collecting and mixing large stool sample quantities in human nutrition intervention studies. PMID:27812352

  14. Conversion of 5-fluorocytosine to 5-fluorouracil by human intestinal microflora

    SciTech Connect

    Harris, B.; Manning, B.; Federle, T.; Diasio, R.

    1986-03-01

    5-Fluorocytosine (FC) is used to treat systemic fungal infections in man. Its clinical effectiveness has been limited by hematologic toxicity which may be secondary to the formation of 5-fluorouracil (FU). It is unclear how FU is formed since human cells lack cytosine deaminase. The present study examined if intestinal microflora (IMF) could convert FC to FU in man. An in vitro semicontinuous culture system was inoculated with human feces and maintained with sterile nutrient suspension. The microbial community was assessed for cell count and anaerobes as well as formation of volatile fatty acids and CH/sub 4/. The system approximated that believed to occur in vivo. The study was initiated with addition of purified (6-/sup 14/C)-FC. Unlabelled FC was then added to the system daily for 2 weeks following which (6-/sup 14/C)-FC was again added. Following each addition of (6-/sup 14/C)-FC, samples were removed at 2,4,8,24,48,72, and 96 hr. Utilizing HPLC, FC and FU could be separated with quantitation of radioactivity in each peak. Following the initial dose, no detectable FU was observed during the first 8 hr, but after 24 hr increasing levels were detected (9.42 ..mu..g FU/ml after 4 days). Following chronic administration of FC, increased levles of FU were noted without an 8 hr lag time in the production of FU (31.86 ..mu..g FU/ml after 4 days). In summary, these studies demonstrate that IMF can convert FC to FU possibly accounting for toxicity observed following administration of FC.

  15. Corneal Endothelial Cell Integrity in Precut Human Donor Corneas Enhanced by Autocrine Vasoactive Intestinal Peptide

    PubMed Central

    Coll, Timothy; Gloria, Dante; Sprehe, Nicholas

    2017-01-01

    Purpose: To demonstrate that vasoactive intestinal peptide (VIP), a corneal endothelial (CE) cell autocrine factor, maintains the integrity of corneal endothelium in human donor corneoscleral explants precut for endothelial keratoplasty. Methods: Twelve paired human donor corneoscleral explants used as control versus VIP-treated explants (10 nM, 30 minutes, 37°C) were shipped (4°C) to the Lions Eye Institute for Transplantation and Research for precutting (Moria CBM-ALTK Keratome), shipped back to the laboratory, and cultured in ciliary neurotrophic factor (CNTF, 0.83 nM, 37°C, 24 hours). Trephined endothelial discs (8–8.5 mm) were analyzed for differentiation markers (N-cadherin, CNTF receptor α subunit [CNTFRα], and connexin 43) by Western blot after a quarter of the discs from 4 paired explants were cut away and stained with alizarin red S for microscopic damage analysis. Two additional paired explants (6 days in culture) were stained for panoramic view of central CE damage. Results: VIP treatment increased N-cadherin and CNTFRα levels (mean ± SEM) to 1.38 ± 0.11-fold (P = 0.003) and 1.46 ± 0.22-fold (P = 0.03) of paired controls, respectively, whereas CE cell CNTF responsiveness in upregulation of connexin 43 increased to 2.02 ± 0.5 (mean ± SEM)-fold of the controls (P = 0.04). CE damage decreased from (mean ± SEM) 10.0% ± 1.2% to 1.6% ± 0.3% (P < 0.0001) and 9.1% ± 1.1% to 2.4% ± 1.0% (P = 0.0006). After 6 days in culture, the damage in whole CE discs decreased from 20.0% (control) to 5.5% (VIP treated). Conclusions: VIP treatment before precut enhanced the preservation of corneal endothelium. PMID:28181929

  16. FMRI analysis of contrast polarity processing in face-selective cortex in humans and monkeys

    PubMed Central

    Yue, Xiaomin; Nasr, Shahin; Devaney, Kathryn J.; Holt, Daphne J.; Tootell, Roger B.H.

    2013-01-01

    Recognition is strongly impaired when the normal contrast polarity of faces is reversed. For instance, otherwise-familiar faces become very difficult to recognize when viewed as photographic negatives. Here, we used fMRI to demonstrate related properties in visual cortex: 1) fMRI responses in the human Fusiform Face Area (FFA) decreased strongly (26%) to contrast-reversed faces across a wide range of contrast levels (5.3-100% RMS contrast), in all subjects tested. In a whole brain analysis, this contrast polarity bias was largely confined to the Fusiform Face Area (FFA; p < 0.0001), with possible involvement of a left occipital face-selective region. 2) It is known that reversing facial contrast affects three image properties in parallel (absorbance, shading, and specular reflection). Here, comparison of FFA responses to those in V1 suggests that the contrast polarity bias is produced in FFA only when all three component properties were reversed simultaneously, which suggests a prominent non-linearity in FFA processing. 3) Across a wide range (180°) of illumination source angles, 3D face shapes without texture produced response constancy in FFA, without a contrast polarity bias. 4) Consistent with psychophysics, analogous fMRI biases for normal contrast polarity were not produced by non-face objects, with image statistics similar to the face stimuli. 5) Using fMRI, we also demonstrated a contrast polarity bias in awake behaving macaque monkeys, in the cortical region considered homologous to human FFA. Thus common cortical mechanisms may underlie facial contrast processing across ~ 25 million years of primate evolution. PMID:23518007

  17. Transport mechanisms of nicotine across the human intestinal epithelial cell line Caco-2.

    PubMed

    Fukada, Atsuko; Saito, Hideyuki; Inui, Ken-Ichi

    2002-08-01

    Ulcerative colitis is a disease more commonly seen in nonsmokers. Because nicotine was postulated to be a beneficial component of tobacco smoke for ulcerative colitis, various formulations of nicotine have been developed to improve the local bioavailability within the gastrointestinal tissue. In the present study, to characterize the disposition of nicotine in the intestines, we investigated intestinal nicotine transport using Caco-2 cells. Nicotine was predominantly transported across Caco-2 cell monolayers in a unidirectional mode, corresponding to intestinal secretion, by pH-dependent specific transport systems. The specific uptake systems appear to be distinct from organic cation transporters and the transport system for tertiary amines, in terms of its substrate specificity and the pattern of the interaction. These transport systems could play a role in the intestinal accumulation of nicotine from plasma and could also be responsible for the topical delivery of nicotine for ulcerative colitis therapy. These findings could provide useful information for the design of effective nicotine delivery.

  18. Predicting human intestinal absorption in the presence of bile salt with micellar liquid chromatography.

    PubMed

    Waters, Laura J; Shokry, Dina S; Parkes, Gareth M B

    2016-10-01

    Understanding intestinal absorption for pharmaceutical compounds is vital to estimate the bioavailability and therefore the in vivo potential of a drug. This study considers the application of micellar liquid chromatography (MLC) to predict passive intestinal absorption with a selection of model compounds. MLC is already known to aid prediction of absorption using simple surfactant systems; however, with this study the focus was on the presence of a more complex, bile salt surfactant, as would be encountered in the in vivo environment. As a result, MLC using a specific bile salt has been confirmed as an ideal in vitro system to predict the intestinal permeability for a wide range of drugs, through the development of a quantitative partition-absorption relationship. MLC offers many benefits including environmental, economic, time-saving and ethical advantages compared with the traditional techniques employed to obtain passive intestinal absorption values. Copyright © 2016 John Wiley & Sons, Ltd.

  19. miR-802 regulates human angiotensin II type 1 receptor expression in intestinal epithelial C2BBe1 cells

    PubMed Central

    Sansom, Sarah E.; Nuovo, Gerard J.; Martin, Mickey M.; Kotha, Sainath R.; Parinandi, Narasimham L.

    2010-01-01

    Studies have demonstrated that angiotensin II (Ang II) can regulate intestinal fluid and electrolyte transport and control intestinal wall muscular activity. Ang II is also a proinflammatory mediator that participates in inflammatory responses such as apoptosis, angiogenesis, and vascular remodeling; accumulating evidence suggests that this hormone may be involved in gastrointestinal (GI) inflammation and carcinogenesis. Ang II binds to two distinct G protein-coupled receptor subtypes, the AT1R and AT2R, which are widely expressed in the GI system. Together these studies suggest that Ang II-AT1R/-AT2R actions may play an important role in GI tract physiology and pathophysiology. Currently it is not known whether miRNAs can regulate the expression of the human AT1R (hAT1R) in the GI system. PCR and in situ hybridization experiments demonstrated that miR-802 was abundantly expressed in human colon and intestine. Luciferase reporter assays demonstrated that miR-802 could directly interact with the bioinformatics-predicted target site harbored within the 3′-untranslated region of the hAT1R mRNA. To validate that the levels of miR-802 were physiologically relevant in the GI system, we demonstrated that miR-802 “loss-of-function” experiments resulted in augmented hAT1R levels and enhanced Ang II-induced signaling in a human intestinal epithelial cell line. These results suggest that miR-802 can modulate the expression of the hAT1R in the GI tract and ultimately play a role in regulating the biological efficacy of Ang II in this system. PMID:20558762

  20. Structural diversity and specific distribution of O-glycans in normal human mucins along the intestinal tract

    PubMed Central

    2004-01-01

    Purified human mucins from different parts of the intestinal tract (ileum, cecum, transverse and sigmoid colon and rectum) were isolated from two individuals with blood group ALeb (A-Lewisb). After alkaline borohydride treatment the released oligosaccharides were structurally characterized by nano-ESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem MS) without prior fractionation or derivatization. More than 100 different oligosaccharides, with up to ten monosaccharide residues, were identified using this technique. Oligosaccharides based on core 3 structures, GlcNAc(β1-3)GalNAc (where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetylgalactosamine), were widely distributed in human intestinal mucins. Core 5 structures, GalNAc(α1-3)GalNAc, were also recovered in all fractions. Moreover, a comparison of the oligosaccharide repertoire, with respect to size, diversity and expression of glycans and terminal epitopes, showed a high level of mucin-specific glycosylation: highly fucosylated glycans, found specifically in the small intestine, were mainly based on core 4 structures, GlcNAc-(β1-3)[GlcNAc(β1-6)]GalNAc, whereas the sulpho-LeX determinant carrying core 2 glycans, Gal(β1-3)[GlcNAc(β1-6)]-GalNAc (where Gal is galactose), was recovered mainly in the distal colon. Blood group H and A antigenic determinants were present exclusively in the ileum and cecum, whereas blood group Sda/Cad related epitopes, GalNAc(β1-4)[NeuAc(α2-3)]Gal (where NeuAc is N-acetylneuraminate), were found to increase along the length of the colon. Our findings suggest that mucins create an enormous repertoire of potential binding sites for micro-organisms that could explain the regio-specific colonization of bacteria in the human intestinal tract. PMID:15361072

  1. The spatial arrangement of the human large intestinal wall blood circulation.

    PubMed

    Kachlik, David; Baca, Vaclav; Stingl, Josef

    2010-03-01

    The aim of the study was to describe and depict the spatial arrangement of the colon microcirculatory bed as a whole. Various parts of the large intestine and terminal ileum were harvested from either cadaver or section material or gained peroperatively. Samples were then injected with India ink or methylmetacrylate Mercox resin for microdissection and corrosion casting for scanning electron microscopy. The results showed that extramural vasa recta ramified to form the subserous plexus, some of them passing underneath the colon taeniae. Branches of both short and long vasa recta merged in the colon wall, pierced the muscular layer and spread out as the submucous plexus, which extended throughout the whole intestine without any interruption. The muscular layer received blood via both the centrifugal branches of the submucous plexus and the minor branches sent off by the subserous plexus. The mucosa was supplied by the mucous plexus, which sent capillaries into the walls of intestinal glands. The hexagonal arrangement of the intestinal glands reflected their vascular bed. All three presumptive critical points are only gross anatomical points of no physiological relevance in healthy individuals. Neither microscopic weak points nor regional differences were proven within the wall of the whole large intestine. The corrosion casts showed a huge density of capillaries under the mucosa of the large intestine. A regular hexagonal pattern of the vascular bed on the inner surface was revealed. No microvascular critical point proofs were confirmed and a correlation model to various pathological states was created.

  2. The spatial arrangement of the human large intestinal wall blood circulation

    PubMed Central

    Kachlik, David; Baca, Vaclav; Stingl, Josef

    2010-01-01

    The aim of the study was to describe and depict the spatial arrangement of the colon microcirculatory bed as a whole. Various parts of the large intestine and terminal ileum were harvested from either cadaver or section material or gained peroperatively. Samples were then injected with India ink or methylmetacrylate Mercox resin for microdissection and corrosion casting for scanning electron microscopy. The results showed that extramural vasa recta ramified to form the subserous plexus, some of them passing underneath the colon taeniae. Branches of both short and long vasa recta merged in the colon wall, pierced the muscular layer and spread out as the submucous plexus, which extended throughout the whole intestine without any interruption. The muscular layer received blood via both the centrifugal branches of the submucous plexus and the minor branches sent off by the subserous plexus. The mucosa was supplied by the mucous plexus, which sent capillaries into the walls of intestinal glands. The hexagonal arrangement of the intestinal glands reflected their vascular bed. All three presumptive critical points are only gross anatomical points of no physiological relevance in healthy individuals. Neither microscopic weak points nor regional differences were proven within the wall of the whole large intestine. The corrosion casts showed a huge density of capillaries under the mucosa of the large intestine. A regular hexagonal pattern of the vascular bed on the inner surface was revealed. No microvascular critical point proofs were confirmed and a correlation model to various pathological states was created. PMID:20447248

  3. Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells.

    PubMed

    Nabokina, Svetlana M; Ramos, Mel Brendan; Valle, Judith E; Said, Hamid M

    2015-05-01

    Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5'-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5'- and 3'-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides -178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

  4. Rapid conversion of the ester prodrug abiraterone acetate results in intestinal supersaturation and enhanced absorption of abiraterone: in vitro, rat in situ and human in vivo studies.

    PubMed

    Stappaerts, Jef; Geboers, Sophie; Snoeys, Jan; Brouwers, Joachim; Tack, Jan; Annaert, Pieter; Augustijns, Patrick

    2015-02-01

    The aim of this study was to evaluate the intestinal disposition of abiraterone acetate, an ester prodrug of the anticancer agent abiraterone. Stability of the prodrug and solubility and dissolution characteristics of both abiraterone and abiraterone acetate were monitored in vitro. Moreover, the in vivo intraluminal concentrations of abiraterone and abiraterone acetate upon intake of one tablet of 250 mg abiraterone acetate were assessed in healthy volunteers. The intestinal absorption resulting from the intraluminal behavior of the ester prodrug was determined using the rat in situ intestinal perfusion technique with mesenteric blood sampling. Simulated and aspirated human intestinal fluids of the fasted state were used as solvent systems. Upon incubation of abiraterone acetate in human intestinal fluids in vitro, rapid hydrolysis of the prodrug was observed, generating abiraterone concentrations largely exceeding the apparent solubility of abiraterone, suggesting the existence of intestinal supersaturation. These findings were confirmed in vivo, by intraluminal sampling of duodenal fluids upon oral intake of an abiraterone acetate tablet by healthy volunteers. Rat in situ intestinal perfusion experiments performed with suspensions of abiraterone and abiraterone acetate in human intestinal fluids of the fasted state revealed significantly higher flux values upon perfusion with the prodrug than with abiraterone. Moreover, rat in situ intestinal perfusion with abiraterone acetate suspensions in simulated fluids of the fasted state in presence or absence of esterases demonstrated that increased hydrolytic activity of the perfusion medium was beneficial to the intestinal absorption of abiraterone. In conclusion, the rapid hydrolysis of abiraterone acetate in the intraluminal environment appears to result in fast and extensive generation of abiraterone supersaturation, creating a strong driving force for abiraterone absorption.

  5. Intestinal Epithelial Barrier Disruption through Altered Mucosal MicroRNA Expression in Human Immunodeficiency Virus and Simian Immunodeficiency Virus Infections

    PubMed Central

    Gaulke, Christopher A.; Porter, Matthew; Han, Yan-Hong; Sankaran-Walters, Sumathi; Grishina, Irina; George, Michael D.; Dang, Angeline T.; Ding, Shou-Wei; Jiang, Guochun; Korf, Ian

    2014-01-01

    ABSTRACT Epithelial barrier dysfunction during human immunodeficiency virus (HIV) infection has largely been attributed to the rapid and severe depletion of CD4+ T cells in the gastrointestinal (GI) tract. Although it is known that changes in mucosal gene expression contribute to intestinal enteropathy, the role of small noncoding RNAs, specifically microRNA (miRNA), has not been investigated. Using the simian immunodeficiency virus (SIV)-infected nonhuman primate model of HIV pathogenesis, we investigated the effect of viral infection on miRNA expression in intestinal mucosa. SIV infection led to a striking decrease in the expression of mucosal miRNA compared to that in uninfected controls. This decrease coincided with an increase in 5′-3′-exoribonuclease 2 protein and alterations in DICER1 and Argonaute 2 expression. Targets of depleted miRNA belonged to molecular pathways involved in epithelial proliferation, differentiation, and immune response. Decreased expression of several miRNA involved in maintaining epithelial homeostasis in the gut was localized to the proliferative crypt region of the intestinal epithelium. Our findings suggest that SIV-induced decreased expression of miRNA involved in epithelial homeostasis, disrupted expression of miRNA biogenesis machinery, and increased expression of XRN2 are involved in the development of epithelial barrier dysfunction and gastroenteropathy. IMPORTANCE MicroRNA (miRNA) regulate the development and function of intestinal epithelial cells, and many viruses disrupt normal host miRNA expression. In this study, we demonstrate that SIV and HIV disrupt expression of miRNA in the small intestine during infection. The depletion of several key miRNA is localized to the proliferative crypt region of the gut epithelium. These miRNA are known to control expression of genes involved in inflammation, cell death, and epithelial maturation. Our data indicate that this disruption might be caused by altered expression of mi

  6. Formation of nitrogen-containing metabolites from the main iridoids of Harpagophytum procumbens and H. zeyheri by human intestinal bacteria.

    PubMed

    Baghdikian, B; Guiraud-Dauriac, H; Ollivier, E; N'Guyen, A; Dumenil, G; Balansard, G

    1999-03-01

    The study of the metabolism of iridoid glycosides from Harpagophytum procumbens and Harpagophytum zeyheri by human intestinal bacteria, was realized in order to elucidate compounds responsible for the pharmacological activities of Harpagophytum. Harpagide, harpagoside and 8-O-p-coumaroyl-harpagide were transformed into the pyridine monoterpene alkaloid aucubinine B by human fecal flora and by bacteria isolated from this flora. Aucubinine B was also prepared from harpagide, harpagoside and 8-O-p-coumaroylharpagide, by beta-glucosidase in the presence of NH4+.

  7. Liquid chromatography/mass spectrometry-based structural analysis of new platycoside metabolites transformed by human intestinal bacteria.

    PubMed

    Ha, Young Wan; Na, Yun-Cheol; Ha, In Jin; Kim, Dong-Hyun; Kim, Yeong Shik

    2010-01-05

    Platycosides, the main active constituents of Platycodi Radix, have been thoroughly studied for the characterization of their potent biological activities. However, metabolism of platycosides has not yet been characterized. A HPLC electrospray ionization-tandem mass spectrometry (LC/ESI-MS(n)) approach was applied to new complex platycoside metabolites transformed by human intestinal bacteria to identify their structures and determine metabolic pathway. The molecular weights of metabolites were identified by LC/ESI-MS analysis in both positive and negative modes. Structures for the platycoside metabolites were proposed by the molecular weights and the expected enzymatic activity of intestinal microbes on platycoside. In the second step, successive LC-MS(n) analysis was used to demonstrate the proposed structures. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M+Na](+) ions exclusively showed signals, consistent with the cleavage of glycoside bonds, rearrangement and some cross-ring cleavage, thus allowing the rapid identification of platycoside metabolites. The metabolites identified in the time-dependent metabolism experiments enable us to propose several microbial pathways for platycosides. Even though the metabolites of some platycosides may have unknown structures and low levels, the analytical tools presented in this study made it possible to obtain a rapid and complete characterization of new metabolites and their metabolism pathway in human intestinal bacteria.

  8. The Metabolic Profiling of Isorhamnetin-3-O-Neohesperidoside Produced by Human Intestinal Flora Employing UPLC-Q-TOF/MS.

    PubMed

    Du, Le-Yue; Zhao, Min; Tao, Jin-Hua; Qian, Da-Wei; Jiang, Shu; Shang, Er-Xin; Guo, Jian-Ming; Liu, Pei; Su, Shu-Lan; Duan, Jin-Ao

    2016-11-23

    Isorhamnetin-3-O-neohesperidoside is the major active substance of Puhuang, a traditional herb medicine widely used in clinical practice to tackle many chronic diseases. However, little is known about the interactions between this ingredient and intestinal flora. In this study, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry together with automated data analysis software (Metabolynx™) was used for analysis of the metabolic profile of isorhamnetin-3-O-neohesperidoside by the isolated human intestinal bacteria. The parent and three metabolites isorhamnetin-3-O-glucoside, isorhamnetin and quercetin were detected and identified based on the characteristics of their deprotonated molecules. These metabolites indicated that isorhamnetin-3-O-neohesperidoside was firstly deglycosylated to isorhamnetin-3-O-glucoside and subsequently to the aglycone isorhamnetin, and the latter was demethylated to quercetin. The majority of bacteria such as Escherichia sp. 23 were capable of converting isorhamnetin-3-O-neohesperidoside to considerable amounts of aglycone isorhamnetin and further to minor amounts of quercetin, while minor amounts of isorhamnetin-3-O-glucoside were detected in minority of bacterial samples such as Enterococcus sp. 30. The metabolic pathway and metabolites of isorhamnetin-3-O-neohesperidoside by the different human intestinal bacteria were firstly investigated. Furthermore, the metabolites of isorhamnetin-3-O-neohesperidoside might influence the effects of traditional herb medicines. Thus, our study is helpful to further unravel how isorhamnetin-3-O-neohesperidoside and Puhuang work in vivo.

  9. Akkermansia muciniphila gen. nov., sp. nov., a human intestinal mucin-degrading bacterium.

    PubMed

    Derrien, Muriel; Vaughan, Elaine E; Plugge, Caroline M; de Vos, Willem M

    2004-09-01

    The diversity of mucin-degrading bacteria in the human intestine was investigated by combining culture and 16S rRNA-dependent approaches. A dominant bacterium, strain MucT, was isolated by dilution to extinction of faeces in anaerobic medium containing gastric mucin as the sole carbon and nitrogen source. A pure culture was obtained using the anaerobic soft agar technique. Strain MucT was a Gram-negative, strictly anaerobic, non-motile, non-spore-forming, oval-shaped bacterium that could grow singly and in pairs. When grown on mucin medium, cells produced a capsule and were found to aggregate. Strain MucT could grow on a limited number of sugars, including N-acetylglucosamine, N-acetylgalactosamine and glucose, but only when a protein source was provided and with a lower growth rate and final density than on mucin. The G + C content of DNA from strain MucT was 47.6 mol%. 16S rRNA gene sequence analysis revealed that the isolate was part of the division Verrucomicrobia. The closest described relative of strain MucT was Verrucomicrobium spinosum (92 % sequence similarity). Remarkably, the 16S rRNA gene sequence of strain MucT showed 99 % similarity to three uncultured colonic bacteria. According to the data obtained in this work, strain MucT represents a novel bacterium belonging to a new genus in subdivision 1 of the Verrucomicrobia; the name Akkermansia muciniphila gen. nov., sp. nov. is proposed; the type strain is MucT (= ATCC BAA-835T = CIP 107961T).

  10. Activation of CFTR trafficking and gating by vasoactive intestinal peptide in human bronchial epithelial cells.

    PubMed

    Qu, Fei; Liu, Hui-Jun; Xiang, Yang; Tan, Yu-Rong; Liu, Chi; Zhu, Xiao-Lin; Qin, Xiao-Qun

    2011-03-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in epithelia and other cell types. The most common cause of cystic fibrosis (CF) is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the cell surface is important. Vasoactive intestinal polypeptide (VIP) plays an important role in CFTR-dependent chloride transport. The present study was designed to observe the affection of VIP on the trafficking of CFTR, and channel gating in human bronchial epithelium cells (HBEC). Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. After VIP treatment, apical extension of CFTR immunofluorescence into the cell was reduced and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP increased cell surface and total CFTR. Compared with the augmented level of total CFTR, the surface CFTR increased more markedly. Immunoprecipitation founded that the mature form of CFTR had a marked increase in HBEC treated with VIP. VIP led to a threefold increase in Cl(-) efflux in HBEC. Glibenclamide-sensitive and DIDS-insensitive CFTR Cl(-) currents were consistently observed after stimulation with VIP (10(-8) mol/L). The augmentation of CFTR Cl(-) currents enhanced by VIP (10(-8) mol/L) was reversed, at least in part, by the protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, H-7, suggesting PKA and PKC participate in the VIP-promoted CFTR Cl(-) currents.

  11. Proteomic responses of human intestinal Caco-2 cells exposed to silver nanoparticles and ionic silver.

    PubMed

    Oberemm, Axel; Hansen, Ulf; Böhmert, Linda; Meckert, Christine; Braeuning, Albert; Thünemann, Andreas F; Lampen, Alfonso

    2016-03-01

    Even although quite a number of studies have been performed so far to demonstrate nanoparticle-specific effects of substances in living systems, clear evidence of these effects is still under debate. The present study was designed as a comparative proteomic analysis of human intestinal cells exposed to a commercial silver nanoparticle reference material and ions from AgNO3. A two-dimensional gel electrophoresis/MALDI mass spectrometry (MS)-based proteomic analysis was conducted after 24-h incubation of differentiated Caco-2 cells with non-cytotoxic and low cytotoxic silver concentrations (2.5 and 25 µg ml(-1) nanosilver, 0.5 and 5 µg ml(-1) AgNO3). Out of an overall number of 316 protein spots differentially expressed at a fold change of ≥ 1.4 or ≤ -1.4 in all treatments, 169 proteins could be identified. In total, 231 spots were specifically deregulated in particle-treated groups compared with 41 spots, which were limited to AgNO3-treatments. Forty-four spots (14 %) were commonly deregulated by both types of treatment. A considerable fraction of the proteins differentially expressed after treatment with nanoparticles is related to protein folding, synthesis or modification of proteins as well as cellular assembly and organization. Overlays of networks obtained for particulate and ionic treatments showed matches, indicating common mechanisms of combined particle and ionic silver exposure and exclusive ionic silver treatment. However, proteomic responses of Caco-2 cells treated with higher concentrations of silver species also showed some differences, for example regarding proteins related to fatty acid and energy metabolism, suggesting an induction of also some different molecular mechanisms for particle exposure and ionic treatment.

  12. High affinity receptors for vasoactive intestinal peptide on a human glioma cell line

    SciTech Connect

    Nielsen, F.C.; Gammeltoft, S.; Westermark, B.; Fahrenkrug, J. )

    1990-11-01

    Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin, PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and (des-His1)VIP bound with 10 and 100 times lower affinity. The fragment VIP(7-28) displaced 25% of the receptor-bound {sup 125}I-VIP whereas VIP(16-28) and VIP(1-22-NH2) were inactive. The binding of {sup 125}I-VIP could be completely inhibited by 10 mumol/l of the antagonists (N-Ac-Tyr1,D-Phe2)GRF(1-29)-NH2, (pCl-D-Phe6,Leu17)VIP and VIP(10-28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated {sup 125}I-VIP was bound to receptors on the cell surface. The internalized {sup 125}I-VIP was completely degraded to {sup 125}I-tyrosine which was released from the cells. Degradation of internalized {sup 125}I-VIP was significantly reduced by chloroquine phenanthroline and pepstatin-A. Surface binding and internalization of {sup 125}I-VIP was increased 3 times by phenanthroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound {sup 125}I-VIP, but caused retention of internalized {sup 125}I-VIP.

  13. Differential diagnosis of human bladder mucosa pathologies in vivo with cross-polarization optical coherence tomography

    PubMed Central

    Kiseleva, Elena; Kirillin, Mikhail; Feldchtein, Felix; Vitkin, Alex; Sergeeva, Ekaterina; Zagaynova, Elena; Streltzova, Olga; Shakhov, Boris; Gubarkova, Ekaterina; Gladkova, Natalia

    2015-01-01

    Quantitative image analysis and parameter extraction using a specific implementation of polarization-sensitive optical coherence tomography (OCT) provides differential diagnosis of mucosal pathologies in in-vivo human bladders. We introduce a cross-polarization (CP) OCT image metric called Integral Depolarization Factor (IDF) to enable automatic diagnosis of bladder conditions (assessment the functional state of collagen fibers). IDF-based diagnostic accuracy of identification of the severe fibrosis of normal bladder mucosa is 79%; recurrence of carcinoma on the post-operative scar is 97%; and differentiation between neoplasia and acute inflammation is 75%. The promising potential of CP OCT combined with image analysis in human urology is thus demonstrated in vivo. PMID:25909028

  14. Degradation in the degree of polarization in human retinal nerve fiber layer

    NASA Astrophysics Data System (ADS)

    Yin, Biwei; Wang, Bingqing; Rylander, Henry G.; Milner, Thomas E.

    2014-01-01

    Using a fiber-based swept-source (SS) polarization-sensitive optical coherence tomography (PS-OCT) system, we investigate the degree of polarization (DOP) of light backscattered from the retinal nerve fiber layer (RNFL) in normal human subjects. Algorithms for processing data were developed to analyze the deviation in phase retardation and intensity of backscattered light in directions parallel and perpendicular to the nerve fiber axis (fast and slow axes of RNFL). Considering superior, inferior, and nasal quadrants, we observe the strongest degradation in the DOP with increasing RNFL depth in the temporal quadrant. Retinal ganglion cell axons in normal human subjects are known to have the smallest diameter in the temporal quadrant, and the greater degradation observed in the DOP suggests that higher polarimetric noise may be associated with neural structure in the temporal RNFL. The association between depth degradation in the DOP and RNFL structural properties may broaden the utility of PS-OCT as a functional imaging technique.

  15. The antifungal antibiotic, clotrimazole, inhibits Cl- secretion by polarized monolayers of human colonic epithelial cells.

    PubMed Central

    Rufo, P A; Jiang, L; Moe, S J; Brugnara, C; Alper, S L; Lencer, W I

    1996-01-01

    Clotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals. PMID:8903326

  16. Polarization sensitive optical low-coherence reflectometry for blood glucose monitoring in human subjects

    NASA Astrophysics Data System (ADS)

    Solanki, Jitendra; Choudhary, Om Prakash; Sen, P.; Andrews, J. T.

    2013-07-01

    A device based on polarization sensitive optical low-coherence reflectometry is developed to monitor blood glucose levels in human subjects. The device was initially tested with tissue phantom. The measurements with human subjects for various glucose concentration levels are found to be linearly dependent on the ellipticity obtainable from the home-made phase-sensitive optical low-coherence reflectometry device. The linearity obtained between glucose concentration and ellipticity are explained with theoretical calculations using Mie theory. A comparison of results with standard clinical methods establishes the utility of the present device for non-invasive glucose monitoring.

  17. Cloning and sequencing of human intestinal alkaline phosphatase cDNA

    SciTech Connect

    Berger, J.; Garattini, E.; Hua, J.C.; Udenfriend, S.

    1987-02-01

    Partial protein sequence data obtained on intestinal alkaline phosphatase indicated a high degree of homology with the reported sequence of the placental isoenzyme. Accordingly, placental alkaline phosphatase cDNA was cloned and used as a probe to clone intestinal alkaline phosphatase cDNA. The latter is somewhat larger (3.1 kilobases) than the cDNA for the placental isozyme (2.8 kilobases). Although the 3' untranslated regions are quite different, there is almost 90% homology in the translated regions of the two isozymes. There are, however, significant differences at their amino and carboxyl termini and a substitution of an alanine in intestinal alkaline phosphatase for a glycine in the active site of the placental isozyme.

  18. Ontogeny of Human Hepatic and Intestinal Transporter Gene Expression during Childhood: Age Matters

    PubMed Central

    Mooij, Miriam G.; Schwarz, Ute I.; de Koning, Barbara A. E.; Leeder, J. Steven; Gaedigk, Roger; Samsom, Janneke N.; Spaans, Edwin; van Goudoever, Johannes B.; Tibboel, Dick; Kim, Richard B.

    2014-01-01

    Many drugs prescribed to children are drug transporter substrates. Drug transporters are membrane-bound proteins that mediate the cellular uptake or efflux of drugs and are important to drug absorption and elimination. Very limited data are available on the effect of age on transporter expression. Our study assessed age-related gene expression of hepatic and intestinal drug transporters. Multidrug resistance protein 2 (MRP2), organic anion transporting polypeptide 1B1 (OATP1B1), and OATP1B3 expression was determined in postmortem liver samples (fetal n = 6, neonatal n = 19, infant n = 7, child n = 2, adult n = 11) and multidrug resistance 1 (MDR1) expression in 61 pediatric liver samples. Intestinal expression of MDR1, MRP2, and OATP2B1 was determined in surgical small bowel samples (neonates n = 15, infants n = 3, adults n = 14). Using real-time reverse-transcription polymerase chain reaction, we measured fetal and pediatric gene expression relative to 18S rRNA (liver) and villin (intestines), and we compared it with adults using the 2−∆∆Ct method. Hepatic expression of MRP2, OATP1B1, and OATP1B3 in all pediatric age groups was significantly lower than in adults. Hepatic MDR1 mRNA expression in fetuses, neonates, and infants was significantly lower than in adults. Neonatal intestinal expressions of MDR1 and MRP2 were comparable to those in adults. Intestinal OATP2B1 expression in neonates was significantly higher than in adults. We provide new data that show organ- and transporter-dependent differences in hepatic and intestinal drug transporter expression in an age-dependent fashion. This suggests that substrate drug absorption mediated by these transporters may be subject to age-related variation in a transporter dependent pattern. PMID:24829289

  19. Central Role of the Gut Epithelial Barrier in the Pathogenesis of Chronic Intestinal Inflammation: Lessons Learned from Animal Models and Human Genetics

    PubMed Central

    Pastorelli, Luca; De Salvo, Carlo; Mercado, Joseph R.; Vecchi, Maurizio; Pizarro, Theresa T.

    2013-01-01

    The gut mucosa is constantly challenged by a bombardment of foreign antigens and environmental microorganisms. As such, the precise regulation of the intestinal barrier allows the maintenance of mucosal immune homeostasis and prevents the onset of uncontrolled inflammation. In support of this concept, emerging evidence points to defects in components of the epithelial barrier as etiologic factors in the pathogenesis of inflammatory bowel diseases (IBDs). In fact, the integrity of the intestinal barrier relies on different elements, including robust innate immune responses, epithelial paracellular permeability, epithelial cell integrity, as well as the production of mucus. The purpose of this review is to systematically evaluate how alterations in the aforementioned epithelial components can lead to the disruption of intestinal immune homeostasis, and subsequent inflammation. In this regard, the wealth of data from mouse models of intestinal inflammation and human genetics are pivotal in understanding pathogenic pathways, for example, that are initiated from the specific loss of function of a single protein leading to the onset of intestinal disease. On the other hand, several recently proposed therapeutic approaches to treat human IBD are targeted at enhancing different elements of gut barrier function, further supporting a primary role of the epithelium in the pathogenesis of chronic intestinal inflammation and emphasizing the importance of maintaining a healthy and effective intestinal barrier. PMID:24062746

  20. Environmental contaminants activate human and polar bear (Ursus maritimus) pregnane X receptors (PXR, NR1I2) differently

    SciTech Connect

    Lille-Langøy, Roger; Goldstone, Jared V.; Rusten, Marte; Milnes, Matthew R.; Male, Rune; Stegeman, John J.; Blumberg, Bruce; Goksøyr, Anders

    2015-04-01

    Background: Many persistent organic pollutants (POPs) accumulate readily in polar bears because of their position as apex predators in Arctic food webs. The pregnane X receptor (PXR, formally NR1I2, here proposed to be named promiscuous xenobiotic receptor) is a xenobiotic sensor that is directly involved in metabolizing pathways of a wide range of environmental contaminants. Objectives: In the present study, we comparably assess the ability of 51 selected pharmaceuticals, pesticides and emerging contaminants to activate PXRs from polar bears and humans using an in vitro luciferase reporter gene assay. Results: We found that polar bear PXR is activated by a wide range of our test compounds (68%) but has a slightly more narrow ligand specificity than human PXR that was activated by 86% of the 51 test compounds. The majority of the agonists identified (70%) produces a stronger induction of the reporter gene via human PXR than via polar bear PXR, however with some notable and environmentally relevant exceptions. Conclusions: Due to the observed differences in activation of polar bear and human PXRs, exposure of each species to environmental agents is likely to induce biotransformation differently in the two species. Bioinformatics analyses and structural modeling studies suggest that amino acids that are not part of the ligand-binding domain and do not interact with the ligand can modulate receptor activation. - Highlights: • Comparative study of ligand activation of human and polar bear PXRs. • Polar bear PXR is a promiscuous ligand-activated nuclear receptor but less so than human PXR. • Environmental contaminants activate human and polar bear PXRs differently. • Expression and ligand promiscuity indicate that PXR is a xenosensor in polar bears.

  1. Persistence and Toxin Production by Clostridium difficile within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function

    PubMed Central

    Leslie, Jhansi L.; Huang, Sha; Opp, Judith S.; Nagy, Melinda S.; Kobayashi, Masayuki; Young, Vincent B.

    2014-01-01

    Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile. PMID:25312952

  2. The human intestinal microbiota of constipated-predominant irritable bowel syndrome patients exhibits anti-inflammatory properties

    PubMed Central

    Gobert, Alain P.; Sagrestani, Giulia; Delmas, Eve; Wilson, Keith T.; Verriere, Thomas G.; Dapoigny, Michel; Del’homme, Christophe; Bernalier-Donadille, Annick

    2016-01-01

    The intestinal microbiota of patients with constipated-predominant irritable bowel syndrome (C-IBS) displays chronic dysbiosis. Our aim was to determine whether this microbial imbalance instigates perturbation of the host intestinal mucosal immune response, using a model of human microbiota-associated rats (HMAR) and dextran sulfate sodium (DSS)-induced experimental colitis. The analysis of the microbiota composition revealed a decrease of the relative abundance of Bacteroides, Roseburia-Eubacterium rectale and Bifidobacterium and an increase of Enterobacteriaceae, Desulfovibrio sp., and mainly Akkermansia muciniphila in C-IBS patients compared to healthy individuals. The bacterial diversity of the gut microbiota of healthy individuals or C-IBS patients was maintained in corresponding HMAR. Animals harboring a C-IBS microbiota had reduced DSS colitis with a decreased expression of pro-inflammatory cytokines from innate, Th1, and Th17 responses. The pre-treatment of conventional C57BL/6 mice or HMAR with A. muciniphila, but not with Escherichia coli, prior exposure to DSS also resulted in a reduction of colitis severity, highlighting that the anti-inflammatory effect of the gut microbiota of C-IBS patients is mediated, in part, by A. muciniphila. This work highlights a novel aspect of the crosstalk between the gut microbiota of C-IBS patients and host intestinal homeostasis. PMID:27982124

  3. SURVEY OF HOUSE RAT INTESTINAL PARASITES FROM SURABAYA DISTRICT, EAST JAVA, INDONESIA THAT CAN CAUSE OPPORTUNISTIC INFECTIONS IN HUMANS.

    PubMed

    Prasetyo, R H

    2016-03-01

    The purpose of this study was to investigate the prevalence of house rat zoonotic intestinal parasites from Surabaya District, East Java, Indonesia that have the potential to cause opportunistic infection in humans. House rat fecal samples were collected from an area of Surabaya District with a dense rat population during May 2015. Intestinal parasites were detected microscopically using direct smear of feces stained with Lugol's iodine and modified Ziehl-Neelsen stains. The fecal samples were also cultured for Strongyloides stercoralis. Ninety-eight house rat fecal samples were examined. The potential opportunistic infection parasite densities found in those samples were Strongyloides stercoralis in 53%, Hymenolepis nana in 42%, Cryptosporidium spp in 33%, and Blastocystis spp in 6%. This is the first report of this kind in Surabaya District. Measures need to be taken to control the house rat population in the study area to reduce the risk of the public health problem. Keywords: zoonotic intestinal parasites, opportunistic infection, house rat, densely populated area, Indonesia

  4. Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function.

    PubMed

    Leslie, Jhansi L; Huang, Sha; Opp, Judith S; Nagy, Melinda S; Kobayashi, Masayuki; Young, Vincent B; Spence, Jason R

    2015-01-01

    Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.

  5. High-speed fiber-based polarization-sensitive optical coherence tomography of in vivo human skin

    SciTech Connect

    Saxer, Christopher E.; Boer, Johannes F. de; Park, B. Hyle; Zhao, Yonghua; Chen, Zhongping; Nelson, J. Stuart

    2000-09-15

    A high-speed single-mode fiber-based polarization-sensitive optical coherence tomography (PS OCT) system was developed. With a polarization modulator, Stokes parameters of reflected flight for four input polarization states are measured as a function of depth. A phase modulator in the reference arm of a Michelson interferometer permits independent control of the axial scan rate and carrier frequency. In vivo PS OCT images of human skin are presented, showing subsurface structures that are not discernible in conventional OCT images. A phase retardation image in tissue is calculated based on the reflected Stokes parameters of the four input polarization states. (c) 2000 Optical Society of America.

  6. The intestine is a blender

    NASA Astrophysics Data System (ADS)

    Yang, Patricia; Lamarca, Morgan; Kravets, Victoria; Hu, David

    According to the U.S. Department of Health and Human Services, digestive disease affects 60 to 70 million people and costs over 140 billion annually. Despite the significance of the gastrointestinal tract to human health, the physics of digestion remains poorly understood. In this study, we ask a simple question: what sets the frequency of intestinal contractions? We measure the frequency of intestinal contractions in rats, as a function of distance down the intestine. We find that intestines Contract radially ten times faster than longitudinally. This motion promotes mixing and, in turn, absorption of food products by the intestinal wall. We calculate viscous dissipation in the intestinal fluid to rationalize the relationship between frequency of intestinal contraction and the viscosity of the intestinal contents. Our findings may help to understand the evolution of the intestine as an ideal mixer.

  7. The intestine is a blender

    NASA Astrophysics Data System (ADS)

    Yang, Patricia; Lamarca, Morgan; Hu, David

    2015-11-01

    According to the U.S. Department of Health and Human Services, digestive disease affects 60 to 70 million people and costs over 140 billion annually. Despite the significance of the gastrointestinal tract to human health, the physics of digestion remains poorly understood. In this study, we ask a simple question: what sets the frequency of intestinal contractions? We measure the frequency of intestinal contractions in rats, as a function of distance down the intestine. We find that intestines contract radially ten times faster than longitudinally. This motion promotes mixing and, in turn, absorption of food products by the intestinal wall. We calculate viscous dissipation in the intestinal fluid to rationalize the relationship between frequency of intestinal contraction and the viscosity of the intestinal contents. Our findings may help to understand the evolution of the intestine as an ideal mixer.

  8. [Intestinal microbiota].

    PubMed

    Debré, Patrice; Le Gall, Jean-Yves

    2014-12-01

    The human body normally lives in symbiosis with a considerable microscopic environment present on all interfaces with the external environment; it hosts ten times more microbes (microbiota) that it has somatic or germ cells, representing a gene diversity (microbiome) 100-150 times higher than the human genome. These germs are located mainly in the gut, where they represent a mass of about one kilogram. The primary colonization of the gastrointestinal tract depends on the delivery route, the bacterial flora rewarding then depending on the environment, food hygiene, medical treatments. The intestinal microbiota plays an important role in the maturation of the immune system and in different physiological functions: digestion of polysaccharides, glycosaminoglycans and glycoproteins, vitamins biosynthesis, bile salt metabolism of some amino acids and xenobiotics. Quantitative and qualitative changes in the microbiota are observed in a wide range of diseases: obesity, colorectal cancer, liver cancer, inflammatory bowel disease, autoimmune diseases, allergies... pharmacobiotics aim to modify the intestinal microbiota in a therapeutic goal and this by various means: prebiotics, probiotics, antibiotics or fecal transplants. Intestinal flora also plays a direct role in the metabolism of certain drugs and the microbiota should be considered as a predictive parameter of response to some chemotherapies.

  9. Thimerosal compromises human dendritic cell maturation, IL-12 production, chemokine release, and T-helper polarization.

    PubMed

    Loison, Emily; Gougeon, Marie-Lise

    2014-01-01

    Thimerosal is a preservative used in multidose vials of vaccine formulations to prevent bacterial and fungal contamination. We recently reported that nanomolar concentrations of thimerosal induce cell cycle arrest of human T cells activated via the TCR and inhibition of proinflammatory cytokine production, thus interfering with T-cell functions. Given the essential role of dendritic cells (DCs) in T-cell polarization and vaccine immunity, we studied the influence of non-toxic concentrations of thimerosal on DC maturation and functions. Ex-vivo exposure of human monocyte-derived DCs to nanomolar concentrations of thimerosal prevented LPS-induced DC maturation, as evidenced by the inhibition of morphological changes and a decreased expression of the maturation markers CD86 and HLA-DR. In addition thimerosal dampened their proinflammatory response, in particular the production of the Th1 polarizing cytokine IL-12, as well as TNF-α and IL-6. DC-dependent T helper polarization was altered, leading to a decreased production of IFN-γ IP10 and GM-CSF and increased levels of IL-8, IL-9, and MIP-1α. Although multi-dose vials of vaccines containing thimerosal remain important for vaccine delivery, our results alert about the ex-vivo immunomodulatory effects of thimerosal on DCs, a key player for the induction of an adaptive response.

  10. Extracellular nucleotides inhibit oxalate transport by human intestinal Caco-2-BBe cells through PKC-δ activation.

    PubMed

    Amin, Ruhul; Sharma, Sapna; Ratakonda, Sireesha; Hassan, Hatim A

    2013-07-01

    Nephrolithiasis remains a major health problem in Western countries. Seventy to 80% of kidney stones are composed of calcium oxalate, and small changes in urinary oxalate affect risk of kidney stone formation. Intestinal oxalate secretion mediated by the anion exchanger SLC26A6 plays an essential role in preventing hyperoxaluria and calcium oxalate nephrolithiasis, indicating that understanding the mechanisms regulating intestinal oxalate transport is critical for management of hyperoxaluria. Purinergic signaling modulates several intestinal processes through pathways including PKC activation, which we previously found to inhibit Slc26a6 activity in mouse duodenal tissue. We therefore examined whether purinergic stimulation with ATP and UTP affects oxalate transport by human intestinal Caco-2-BBe (C2) cells. We measured [¹⁴C]oxalate uptake in the presence of an outward Cl⁻ gradient as an assay of Cl⁻/oxalate exchange activity, ≥50% of which is mediated by SLC26A6. We found that ATP and UTP significantly inhibited oxalate transport by C2 cells, an effect blocked by the PKC inhibitor Gö-6983. Utilizing pharmacological agonists and antagonists, as well as PKC-δ knockdown studies, we observed that ATP inhibits oxalate transport through the P2Y₂ receptor, PLC, and PKC-δ. Biotinylation studies showed that ATP inhibits oxalate transport by lowering SLC26A6 surface expression. These findings are of potential relevance to pathophysiology of inflammatory bowel disease-associated hyperoxaluria, where supraphysiological levels of ATP/UTP are expected and overexpression of the P2Y₂ receptor has been reported. We conclude that ATP and UTP inhibit oxalate transport by lowering SLC26A6 surface expression in C2 cells through signaling pathways including the P2Y₂ purinergic receptor, PLC, and PKC-δ.

  11. Ecological Effect of Solithromycin on Normal Human Oropharyngeal and Intestinal Microbiota

    PubMed Central

    Rashid, Mamun-Ur; Rosenborg, Staffan; Panagiotidis, Georgios; Holm, Johan; Söderberg Löfdal, Karin; Nord, Carl Erik

    2016-01-01

    Solithromycin is a new fluoroketolide. The purpose of the present study was to investigate the effect of orally administered solithromycin on the human oropharyngeal and intestinal microbiota. Thirteen healthy volunteers (median age, 27.3 years) received oral solithromycin at 800 mg on day 1 followed by 400 mg daily on days 2 to 7. Fecal and saliva samples were collected at baseline and on days 2, 5, 7, 9, 14, and 21 for pharmacokinetic and microbiological analyses. Plasma samples were collected predose on days 2, 5, and 7 as proof of exposure, and solithromycin concentration ranges were 21.9 to 258 ng/ml, 18.0 to 386 ng/ml, and 16.9 to 417 ng/ml, respectively. The solithromycin concentrations in feces were 15.8 to 65.4 mg/kg, 24.5 to 82.7 mg/kg, 21.4 to 82.7 mg/kg, 12.1 to 72.4 mg/kg, 0.2 to 25.6 mg/kg, and 0 to 0.5 mg/kg on days 2, 5, 7, 9, 14, and 21, respectively. The numbers of enterobacteria and enterococci decreased and were normalized on day 14. The numbers of lactobacilli and bifidobacteria decreased from day 2 to day 14 and were normalized on day 21. The clostridia decreased on days 2, 7, and 14 and were normalized on day 21. No Clostridium difficile strains or toxins were detected during the study period. The number of Bacteroides strains was not significantly changed. The solithromycin concentrations in saliva were 0 to 1.2 mg/liter, 0 to 0.5 mg/liter, 0 to 0.5 mg/liter, and 0 to 0.1 mg/liter on days 2, 5, 7, and 9, respectively. The numbers of streptococci decreased on day 2 and were normalized on day 5. The numbers of lactobacilli, prevotellae, fusobacteria, and leptotrichiae decreased from day 2 and were normalized on day 21. PMID:27139483

  12. Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9.

    PubMed

    Kishi, Naoki; Takasuka, Akane; Kokawa, Yuki; Isobe, Takashi; Taguchi, Maho; Shigeyama, Masato; Murata, Mikio; Suno, Manabu; Hanioka, Nobumitsu

    2016-01-01

    1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the K(m) and CL(int) values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans < monkeys) were observed, no significant differences were noted in the K(m) or S50, Vmax and CL(int) or CLmax values for the 4'-glucuronidation of liver and intestinal microsomes between humans and monkeys. 3. The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1 > UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

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

    PubMed Central

    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

    2017-01-01

    ABSTRACT 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

  14. Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

    PubMed Central

    Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh M. B.; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B.; Claesson, Marcus J.; Ross, R. Paul; Scott, Karen P.; O'Toole, Paul W.

    2013-01-01

    Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved

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

    PubMed

    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

  16. Expression of thirty-six drug transporter genes in human intestine, liver, kidney, and organotypic cell lines.

    PubMed

    Hilgendorf, Constanze; Ahlin, Gustav; Seithel, Annick; Artursson, Per; Ungell, Anna-Lena; Karlsson, Johan

    2007-08-01

    This study was designed to quantitatively assess the mRNA expression of 36 important drug transporters in human jejunum, colon, liver, and kidney. Expression of these transporters in human organs was compared with expression in commonly used cell lines (Caco-2, HepG2, and Caki-1) originating from these organs to assess their value as in vitro transporter system models, and was also compared with data obtained from the literature on expression in rat tissues to assess species differences. Transporters that were highly expressed in the intestine included HPT1, PEPT1, BCRP, MRP2, and MDR1, whereas, in the liver, OCT1, MRP2, OATP-C, NTCP and BSEP were the main transporters. In the kidney, OAT1 was expressed at the highest levels, followed by OAT3, OAT4, MCT5, MDR1, MRP2, OCT2, and OCTN2. The best agreement between human tissue and the representative cell line was observed for human jejunum and Caco-2 cells. Expression in liver and kidney ortholog cell lines was not correlated with that in the associated tissue. Comparisons with rat transporter gene expression revealed significant species differences. Our results allowed a comprehensive quantitative comparison of drug transporter expression in human intestine, liver, and kidney. We suggest that it would be beneficial for predictive pharmacokinetic research to focus on the most highly expressed transporters. We hope that our comparison of rat and human tissue will help to explain the observed species differences in in vivo models, increase understanding of the impact of active transport processes on pharmacokinetics and distribution, and improve the quality of predictions from animal studies to humans.

  17. L-Arginine modulates CXC chemokines in the human intestinal epithelial cell line HCT-8 by the NO pathway.

    PubMed

    Marion, Rachel; Coëffier, Moïse; Lemoulan, Sabrina; Gargala, Gilles; Ducrotté, Philippe; Déchelotte, Pierre

    2005-12-01

    Arginine has immunomodulating properties in different animal models but its effects in human intestine remain unknown. This study examined whether arginine modulates inflammatory mediators as chemokines and nitric oxide (NO) in the human intestinal epithelial cell line HCT-8 induced by cytokines. Under basal conditions, arginine did not influence iNOS protein expression, NO and chemokine production and mRNA levels (P>0.05 for all). Stimulation with cytokines-induced a significant increase of NO and chemokine production, iNOS and chemokine mRNA level and iNOS protein expression. Under inflammatory conditions, arginine increased 30% NO production (P<0.05) but did not influence iNOS mRNA level or iNOS protein expression. Under stimulated conditions, arginine decreased IL-8 and Mig mRNA level (57% and 39%, for 0.1 vs. 2 mmol/l l-arginine, P<0.05, respectively), and production (respectively, 28 and 23%, both P<0.05). IP-10 and I-TAC mRNA level and production were not significantly influenced by arginine. Under inflammatory conditions, l-arginine as well as a NO donor (sodium nitroprusside (SNP)) increased NO production, which was inversely correlated with IL-8 production (r'=-0.66, P=0.007 for arginine; r'=-0.79, P<0.0001 for SNP). Use of NG-Methyl-l-arginine acetate, a NOS inhibitor which prevents arginine-induced NO production, suppressed the arginine-induced IL-8 inhibition (P<0.05). In HCT-8 cells, arginine enhanced cytokine-induced NO production, reduced IL-8 and Mig production and mRNA level and had no effects on other assessed chemokines. In conclusion, arginine-induced IL-8 inhibition in HCT-8 cells involves NO pathway under inflammatory conditions. These data suggest that arginine-enriched enteral nutrition may have significant influence on inflammatory response in human intestine.

  18. Commensal gut bacteria modulate phosphorylation-dependent PPARγ transcriptional activity in human intestinal epithelial cells

    PubMed Central

    Nepelska, Malgorzata; de Wouters, Tomas; Jacouton, Elsa; Béguet-Crespel, Fabienne; Lapaque, Nicolas; Doré, Joël; Arulampalam, Velmurugesan; Blottière, Hervé M.

    2017-01-01

    In healthy subjects, the intestinal microbiota interacts with the host’s epithelium, regulating gene expression to the benefit of both, host and microbiota. The underlying mechanisms remain poorly understood, however. Although many gut bacteria are not yet cultured, constantly growing culture collections have been established. We selected 57 representative commensal bacterial strains to study bacteria-host interactions, focusing on PPARγ, a key nuclear receptor in colonocytes linking metabolism and inflammation to the microbiota. Conditioned media (CM) were harvested from anaerobic cultures and assessed for their ability to modulate PPARγ using a reporter cell line. Activation of PPARγ transcriptional activity was linked to the presence of butyrate and propionate, two of the main metabolites of intestinal bacteria. Interestingly, some stimulatory CMs were devoid of these metabolites. A Prevotella and an Atopobium strain were chosen for further study, and shown to up-regulate two PPARγ-target genes, ANGPTL4 and ADRP. The molecular mechanisms of these activations involved the phosphorylation of PPARγ through ERK1/2. The responsible metabolites were shown to be heat sensitive but markedly diverged in size, emphasizing the diversity of bioactive compounds found in the intestine. Here we describe different mechanisms by which single intestinal bacteria can directly impact their host’s health through transcriptional regulation. PMID:28266623

  19. Commensal gut bacteria modulate phosphorylation-dependent PPARγ transcriptional activity in human intestinal epithelial cells.

    PubMed

    Nepelska, Malgorzata; de Wouters, Tomas; Jacouton, Elsa; Béguet-Crespel, Fabienne; Lapaque, Nicolas; Doré, Joël; Arulampalam, Velmurugesan; Blottière, Hervé M

    2017-03-07

    In healthy subjects, the intestinal microbiota interacts with the host's epithelium, regulating gene expression to the benefit of both, host and microbiota. The underlying mechanisms remain poorly understood, however. Although many gut bacteria are not yet cultured, constantly growing culture collections have been established. We selected 57 representative commensal bacterial strains to study bacteria-host interactions, focusing on PPARγ, a key nuclear receptor in colonocytes linking metabolism and inflammation to the microbiota. Conditioned media (CM) were harvested from anaerobic cultures and assessed for their ability to modulate PPARγ using a reporter cell line. Activation of PPARγ transcriptional activity was linked to the presence of butyrate and propionate, two of the main metabolites of intestinal bacteria. Interestingly, some stimulatory CMs were devoid of these metabolites. A Prevotella and an Atopobium strain were chosen for further study, and shown to up-regulate two PPARγ-target genes, ANGPTL4 and ADRP. The molecular mechanisms of these activations involved the phosphorylation of PPARγ through ERK1/2. The responsible metabolites were shown to be heat sensitive but markedly diverged in size, emphasizing the diversity of bioactive compounds found in the intestine. Here we describe different mechanisms by which single intestinal bacteria can directly impact their host's health through transcriptional regulation.

  20. Polarity and cell division orientation in the cleavage embryo: from worm to human

    PubMed Central

    Ajduk, Anna; Zernicka-Goetz, Magdalena

    2016-01-01

    Cleavage is a period after fertilization, when a 1-cell embryo starts developing into a multicellular organism. Due to a series of mitotic divisions, the large volume of a fertilized egg is divided into numerous smaller, nucleated cells—blastomeres. Embryos of different phyla divide according to different patterns, but molecular mechanism of these early divisions remains surprisingly conserved. In the present paper, we describe how polarity cues, cytoskeleton and cell-to-cell communication interact with each other to regulate orientation of the early embryonic division planes in model animals such as Caenorhabditis elegans, Drosophila and mouse. We focus particularly on the Par pathway and the actin-driven cytoplasmic flows that accompany it. We also describe a unique interplay between Par proteins and the Hippo pathway in cleavage mammalian embryos. Moreover, we discuss the potential meaning of polarity, cytoplasmic dynamics and cell-to-cell communication as quality biomarkers of human embryos. PMID:26660321

  1. Automated measurement of choroidal thickness in the human eye by polarization sensitive optical coherence tomography

    PubMed Central

    Torzicky, Teresa; Pircher, Michael; Zotter, Stefan; Bonesi, Marco; Götzinger, Erich; Hitzenberger, Christoph K.

    2015-01-01

    We present a new method to automatically segment the thickness of the choroid in the human eye by polarization sensitive optical coherence tomography (PS-OCT). A swept source PS-OCT instrument operating at a center wavelength of 1040 nm is used. The segmentation method is based entirely on intrinsic, tissue specific polarization contrast mechanisms. In a first step, the anterior boundary of the choroid, the retinal pigment epithelium, is segmented based on depolarization. In a second step, the choroid-sclera interface is found by using the birefringence of the sclera. The method is demonstrated in five healthy eyes. The mean repeatability (standard deviation) of thickness measurement was found to be 18.3 μm. PMID:22453435

  2. Chemical Synthesis of O-Glycosylated Human Interleukin-2 by the Reverse Polarity Protection Strategy.

    PubMed

    Asahina, Yuya; Komiya, Shinobu; Ohagi, Ami; Fujimoto, Rina; Tamagaki, Hiroko; Nakagawa, Katsuhiro; Sato, Takashi; Akira, Shizuo; Takao, Toshifumi; Ishii, Akira; Nakahara, Yoshiaki; Hojo, Hironobu

    2015-07-06

    The chemical synthesis of human interleukin-2 (IL-2) , having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity.

  3. VESGEN Mapping of Bioactive Protection against Intestinal Inflammation: Application to Human Spaceflight and ISS Experiments

    NASA Technical Reports Server (NTRS)

    Parsons-Wingerter, P. A.; Chen, X.; Kelly, C. P.; Reinecker, H. C.

    2011-01-01

    Challenges to successful space exploration and colonization include adverse physiological reactions to micro gravity and space radiation factors. Constant remodeling of the microvasculature is critical for tissue preservation, wound healing, and recovery after ischemia. Regulation of the vascular system in the intestine is particularly important to enable nutrient absorption while maintaining barrier function and mucosal defense against micro biota. Although tremendous progress has been made in understanding the molecular circuits regulating neovascularization, our knowledge of the adaptations of the vascular system to environmental challenges in the intestine remains incomplete. This is in part because of the lack of methods to observe and quantify the complex processes associated with vascular responses in vivo. Developed by GRC as a mature beta version, pre-release research software, VESsel GENeration Analysis (VESGEN) maps and quantifies the fractal-based complexity of vascular branching for novel insights into the cytokine, transgenic and therapeutic regulation of angiogenesis, lymphangiogenesis and microvascular remodeling. Here we demonstrate that VESGEN can be used to characterize the dynamic vascular responses to acute intestinal inflammation and mucosal recovery from in vivo confocal microscopic 3D image series. We induced transient intestinal inflammation in mice by DSS treatment and investigated whether the ability of the pro biotic yeast Saccharomyces boulardii (Sb) to protect against intestinal inflammation was due to regulation of vascular remodeling. A primary characteristic of inflammation is excessive neovascularization (angiogenesis) resulting in fragile vessels prone to bleeding. Morphological parameters for triplicate specimens revealed that Sb treatment greatly reduced the inflammatory response of vascular networks by an average of 78%. This resulted from Sb inhibition of vascular endothelial growth factor receptor signaling, a major

  4. Dietary fibers affect viscosity of solutions and simulated human gastric and small intestinal digesta.

    PubMed

    Dikeman, Cheryl L; Murphy, Michael R; Fahey, George C

    2006-04-01

    Two experiments were conducted to determine the viscosities of both soluble and insoluble dietary fibers. In Expt. 1, corn bran, defatted rice bran, guar gum, gum xanthan, oat bran, psyllium, soy hulls, stabilized rice bran, wheat bran, wood cellulose, and 2 methylcellulose controls (Ticacel 42, Ticacel 43) were hydrated in water overnight at 0.5, 1, 1.5, or 2% concentrations. In Expt. 2, guar gum, oat bran, psyllium, rice bran, wheat bran, and wood cellulose were subjected to a 2-stage in vitro gastric and small intestinal digestion simulation model. Viscosity was measured every 2 and 3 h during gastric and small intestinal simulation, respectively. Viscosities in both experiments were measured at multiple shear rates. Viscosities of all fiber solutions were concentration- and shear rate-dependent. Rice brans, soy hulls, and wood cellulose had the lowest viscosities, whereas guar gum, psyllium, and xanthan gum had the highest viscosities, regardless of concentration. During gastric simulation, viscosity was higher (P < 0.05) at 4 h than at 0 h for guar gum, psyllium, rice bran, and wheat bran. During small intestinal simulation, viscosities were higher (P < 0.05) between 3 and 9 h compared with 18 h for guar gum, oat bran, and rice bran. Guar gum, psyllium, and oat bran exhibited viscous characteristics throughout small intestinal simulation, indicating potential for these fibers to elicit blood glucose and lipid attenuation. Wheat and rice brans and wood cellulose did not exhibit viscous characteristics throughout small intestinal digestion; thus, they may be beneficial for laxation.

  5. Preterm infant gut microbiota affects intestinal epithelial development in a humanized microbiome gnotobiotic mouse model.

    PubMed

    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.

  6. Transcriptional regulation of the human Na{sup +}/H{sup +} exchanger NHE3 by serotonin in intestinal epithelial cells

    SciTech Connect

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

    2009-05-08

    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 {mu}M) resulted in a significant decrease in NHE3 mRNA and protein expression. In transient transfection studies, 5-HT repressed the NHE3 promoter activity by {approx}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 PKC{alpha} and modulation of DNA-binding affinities of Sp1 and Sp3.

  7. Intestinal Cancer

    MedlinePlus

    ... connects your stomach to your large intestine. Intestinal cancer is rare, but eating a high-fat diet ... increase your risk. Possible signs of small intestine cancer include Abdominal pain Weight loss for no reason ...

  8. Intestinal leiomyoma

    MedlinePlus

    Leiomyoma - intestine ... McLaughlin P, Maher MM. The duodenum and small intestine. In: Adam A, Dixon AK, Gillard JH, Schaefer- ... Roline CE, Reardon RF. Disorders of the small intestine. In: Marx JA, Hockberger RS, Walls RM, et ...

  9. Intestinal obstruction

    MedlinePlus

    Paralytic ileus; Intestinal volvulus; Bowel obstruction; Ileus; Pseudo-obstruction - intestinal; Colonic ileus ... objects that are swallowed and block the intestines) Gallstones (rare) Hernias Impacted stool Intussusception (telescoping of 1 ...

  10. Intestinal Obstruction

    MedlinePlus

    An intestinal obstruction occurs when food or stool cannot move through the intestines. The obstruction can be complete or partial. ... abdomen Inability to pass gas Constipation A complete intestinal obstruction is a medical emergency. It often requires surgery. ...

  11. The rule of unity for human intestinal absorption 2: application to pharmaceutical drugs that are marketed as salts.

    PubMed

    Patel, Raj B; Admire, Brittany; Yalkowsky, Samuel H

    2015-01-01

    The efficiency of the human intestinal absorption (HIA) of the 59 drugs which are marketed as salts is predicted using the rule of unity. Intrinsic aqueous solubilities and partition coefficients along with the drug dose are used to calculate modified absorption potential (MAP) values. These values are shown to be related to the fraction of the dose that is absorbed upon oral administration in humans (FA). It is shown that the MAP value can distinguish between drugs that are poorly absorbed (FA <0.5) and those that are well absorbed (FA ≥ 0.5). Inspection of the data as well as a receiver operative characteristic (ROC) plot shows that a single critical MAP value can be used for predicting efficient human absorption of drugs. This forms the basis of a simple rule of unity based solely on in vitro data for predicting whether or not a drug will be well absorbed at a given dose.

  12. Polarization-sensitive optical coherence tomography-based imaging, parameterization, and quantification of human cartilage degeneration

    NASA Astrophysics Data System (ADS)

    Brill, Nicolai; Wirtz, Mathias; Merhof, Dorit; Tingart, Markus; Jahr, Holger; Truhn, Daniel; Schmitt, Robert; Nebelung, Sven

    2016-07-01

    Polarization-sensitive optical coherence tomography (PS-OCT) is a light-based, high-resolution, real-time, noninvasive, and nondestructive imaging modality yielding quasimicroscopic cross-sectional images of cartilage. As yet, comprehensive parameterization and quantification of birefringence and tissue properties have not been performed on human cartilage. PS-OCT and algorithm-based image analysis were used to objectively grade human cartilage degeneration in terms of surface irregularity, tissue homogeneity, signal attenuation, as well as birefringence coefficient and band width, height, depth, and number. Degeneration-dependent changes were noted for the former three parameters exclusively, thereby questioning the diagnostic value of PS-OCT in the assessment of human cartilage degeneration.

  13. Human ex-vivo oral tissue imaging using spectral domain polarization sensitive optical coherence tomography.

    PubMed

    Sharma, Priyanka; Verma, Yogesh; Sahu, Khageswar; Kumar, Sudhir; Varma, Amit V; Kumawat, Jyoti; Gupta, Pradeep Kumar

    2017-01-01

    We report the use of spectral domain polarization sensitive optical coherence tomography for ex-vivo imaging of human oral mandibular tissue samples. Our results show that compared to the changes observed in the epithelium thickness and the decay constant of A-scan intensity profile, a much larger degree of change was observed in the phase retardation for tissue sites progressing from normal to the malignant state. These results suggest that monitoring of tissue retardance can help in better differentiation of normal and cancerous oral tissue sites.

  14. Fluorescence in situ hybridization and spectral imaging analysisof human oocytes and first polar bodies

    SciTech Connect

    Weier, Heinz-Ulli G.; Weier, Jingly F.; Oter Renom, Maria; Zheng,Xuezhong; Colls, Pere; Nureddin, Aida; Pham, Chau D.; Chu, Lisa W.; Racowsky, Catherine; Munne, Santiago

    2004-10-06

    We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18 or 21 in failed-fertilized human oocytes.While abnormalities involving chromosome 16 showed an age-dependant increase, results for the other chromosomes did not show statistically significant differences between the three age groups <35 yrs, 35-39 yrs, and >39 yrs. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Thus, for a pilot study investigating a more comprehensive analysis of oocytes and their corresponding first polar bodies (1PBs), we developed a novel 8-probe chromosome enumeration scheme using FISH and SIm.

  15. Purification of human adult and foetal intestinal alkaline phosphatases by monoclonal antibody immunoaffinity chromatography.

    PubMed Central

    Vockley, J; Harris, H

    1984-01-01

    We have used the technique of monoclonal antibody immunoaffinity chromatography to purify adult and foetal intestinal alkaline phosphatases. Pure adult intestinal enzyme was obtained from a crude tissue extract with a single immunoaffinity chromatographic step in yields exceeding 95%. An additional ion-exchange chromatographic step was necessary for purification of the foetal enzyme, but yields still exceeded 70%. Experiments to optimize the efficiency of the monoclonal antibody immunoaffinity chromatography procedure suggest that the relative strength of binding of an antibody to its antigen is the most important factor to consider when constructing such columns. A column made from an antibody of too low an avidity will not retain the enzyme, while one of too high an avidity will make elution of enzyme in the active state difficult. A scheme is suggested for the application of this technique to a general approach to enzyme purification. Images Fig. 2. PMID:6365087

  16. Impairments in enzyme activity and biosynthesis of brush border-associated hydrolases in human intestinal Caco-2/TC7 cells infected by members of the Afa/Dr family of diffusely adhering Escherichia coli.

    PubMed

    Peiffer, I; Bernet-Camard, M F; Rousset, M; Servin, A L

    2001-05-01

    Wild-type diffusely adhering Escherichia coli (DAEC) harbouring afimbrial adhesin (Afa) or fimbrial Dr and F1845 adhesins (Afa/Dr DAEC) apically infecting the human intestinal epithelial cells promote injuries in the brush border of the cells. We report here that infection by Afa/Dr DAEC wild-type strains C1845 and IH11128 in polarized human fully differentiated Caco-2/TC7 cells dramatically impaired the enzyme activity of functional brush border-associated proteins sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPP IV). Blockers of the transduction signal molecules, previously found to be active against the Afa/Dr DAEC-induced cytoskeleton injury, were inactive against the Afa/Dr-induced decrease in sucrase enzyme activity. In parallel, Afa/Dr DAEC infection promotes the blockade of the biosynthesis of SI and DPP IV without affection enzyme stability. The observation that no changes occurred in mRNA levels of SI and DPP IV upon infection suggested that the decrease in biosynthesis probably resulted from a decrease in the translation rate. When the cells were infected with recombinant E. coli strains expressing homologous adhesins of the wild-type strains, neither a decrease in sucrase and DPP IV enzyme activities nor an inhibition of enzyme biosynthesis were observed. In conclusion, taken together, these data give new insights into the mechanisms by which the wild-type Afa/Dr DAEC strains induce functional injuries in polarized fully differentiated human intestinal cells. Moreover, the results revealed that other pathogenic factor(s) distinct from the Afa/Dr adhesins may play(s) a crucial role in this mechanism of pathogenicity.

  17. Concurrent profiling of polar metabolites and lipids in human plasma using HILIC-FTMS

    NASA Astrophysics Data System (ADS)

    Cai, Xiaoming; Li, Ruibin

    2016-11-01

    Blood plasma is the most popularly used sample matrix for metabolite profiling studies, which aim to achieve global metabolite profiling and biomarker discovery. However, most of the current studies on plasma metabolite profiling focused on either the polar metabolites or lipids. In this study, a comprehensive analysis approach based on HILIC-FTMS was developed to concurrently examine polar metabolites and lipids. The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC mode than in C5 and C18 reversed phase (RP) chromatography. This method exhibits good reproducibility in retention times (CVs < 3.43%) and high mass accuracy (<3.5 ppm). In addition, we found MeOH/ACN/Acetone (1:1:1, v/v/v) as extraction cocktail could achieve desirable gathering of demanded extracts from plasma samples. We further integrated the MeOH/ACN/Acetone extraction with the HILIC-FTMS method for metabolite profiling and smoking-related biomarker discovery in human plasma samples. Heavy smokers could be successfully distinguished from non smokers by univariate and multivariate statistical analysis of the profiling data, and 62 biomarkers for cigarette smoke were found. These results indicate that our concurrent analysis approach could be potentially used for clinical biomarker discovery, metabolite-based diagnosis, etc.

  18. Analysis of human knee osteoarthritic cartilage using polarization sensitive second harmonic generation microscopy

    NASA Astrophysics Data System (ADS)

    Kumar, Rajesh; Grønhaug, Kirsten M.; Romijn, Elisabeth I.; Drogset, Jon O.; Lilledahl, Magnus B.

    2014-05-01

    Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

  19. Surface hydride on titanium by cathodic polarization promotes human gingival fibroblast growth.

    PubMed

    Xing, Rui; Salou, Laëtitia; Taxt-Lamolle, Sébastien; Reseland, Janne E; Lyngstadaas, Ståle P; Haugen, Håvard J

    2014-05-01

    Connective tissue seal to dental abutment is crucial for peri-implant health. Several efforts have been made previously to optimize abutment surfaces, but no consensus has been reached regarding the optimal surface architecture and/or composition for soft tissue seal. Here, we report on experiments using cathodic polarization in organic acids to optimize titanium (Ti) surfaces for use as abutments. The three main factors affecting surface topography and chemistry were electrolyte composition, current density, and polarization time. Under identical conditions, oxalic acid created rougher surfaces than tartaric acid and acetic acid, and acetic acid produced more surface hydride. Surface hydride amount was suggested to first increase and then decrease with current density from 1 mA/cm(2) to 15 mA/cm(2) . The complexity of the surface topography and hydride production both increased with polarization time. Proliferation rate of human gingival fibroblasts (HGFs) was positively correlated with surface hydride content, suggesting the positive effect of surface hydride on connective tissue growth around dental abutment. Changes in surface topography and hydrophilicity did not significantly influence HGF growth.

  20. Concurrent profiling of polar metabolites and lipids in human plasma using HILIC-FTMS

    PubMed Central

    Cai, Xiaoming; Li, Ruibin

    2016-01-01

    Blood plasma is the most popularly used sample matrix for metabolite profiling studies, which aim to achieve global metabolite profiling and biomarker discovery. However, most of the current studies on plasma metabolite profiling focused on either the polar metabolites or lipids. In this study, a comprehensive analysis approach based on HILIC-FTMS was developed to concurrently examine polar metabolites and lipids. The HILIC-FTMS method was developed using mixed standards of polar metabolites and lipids, the separation efficiency of which is better in HILIC mode than in C5 and C18 reversed phase (RP) chromatography. This method exhibits good reproducibility in retention times (CVs < 3.43%) and high mass accuracy (<3.5 ppm). In addition, we found MeOH/ACN/Acetone (1:1:1, v/v/v) as extraction cocktail could achieve desirable gathering of demanded extracts from plasma samples. We further integrated the MeOH/ACN/Acetone extraction with the HILIC-FTMS method for metabolite profiling and smoking-related biomarker discovery in human plasma samples. Heavy smokers could be successfully distinguished from non smokers by univariate and multivariate statistical analysis of the profiling data, and 62 biomarkers for cigarette smoke were found. These results indicate that our concurrent analysis approach could be potentially used for clinical biomarker discovery, metabolite-based diagnosis, etc. PMID:27819279

  1. Effects of Clindamycin on Adherence of Clostridium difficile to Human Embryonic Intestinal Cells

    DTIC Science & Technology

    1991-11-01

    difficile, such an adhesin may play a role in disease production , by serving as the initial anchor to the intestinal epithelium. Response of the...endorsement of any commercial products . This report may not be cited for purposes of advertisement. Reproduction of this document in whole or in part is...factors help this microorqanism colonize and proliferate prior to toxin production .3,4,5, 6.7 Previous studies using the hamster model clarified some

  2. Calcitonin receptor-mediated CFTR activation in human intestinal epithelial cells

    PubMed Central

    Liu, Hongguang; Singla, Amika; Ao, Mei; Gill, Ravinder K; Venkatasubramanian, Jayashree; Rao, Mrinalini C; Alrefai, Waddah A; Dudeja, Pradeep K

    2011-01-01

    Abstract High levels of calcitonin (CT) observed in medullary thyroid carcinoma and other CT-secreting tumours cause severe diarrhoea. Previous studies have suggested that CT induces active chloride secretion. However, the involvement of CT receptor (CTR) and the molecular mechanisms underlying the modulation of intestinal electrolyte secreting intestinal epithelial cells have not been investigated. Therefore, current studies were undertaken to investigate the direct effects of CT on ion transport in intestinal epithelial cells. Real time quantitative RT-PCR and Western blot analysis demonstrated the expression of CTR in intestinal epithelial T84 cells. Exposure of T84 cells to CT from the basolateral but not from apical side significantly increased short circuit current (ISC) in a dose-dependent manner that was blocked by 1 μM of CTR antagonist, CT8–32. CT-induced ISC was blocked by replacing chloride in the bath solutions with equimolar gluconate and was significantly inhibited by the specific cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, CFTR127inh. Further, biotinylation studies showed that CT increased CFTR levels on the apical membrane. The presence of either the Ca2+ chelator, bis(2-aminophenoxy)ethane tetraacetic acid-acetoxymethyl (BAPTA-AM) ester or the protein kinase A (PKA) inhibitor, H89, significantly inhibited ISC induced by CT (∼32–58% reduction). Response to CT was retained after permeabilization of the basolateral or the apical membranes of T84 cells with nystatin. In conclusion, the activation of CTR by CT induced chloride secretion across T84 monolayers via CFTR channel and the involvement of PKA- and Ca2+-dependent signalling pathways. These data elucidate the molecular mechanisms underlying CT-induced diarrhoea. PMID:21251218

  3. Farnesoid X receptor signal is involved in deoxycholic acid-induced intestinal metaplasia of normal human gastric epithelial cells.

    PubMed

    Li, Shu; Chen, Xin; Zhou, Lu; Wang, Bang-Mao

    2015-11-01

    The farnesoid X receptor (FXR) signaling pathway is known to be involved in the metabolism of bile acid, glucose and lipid. In the present study, we demonstrated that 400 µmol/l deoxycholic acid (DCA) stimulation promotes the proliferation of normal human gastric epithelial cells (GES-1). In addition, DCA activated FXR and increased the expression of intestinal metaplasia genes, including caudal-related homeobox transcription factor 2 (Cdx2) and mucin 2 (MUC2). The treatment of FXR agonist GW4064/antagonist guggulsterone (Gug.) significantly increased/decreased the expression levels of FXR, Cdx2 and MUC2 protein in DCA-induced GES-1 cells. GW4064/Gug. also enhanced/reduced the nuclear factor-κB (NF-κB) activity and binding of the Cdx2 promoter region and NF-κB, the most common subunit p50 protein. Taken together, the results indicated that DCA is capable of modulating the expression of Cdx2 and the downstream MUC2 via the nuclear receptor FXR-NF-κB activity in normal gastric epithelial cells. FXR signaling pathway may therefore be involved in the intestinal metaplasia of human gastric mucosa.

  4. Study on human intestinal bacterium Blautia sp. AUH-JLD56 for the conversion of arctigenin to (-)-3'-desmethylarctigenin.

    PubMed

    Liu, Ming-Yue; Li, Meng; Wang, Xiu-Ling; Liu, Peng; Hao, Qing-Hong; Yu, Xiu-Mei

    2013-12-11

    Arctium lappa L. (A. lappa) is a popularly used vegetable as well as herbal medicine. Human intestinal microflora was reported to convert arctiin, the lignan compound with highest content in the dried fruits of Arctium lappa, to a series of metabolites. However, the specific bacterium responsible for the formation of 3'-desmethylarctigenin (3'-DMAG), the most predominant metabolite of arctiin by rat or human intestinal microflora, has not been isolated yet. In the present study, we isolated one single bacterium, which we named Blautia sp. AUH-JLD56, capable of solely biotransforming arctiin or arctigenin to (-)-3'-DMAG. The structure of the metabolite 3'-DMAG was elucidated by electrospray ionization mass spectrometry (ESI-MS) and (1)H and (13)C nuclear magnetic resonance spectroscopy. The biotransforming kinetics and maximum biotransforming capacity of strain AUH-JLD56 was investigated. In addition, the metabolite 3'-DMAG showed significantly higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity than that of the substrate arctigenin at the concentrations tested.

  5. Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease.

    PubMed

    Holmberg, Fredrik Eo; Seidelin, Jakob B; Yin, Xiaolei; Mead, Benjamin E; Tong, Zhixiang; Li, Yuan; Karp, Jeffrey M; Nielsen, Ole H

    2017-03-10

    Both the incidence and prevalence of inflammatory bowel disease (IBD) is increasing globally; in the industrialized world up to 0.5% of the population are affected and around 4.2 million individuals suffer from IBD in Europe and North America combined. Successful engraftment in experimental colitis models suggests that intestinal stem cell transplantation could constitute a novel treatment strategy to re-establish mucosal barrier function in patients with severe disease. Intestinal stem cells can be grown in vitro in organoid structures, though only a fraction of the cells contained are stem cells with regenerative capabilities. Hence, techniques to enrich stem cell populations are being pursued through the development of multiple two-dimensional and three-dimensional culture protocols, as well as co-culture techniques and multiple growth medium compositions. Moreover, research in support matrices allowing for efficient clinical application is in progress. In vitro culture is accomplished by modulating the signaling pathways fundamental for the stem cell niche with a suitable culture matrix to provide additional contact-dependent stimuli and structural support. The aim of this review was to discuss medium compositions and support matrices for optimal intestinal stem cell culture, as well as potential modifications to advance clinical use in IBD.

  6. Resolvin D1 Polarizes Primary Human Macrophages toward a Proresolution Phenotype through GPR32.

    PubMed

    Schmid, Mattia; Gemperle, Claudio; Rimann, Nicole; Hersberger, Martin

    2016-04-15

    Resolvin D1 (RvD1) was shown to be a potent anti-inflammatory and proresolution lipid mediator in several animal models of inflammation, but its mechanism of action in humans is not clear. We show that the RvD1 receptor GPR32 is present on resting, proinflammatory M(LPS) and alternatively activated primary human M(IL-4) macrophages, whereas TGF-β and IL-6 reduce its membrane expression. Accordingly, stimulation of resting primary human macrophages with 10 nM RvD1 for 48 h maximally reduced the secretion of the proinflammatory cytokines IL-1β and IL-8; abolished chemotaxis to several chemoattractants like chemerin, fMLF, and MCP-1; and doubled the phagocytic activity of these macrophages toward microbial particles. In contrast, these functional changes were not accompanied by surface expression of markers specific for alternatively activated M(IL-4) macrophages. Similar proresolution effects of RvD1 were observed when proinflammatory M(LPS) macrophages were treated with RvD1. In addition, we show that these RvD1-mediated effects are GPR32 dependent because reduction of GPR32 expression by small interfering RNA, TGF-β, and IL-6 treatment ablated these proresolution effects in primary human macrophages. Taken together, our results indicate that in humans RvD1 triggers GPR32 to polarize and repolarize macrophages toward a proresolution phenotype, supporting the role of this mediator in the resolution of inflammation in humans.

  7. Human Lactobacillus Strains from the Intestine can Suppress IgE-Mediated Degranulation of Rat Basophilic Leukaemia (RBL-2H3) Cells

    PubMed Central

    Harata, Gaku; He, Fang; Takahashi, Kyoko; Hosono, Akira; Miyazawa, Kenji; Yoda, Kazutoyo; Hiramatsu, Masaru; Kaminogawa, Shuichi

    2016-01-01

    Mast cells play a critical role in immunoglobulin E (IgE)-mediated allergic diseases, and the degranulation of mast cells is important in the pathogenesis of these diseases. A disturbance of the intestinal microflora, especially of endogenous lactic acid bacteria, might be a contributing factor for IgE-mediated allergic diseases. Additional knowledge regarding the interaction of human intestinal Lactobacilli with mast cells is still necessary. Twenty-three strains of Lactobacilli, including commercial and reference strains and strains from the human intestine, were tested for their ability to regulate degranulation of cells from rat basophilic leukemia RBL-2H3 cells (RBL-2H3) in vitro based on a β-hexosaminidase release assay. Each of the tested Lactobacilli characteristically suppressed IgE-mediated degranulation of RBL-2H3 cells, and Lactobacillus GG showed the strongest inhibitory effect on the cells. Furthermore, the bacteria isolated from the human intestine significantly suppressed degranulation of RBL-2H3 cellsin comparison with the reference strains. These results suggest that Lactobacilli, particularly those from the human intestine, can affect the activation of mast cells in a strain-dependent manner. Further study should be conducted to analyse the understanding mechanism. PMID:27801804

  8. Aeromonas hydrophila isolated from food and drinking water: hemagglutination, hemolysis, and cytotoxicity for a human intestinal cell line (HT-29).

    PubMed Central

    Handfield, M; Simard, P; Couillard, M; Letarte, R

    1996-01-01

    Aeromonas hydrophila isolated from food and drinking water was tested for pathogenicity by studying its hemolysis, hemagglutination, and cytotoxicity. Hemolysis, tested on erythrocytes from six different species, was more frequently seen with water isolates (64%) than with food isolates (48%). Hemagglutination was more frequently encountered with food isolates (92%) than with water isolates (73%). Cytotoxicity, evaluated on seven cell lines, was frequently observed with food isolates (92%) and with water isolates (73%). Heat treatment (56 degrees C for 10 min) of culture supernatant fluids inhibited the toxicity of some but not all toxin-producing isolates. Our results suggest that the human intestinal cell line HT-29 could be a useful complement for testing A. hydrophila exotoxins and for studying the enteropathogenicity of this species for humans. PMID:8795237

  9. Functional evidence for a 5-HT2B receptor mediating contraction of longitudinal muscle in human small intestine.

    PubMed Central

    Borman, R A; Burleigh, D E

    1995-01-01

    Application of 5-hydroxytryptamine induces contraction of longitudinal muscle strips from human terminal ileum. The response was resistant to antagonism by ketanserin, ondansetron or DAU6285, but was non-surmountably antagonized by methysergide. The selective 5-HT2B/2C receptor antagonist, SB 200646A evoked a concentration-dependent, parallel and dextral displacement of the concentration-response curve to 5-HT, yielding a pA2 estimate of 7.17. Application of yohimbine, a 5-HT1 and 5-HT2B receptor antagonist, also induced a rightward shift of the response curve to 5-HT, yielding a pA2 estimate of 8.10. In conclusion, it appears that a 5-HT2B receptor mediates the contractile response of the longitudinal muscle of human small intestine to 5-HT. PMID:7599919

  10. Determination using synchrotron radiation-based Fourier transform infrared microspectroscopy of putative stem cells in human adenocarcinoma of the intestine: corresponding benign tissue as a template.

    PubMed

    Ahmadzai, Abdullah A; Patel, Imran I; Veronesi, Giulia; Martin-Hirsch, Pierre L; Llabjani, Valon; Cotte, Marine; Stringfellow, Helen F; Martin, Francis L

    2014-01-01

    The epithelial-cell layer lining the two morphologically and functionally distinct segments of the mammalian intestinal tract, small intestine, and colon is constantly being renewed. This renewal is necessitated by a harsh lumen environment and is hypothesized to be driven by a small population of stem cells (SCs) that are believed to reside at the base of intestinal crypts. A lack of specific markers has hampered previous attempts to identify their exact location. We obtained tissue sections containing small intestine and colon crypts derived from normal (benign) or adenocarcinoma (AC) human intestine. The samples were floated onto BaF2 windows and analyzed using synchrotron radiation-based Fourier transform infrared microspectroscopy via an aperture size of 10 × 10 μm. Derived infrared (IR) spectral data was then analyzed using principal component analysis and/or linear discriminant analysis. Hypothesized cell types (as a function of aperture location along the length of individual crypts) within benign crypts were classed based on exploratory unsupervised IR spectral point clustering. Scores plots derived from individual small intestine crypts consistently generated one or two distinct spectra that clustered away from the remaining cell categories; these were retrospectively classed as "distinct base region" spectra. In these plots, a clear progression of locations along crypt lengths designated as from putative stem cells (SCs) to transit-amplifying (TA) cells to terminally differentiated (TD) cells was observed in benign small intestine and colon crypts. This progression of spectral points was crypt specific, pointing away from a unifying cell lineage model in human intestinal crypts. On comparison of AC-derived spectra versus corresponding benign, a subpopulation of AC-derived spectra suggested a putative SC-like spectral fingerprint; remaining IR spectra were classed as exhibiting TA cell-like or TD cell-like spectral characteristics. These observations

  11. Intestinal microbiota and ulcerative colitis.

    PubMed

    Ohkusa, Toshifumi; Koido, Shigeo

    2015-11-01

    There is a close relationship between the human host and the intestinal microbiota, which is an assortment of microorganisms, protecting the intestine against colonization by exogenous pathogens. Moreover, the intestinal microbiota play a critical role in providing nutrition and the modulation of host immune homeostasis. Recent reports indicate that some strains of intestinal bacteria are responsible for intestinal ulceration and chronic inflammation in inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). Understanding the interaction of the intestinal microbiota with pathogens and the human host might provide new strategies treating patients with IBD. This review focuses on the important role that the intestinal microbiota plays in maintaining innate immunity in the pathogenesis and etiology of UC and discusses new antibiotic therapies targeting the intestinal microbiota.

  12. Actin-interacting protein 1 controls assembly and permeability of intestinal epithelial apical junctions

    PubMed Central

    Baranwal, Somesh

    2015-01-01

    Adherens junctions (AJs) and tight junctions (TJs) are crucial regulators of the integrity and restitution of the intestinal epithelial barrier. The structure and function of epithelial junctions depend on their association with the cortical actin cytoskeleton that, in polarized epithelial cells, is represented by a prominent perijunctional actomyosin belt. The assembly and stability of the perijunctional cytoskeleton is controlled by constant turnover (disassembly and reassembly) of actin filaments. Actin-interacting protein (Aip) 1 is an emerging regulator of the actin cytoskeleton, playing a critical role in filament disassembly. In this study, we examined the roles of Aip1 in regulating the structure and remodeling of AJs and TJs in human intestinal epithelium. Aip1 was enriched at apical junctions in polarized human intestinal epithelial cells and normal mouse colonic mucosa. Knockdown of Aip1 by RNA interference increased the paracellular permeability of epithelial cell monolayers, decreased recruitment of AJ/TJ proteins to steady-state intercellular contacts, and attenuated junctional reassembly in a calcium-switch model. The observed defects of AJ/TJ structure and functions were accompanied by abnormal organization and dynamics of the perijunctional F-actin cytoskeleton. Moreover, loss of Aip1 impaired the apico-basal polarity of intestinal epithelial cell monolayers and inhibited formation of polarized epithelial cysts in 3-D Matrigel. Our findings demonstrate a previously unanticipated role of Aip1 in regulating the structure and remodeling of intestinal epithelial junctions and early steps of epithelial morphogenesis. PMID:25792565

  13. Anti inflammatory and anti angiogenic effect of black raspberry extract on human esophageal and intestinal microvascular endothelial cells

    PubMed Central

    Medda, Rituparna; Lyros, Orestis; Schmidt, Jamie L.; Jovanovic, Nebojsa; Nie, Linghui; Link, Benjamin J.; Otterson, Mary F.; Stoner, Gary D.; Shaker, Reza; Rafiee, Parvaneh

    2014-01-01

    Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat’s digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer. PMID:25446010

  14. Anti inflammatory and anti angiogenic effect of black raspberry extract on human esophageal and intestinal microvascular endothelial cells.

    PubMed

    Medda, Rituparna; Lyros, Orestis; Schmidt, Jamie L; Jovanovic, Nebojsa; Nie, Linghui; Link, Benjamin J; Otterson, Mary F; Stoner, Gary D; Shaker, Reza; Rafiee, Parvaneh

    2015-01-01

    Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat's digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial cell-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer.

  15. Measurement of parameters of polarization in the living human eye using imaging polarimetry.

    PubMed

    Bueno, J M

    2000-01-01

    An imaging polarimeter using liquid-crystal variable retarders (Bueno, J. M., Artal, P. (1999). Double-pass imaging polarimetry in the human eye. Optics Letters, 24, 64-66) has been used to study the parameters of polarization in the living human eye. Retardation introduced by birefringent structures of the eye has been calculated by using a spatially resolved collection of Mueller matrices obtained from series of 16 double-pass retinal images. Results for images with a 2-mm pupil diameter show that although the retardation introduced by the eye in a double-pass varies among individuals, at the central cornea the slow axis is directed along the upper-temporal to lower-nasal line and the ellipticity is close to zero, which indicates the presence of linear birefringence. As pupil size increased, the measured retardation also increased, while ocular birefringence remained linear and azimuthal angle changed without a clear tendency.

  16. Degradation in the degree of polarization in human retinal nerve fiber layer

    PubMed Central

    Yin, Biwei; Wang, Bingqing; Rylander, Henry G.; Milner, Thomas E.

    2014-01-01

    Abstract. Using a fiber-based swept-source (SS) polarization-sensitive optical coherence tomography (PS-OCT) system, we investigate the degree of polarization (DOP) of light backscattered from the retinal nerve fiber layer (RNFL) in normal human subjects. Algorithms for processing data were developed to analyze the deviation in phase retardation and intensity of backscattered light in directions parallel and perpendicular to the nerve fiber axis (fast and slow axes of RNFL). Considering superior, inferior, and nasal quadrants, we observe the strongest degradation in the DOP with increasing RNFL depth in the temporal quadrant. Retinal ganglion cell axons in normal human subjects are known to have the smallest diameter in the temporal quadrant, and the greater degradation observed in the DOP suggests that higher polarimetric noise may be associated with neural structure in the temporal RNFL. The association between depth degradation in the DOP and RNFL structural properties may broaden the utility of PS-OCT as a functional imaging technique. PMID:24390374

  17. Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion

    PubMed Central

    1993-01-01

    Neutrophil, or polymorphonuclear leukocyte (PMN), migration across intestinal epithelial barriers, such as occurs in many disease states, appears to result in modifications of epithelial barrier and ion transport functions (Nash, S., J. Stafford, and J. L. Madara. 1987. J. Clin. Invest. 80:1104-1113; Madara, J. L., C. A. Parkos, S. P. Colgan, R. J. MacLeod, S. Nash, J. B. Matthews, C. Delp, and W. I. Lencer. 1992. J. Clin. Invest. 89:1938-1944). Here we investigate the effects of epithelial exposure to IFN-gamma on PMN migration across cultured monolayers of the human intestinal epithelial cell line T84. Transepithelial migration of PMN was initially assessed in the apical- to-basolateral direction, since previous studies indicate general qualitative similarities between PMN migration in the apical-to- basolateral and in the basolateral-to-apical directions. In the apical- to-basolateral direction, epithelial exposure to IFN-gamma markedly upregulated transepithelial migration of PMN in a dose- and time- dependent fashion as measured by both electrical and myeloperoxidase assays. This IFN-gamma-elicited effect on transmigration was specifically due to a IFN-gamma effect on epithelial cells and was not secondary to IFN-gamma effects on epithelial tight junction permeability. Moreover, this IFN-gamma effect was dependent on epithelial protein synthesis, and involved a pathway in which CD11b/18, but not ICAM-1 or CD11a/18, appeared to play a crucial role in PMN- epithelial adhesion. IFN-gamma also substantially modified PMN transepithelial migration in the natural, basolateral-to-apical direction. The IFN-gamma effect on naturally directed transmigration was also specifically due to an IFN-gamma effect on epithelial cells, showed comparable time and dose dependency to that of oppositely directed migration, was CD11b/18 dependent, and required epithelial protein synthesis. Additionally, however, important qualitative differences existed in how IFN-gamma affected

  18. Altered expression of intestinal human leucocyte antigen D-related and immune signalling molecules in juvenile idiopathic arthritis

    PubMed Central

    Arvonen, M; Vähäsalo, P; Turunen, S; Salo, H M; Mäki, M; Laurila, K; Vaarala, O; Karttunen, T J

    2012-01-01

    We aimed to study intestinal immune activation status in juvenile idiopathic arthritis (JIA) by assessing intestinal human leucocyte antigen (HLA) class II expression and the mRNA expression levels of the pro- and anti-inflammatory mediators and pattern recognition receptors. HLA-D-related (HLA-DR) expression was assessed using immunohistochemical staining of frozen sections in 11 children with JIA and 17 controls. The gene expression levels of the anti- and proinflammatory cytokines, lymphocyte recognition receptors and pattern recognition receptors were studied with reverse transcription–polymerase chain reaction (RT–PCR) in 14 children with JIA and 12 controls. All subjects had various gastrointestinal (GI) symptoms indicating endoscopic examinations, but eventually were not diagnosed with GI disease. In JIA patients, the expression of HLA-DR was increased in the crypt epithelial cells and in the epithelial basement membrane of the ileum when compared with the controls. Positive HLA-DR staining in the ileal mucosa was associated with the presence of high clinical disease activity of JIA and low mRNA expression of anti-inflammatory mediators, such as forkhead box protein P3 (FoxP3), glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR) and transforming growth factor (TGF)-beta. Low ileal expression of interleukin (IL)-10, TGF-β, FoxP3, Toll-like receptor 2 (TLR-2) and TLR-4 transcripts correlated significantly with a high clinical disease activity in the JIA patients. The increased HLA-DR expression suggests enhanced intestinal antigen presentation in JIA. A correlation between clinical disease activity and low gene expression of tolerogenic mediators in the ileum supports the hypothesis that a link exists between the gut immune system and JIA. PMID:23121667

  19. Active intestinal chloride secretion in human carriers of cystic fibrosis mutations: an evaluation of the hypothesis that heterozygotes have subnormal active intestinal chloride secretion.

    PubMed

    Högenauer, C; Santa Ana, C A; Porter, J L; Millard, M; Gelfand, A; Rosenblatt, R L; Prestidge, C B; Fordtran, J S

    2000-12-01

    To explain the very high frequency of cystic fibrosis (CF) mutations in most populations of European descent, it has been proposed that CF heterozygotes have a survival advantage when infected with Vibrio cholerae or Escherichia coli, the toxins of which induce diarrhea by stimulation of active intestinal chloride secretion. Two assumptions underlie this hypothesis: (1) chloride conductance by the CF transmembrane conductance regulator (CFTR) is the rate-limiting step for active intestinal chloride secretion at all levels of expression, from approximately zero in patients with CF to normal levels in people who are not carriers of a mutation; and (2) heterozygotes have smaller amounts of functional intestinal CFTR than do people who are not carriers, and heterozygotes therefore secrete less chloride when exposed to secretagogues. The authors used an intestinal perfusion technique to measure in vivo basal and prostaglandin-stimulated jejunal chloride secretion in normal subjects, CF heterozygotes, and patients with CF. Patients with CF had essentially no active chloride secretion in the basal state, and secretion was not stimulated by a prostaglandin analogue. However, CF heterozygotes secreted chloride at the same rate as did people without a CF mutation. If heterozygotes are assumed to have less-than-normal intestinal CFTR function, these results mean that CFTR expression is not rate limiting for active chloride secretion in heterozygotes. The results do not support the theory that the very high frequency of CF mutations is due to a survival advantage that is conferred on heterozygotes who contract diarrheal illnesses mediated by intestinal hypersecretion of chloride.

  20. Comparison of the Flotac-400 dual technique and the formalin-ether concentration technique for diagnosis of human intestinal protozoon infection.

    PubMed

    Becker, Sören L; Lohourignon, Laurent K; Speich, Benjamin; Rinaldi, Laura; Knopp, Stefanie; N'goran, Eliézer K; Cringoli, Giuseppe; Utzinger, Jürg

    2011-06-01

    There is a need for accurate diagnosis of intestinal parasite infections in humans, but currently available copromicroscopic techniques have shortcomings, such as low sensitivity. We compared the diagnostic accuracy of a further modified version of the recently developed Flotac technique with that of the widely used formalin-ether concentration technique (FECT) for the detection of intestinal protozoa in human stool samples. Formaldehyde-preserved stool samples from 108 individuals in Côte d'Ivoire were subjected to the Flotac-400 dual technique, using two different flotation solutions (FS), and to the FECT. Stool samples were examined according to computer-generated random lists by an experienced laboratory technician blinded for the results of each method. Both methods detected the same eight intestinal protozoon species. While the Flotac-400 dual technique (results from both FS combined) found higher prevalences of Entamoeba coli (77.8% versus 71.3%, P < 0.001), Blastocystis hominis (20.4% versus 13.0%, P = 0.458), and Giardia intestinalis (8.3% versus 6.5%, P < 0.001), the FECT detected higher prevalences of Entamoeba histolytica/Entamoeba dispar (27.8% versus 20.4%, P = 0.019) and four species of nonpathogenic intestinal protozoa. The diagnostic agreement between the two methods differed considerably depending on the intestinal protozoon investigated (Cohen's kappa measures; range, 0.01 to 0.46). Our study confirmed that the Flotac-400 dual technique can be utilized for the diagnosis of intestinal protozoon infections in humans. Since Flotac is a sensitive technique for the detection of soil-transmitted helminths and Schistosoma mansoni, it might become a viable copromicroscopic technique for the concurrent diagnosis of helminths and intestinal protozoon infections.

  1. Expression Quantitative Trait Loci Analysis Identifies Associations Between Genotype and Gene Expression in Human Intestine

    PubMed Central

    KABAKCHIEV, BOYKO; SILVERBERG, MARK S.

    2013-01-01

    BACKGROUND & AIMS Genome-wide association studies have greatly increased our understanding of intestinal disease. However, little is known about how genetic variations result in phenotypic changes. Some polymorphisms have been shown to modulate quantifiable phenotypic traits; these are called quantitative trait loci. Quantitative trait loci that affect levels of gene expression are called expression quantitative trait loci (eQTL), which can provide insight into the biological relevance of data from genome-wide association studies. We performed a comprehensive eQTL scan of intestinal tissue. METHODS Total RNA was extracted from ileal biopsy specimens and genomic DNA was obtained from whole-blood samples from the same cohort of individuals. Cis- and trans-eQTL analyses were performed using a custom software pipeline for samples from 173 subjects. The analyses determined the expression levels of 19,047 unique autosomal genes listed in the US National Center for Biotechnology Information database and more than 580,000 variants from the Single Nucleotide Polymorphism database. RESULTS The presence of more than 15,000 cis- and trans-eQTL was detected with statistical significance. eQTL associated with the same expression trait were in high linkage disequilibrium. Comparative analysis with previous eQTL studies showed that 30% to 40% of genes identified as eQTL in monocytes, liver tissue, lymphoblastoid cell lines, T cells, and fibroblasts are also eQTL in ileal tissue. Conversely, most of the significant eQTL have not been previously identified and could be tissue specific. These are involved in many cell functions, including division and antigen processing and presentation. Our analysis confirmed that previously published cis-eQTL are single nucleotide polymorphisms associated with inflammatory bowel disease: rs2298428/UBE2L3, rs1050152/SLC22A4, and SLC22A5. We identified many new associations between inflammatory bowel disease susceptibility loci and gene expression

  2. An update discussion on the current assessment of the safety of veterinary antimicrobial drug residues in food with regard to their impact on the human intestinal microbiome.

    PubMed

    Cerniglia, Carl E; Pineiro, Silvia A; Kotarski, Susan F

    2016-05-01

    The human gastrointestinal tract ecosystem consists of complex and diverse microbial communities that have now been collectively termed the intestinal microbiome. Recent scientific breakthroughs and research endeavours have increased our understanding of the important role the intestinal microbiome plays in human health and disease. The use of antimicrobial new animal drugs in food-producing animals may result in the presence of low levels of drug residues in edible foodstuffs. There is concern that antimicrobial new animal drugs in or on animal-derived food products at residue-level concentrations could disrupt the colonization barrier and/or modify the antimicrobial resistance profile of human intestinal bacteria. Therapeutic doses of antimicrobial drugs have been shown to promote shifts in the intestinal microbiome, and these disruptions promote the emergence of antimicrobial-resistant bacteria. To assess the effects of antimicrobial new animal drug residues in food on human intestinal bacteria, many national regulatory agencies and international committees follow a harmonized process, VICH GL36(R), which was issued by a trilateral organization of the European Union, the USA, and Japan called the International Cooperation on Harmonization of Technical Requirements for Veterinary Medicinal Products (VICH). The guidance describes a general approach currently used by national regulatory agencies and international committees to assess the effects of antimicrobial new animal drug residues in animal-derived food on human intestinal bacteria. The purpose of this review is to provide an overview of this current approach as part of the antimicrobial new animal drug approval process in participating countries, give insights on the microbiological endpoints used in this safety evaluation, and discuss the availability of new information. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Cytokine modulation (IL-6, IL-8, IL-10) by human breast milk lipids on intestinal epithelial cells (Caco-2).

    PubMed

    Barrera, Girolamo J; Sánchez, Gabriela

    2016-01-01

    Human breast milk is the best form of nourishment for infants during the first year of life. It is composed by a complex mixture of carbohydrates, proteins and fats. Breast milk provides nutrients and bioactive factors that themselves modulate maturation and development of the gastrointestinal tract. Many studies have shown that it provides protection against gastrointestinal tract inflammation. In this sense, this study aimed to evaluate the effect of human breast milk lipids on epithelial intestinal cells (Caco-2) cytokine regulation and the fatty acid transporter protein (FATP) involved in this process. Caco-2 cells were cultivated and stimulated with different concentration of human milk lipids from healthy human mothers (18-30-year-olds) or single commercial lipids for 48 h. We measured the concentrations and mRNA levels of IL-6, IL-8 and IL-10 cytokines by immunoassay (ELISA) and quantitative-PCR (qRT-PCR) technique, respectively. We observed a two to three times decrease in pro-inflammatory cytokine levels (p < 0.01) as well as an increase in anti-inflammatory IL-10 levels in cells stimulated with increasing concentrations of breast milk lipids. These results suggest that human breast milk lipids could have an important role on the cytokine modulation in the newborn bowel.

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

    SciTech Connect

    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

    2014-01-17

    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.

  5. Nutraceutical Improvement Increases the Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation.

    PubMed

    Ferruzza, Simonetta; Natella, Fausta; Ranaldi, Giulia; Murgia, Chiara; Rossi, Carlotta; Trošt, Kajetan; Mattivi, Fulvio; Nardini, Mirella; Maldini, Mariateresa; Giusti, Anna Maria; Moneta, Elisabetta; Scaccini, Cristina; Sambuy, Yula; Morelli, Giorgio; Baima, Simona

    2016-08-12

    Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts' juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids.

  6. Phytosterol ester processing in the small intestine: impact on cholesterol availability for absorption and chylomicron cholesterol incorporation in healthy humans.

    PubMed

    Amiot, Marie Josèphe; Knol, Diny; Cardinault, Nicolas; Nowicki, Marion; Bott, Romain; Antona, Claudine; Borel, Patrick; Bernard, Jean-Paul; Duchateau, Guus; Lairon, Denis

    2011-06-01

    Phytosterols (plant sterols and stanols) can lower intestinal cholesterol absorption, but the complex dynamics of the lipid digestion process in the presence of phytosterol esters (PEs) are not fully understood. We performed a clinical experiment in intubated healthy subjects to study the time course of changes in the distribution of all lipid moieties present in duodenal phases during 4 h of digestion of meals with 3.2 g PE (PE meal) or without (control meal) PE. In vitro experiments under simulated gastrointestinal conditions were also performed. The addition of PE did not alter triglyceride (TG) hydrolysis in the duodenum or subsequent chylomicron TG occurrence in the circulation. In contrast, cholesterol accumulation in the duodenum aqueous phase was markedly reduced in the presence of PE (-32%, P < 0.10). In vitro experiments confirmed that PE reduces cholesterol transfer into the aqueous phase. The addition of PE resulted in a markedly reduced presence of meal-derived hepta-deuterated cholesterol in the circulation, i.e., in chylomicrons (-43%, PE meal vs. control; P < 0.0001) and plasma (-54%, PE meal vs. control; P < 0.0001). The present data show that addition of PE to a meal does not alter TG hydrolysis but displaces cholesterol from the intestinal aqueous phase and lowers chylomicron cholesterol occurrence in humans.

  7. The transepithelial transport mechanism of polybrominated diphenyl ethers in human intestine determined using a Caco-2 cell monolayer.

    PubMed

    Yu, Yingxin; Wang, Mengmeng; Zhang, Kaiqiong; Yang, Dan; Zhong, Yufang; An, Jing; Lei, Bingli; Zhang, Xinyu

    2017-04-01

    Oral ingestion plays an important role in human exposure to polybrominated diphenyl ethers (PBDEs). The uptake of PBDEs primarily occurs in the small intestine. The aim of the present study is to investigate the transepithelial transport characteristics and mechanisms of PBDEs in the small intestine using a Caco-2 cell monolayer model. The apparent permeability coefficients of PBDEs indicated that tri- to hepta-BDEs were poorly absorbed compounds. A linear increase in transepithelial transport was observed with various concentrations of PBDEs, which suggested that passive diffusion dominated their transport at the concentration range tested. In addition, the pseudo-first-order kinetics equation can be applied to the transepithelial transport of PBDEs. The rate-determining step in transepithelial transport of PBDEs was trans-cell transport including the trans-pore process. The significantly lower transepithelial transport rates at low temperature for bidirectional transepithelial transport suggested that an energy-dependent transport mechanism was involved. The efflux transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) and influx transporters (organic cation transporters) participated in the transepithelial transport of PBDEs. In addition, the transepithelial transport of PBDEs was pH sensitive; however, more information is required to understand the influence of pH.

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

    PubMed Central

    Lodemann, Ulrike; Strahlendorf, Julia; Schierack, Peter; Klingspor, Shanti; Aschenbach, Jörg R.

    2015-01-01

    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. PMID:25883829

  9. Nutraceutical Improvement Increases the Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation

    PubMed Central

    Ferruzza, Simonetta; Natella, Fausta; Ranaldi, Giulia; Murgia, Chiara; Rossi, Carlotta; Trošt, Kajetan; Mattivi, Fulvio; Nardini, Mirella; Maldini, Mariateresa; Giusti, Anna Maria; Moneta, Elisabetta; Scaccini, Cristina; Sambuy, Yula; Morelli, Giorgio; Baima, Simona

    2016-01-01

    Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts’ juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids. PMID:27529258

  10. Human small-intestinal β-galactosidases. Separation and characterization of three forms of an acid β-galactosidase

    PubMed Central

    Asp, Nils-Georg

    1971-01-01

    1. An acid β-galactosidase, optimum pH4.0–4.5, in the human small-intestinal mucosa was separated and characterized. 2. Autolysis of mucosal homogenates at acid pH inactivated the lactase and hetero β-galactosidase; the total activity of the acid β-galactosidase was only slightly depleted, but a greater proportion of the enzyme was solubilized by this treatment. 3. Separation on a Sephadex G-200 column revealed that the acid β-galactosidase could occur in at least three different forms, probably representing monomer, dimer and octamer or polymer of the enzyme. 4. The properties of the different forms of the acid β-galactosidase were studied with regard to pH optimum, Km, rate of hydrolysis of different substrates, and sensitivity to p-chloromercuribenzoate and tris as inhibitors. All these properties were the same for the different forms of the enzyme. 5. The acid β-galactosidase hydrolyses lactose as well as hetero β-galactosides and contributes to the lactase activity of intestinal biopsies also when measured at pH 6. This enzyme may therefore be responsible for a considerable part of the residual lactase activity found in lactose-intolerant patients. PMID:5117032

  11. Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

    PubMed

    Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

    2012-01-01

    A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

  12. The human intestinal fatty acid binding protein (hFABP2) gene is regulated by HNF-4{alpha}

    SciTech Connect

    Klapper, Maja . E-mail: klapper@molnut.uni-kiel.de; Boehme, Mike; Nitz, Inke; Doering, Frank

    2007-04-27

    The cytosolic human intestinal fatty acid binding protein (hFABP2) is proposed to be involved in intestinal absorption of long-chain fatty acids. The aim of this study was to investigate the regulation of hFABP2 by the endodermal hepatocyte nuclear factor 4{alpha} (HNF-4{alpha}), involved in regulation of genes of fatty acid metabolism and differentiation. Electromobility shift assays demonstrated that HNF-4{alpha} binds at position -324 to -336 within the hFABP2 promoter. Mutation of this HNF-4 binding site abolished the luciferase reporter activity of hFABP2 in postconfluent Caco-2 cells. In HeLa cells, this mutation reduced the activation of the hFABP2 promoter by HNF-4{alpha} by about 50%. Thus, binding element at position -336/-324 essentially determines the transcriptional activity of promoter and may be important in control of hFABP2 expression by dietary lipids and differentiation. Studying genotype interactions of hFABP2 and HNF-4{alpha}, that are both candidate genes for diabetes type 2, may be a powerful approach.

  13. Structural Stability of Human Fibroblast Growth Factor-1 Is Essential for Protective Effects Against Radiation-Induced Intestinal Damage

    SciTech Connect

    Nakayama, Fumiaki; Umeda, Sachiko; Yasuda, Takeshi; Asada, Masahiro; Motomura, Kaori; Suzuki, Masashi; Zakrzewska, Malgorzata; Imamura, Toru; Imai, Takashi

    2013-02-01

    Purpose: Human fibroblast growth factor-1 (FGF1) has radioprotective effects on the intestine, although its structural instability limits its potential for practical use. Several stable FGF1 mutants were created increasing stability in the order, wild-type FGF1, single mutants (Q40P, S47I, and H93G), Q40P/S47I, and Q40P/S47I/H93G. This study evaluated the contribution of the structural stability of FGF1 to its radioprotective effect. Methods and Materials: Each FGF1 mutant was administered intraperitoneally to BALB/c mice in the absence of heparin 24 h before or after total body irradiation (TBI) with {gamma}-rays at 8-12 Gy. Several radioprotective effects were examined in the jejunum. Results: Q40P/S47I/H93G could activate all subtypes of FGF receptors in vitro much more strongly than the wild-type without endogenous or exogenous heparin. Preirradiation treatment with Q40P/S47I/H93G significantly increased crypt survival more than wild-type FGF1 after TBI at 10 or 12 Gy, and postirradiation treatment with Q40P/S47I/H93G was effective in promoting crypt survival after TBI at 10, 11, or 12 Gy. In addition, crypt cell proliferation, crypt depth, and epithelial differentiation were significantly promoted by postirradiation treatment with Q40P/S47I/H93G. The level of stability of FGF1 mutants correlated with their mitogenic activities in vitro in the absence of heparin; however, preirradiation treatment with the mutants increased the crypt number to almost the same level as Q40P/S47I/H93G. When given 24 h after TBI at 10 Gy, all FGF1 mutants increased crypt survival more than wild-type FGF1, and Q40P/S47I/H93G had the strongest mitogenic effects in intestinal epithelial cells after radiation damage. Moreover, Q40P/S47I/H93G prolonged mouse survival after TBI because of the repair of intestinal damage. Conclusion: These findings suggest that the structural stability of FGF1 can contribute to the enhancement of protective effects against radiation-induced intestinal

  14. Pasteurization Procedures for Donor Human Milk Affect Body Growth, Intestinal Structure, and Resistance against Bacterial Infections in Preterm Pigs.

    PubMed

    Li, Yanqi; Nguyen, Duc Ninh; de Waard, Marita; Christensen, Lars; Zhou, Ping; Jiang, Pingping; Sun, Jing; Bojesen, Anders Miki; Lauridsen, Charlotte; Lykkesfeldt, Jens; Dalsgaard, Trine Kastrup; Bering, Stine Brandt; Sangild, Per Torp

    2017-03-15

    Background: Holder pasteurization (HP) destroys multiple bioactive factors in donor human milk (DM), and UV-C irradiation (UVC) is potentially a gentler method for pasteurizing DM for preterm infants.Objective: We investigated whether UVC-treated DM improves gut maturation and resistance toward bacterial infections relative to HP-treated DM.Methods: Bacteria, selected bioactive components, and markers of antioxidant capacity were measured in unpasteurized donor milk (UP), HP-treated milk, and UVC-treated milk (all from the same DM pool). Fifty-seven cesarean-delivered preterm pigs (91% gestation; ratio of males to females, 30:27) received decreasing volumes of parental nutrition (average 69 mL ⋅ kg(-1) ⋅ d(-1)) and increasing volumes of the 3 DM diets (n = 19 each, average 89 mL ⋅ kg(-1) ⋅ d(-1)) for 8-9 d. Body growth, gut structure and function, and systemic bacterial infection were evaluated.Results: A high bacterial load in the UP (6×10(5) colony forming units/mL) was eliminated similarly by HP and UVC treatments. Relative to HP-treated milk, both UVC-treated milk and UP showed greater activities of lipase and alkaline phosphatase and concentrations of lactoferrin, secretory immunoglobulin A, xanthine dehydrogenase, and some antioxidant markers (all P < 0.05). The pigs fed UVC-treated milk and pigs fed UP showed higher relative weight gain than pigs fed HP-treated milk (5.4% and 3.5%), and fewer pigs fed UVC-treated milk had positive bacterial cultures in the bone marrow (28%) than pigs fed HP-treated milk (68%) (P < 0.05). Intestinal health was also improved in pigs fed UVC-treated milk compared with those fed HP-treated milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%) (P < 0.05) and a tendency for higher aminopeptidase N (48%) and claudin-4 (26%) concentrations in the distal intestine (P < 0.08). The gut microbiota composition was similar among groups except for greater proportions of Enterococcus in pigs

  15. STORM: A General Model to Determine the Number and Adaptive Changes of Epithelial Stem Cells in Teleost, Murine and Human Intestinal Tracts

    PubMed Central

    Wang, Zhengyuan; Matsudaira, Paul; Gong, Zhiyuan

    2010-01-01

    Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0–4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0–4.1 stem cells on each crypt section of murine small intestine and 1.8–3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions. PMID:21124758

  16. How to make an intestine

    PubMed Central

    Wells, James M.; Spence, Jason R.

    2014-01-01

    With the high prevalence of gastrointestinal disorders, there is great interest in establishing in vitro models of human intestinal disease and in developing drug-screening platforms that more accurately represent the complex physiology of the intestine. We will review how recent advances in developmental and stem cell biology have made it possible to generate complex, three-dimensional, human intestinal tissues in vitro through directed differentiation of human pluripotent stem cells. These are currently being used to study human development, genetic forms of disease, intestinal pathogens, metabolic disease and cancer. PMID:24496613

  17. Human small intestinal contractions and aboral traction forces during fasting and after feeding.

    PubMed Central

    Ahluwalia, N K; Thompson, D G; Barlow, J; Heggie, L

    1994-01-01

    Small intestinal intraluminal pressure activity and aboral traction forces were explored in 19 healthy volunteers using a combined manometry and traction force detecting assembly sited in the upper small intestine. Each aboral traction event was classified as being associated with either a propagating or a stationary contraction and its force measured. During phase I no contractions or traction events were seen. During phase II, traction events related to propagating contractions mean (SEM) (2.2 (0.2)/min) and to stationary contractions (0.3 (0.1)/min) generated similar force/event (7.5(0.9 g v 8.7 (1.4) g, p > 0.05). During phase III, all traction events were related to propagating contractions and generated 9.3 (2.4) g force/event (p > 0.05 v phase II). After feeding, traction events related to propagating contractions generated similar force/event to those related to stationary contractions (5.9 (1.0) g v 9.3 (2.7) g, p > 0.05 v each other and v fasting). No consistent pattern was seen in the temporal distribution of the traction events or in the pattern of the amplitude of the force of successive traction events. PMID:8200554

  18. Chromosomes and causation of human cancer and leukemia. XXX. Banding studies of primary intestinal tumors.

    PubMed

    Sonta, S; Sandberg, A A

    1978-01-01

    The chromosomes of 15 primary intestinal tumors were analyzed with a banding technique. Of the 15 tumors, 12 had some chromosomal abnormalities (8 with numerical changes and 4 with both numerical and structural abnormalities) and in the remaining three no karyotypic abnormalities were found. No common marker chromosomes were seen among the various tumors and no two tumors with chromosomal changes and identical karyotypes, though some chromosomes were involved more often than others. Excessive chromosomes in the primary tumors were usually due to extra chromosomes in the following groups (numbers of tumors involved are shown in parenthesis): No. 8 (7), No. 13 (4), No. 15 (4), No. 17 (6) and No. 21 (6). On the other hand, chromosomes losses, though much less frequent, involved chromosomes No. 5, No. 6, No. 7, No. 10 and No. 16. Most of the tumor cells with chromosomal changes were hyperdiploid and usually contained less than 60 chromosomes. Only one tumor contained hypodiploid cells. The cytogenetic data presented on primary intestinal tumors indicate that they consist primarily of numerical changes, relative infrequency (when compared to metastases) and small number (1-4) of markers.

  19. Specific vaginal lactobacilli suppress the inflammation induced by lipopolysaccharide stimulation through downregulation of toll-like receptor 4 expression in human embryonic intestinal epithelial cells

    PubMed Central

    TOBITA, Keisuke; WATANABE, Itsuki; SAITO, Masanori

    2016-01-01

    Vaginal lactobacilli (VLB) spread from the mother to the infant during vaginal delivery. However, the effects of VLB on infant intestinal function remain unclear. We investigated the probiotic function and immune effects of VLB on the human embryonic intestinal epithelial cell line INT-407. VLB survived artificial gastric juice and adhered to INT-407 cells. Exposure of INT-407 cells to VLB attenuated both the lipopolysaccharide (LPS)-induced stimulation of interleukin-8 and tumor necrosis factor alpha production and the LPS-stimulated upregulation of TLR4 expression. These results suggest that specific VLB suppresses the inflammation induced by LPS stimulation through downregulation of TLR4 expression in human embryonic intestinal epithelial cells. PMID:28243550

  20. Involvement of UDP-glucuronosyltranferases and sulfotransferases in the liver and intestinal first-pass metabolism of seven flavones in C57 mice and humans in vitro.

    PubMed

    Tang, Lan; Feng, Qian; Zhao, Jie; Dong, Lingna; Liu, Wei; Yang, Caihua; Liu, Zhongqiu

    2012-05-01

    The present study investigated the involvement of UDP-glucuronosyltransferase and sulfotransferase in the extensive liver and intestinal first-pass glucuronidation and sulfation of flavones in both mice and humans. Seven structurally similar mono- and di-hydroxyflavones were chosen as model compounds. Human liver, C57 mouse liver and intestinal S9 fraction, as well as C57 intestinal perfusion model were used. In human and C57 mouse, all selected flavones were found to be glucuronidated with the highest rates at the 7-OH group. In contrast, flavones with 3-OH group were not sulfated at all. Both glucuronidation and sulfation preferred 4'- and 7-OH in human and mouse in vitro and in situ. There were differences in glucuronidation and sulfation in human and mouse observed for all flavones and it is based on substitutional positions of the hydroxyl groups. The S9 fractions could accurately model glucuronidation (as the slope of correlation curve was 0.7988 for those flavones with 4'- or 7-OH) and sulfation (as the slope of correlation curve was 0.9834) in situ. Conclusively, the sulfation and glucuronidation of the flavones was regiospecific- and speciesdependent. Sulfation and glucuronidation in the mouse intestine in vitro were correlated well with those in situ.

  1. An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

    PubMed Central

    Erbel, Christian; Rupp, Gregor; Helmes, Christian M.; Tyka, Mirjam; Linden, Fabian; Doesch, Andreas O.; Katus, Hugo A.; Gleissner, Christian A.

    2013-01-01

    Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases. PMID:23792882

  2. The magnitude and risk factors of intestinal parasitic infection in relation to Human Immunodeficiency Virus infection and immune status, at ALERT Hospital, Addis Ababa, Ethiopia.

    PubMed

    Taye, Biruhalem; Desta, Kassu; Ejigu, Selamawit; Dori, Geme Urge

    2014-06-01

    Human Immunodeficiency Virus (HIV) and intestinal parasitic infections are among the main health problems in developing countries like Ethiopia. Particularly, co-infections of these diseases would worsen the progression of HIV to Acquired Immunodeficiency Syndrome (AIDS). The purpose of this study was to determine the magnitude and risk factors for intestinal parasites in relation to HIV infection and immune status. The study was conducted in (1) HIV positive on antiretroviral therapy (ART) and (2) ART naïve HIV positive patients, and (3) HIV-negative individuals, at All African Leprosy and Tuberculosis (TB) Eradication and Rehabilitation Training Center (ALERT) hospital in Addis Ababa, Ethiopia. Study participants were interviewed using structured questionnaires to obtain socio-demographic characteristics and assess risk factors associated with intestinal parasitic infection. Intestinal parasites were identified from fecal samples by direct wet mount, formol ether concentration, and modified Ziehl-Neelsen staining techniques. The immune status was assessed by measuring whole blood CD4 T-cell count. The overall magnitude of intestinal parasite was 35.08%. This proportion was different among study groups with 39.2% (69/176), 38.83% (40/103) and 27.14% (38/140) in ART naïve HIV positives patients, in HIV negatives, and in HIV positive on ART patients respectively. HIV positive patients on ART had significantly lower magnitude of intestinal parasitic infection compared to HIV negative individuals. Intestinal helminths were significantly lower in HIV positive on ART and ART naïve patients than HIV negatives. Low monthly income, and being married, divorced or widowed were among the socio-demographic characteristics associated with intestinal parasitic infection. No association was observed between the magnitude of intestinal parasites and CD4 T-cell count. However, Cryptosporidium parvum, and Isospora belli were exclusively identified in individuals with CD4 T

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

    PubMed Central

    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.

    2017-01-01

    ABSTRACT 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. PMID:28292985

  4. Polar Bear

    USGS Publications Warehouse

    Amstrup, S.D.; ,; Lentfer, J.W.

    1988-01-01

    Polar bears are long-lived, late-maturing carnivores that have relatively low rates of reproduction and natural mortality. Their populations are susceptible to disturbance from human activities, such as the exploration and development of mineral resources or hunting. Polar bear populations have been an important renewable resource available to coastal communities throughout the Arctic for thousands of years.

  5. Non-polar extracts of serum from human males contain covert radioimmunoassayable testosterone

    SciTech Connect

    Addo, S.B.

    1989-01-01

    A form of testosterone that cannot be detected by standard radioimmunoassay procedures was identified in the sera of healthy men. The sera were extracted with petroleum ether, subjected to mild alkali hydrolysis, purified by reverse phase high pressure liquid chromatography, and assayed by radioimmunoassay. Hydrolyzed non-polar serum extracts from ten adult male volunteers contained 2.0 {plus minus} 0.8 ng/ml testosterone. In contrast, no testosterone was found in hydrolyzed petroleum ether extracts of sera from eight women or in any of the unhydrolyzed samples. Two other women had values of 0.7 ng/ml and 0.8 ng/ml. Testosterone palmitate, used as a model compound, was hydrolyzable by the procedure and gave recoveries of more than 90%. These findings reveal the presence of substantial quantities of novel naturally-occurring non-polar conjugates of testosterone (NPT) in sera of human males. The compounds may be analogous to long-chain fatty acid conjugates of estradiol identified in female sera. The lipoidal estrogens exert long-lasting biological actions attributed to slow release of free hormone. NPT may therefore act physiologically as hormone reservoirs when little or no androgen is released by the testis. They may also contribute to abnormal stimulation of androgen-responsive tissues. Some evidence for their presence in the sera of patients with prostatic cancer receiving androgen-supression therapy has been obtained.

  6. Environmental contaminants activate human and polar bear (Ursus maritimus) pregnane X receptors (PXR, NR1I2) differently

    PubMed Central

    Roger, Lille-Langøy; V, Goldstone Jared; Marte, Rusten; R, Milnes Matthew; Rune, Male; J, Stegeman John; Bruce, Blumberg; Anders, Goksøyr

    2015-01-01

    BACKGROUND Many persistent organic pollutants (POPs) accumulate readily in polar bears because of their position as apex predators in Arctic food webs. The pregnane X receptor (PXR, formally NR1I2, here proposed to be named promiscuous xenobiotic receptor) is a xenobiotic sensor that is directly involved in metabolizing pathways of a wide range of environmental contaminants. OBJECTIVES In the present study, we comparably assess the ability of 51 selected pharmaceuticals, pesticides and emerging contaminants to activate PXRs from polar bears and humans using an in vitro luciferase reporter gene assay. RESULTS We found that polar bear PXR is activated by a wide range of our test compounds (68%) but has a slightly more narrow ligand specificity than human PXR that was activated by 86% of the 51 test compounds. The majority of the agonists identified (70%) produces a stronger induction of the reporter gene via human PXR than via polar bear PXR, however with some notable and environmentally relevant exceptions. CONCLUSIONS Due to the observed differences in activation of polar bear and human PXRs, exposure of each species to environmental agents is likely to induce biotransformation differently in the two species. Bioinformatics analyses and structural modelling studies suggests that amino acids that are not part of the ligand-binding domain and do not interact with the ligand can modulate receptor activation. PMID:25680588

  7. The prenylflavonoid isoxanthohumol from hops (Humulus lupulus L.) is activated into the potent phytoestrogen 8-prenylnaringenin in vitro and in the human intestine.

    PubMed

    Possemiers, Sam; Bolca, Selin; Grootaert, Charlotte; Heyerick, Arne; Decroos, Karel; Dhooge, Willem; De Keukeleire, Denis; Rabot, Sylvie; Verstraete, Willy; Van de Wiele, Tom

    2006-07-01

    Hops, an essential beer ingredient, are a source of prenylflavonoids, including 8-prenylnaringenin (8-PN), one of the most potent phytoestrogens. Because 8-PN concentrations in beers are generally low, its health effects after moderate beer consumption were considered negligible. However, human intestinal microbiota may activate up to 4 mg/L isoxanthohumol (IX) in beer into 8-PN. Depending on interindividual differences in the intestinal transformation potential, this conversion could easily increase the 8-PN exposure 10-fold upon beer consumption. Here, we present a further investigation of the process both in vitro and in vivo. In vitro experiments with the dynamic SHIME model showed that hop prenylflavonoids pass unaltered through the stomach and small intestine and that activation of IX into 8-PN (up to 80% conversion) occurs only in the distal colon. In vitro incubations of 51 fecal samples from female volunteers with IX enabled us to separate the fecal microbiota into high (8 of 51), moderate (11 of 51) and slow (32 of 51) 8-PN producers, clearly illustrating an interindividual variability. Three women, selected from the respective groups, received a daily dose of 5.59 mg IX for 4 d. Intestinal IX activation and urinary 8-PN excretion were correlated (R(2) = 0.6417, P < 0.01). These data show that intestinal conversion of IX upon moderate beer consumption can lead to 8-PN exposure values that might fall within the range of human biological activity.

  8. Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs.

    PubMed

    Inoue, M; Morikawa, M; Tsuboi, M; Ito, Y; Sugiura, M

    1980-08-01

    In attempts to determine the exact role of intestinal esterase in the body, we purified esterases from human intestinal mucosa and liver, and compared the enzymatic properties and substrate specificities with those of purified esterases. Esterase from human liver was purified 58-fold, by treatment with butanol, DE-52 and DEAE Sephadex A-50 column chromatographies, Sephadex G-200 gel filtration, and isoelectric focusing. The purified preparation showed a single band by polyacylamide gel electrophoresis. The molecular weights of intestinal and hepatic esterases were determined to be 53,000-55,000 and 180,000, respectively, by gel filtration on Sephadex G-200. The activity of the purified intestinal and hepatic esterases was strongly inhibited by diethyl-p-nitrophenyl phosphate and diisopropyl fluorophosphate, and was not inhibited by eserine sulfate and p-chloromercuribenzoate. Moreover, the purified esterases hydrolyzed ester-type drugs such as aspirin, clofibrate, indanyl carbenicillin and procaine. Hepatic esterase had properties similar to those of intestinal esterase with respect to the sensitivity to organophosphate and the substrate specificity. However, the two purified esterases differed in properties such as molecular weight, isoelectric point, thermostability and optimal pH.

  9. Inulin-type fructan fermentation by bifidobacteria depends on the strain rather than the species and region in the human intestine.

    PubMed

    Selak, Marija; Rivière, Audrey; Moens, Frédéric; Van den Abbeele, Pieter; Geirnaert, Annelies; Rogelj, Irena; Leroy, Frédéric; De Vuyst, Luc

    2016-05-01

    Inulin-type fructans (ITF) are known to cause a health-promoting bifidogenic effect, although the ITF degradation capacity of bifidobacteria in different intestinal regions remains unclear. The present study aims at offering new insights into this link, making use of a collection of 190 bifidobacterial strains, encompassing strains from gut biopsies (terminal ileum and proximal colon; mucosa-associated strains) and the simulator of the human intestinal microbial ecosystem (SHIME®; proximal and distal colon vessels; lumen-associated strains). A multivariate data analysis of all fermentation data revealed four clusters corresponding with different types of ITF degradation fingerprints, which were not correlated with the region in the intestine, suggesting that the degradation of ITF is uniform along the human intestine. Strains from cluster 1 consumed fructose, while strains from cluster 2 consumed more oligofructose than fructose. Higher fructose and oligofructose consumption was characteristic for clusters 3 and 4 strains, which degraded inulin too. In general, the mucosa-associated strains from biopsy origin seemed to be more specialized in the consumption of fructose and oligofructose, while the lumen-associated strains from SHIME origin displayed a higher degradation degree of inulin. Further, intra-species variability in ITF degradation was found, indicating strain-specific variations. The coexistence of different bifidobacterial strains with different ITF degradation fingerprints within the same intestinal region suggests cooperation for the degradation of ITF, with opportunities for cross-feeding on strain and/or species level.

  10. DNA–Methylome Analysis of Mouse Intestinal Adenoma Identifies a Tumour-Specific Signature That Is Partly Conserved in Human Colon Cancer

    PubMed Central

    Grimm, Christina; Chavez, Lukas; Vilardell, Mireia; Farrall, Alexandra L.; Tierling, Sascha; Böhm, Julia W.; Grote, Phillip; Lienhard, Matthias; Dietrich, Jörn; Timmermann, Bernd; Walter, Jörn; Schweiger, Michal R.; Lehrach, Hans; Herwig, Ralf; Herrmann, Bernhard G.; Morkel, Markus

    2013-01-01

    Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers. PMID:23408899

  11. Effects of Japanese mistletoe lectin on cytokine gene expression in human colonic carcinoma cells and in the mouse intestine.

    PubMed

    Monira, Pervin; Koyama, Yu; Fukutomi, Ryuuta; Yasui, Kensuke; Isemura, Mamoru; Yokogoshi, Hidehiko

    2009-10-01

    Mistletoe lectins have various biological activities including anti-cancer and immunomodulatory effects. We previously isolated a lectin (ML-J) from Japanese mistletoe. In the present study, we examined the effects of ML-J on cytokine gene expression in human colon adenocarcinoma Caco-2 cells and in the mouse intestine. The results of reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction indicated that ML-J caused an upregulation of the gene expression of the proinflammatory cytokines interleukin (IL)-8, tumor necrosis factor-alpha (TNF-alpha) and IL-6 in Caco-2 cells and TNF-alpha and IL-6 in the duodenum. This study provides the first example to show that a perorally administered plant lectin affects gene expression in the duodenum.

  12. Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells

    PubMed Central

    Brancaccio, Mariarita; Coretti, Lorena; Florio, Ermanno; Pezone, Antonio; Calabrò, Viola; Falco, Geppino; Keller, Simona; Lembo, Francesca; Avvedimento, Vittorio Enrico; Chiariotti, Lorenzo

    2016-01-01

    Bacterial lipopolysaccharide (LPS) induces release of inflammatory mediators both in immune and epithelial cells. We investigated whether changes of epigenetic marks, including selected histone modification and DNA methylation, may drive or accompany the activation of COX-2 gene in HT-29 human intestinal epithelial cells upon exposure to LPS. Here we describe cyclical histone acetylation (H3), methylation (H3K4, H3K9, H3K27) and DNA methylation changes occurring at COX-2 gene promoter overtime after LPS stimulation. Histone K27 methylation changes are carried out by the H3 demethylase JMJD3 and are essential for COX-2 induction by LPS. The changes of the histone code are associated with cyclical methylation signatures at the promoter and gene body of COX-2 gene. PMID:27253528

  13. Biotransformation of prim-O-glucosylcimifugin by human intestinal flora and its inhibition on NO production and DPPH free radical.

    PubMed

    Zhao, Bo; Yang, Xin-Bao; Yang, Xiu-Wei; Liu, Jian-Xun

    2012-01-01

    prim-O-Glucosylcimifugin (PGCN), a highest content chromone in the roots of Saposhnikovia divaricata, was incubated with human intestinal flora (HIF), and two biotransformation products were obtained from the incubated solution by chromatographic methods. The chemical structures of the two biotransformation products were elucidated as cimifugin (CN) and 5-O-methylvisamminol (MVL), respectively, on the basis of NMR and MS data. The biotransformation product CN was formed through a deglucosylation of PGCN by β-glucosidase secreted from the HIF, and then the hydroxymethyl group of CN was reduced to lead to occurrence of MVL. All of these compounds were evaluated for their effect on the inhibition of nitric oxide production induced by lipopolysaccharide in macrophage cell line RAW 264.7 and for 1,1-diphenyl-2-picrylhydrazyl free-radical scavenging activity in cell-free bioassay system.

  14. Semi-automated solid-phase extraction method for studying the biodegradation of ochratoxin A by human intestinal microbiota.

    PubMed

    Camel, Valérie; Ouethrani, Minale; Coudray, Cindy; Philippe, Catherine; Rabot, Sylvie

    2012-04-15

    A simple and rapid semi-automated solid-phase (SPE) extraction method has been developed for the analysis of ochratoxin A in aqueous matrices related to biodegradation experiments (namely digestive contents and faecal excreta), with a view of using this method to follow OTA biodegradation by human intestinal microbiota. Influence of extraction parameters that could affect semi-automated SPE efficiency was studied, using C18-silica as the sorbent and water as the simplest matrix, being further applied to the matrices of interest. Conditions finally retained were as follows: 5-mL aqueous samples (pH 3) containing an organic modifier (20% ACN) were applied on 100-mg cartridges. After drying (9 mL of air), the cartridge was rinsed with 5-mL H(2)O/ACN (80:20, v/v), before eluting the compounds with 3 × 1 mL of MeOH/THF (10:90, v/v). Acceptable recoveries and limits of quantification could be obtained considering the complexity of the investigated matrices and the low volumes sampled; this method was also suitable for the analysis of ochratoxin B in faecal extracts. Applicability of the method is illustrated by preliminary results of ochratoxin A biodegradation studies by human intestinal microbiota under simple in vitro conditions. Interestingly, partial degradation of ochratoxin A was observed, with efficiencies ranging from 14% to 47% after 72 h incubation. In addition, three phase I metabolites could be identified using high resolution mass spectrometry, namely ochratoxin α, open ochratoxin A and ochratoxin B.

  15. Tick-Borne Encephalitis Virus Replication, Intracellular Trafficking, and Pathogenicity in Human Intestinal Caco-2 Cell Monolayers

    PubMed Central

    Möller, Lars; Schulzke, Joerg D.; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route. PMID

  16. Enhanced uptake and transport of (+)-catechin and (-)-epigallocatechin gallate in niosomal formulation by human intestinal Caco-2 cells.

    PubMed

    Song, Qinxin; Li, Danhui; Zhou, Yongzhi; Yang, Jie; Yang, Wanqi; Zhou, Guohua; Wen, Jingyuan

    2014-01-01

    The aim of this study was to evaluate (+)-catechin and (-)-epigallocatechin gallate (EGCG) cellular uptake and transport across human intestinal Caco-2 cell monolayer in both the absence and presence of niosomal carrier in variable conditions. The effect of free drugs and drug-loaded niosomes on the growth of Caco-2 cells was studied. The effects of time, temperature, and concentration on drug cellular uptake in the absence or presence of its niosomal delivery systems were investigated. The intestinal epithelial membrane transport of the drug-loaded niosomes was examined using the monolayer of the human Caco-2 cells. The kinetics of transport, and the effect of temperature, adenosine triphosphate inhibitor, permeability glycoprotein inhibitor, multidrug resistance-associated protein 2 inhibitor, and the absorption enhancer on transport mechanism were investigated. It was found that the uptake of catechin, EGCG, and their niosomes by Caco-2 cells was 1.22 ± 0.16, 0.90 ± 0.14, 3.25 ± 0.37, and 1.92 ± 0.22 μg/mg protein, respectively (n=3). The apparent permeability coefficient values of catechin, EGCG, and their niosomes were 1.68 ± 0.16, 0.88 ± 0.09, 2.39 ± 0.31, and 1.42 ± 0.24 cm/second (n=3) at 37°C, respectively. The transport was temperature- and energy-dependent. The inhibitors of permeability glycoprotein and multidrug resistance-associated protein 2 and the absorption enhancer significantly enhanced the uptake amount. Compared with the free drugs, niosomal formulation significantly enhanced drug absorption. Additionally, drug-loaded niosomes exhibited stronger stability and lower toxicity. These findings showed that the oral absorption of tea flavonoids could be improved by using the novel drug delivery systems.

  17. Tick-borne encephalitis virus replication, intracellular trafficking, and pathogenicity in human intestinal Caco-2 cell monolayers.

    PubMed

    Yu, Chao; Achazi, Katharina; Möller, Lars; Schulzke, Joerg D; Niedrig, Matthias; Bücker, Roland

    2014-01-01

    Tick-borne encephalitis virus (TBEV) is one of the most important vector-borne viruses in Europe and Asia. Its transmission mainly occurs by the bite of an infected tick. However, consuming milk products from infected livestock animals caused TBEV cases. To better understand TBEV transmission via the alimentary route, we studied viral infection of human intestinal epithelial cells. Caco-2 cells were used to investigate pathological effects of TBEV infection. TBEV-infected Caco-2 monolayers showed morphological changes including cytoskeleton rearrangements and cytoplasmic vacuolization. Ultrastructural analysis revealed dilatation of the rough endoplasmic reticulum and further enlargement to TBEV containing caverns. Caco-2 monolayers maintained an intact epithelial barrier with stable transepithelial electrical resistance (TER) during early stage of infection. Concomitantly, viruses were detected in the basolateral medium, implying a transcytosis pathway. When Caco-2 cells were pre-treated with inhibitors of cellular pathways of endocytosis TBEV cell entry was efficiently blocked, suggesting that actin filaments (Cytochalasin) and microtubules (Nocodazole) are important for PI3K-dependent (LY294002) virus endocytosis. Moreover, experimental fluid uptake assay showed increased intracellular accumulation of FITC-dextran containing vesicles. Immunofluorescence microscopy revealed co-localization of TBEV with early endosome antigen-1 (EEA1) as well as with sorting nexin-5 (SNX5), pointing to macropinocytosis as trafficking mechanism. In the late phase of infection, further evidence was found for translocation of virus via the paracellular pathway. Five days after infection TER was slightly decreased. Epithelial barrier integrity was impaired due to increased epithelial apoptosis, leading to passive viral translocation. These findings illuminate pathomechanisms in TBEV infection of human intestinal epithelial cells and viral transmission via the alimentary route.

  18. Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity.

    PubMed

    Salerno-Goncalves, Rosangela; Fasano, Alessio; Sztein, Marcelo B

    2016-07-25

    Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA's Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

  19. Drug resistance and adherence to human intestines of enteroaggregative Escherichia coli.

    PubMed

    Yamamoto, T; Echeverria, P; Yokota, T

    1992-04-01

    Clinical isolates of enteroaggregative Escherichia coli (EAggEC) were tested for their in vitro susceptibilities to 27 antimicrobial agents. Marked drug resistance was observed with sulfamethoxazole, ampicillin, and chloramphenicol in contrast to such antimicrobial agents as cefixime, sparfloxacin, and ciprofloxacin. One of the EAggEC strains carried a plasmid that conferred on its host resistance to ampicillin, tetracycline, sulfamethoxazole, streptomycin, and spectinomycin and an ability to adhere to child ileal villi or HeLa cells in the characteristic aggregative pattern. This plasmid also mediated D-mannose-resistant hemagglutinin production and bacterial clump formation (autoagglutination). The data demonstrate appearance of marked drug resistance and an intestine-adherence and drug-resistance plasmid in the newest category of diarrheagenic E. coli.

  20. Differences between human and rat intestinal and hepatic bisphenol A glucuronidation and the influence of alamethicin on in vitro kinetic measurements.

    PubMed

    Mazur, Christopher S; Kenneke, John F; Hess-Wilson, Janet K; Lipscomb, John C

    2010-12-01

    The extent to which membrane-disrupting agents, such as alamethicin, may alter cofactor transport and influence in vitro kinetic measurements of glucuronidation is a major concern regarding the characterization and extrapolation of inter- and intraspecies pharmacokinetics of bisphenol A (BPA). An additional concern is the omission of a BPA intestinal metabolism component in current pharmacokinetic models used to assess oral exposure. In this study, BPA glucuronidation in native hepatic microsomes from female rat and female human liver displayed higher V(max) values than that in males. In the presence of alamethicin, all hepatic V(max) values increased; however, this increase was disproportionately greater in males and gender differences were no longer observed. Female rats exhibited a much higher K(m) than all other species and genders; the addition of alamethicin had little influence on K(m) values for any of the test systems. The dissimilar K(m) measured for female rat suggests that different UDP-glucuronosyltransferase (UGT) enzyme(s) are involved in BPA glucuronidation. The presence of different UGTs in female rat was confirmed using Hill coefficients measured from diclofenac-mediated chemical inhibition assays within hepatic microsomes and purified human UGT2B7 and UGT2B15. Mixed-gender human intestinal microsomes showed little BPA glucuronidation reactivity compared with those from male rat intestine. Male rat intestinal microsomes in the presence of alamethicin exhibited a V(max) that was nearly 30-fold higher than that for mixed human microsomes. The species and gender metabolic differences we observed between rat and human liver and intestine provide key information for delineating BPA pharmacokinetics needed for human health risk assessment.

  1. Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial Cells to Galectin-1.

    PubMed

    Muglia, Cecilia I; Gobbi, Rodrigo Papa; Smaldini, Paola; Delgado, María Lucía Orsini; Candia, Martín; Zanuzzi, Carolina; Sambuelli, Alicia; Rocca, Andrés; Toscano, Marta A; Rabinovich, Gabriel A; Docena, Guillermo H

    2016-07-01

    Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin-1 (Gal-1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro-inflammatory cytokines, and Gal-1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal-1 binding and modulation of IECs. We found an enhanced binding of Gal-1 to: (a) murine IECs exposed to IL-1β, TNF, and IL-13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal-1 favored a tolerogenic micro-environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase-9 and modulation of Bcl-2 protein family members. Our results showed that, when added in the presence of diverse pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-13 and IL-5, Gal-1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL-10, IL-25, and transforming growth factor (TGF-β1 ). In conclusion, we found an augmented binding of Gal-1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal-1 concentration. These findings highlight the importance of the inflammatory micro-environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal-1 and its role in epithelial cell homeostasis.

  2. Grapefruit juice increases felodipine oral availability in humans by decreasing intestinal CYP3A protein expression.

    PubMed Central

    Lown, K S; Bailey, D G; Fontana, R J; Janardan, S K; Adair, C H; Fortlage, L A; Brown, M B; Guo, W; Watkins, P B

    1997-01-01

    The increase in oral availability of felodipine and other commonly used medications when taken with grapefruit juice has been assumed to be due to inhibition of CYP3A4, a cytochrome P450 that is present in liver and intestine. To evaluate the effect of repeated grapefruit juice ingestion on CYP3A4 expression, 10 healthy men were given 8 oz of grapefruit juice three times a day for 6 d. Before and after receiving grapefruit juice, small bowel and colon mucosal biopsies were obtained endoscopically, oral felodipine kinetics were determined, and liver CYP3A4 activity was measured with the [14C N-methyl] erythromycin breath test in each subject. Grapefruit juice did not alter liver CYP3A4 activity, colon levels of CYP3A5, or small bowel concentrations of P-glycoprotein, villin, CYP1A1, and CYP2D6. In contrast, the concentration of CYP3A4 in small bowel epithelia (enterocytes) fell 62% (P = 0.0006) with no corresponding change in CYP3A4 mRNA levels. In addition, enterocyte concentrations of CYP3A4 measured before grapefruit juice consumption correlated with the increase in Cmax when felodipine was taken with either the 1st or the 16th glass of grapefruit juice relative to water (r = 0. 67, P = 0.043, and r = 0.71, P = 0.022, respectively). We conclude that a mechanism for the effect of grapefruit juice on oral felodipine kinetics is its selective downregulation of CYP3A4 in the small intestine. PMID:9153299

  3. Pressure and frequency dependent linkage between motility and epithelial secretion in human proximal small intestine

    PubMed Central

    Mellander, A; Jarbur, K; Sjovall, H

    2000-01-01

    BACKGROUND—Motor disturbances are sometimes associated with diarrhoea by unknown mechanisms.
AIM—To determine if there is a quantitative link between intestinal motility and epithelial secretion.
SUBJECTS—Experiments were performed in 21 healthy volunteers and three patients with villus atrophy.
METHODS—Duodenal and jejunal motor activities were registered in the fasted state by open tip manometry. Secretion was measured directly by marker perfusion and indirectly by recording transmural potential difference (PD).
RESULTS—A significant correlation was found between "low pass filtered" pressure and PD, but no correlation was found between amplitudes of isolated contractions and PD changes. During repeated phasic contractions (phase III of migrating motor complex), PD increased at a rate that was higher in the duodenum than in the jejunum, and higher in patients with villus atrophy than in healthy controls. After reaching a peak, PD decreased despite continuing phasic motor activity, provided that there was no concomitant increase in mean pressure. Fluid secretion increased roughly in parallel with PD, except at the very end of the cycle.
CONCLUSIONS—To explain these findings, one has to postulate participation of at least two types of receptor: a slowly adapting pressure sensitive receptor and another mechanoreceptor, possibly a mucosal touch receptor, to account for the run down phenomenon. This model predicts that short lasting trains of contractions, so called discrete clusters, will be a particularly potent stimulus for activation of mucosal secretion.


Keywords: enteric nervous system; intestinal mucosa; migrating motor complex; receptor; chemoreceptor; bile PMID:10673300

  4. Differentiation of ex vivo human breast tissue using polarization-sensitive optical coherence tomography

    PubMed Central

    South, Fredrick A.; Chaney, Eric J.; Marjanovic, Marina; Adie, Steven G.; Boppart, Stephen A.

    2014-01-01

    Successful treatment of breast cancer typically requires surgical removal of the tumor. Optical coherence tomography (OCT) has been previously developed for real-time imaging of the surgical margin. However, it can be difficult to distinguish between normal stromal tissue and cancer tissue based on scattering intensity and structure alone. Polarization-sensitive optical coherence tomography (PS-OCT) is sensitive to form birefringence of biological tissue. We report on the development of a high-speed PS-OCT system and imaging of ex vivo human breast tissue, showing enhanced contrast between healthy and cancerous tissues based upon collagen content confirmed with corresponding histology. These results demonstrate the feasibility of using PS-OCT to supplement structural OCT as a possible method for intraoperative tumor margin evaluation. PMID:25360360

  5. In vitro study of soil arsenic release by human gut microbiota and its intestinal absorption by Caco-2 cells.

    PubMed

    Yin, Naiyi; Cai, Xiaolin; Du, Huili; Zhang, Zhennan; Li, Zejiao; Chen, Xiaochen; Sun, Guoxin; Cui, Yanshan

    2017-02-01

    Arsenic (As) speciation is essential in assessing health risks from As-contaminated soil. Release of soil-bound arsenic, As transformation by human gut microbiota, and the subsequent intestinal absorption of soil As metabolites were evaluated. A colon microbial community in a dynamic human gut model and the intestinal epithelial cell line Caco-2 were cultured. Arsenic speciation analysis and absorption of different As species were undertaken. In this study, soil As release (3.7-581.2 mg kg(-1)) was observed in the colon. Arsenic in the colon digests was transformed more quickly than that in the soil solid phase. X-ray absorption near-edge spectroscopy (XANES) analysis showed that 44.2-97.6% of arsenite [As(III)] generated due to arsenate [As(V)] reduction was in the soil solid phase after the colon phase. We observed a high degree of cellular absorption of soil As metabolites, exhibiting that the intestinal absorption of monomethylarsonic acid and As(III) (33.6% and 30.2% resp.) was slightly higher than that of dimethylarsinic acid and As(V) (25.1% and 21.7% resp.). Our findings demonstrate that human gut microbiota can directly release soil-bound arsenic, particularly As-bearing amorphous Fe/Al-oxides. Determining As transformation and intestinal absorption simultaneously will result in an accurate risk assessment of human health with soil As exposures.

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

    PubMed

    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.

  7. Genome sequence of Victivallis vadensis ATCC BAA-548, an anaerobic bacterium from the phylum Lentisphaerae, isolated from the human gastro-intestinal tract

    SciTech Connect

    Van Passel, Mark W.J.; Kant, Ravi; Palva, Airi; Lucas, Susan; Copeland, A; Lapidus, Alla L.; Glavina Del Rio, Tijana; Dalin, Eileen; Tice, Hope; Bruce, David; Goodwin, Lynne A.; Pitluck, Sam; Davenport, Karen W.; Sims, David; Detter, J. Chris; Han, Cliff; Larimer, Frank W; Land, Miriam L; Hauser, Loren John; Kyrpides, Nikos C; Ovchinnikova, Galina; Richardson, Paul; De Vos, Willem M.; Smidt, Hauke; Zoetendal, Erwin G.

    2011-01-01

    Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastro-intestinal tract.

  8. Acute Small-Bowel Obstruction From Intestinal Anisakiasis After the Ingestion of Raw Clams; Documenting a New Method of Marine-to-Human Parasitic Transmission

    PubMed Central

    Shweiki, Ehyal; Rittenhouse, David W.; Ochoa, Joana E.; Punja, Viren P.; Zubair, Muhammad H.; Baliff, Jeffrey P.

    2014-01-01

    Enteric anisakiasis is a known parasitic infection. To date, human infection has been reported as resulting from the inadvertent ingestion of the anisakis larvae when eating raw/undercooked fish, squid, or eel. We present a first reported case of intestinal obstruction caused by anisakiasis, after the ingestion of raw clams. PMID:25734153

  9. Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

    PubMed

    Barrera, G J; Tortolero, G Sanchez

    2016-01-01

    Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide