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  1. Autonomic Modification of Intestinal Smooth Muscle Contractility

    ERIC Educational Resources Information Center

    Montgomery, Laura E. A.; Tansey, Etain A.; Johnson, Chris D.; Roe, Sean M.; Quinn, Joe G.

    2016-01-01

    Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe…

  2. Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

    PubMed Central

    Halim, Danny; Wilson, Michael P.; Oliver, Daniel; Brosens, Erwin; Verheij, Joke B. G. M.; Han, Yu; Nanda, Vivek; Lyu, Qing; Doukas, Michael; Stoop, Hans; Brouwer, Rutger W. W.; van IJcken, Wilfred F. J.; Slivano, Orazio J.; Burns, Alan J.; Christie, Christine K.; de Mesy Bentley, Karen L.; Brooks, Alice S.; Tibboel, Dick; Xu, Suowen; Jin, Zheng Gen; Djuwantono, Tono; Yan, Wei; Alves, Maria M.; Hofstra, Robert M. W.; Miano, Joseph M.

    2017-01-01

    Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal–contractile coupling. PMID:28292896

  3. Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice.

    PubMed

    Halim, Danny; Wilson, Michael P; Oliver, Daniel; Brosens, Erwin; Verheij, Joke B G M; Han, Yu; Nanda, Vivek; Lyu, Qing; Doukas, Michael; Stoop, Hans; Brouwer, Rutger W W; van IJcken, Wilfred F J; Slivano, Orazio J; Burns, Alan J; Christie, Christine K; de Mesy Bentley, Karen L; Brooks, Alice S; Tibboel, Dick; Xu, Suowen; Jin, Zheng Gen; Djuwantono, Tono; Yan, Wei; Alves, Maria M; Hofstra, Robert M W; Miano, Joseph M

    2017-03-28

    Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a congenital visceral myopathy characterized by severe dilation of the urinary bladder and defective intestinal motility. The genetic basis of MMIHS has been ascribed to spontaneous and autosomal dominant mutations in actin gamma 2 (ACTG2), a smooth muscle contractile gene. However, evidence suggesting a recessive origin of the disease also exists. Using combined homozygosity mapping and whole exome sequencing, a genetically isolated family was found to carry a premature termination codon in Leiomodin1 (LMOD1), a gene preferentially expressed in vascular and visceral smooth muscle cells. Parents heterozygous for the mutation exhibited no abnormalities, but a child homozygous for the premature termination codon displayed symptoms consistent with MMIHS. We used CRISPR-Cas9 (CRISPR-associated protein) genome editing of Lmod1 to generate a similar premature termination codon. Mice homozygous for the mutation showed loss of LMOD1 protein and pathology consistent with MMIHS, including late gestation expansion of the bladder, hydronephrosis, and rapid demise after parturition. Loss of LMOD1 resulted in a reduction of filamentous actin, elongated cytoskeletal dense bodies, and impaired intestinal smooth muscle contractility. These results define LMOD1 as a disease gene for MMIHS and suggest its role in establishing normal smooth muscle cytoskeletal-contractile coupling.

  4. Autonomic modification of intestinal smooth muscle contractility.

    PubMed

    Montgomery, Laura E A; Tansey, Etain A; Johnson, Chris D; Roe, Sean M; Quinn, Joe G

    2016-03-01

    Intestinal smooth muscle contracts rhythmically in the absence of nerve and hormonal stimulation because of the activity of pacemaker cells between and within the muscle layers. This means that the autonomic nervous system modifies rather than initiates intestinal contractions. The practical described here gives students an opportunity to observe this spontaneous activity and its modification by agents associated with parasympathetic and sympathetic nerve activity. A section of the rabbit small intestine is suspended in an organ bath, and the use of a pressure transducer and data-acquisition software allows the measurement of tension generated by the smooth muscle of intestinal walls. The application of the parasympathetic neurotransmitter ACh at varying concentrations allows students to observe an increase in intestinal smooth muscle tone with increasing concentrations of this muscarinic receptor agonist. Construction of a concentration-effect curve allows students to calculate an EC50 value for ACh and consider some basic concepts surrounding receptor occupancy and activation. Application of the hormone epinephrine to the precontracted intestine allows students to observe the inhibitory effects associated with sympathetic nerve activation. Introduction of the drug atropine to the preparation before a maximal concentration of ACh is applied allows students to observe the inhibitory effect of a competitive antagonist on the physiological response to a receptor agonist. The final experiment involves the observation of the depolarizing effect of K(+) on smooth muscle. Students are also invited to consider why the drugs atropine, codeine, loperamide, and botulinum toxin have medicinal uses in the management of gastrointestinal problems.

  5. In a non-human primate model, aging disrupts the neural control of intestinal smooth muscle contractility in a region-specific manner

    PubMed Central

    Tran, L; Greenwood-Van Meerveld, B

    2014-01-01

    Background Incidences of gastrointestinal (GI) motility disorders increase with age. However, there is a paucity of knowledge about the aging mechanisms leading to GI dysmotility. Motility in the GI tract is a function of smooth muscle contractility, which is modulated in part by the enteric nervous system (ENS). Evidence suggests that aging impairs the ENS, thus we tested the hypothesis that senescence in the GI tract precipitates abnormalities in smooth muscle and neurally mediated contractility in a region-specific manner. Methods Jejunal and colonic circular muscle strips were isolated from young (4–10 years) and old (18+ years) baboons. Myogenic responses were investigated using potassium chloride (KCl) and carbachol (CCh). Neurally mediated contractile responses were evoked by electrical field stimulation (EFS) and were recorded in the absence and presence of atropine (1 μM) or NG-Nitro-l-arginine methyl ester (l-NAME; 100 μM). Key Results The myogenic responses to KCl in the jejunum and colon were unaffected by age. In the colon, but not the jejunum, CCh-induced contractile responses were reduced in aged animals. Compared to young baboons, there was enhanced EFS-induced contractility of old baboon jejunal smooth muscle in contrast to the reduced contractility in the colon. The effect of atropine on the EFS response was lower in aged colonic tissue, suggesting reduced participation of acetylcholine. In aged jejunal tissue, higher contractile responses to EFS were found to be due to reduced nitregic inhibition. Conclusions & Inferences These findings provide key evidence for the importance of intestinal smooth muscle and ENS senescence in age-associated GI motility disorders. PMID:24548258

  6. Intestinal smooth muscle cell maintenance by basic fibroblast growth factor.

    PubMed

    Lee, Min; Wu, Benjamin M; Stelzner, Matthias; Reichardt, Holger M; Dunn, James C Y

    2008-08-01

    Intestinal tissue engineering is a potential therapy for patients with short bowel syndrome. Tissue engineering scaffolds that promote smooth muscle cell proliferation and angiogenesis are essential toward the regeneration of functional smooth muscles for peristalsis and motility. Since basic fibroblast growth factor (bFGF) can stimulate smooth muscle proliferation and angiogenesis, the delivery of bFGF was employed to stimulate proliferation and survival of primary intestinal smooth muscle cells. Two methods of local bFGF delivery were examined: the incorporation of bFGF into the collagen coating and the encapsulation of bFGF into poly(D,L-lactic-co-glycolic acid) microspheres. Cell-seeded scaffolds were implanted into the omentum and were retrieved after 4, 14, and 28 days. The seeded cells proliferated from day 4 to day 14 in all implants; however, at 28 days, significantly higher density of implanted cells and blood vessels was observed, when 10 microg of bFGF was incorporated into the collagen coating of scaffolds as compared to scaffolds with either no bFGF or 1 microg of bFGF in collagen. Microsphere encapsulation of 1 microg of bFGF produced similar effects as 10 microg of bFGF mixed in collagen and was more effective than the delivery of 1 microg of bFGF by collagen incorporation. The majority of the implanted cells also expressed alpha-smooth muscle actin. Scaffolds coated with microsphere-encapsulated bFGF and seeded with smooth muscle cells may be a useful platform for the regeneration of the intestinal smooth muscle.

  7. Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression.

    PubMed

    Han, T Y; Lourenssen, S; Miller, K G; Blennerhassett, M G

    2015-04-02

    Intestinal inflammation causes initial axonal degeneration and neuronal death, as well as the proliferation of intestinal smooth muscle cells (ISMC), but subsequent axonal outgrowth leads to re-innervation. We recently showed that expression of glial cell-derived neurotrophic factor (GDNF), the critical neurotrophin for the post-natal enteric nervous system (ENS) is upregulated in ISMC by inflammatory cytokines, leading us to explore the relationship between ISMC growth and GDNF expression. In co-cultures of myenteric neurons and ISMC, GDNF or fetal calf serum (FCS) was equally effective in supporting neuronal survival, with neurons forming extensive axonal networks among the ISMC. However, only GDNF was effective in low-density cultures where neurons lacked contact with ISMC. In early-passage cultures of colonic circular smooth muscle cells (CSMC), polymerase chain reaction (PCR) and western blotting showed that proliferation was associated with expression of GDNF, and the successful survival of neonatal neurons co-cultured on CSMC was blocked by vandetanib or siGDNF. In tri-nitrobenzene sulfonic acid (TNBS)-induced colitis, immunocytochemistry showed the selective expression of GDNF in proliferating CSMC, suggesting that smooth muscle proliferation supports the ENS in vivo as well as in vitro. However, high-passage CSMC expressed significantly less GDNF and failed to support neuronal survival, while expressing reduced amounts of smooth muscle marker proteins. We conclude that in the inflamed intestine, smooth muscle proliferation supports the ENS, and thus its own re-innervation, by expression of GDNF. In chronic inflammation, a compromised smooth muscle phenotype may lead to progressive neural damage. Intestinal stricture formation in human disease, such as inflammatory bowel disease (IBD), may be an endpoint of failure of this homeostatic mechanism.

  8. A comprehensive mathematical framework for modeling intestinal smooth muscle cell contraction with applications to intestinal edema.

    PubMed

    Young, Jennifer; Ozisik, Sevtap; Riviere, Beatrice; Shamim, Muhammad

    2015-04-01

    The contraction of intestinal smooth muscle cells (ISMCs) involves many coordinated biochemical and mechanical processes. In this work, we present a framework for modeling ISMC contractility that begins with chemical models of calcium dynamics, continues with myosin light chain phosphorylation and force generation, and ends with a cell model of the ISMC undergoing contraction-relaxation. The motivation for developing this comprehensive framework is to study the effects of edema (excess fluid build-up in the muscle tissue) on ISMC contractility. The hypothesis is that more fluid equates to dilution of an external stimulis, eventually leading to reduced contractility. We compare our results to experimental data collected from normal versus edematous intestinal muscle tissue.

  9. Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

    PubMed

    Chu, Ji; Pham, Ngoc T; Olate, Nicole; Kislitsyna, Karina; Day, Mary-Clare; LeTourneau, Phillip A; Kots, Alexander; Stewart, Randolph H; Laine, Glen A; Cox, Charles S; Uray, Karen

    2013-01-11

    Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

  10. Nervous factors influencing the membrane activity of intestinal smooth muscle

    PubMed Central

    Kuriyama, H.; Osa, T.; Toida, N.

    1967-01-01

    The effects of various chemical agents on the spontaneous membrane activities and those electrically elicited in the smooth muscles of small intestine were investigated. 1. The effects of various chemicals on the spontaneously active membrane might be summarized as follows. (a) Cholinergic agents; atropine slightly hyperpolarized the membrane and reduced the amplitude of slow potential changes even in aged preparations. Prostigmine depolarized the membrane, and enhanced the amplitude and prolonged the duration of the slow potential changes. Atropine prevented the actions of prostigmine on the membrane. (b) Ba2+ depolarized the membrane, and enhanced the amplitude and prolonged the duration of the slow potential changes. The spike frequency was initially increased, then reduced. Atropine and tetrodotoxin partially prevented the action of Ba2+ on the membrane activities. 2. Effects of chemical agents on the membrane activity elicited by electrical stimulation might be summarized as follows. (a) Short pulse stimulation (0·5-1 msec) generated the spike as a direct response of the muscle cell membrane, then it was followed by slow depolarization, delayed hyperpolarization, i.e. the `inhibitory potential', and post-inhibitory rebound successively. (b) The slow depolarization and the post-inhibitory rebound were reduced in amplitude by treatment with atropine, and enhanced by treatments with prostigmine and Ba2+. Tetrodotoxin blocked all activities except the spike. 3. When repetitive stimulation (20 c/s) was applied to the membrane, the membrane hyperpolarized; then, after 3-5 sec, it gradually depolarized even if the stimulation was continued, and triggered spikes. The hyperpolarization always preceded depolarization. The duration and the amplitude of the delayed depolarization was proportionally increased by the increased intensity and duration of stimulation. Atropine and tetrodotoxin blocked the generation of the post-inhibitory rebound. 4. Effects of repetitive

  11. Regeneration and Maintenance of Intestinal Smooth Muscle Phenotypes

    NASA Astrophysics Data System (ADS)

    Walthers, Christopher M.

    Tissue engineering is an emerging field of biomedical engineering that involves growing artificial organs to replace those lost to disease or injury. Within tissue engineering, there is a demand for artificial smooth muscle to repair tissues of the digestive tract, bladder, and vascular systems. Attempts to develop engineered smooth muscle tissues capable of contracting with sufficient strength to be clinically relevant have so far proven unsatisfactory. The goal of this research was to develop and sustain mature, contractile smooth muscle. Survival of implanted SMCs is critical to sustain the benefits of engineered smooth muscle. Survival of implanted smooth muscle cells was studied with layered, electrospun polycaprolactone implants with lasercut holes ranging from 0--25% porosity. It was found that greater angiogenesis was associated with increased survival of implanted cells, with a large increase at a threshold between 20% and 25% porosity. Heparan sulfate coatings improved the speed of blood vessel infiltration after 14 days of implantation. With these considerations, thicker engineered tissues may be possible. An improved smooth muscle tissue culture technique was utilized. Contracting smooth muscle was produced in culture by maintaining the native smooth muscle tissue organization, specifically by sustaining intact smooth muscle strips rather than dissociating tissue in to isolated smooth muscle cells. Isolated cells showed a decrease in maturity and contained fewer enteric neural and glial cells. Muscle strips also exhibited periodic contraction and regular fluctuation of intracellular calclium. The muscle strip maturity persisted after implantation in omentum for 14 days on polycaprolactone scaffolds. A low-cost, disposable bioreactor was developed to further improve maturity of cultured smooth muscle cells in an environment of controlled cyclical stress.The bioreactor consistently applied repeated mechanical strain with controllable inputs for strain

  12. Contribution of Intestinal Smooth Muscle to Crohn’s Disease Fibrogenesis

    PubMed Central

    Severi, C.; Sferra, R.; Scirocco, A.; Vetuschi, A.; Pallotta, N.; Pronio, A.; Caronna, R.; Di Rocco, G.; Gaudio, E.; Corazziari, E.; Onori, P.

    2014-01-01

    Mesenchymal cells transdifferentiation and extracellular matrix deposition are involved in the fibrotic process of Crohn’s disease (CD). Mesenchymal smooth muscle cells (SMCs) de-differentiation, driven by Platelet-derived growth factor (PDGF) that counteracts Transforming growth factor (TGF-β) has been studied in vascular muscle. The role of SMCs in intestinal fibrogenesis is still not clearly elucidated. Aim of the study was to evaluate the possible myogenic contribution to CD fibrotic process through the comparative analysis of histological, morphometric and molecular alterations occurring in human smooth muscle. Full thickness specimens were obtained from CD (non-involved and stenotic tracts) and healthy (control) ileum. Tissues were processed for histological and immunohistochemical (IHC) analyses and SMCs were isolated from the muscularis propria for morphofunctional and molecular (qPCR) analyses. CD stenotic ileum showed a significant increased thickness of all layers compared to CD non-involved and control ileum. IHC revealed an overexpression of α-smooth muscle actin and collagens I-III throughout all intestinal layers only in stenotic tracts. The two growth factors, PDGF and TGF-β, showed a progressive increase in expression in the muscle layer from CD non-involved to stenotic tracts. Freshly isolated SMCs presented alterations in CD non-involved tracts that progressively increased in the stenotic tracts consisting in a statistical increase in mRNA encoding for PDGF-β and collagen III, paralleled to a decrease in TGF-β and Tribbles-like protein-3 mRNA, and altered morphofunctional parameters consisting in progressive decreases in cell length and contraction to acetylcholine. These findings indicate that intrinsic myogenic alterations occur in CD ileum, that they likely precede stricture formation, and might represent suitable new targets for anti-fibrotic interventions. PMID:25578979

  13. Voltage dependent potassium channel remodeling in murine intestinal smooth muscle hypertrophy induced by partial obstruction.

    PubMed

    Liu, Dong-Hai; Huang, Xu; Guo, Xin; Meng, Xiang-Min; Wu, Yi-Song; Lu, Hong-Li; Zhang, Chun-Mei; Kim, Young-chul; Xu, Wen-Xie

    2014-01-01

    Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.

  14. Increased IGF-IEc expression and mechano-growth factor production in intestinal muscle of fibrostenotic Crohn's disease and smooth muscle hypertrophy.

    PubMed

    Li, Chao; Vu, Kent; Hazelgrove, Krystina; Kuemmerle, John F

    2015-12-01

    The igf1 gene is alternatively spliced as IGF-IEa and IGF-IEc variants in humans. In fibrostenotic Crohn's disease, the fibrogenic cytokine TGF-β1 induces IGF-IEa expression and IGF-I production in intestinal smooth muscle and results in muscle hyperplasia and collagen I production that contribute to stricture formation. Mechano-growth factor (MGF) derived from IGF-IEc induces skeletal and cardiac muscle hypertrophy following stress. We hypothesized that increased IGF-IEc expression and MGF production mediated smooth muscle hypertrophy also characteristic of fibrostenotic Crohn's disease. IGF-IEc transcripts and MGF protein were increased in muscle cells isolated from fibrostenotic intestine under regulation by endogenous TGF-β1. Erk5 and MEF2C were phosphorylated in vivo in fibrostenotic muscle; both were phosphorylated and colocalized to nucleus in response to synthetic MGF in vitro. Smooth muscle-specific protein expression of α-smooth muscle actin, γ-smooth muscle actin, and smoothelin was increased in affected intestine. Erk5 inhibition or MEF2C siRNA blocked smooth muscle-specific gene expression and hypertrophy induced by synthetic MGF. Conditioned media of cultured fibrostenotic muscle induced muscle hypertrophy that was inhibited by immunoneutralization of endogenous MGF or pro-IGF-IEc. The results indicate that TGF-β1-dependent IGF-IEc expression and MGF production in patients with fibrostenotic Crohn's disease regulates smooth muscle cell hypertrophy a critical factor that contributes to intestinal stricture formation.

  15. Control of stomach smooth muscle development and intestinal rotation by transcription factor BARX1.

    PubMed

    Jayewickreme, Chenura D; Shivdasani, Ramesh A

    2015-09-01

    Diverse functions of the homeodomain transcription factor BARX1 include Wnt-dependent, non-cell autonomous specification of the stomach epithelium, tracheo-bronchial septation, and Wnt-independent expansion of the spleen primordium. Tight spatio-temporal regulation of Barx1 levels in the mesentery and stomach mesenchyme suggests additional roles. To determine these functions, we forced constitutive BARX1 expression in the Bapx1 expression domain, which includes the mesentery and intestinal mesenchyme, and also examined Barx1(-/)(-) embryos in further detail. Transgenic embryos invariably showed intestinal truncation and malrotation, in part reflecting abnormal left-right patterning. Ectopic BARX1 expression did not affect intestinal epithelium, but intestinal smooth muscle developed with features typical of the stomach wall. BARX1, which is normally restricted to the developing stomach, drives robust smooth muscle expansion in this organ by promoting proliferation of myogenic progenitors at the expense of other sub-epithelial cells. Undifferentiated embryonic stomach and intestinal mesenchyme showed modest differences in mRNA expression and BARX1 was sufficient to induce much of the stomach profile in intestinal cells. However, limited binding at cis-regulatory sites implies that BARX1 may act principally through other transcription factors. Genes expressed ectopically in BARX1(+) intestinal mesenchyme and reduced in Barx1(-/-) stomach mesenchyme include Isl1, Pitx1, Six2 and Pitx2, transcription factors known to control left-right patterning and influence smooth muscle development. The sum of evidence suggests that potent BARX1 functions in intestinal rotation and stomach myogenesis occur through this small group of intermediary transcription factors.

  16. Neo-innervation of a bioengineered intestinal smooth muscle construct around chitosan scaffold.

    PubMed

    Zakhem, Elie; Raghavan, Shreya; Bitar, Khalil N

    2014-02-01

    Neuromuscular disorders of the gut result in disturbances in gastrointestinal transit. The objective of this study was to evaluate the neo-innervation of smooth muscle in an attempt to restore lost innervation. We have previously shown the potential use of composite chitosan scaffolds as support for intestinal smooth muscle constructs. However, the constructs lacked neuronal component. Here, we bioengineered innervated colonic smooth muscle constructs using rabbit colon smooth muscle and enteric neural progenitor cells. We also bioengineered smooth muscle only tissue constructs using colonic smooth muscle cells. The constructs were placed next to each other around tubular chitosan scaffolds and left in culture. Real time force generation conducted on the intrinsically innervated smooth muscle constructs showed differentiated functional neurons. The bioengineered smooth muscle only constructs became neo-innervated. The neo-innervation results were confirmed by immunostaining assays. Chitosan supported (1) the differentiation of neural progenitor cells in the constructs and (2) the neo-innervation of non-innervated smooth muscle around the same scaffold.

  17. Intestinal smooth muscle cells locally enhance stem cell factor (SCF) production against gastrointestinal nematode infections.

    PubMed

    Morimoto, Masahiro

    2011-06-01

    Smooth muscle cells can produce stem cell factor (SCF) in the normal state for the preservation of mast cells, but it is still unknown whether smooth muscle cells can enhance SCF production in response to the pathological stimuli. The present study showed that smooth muscle cells in mast cell-increased regions around worm cysts of intestinal nematodes significantly enhanced SCF gene expression compared with mast cell non-increased regions in same sample. SCF gene expression in mast cell non-increased regions in nematode-infected mice showed almost the same level as in non-infected control groups. These results indicate that smooth muscle cells can locally enhance SCF gene expression, and may have a role in local immunological reactions as growth factor-producing cells.

  18. Immortalization of primary human smooth muscle cells.

    PubMed Central

    Perez-Reyes, N; Halbert, C L; Smith, P P; Benditt, E P; McDougall, J K

    1992-01-01

    Primary human aortic and myometrial smooth muscle cells (SMCs) were immortalized using an amphotropic recombinant retroviral construct containing the E6 and E7 open reading frames (ORFs) of human papillomavirus type 16. The SMCs expressing the E6/E7 ORFs have considerably elevated growth rates when compared with nonimmortalized control cells and show no signs of senescence with long-term passage. The first SMC line derived in this study has been maintained in continuous tissue culture for greater than 1 year (greater than 180 population doublings). The immortalized SMCs have decreased cell size and decreased content of muscle-specific alpha-actin filaments as determined by indirect immunofluorescence. Southern blot analysis has demonstrated the stable integration of the E6/E7 ORFs in the retrovirally infected cells, and radioimmunoprecipitation has confirmed the continued expression of the E6 and E7 genes. Cytogenetic studies of the SMC lines have revealed essentially diploid populations except for the myometrial clonal line, which became aneuploid at late passage (greater than 125 doublings). These cell lines were not tumorigenic in nude mice. Images PMID:1311088

  19. Suppression of intestinal smooth muscle contraction by 4-ethylguaiacol, a constituent of wood creosote.

    PubMed

    Toyoda, M; Ogata, N; Shibata, T

    1993-11-01

    Wood creosote, a mixture of phenolic compounds, suppresses in vitro contractions of rat intestine. To identify a compound in wood creosote able to inhibit intestinal motility, we screened its constituent phenolic compounds and found 4-ethylguaiacol (4-EG) as an active compound. It suppressed the spontaneous phasic (IC50 = 513 +/- 48 mumol/l) as well as spasmogenic-agent-induced tonic longitudinal contractions of isolated rat ileum in a reversible and concentration-dependent manner. KCl-depolarization-induced tonic contraction, which was susceptible to a calcium channel blocking agent, was also suppressed by 4-EG with an IC50 of 433 +/- 41 mumol/l. Furthermore, calcium-ionophore-induced contraction, which was affected by an influx of extracellular calcium ion that bypassed calcium channels, was suppressed by 4-EG with an IC50 of 97 +/- 18 mumol/l. These results support the concept that the effect of wood creosote to suppress intestinal motility is attributable, partially or entirely, to its component 4-EG and that this effect of 4-EG on the intestinal muscle is produced at some stage(s) of the muscle contraction process after influx of extracellular calcium into the cytosol of smooth muscle.

  20. 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

  1. 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.

  2. Visceral smooth muscle α-actin deficiency associated with chronic intestinal pseudo-obstruction in a Bengal cat (Felis catus x Prionailurus bengalensis).

    PubMed

    Imai, D M; Miller, J L; Leonard, B C; Bach, J; Drees, R; Steinberg, H; Teixeira, L B C

    2014-05-01

    An adult Bengal cat (Felis catus × Prionailurus bengalensis) with a prolonged history of partial anorexia, regurgitation, and weight loss and a clinical, radiographic, and ultrasonographic diagnosis of persistent megaesophagus and gastrointestinal ileus was submitted for necropsy. The intestinal tract was diffusely distended by gas and fluid with appreciable loss of muscle tone and an absence of luminal obstruction, consistent with the clinical history of chronic intestinal pseudo-obstruction. Histologically, the autonomic nervous system was intact, but the smooth muscle within the gastrointestinal wall exhibited a marked basophilia that was most pronounced in the jejunum. Immunohistochemistry for neurofilament, synaptophysin, CD117, and desmin demonstrated that the number of myenteric ganglia, number of interstitial cells, and leiomyocyte desmin content were similar when compared with the unaffected age- and species-matched control. Immunohistochemistry for smooth muscle α-actin demonstrated a striking loss of immunoreactivity, predominantly in the circular layer of the jejunum, that corresponded with the tinctorial change in leiomyocytes. Transmission electron microscopy revealed loss of myofibrils, loss of organelle polarity, and significantly larger central mitochondria (megamitochondria) in affected leiomyocytes, as well as nonspecific degenerative changes. Although the presence of a primary leiomyopathy and a causal relationship could not be confirmed in this case, leiomyopathies are considered a cause of chronic intestinal pseudo-obstruction in human medicine, and loss of smooth muscle α-actin immunoreactivity is one recognized marker for intestinal dysmotility.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. IL-17A induces hypo-contraction of intestinal smooth muscle via induction of iNOS in muscularis macrophages.

    PubMed

    Mori, Daisuke; Watanabe, Nobumasa; Kaminuma, Osamu; Murata, Takahisa; Hiroi, Takachika; Ozaki, Hiroshi; Hori, Masatoshi

    2014-01-01

    Intestinal inflammation causes disorder in bowel motility. Th17 cytokines are involved in intestinal inflammation. To understand the role of interleukin (IL)-17 in intestinal motility, we examined effects of IL-17A on contractile activities of organ-cultured ileum. Rat ileal smooth muscle strips were organ cultured with IL-17A. Muscle contraction was measured, and cells expressing inducible nitric oxide synthase (iNOS) were identified with immunohistochemistry. Creating Th17-transferred colitis model mice, in vivo effects of IL-17 on contractile activities, and iNOS mRNA expression in colonic smooth muscle were investigated. Treatment with IL-17A for 12 h and 3 days attenuated carbachol- and membrane depolarization-induced contractions in organ-cultured rat ileum. N(G)-Nitro-l-arginine methyl ester (100 μM), a nitric oxide synthase inhibitor, completely reversed the IL-17A-induced inhibition of contractile force. Ileal tissue cultured in the presence of IL-17A showed increased expression of iNOS mRNA and protein. Immunohistochemical analysis using an iNOS antibody revealed that iNOS protein was expressed on ED2-positive muscularis macrophages. The level of iNOS mRNA was also increased in inflamed colonic smooth muscle of Th17-transferred colitis model mice. In intestinal inflammation, IL-17A induces an intestinal motility disorder through iNOS expression in muscularis macrophages.

  10. Electrophysiological study of the intestinal smooth muscle of the guinea-pig

    PubMed Central

    Kuriyama, H.; Osa, T.; Toida, N.

    1967-01-01

    The membrane properties of single cells of intestinal smooth muscle of duodenum, jejunum, ileum, caecum and rectum of guinea-pig have been studied. 1. The membrane potentials of longitudinal muscles of the duodenum, jejunum, ileum, caecum and rectum varied from 54 to 56 mV and those of circular muscles of jejunum, ileum, caecum and rectum varied from 57 to 60 mV. The ablated longitudinal muscle had a slightly lower value (50 mV) than the intact one. 2. The longitudinal muscle generated spontaneous discharges but these were rare in the circular muscles of the intestine except for the caecum. Overshoot potentials could be observed in all regions of the intestine. The maximum rate of rise of the spontaneously discharging longitudinal muscles varied from 11 to 18 V/sec. 3. Not all of the slow potential changes (but at least some) were generated by the nervous elements distributed between the muscle layers and in them. 4. The conduction velocities measured from the longitudinal muscles of jejunum and rectum in the presence of tetrodotoxin were 2·1 cm/sec and 4·0 cm/sec respectively. 5. Chronaxies of the longitudinal muscles of jejunum and rectum were 2-5 msec and 5-18 msec respectively. 6. Intracellular stimulation of the single cells of the duodenum and caecum could trigger a spike, similar to that observed in the taenia coli. The spikes were mostly graded ones; a spike of full size was rarely elicited. When the spikes were triggered, the after-hyperpolarization appeared consistently presumably caused by the increased potassium conductance. 7. The effective membrane resistance and the time constant were measured for the longitudinal muscles of the jejunum and rectum. When spikes were generated by intracellular stimulation the observed values were 40-50 MΩ and 3-5 msec in both tissues. These values were the same as those observed in the taenia coli. 8. When the time constant of the membrane was measured by the extracellular polarizing method, the longitudinal muscles

  11. Bioengineering functional human sphincteric and non-sphincteric gastrointestinal smooth muscle constructs.

    PubMed

    Rego, Stephen L; Zakhem, Elie; Orlando, Giuseppe; Bitar, Khalil N

    2016-04-15

    Digestion and motility of luminal content through the gastrointestinal (GI) tract are achieved by cooperation between distinct cell types. Much of the 3 dimensional (3D) in vitro modeling used to study the GI physiology and disease focus solely on epithelial cells and not smooth muscle cells (SMCs). SMCs of the gut function either to propel and mix luminal contents (phasic; non-sphincteric) or to act as barriers to prevent the movement of luminal materials (tonic; sphincteric). Motility disorders including pyloric stenosis and chronic intestinal pseudoobstruction (CIPO) affect sphincteric and non-sphincteric SMCs, respectively. Bioengineering offers a useful tool to develop functional GI tissue mimics that possess similar characteristics to native tissue. The objective of this study was to bioengineer 3D human pyloric sphincter and small intestinal (SI) constructs in vitro that recapitulate the contractile phenotypes of sphincteric and non-sphincteric human GI SMCs. Bioengineered 3D human pylorus and circular SI SMC constructs were developed and displayed a contractile phenotype. Constructs composed of human pylorus SMCs displayed tonic SMC characteristics, including generation of basal tone, at higher levels than SI SMC constructs which is similar to what is seen in native tissue. Both constructs contracted in response to potassium chloride (KCl) and acetylcholine (ACh) and relaxed in response to vasoactive intestinal peptide (VIP). These studies provide the first bioengineered human pylorus constructs that maintain a sphincteric phenotype. These bioengineered constructs provide appropriate models to study motility disorders of the gut or replacement tissues for various GI organs.

  12. Inflammatory cytokines promote growth of intestinal smooth muscle cells by induced expression of PDGF-Rβ.

    PubMed

    Nair, Dileep G; Miller, Kurtis G; Lourenssen, Sandra R; Blennerhassett, Michael G

    2014-03-01

    Thickening of the inflamed intestinal wall involves growth of smooth muscle cells (SMC), which contributes to stricture formation. Earlier, the growth factor platelet-derived growth factor (PDGF)-BB was identified as a key mitogen for SMC from the rat colon (CSMC), and CSMC growth in colitis was associated with both appearance of its receptor, PDGF-Rβ and modulation of phenotype. Here, we examined the role of inflammatory cytokines in inducing and modulating the growth response to PDGF-BB. CSMC were enzymatically isolated from Sprague-Dawley rats, and the effect of tumour necrosis factor (TNF)-α, interleukin (IL)-1β, transforming growth factor (TGF), IL-17A and IL-2 on CSMC growth and responsiveness to PDGF-BB were assessed using proliferation assays, PCR and western blotting. Conditioned medium (CM) was obtained at 48 hrs of trinitrobenzene sulphonic acid-induced colitis. Neither CM alone nor cytokines caused proliferation of early-passage CSMC. However, CM from inflamed, but not control colon significantly promoted the effect of PDGF-BB. IL-1β, TNF-α and IL-17A, but not other cytokines, increased the effect of PDGF-BB because of up-regulation of mRNA and protein for PDGF-Rβ without change in receptor phosphorylation. PDGF-BB was identified in adult rat serum (RS) and RS-induced CSMC proliferation was inhibited by imatinib, suggesting that blood-derived PDGF-BB is a local mitogen in vivo. In freshly isolated CSMC, CM from the inflamed colon as well as IL-1β and TNF-α induced the early expression of PDGF-Rβ, while imatinib blocked subsequent RS-induced cell proliferation. Thus, pro-inflammatory cytokines both initiate and maintain a growth response in CSMC via PDGF-Rβ and serum-derived PDGF-BB, and control of PDGF-Rβ expression may be beneficial in chronic intestinal inflammation.

  13. [Electrophysiology and calcium signalling in human bronchial smooth muscle].

    PubMed

    Marthan, R; Hyvelin, J M; Roux, E; Savineau, J P

    1999-01-01

    Recently, cells isolated from airways have been used to characterize precisely the electrophysiological properties of this smooth muscle and to describe the changes in cytosolic calcium concentration ([Ca2+]i) occurring upon agonist stimulation. Although most studies have produced consistent results in terms of types of ion channel and pathways of calcium signalling implicated in the mechanical activity of airways, there are differences according to (i) the site along the bronchial tree (trachea vs. bronchi); (ii) the proliferating status of the cells (freshly isolated vs. cultured) and (iii) the species (human vs. animals). With regard to the electrophysiological properties of airway smooth muscle, the contribution to [Ca2+]i rise of Ca2+ influx through L-type voltage-dependent calcium channels depends on the balance between depolarization related to non-specific cation channel and/or chloride channel activation and hyperpolarization related to activation of a variety of potassium channels. Most of the above-mentioned channels appear to be controlled, directly or indirectly, by agonists in human bronchial smooth muscle. With regard to calcium signalling, the pattern of agonist-induced [Ca2+]i responses, the so-called [Ca2+]i oscillations, has been observed recently in freshly isolated airway smooth muscle cells. The role and the calcium sources involved in these oscillations in human bronchial smooth muscle are currently being investigated.

  14. 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

  15. 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

  16. 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.

  17. Inhibitory action of relaxin on human cervical smooth muscle.

    PubMed

    Norström, A; Bryman, I; Wiqvist, N; Sahni, S; Lindblom, B

    1984-09-01

    The influence of purified porcine relaxin on contractility of human cervical smooth muscle was investigated in vitro. Strips of cervical tissue were obtained by needle biopsy from pregnant and nonpregnant women and were mounted in a superfused organ chamber for isometric measurement of contractile activity. Relaxin (0.005-25 micrograms/ml) inhibited the spontaneous contractions in cervical strips from 18% of nonpregnant, 68% of early pregnant, and in 100% of term pregnant women. These results indicate that relaxin has an inhibitory action on cervical smooth muscle and that this effect is more constantly detected as pregnancy proceeds.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment.

    PubMed

    Zhang, Rong; Jack, Gregory S; Rao, Nagesh; Zuk, Patricia; Ignarro, Louis J; Wu, Benjamin; Rodríguez, Larissa V

    2012-03-01

    Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion.

  3. Transdifferentiation of human endothelial progenitors into smooth muscle cells.

    PubMed

    Ji, HaYeun; Atchison, Leigh; Chen, Zaozao; Chakraborty, Syandan; Jung, Youngmee; Truskey, George A; Christoforou, Nicolas; Leong, Kam W

    2016-04-01

    Access to smooth muscle cells (SMC) would create opportunities for tissue engineering, drug testing, and disease modeling. Herein we report the direct conversion of human endothelial progenitor cells (EPC) to induced smooth muscle cells (iSMC) by induced expression of MYOCD. The EPC undergo a cytoskeletal rearrangement resembling that of mesenchymal cells within 3 days post initiation of MYOCD expression. By day 7, the reprogrammed cells show upregulation of smooth muscle markers ACTA2, MYH11, and TAGLN by qRT-PCR and ACTA2 and MYH11 expression by immunofluorescence. By two weeks, they resemble umbilical artery SMC in microarray gene expression analysis. The iSMC, in contrast to EPC control, show calcium transients in response to phenylephrine stimulation and a contractility an order of magnitude higher than that of EPC as determined by traction force microscopy. Tissue-engineered blood vessels constructed using iSMC show functionality with respect to flow- and drug-mediated vasodilation and vasoconstriction.

  4. Muscarinic receptor subtypes in human and rat colon smooth muscle.

    PubMed

    Gómez, A; Martos, F; Bellido, I; Marquez, E; Garcia, A J; Pavia, J; Sanchez de la Cuesta, F

    1992-06-09

    Muscarinic receptor subtypes in human and rat colon smooth muscle homogenates were characterized with [3H]N-methylscopolamine ([3H]NMS) by ligand binding studies. [3H]NMS saturation experiments show the existence of a homogeneous population of non-interacting binding sites with similar affinity (KD values of 1.38 +/- 0.20 nM in human colon smooth muscle and 1.48 +/- 0.47 nM in rat colon smooth muscle) and with Hill slopes close to unity in both samples of tissue. However, a significant (P less than 0.01) increase in muscarinic receptor density (Bmax) is found in human colon (29.9 +/- 2.9 fmol/mg protein) compared with rat colon (17.2 +/- 1.5 fmol/mg protein). Inhibition of [3H]NMS binding by non-labelled compounds shows the following order in human colon: atropine greater than AF-DX 116 greater than pirenzepine. Whereas in rat colon the rank order obtained is atropine greater than pirenzepine greater than AF-DX 116. Atropine and pirenzepine bind to a homogeneous population of binding sites, although pirenzepine shows higher affinity to bind to the sites present in rat colon (Ki = 1.08 +/- 0.08 microM) than those in human colon (Ki = 1.74 +/- 0.02 microM) (P less than 0.05). Similarly, IC50 values obtained in AF-DX 116 competition experiments were significantly different (P less than 0.01) in human colon (IC50 = 1.69 +/- 0.37 microM) than in rat colon (IC50 = 3.78 +/- 0.75 microM). Unlike atropine and pirenzepine, the inhibition of [3H]NMS binding by AF-DX 116 did not yield a simple mass-action binding curve (nH less than 1, P less than 0.01) suggesting the presence of more than one subtype of muscarinic receptor in both species. Computer analysis of these curves with a two binding site model suggests the presence of two populations of receptor. The apparent Ki1 value for the high affinity binding site is 0.49 +/- 0.07 microM for human colon smooth muscle and 0.33 +/- 0.05 microM for rat colon smooth muscle. The apparent Ki2 for the low affinity binding site is 8

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. Effects of lubiprostone on human uterine smooth muscle cells.

    PubMed

    Cuppoletti, John; Malinowska, Danuta H; Chakrabarti, Jayati; Ueno, Ryuji

    2008-06-01

    Lubiprostone, a bicyclic fatty acid derivative and member of a new class of compounds called prostones, locally activates ClC-2 Cl(-) channels without activation of prostaglandin receptors. The present study was specifically designed to test and compare lubiprostone and prostaglandin effects at the cellular level using human uterine smooth muscle cells. Effects on [Ca(2+)](i), membrane potential and [cAMP](i) in human uterine smooth muscle cells were measured. 10 nM lubiprostone significantly decreased [Ca(2+)](i) from 188 to 27 nM, which was unaffected by 100 nM SC-51322, a prostaglandin EP receptor antagonist. In contrast 10nM PGE(2) and PGE(1) both increased [Ca(2+)](i) 3-5-fold which was blocked by SC-51322. Similarly, lubiprostone and prostaglandins had opposite/different effects on membrane potential and [cAMP](i). Lubiprostone caused SC-51322-insensitive membrane hyperpolarization and no effect on [cAMP](i). PGE(2) and PGE(1) both caused SC-51322-sensitive membrane depolarization and increased [cAMP](i). Lubiprostone has fundamentally different cellular effects from prostaglandins that are not mediated by EP receptors.

  10. 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.

  11. Neurophysiology and Neuroanatomy of Smooth Pursuit in Humans

    ERIC Educational Resources Information Center

    Lencer, Rebekka; Trillenberg, Peter

    2008-01-01

    Smooth pursuit eye movements enable us to focus our eyes on moving objects by utilizing well-established mechanisms of visual motion processing, sensorimotor transformation and cognition. Novel smooth pursuit tasks and quantitative measurement techniques can help unravel the different smooth pursuit components and complex neural systems involved…

  12. 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).

  13. Relaxant effect of ethanol extract of Carum carvi on dispersed intestinal smooth muscle cells of the guinea pig.

    PubMed

    Al-Essa, Mohammed K; Shafagoj, Yanal A; Mohammed, Faysal I; Afifi, Fatma U

    2010-01-01

    The present study investigates the direct effects of Carum carvi L. (Apiaceae) ethanol extract on dispersed intestinal smooth muscle cells (SMC) of guinea pigs. Effects of the plant extract on SMC and of acetylcholine (Ach) on extract pretreated SMC were measured by micrometric scanning technique. Three different extract concentrations (2.5 mg/mL, 250 mug/mL, and 25 mug/mL) were used. Ethanol extract of C. carvi reduced significantly the response of dispersed SMC to Ach. Pretreatment of SMC with the highest concentration of C. carvi ethanol extract (2.5 mg/mL) has significantly inhibited the response of SMC to Ach. The data obtained indicate a dose-dependent inhibition of the contraction induced by Ach. This response may explain, in part, the beneficial effect of caraway in relieving gastrointestinal symptoms associated with dyspepsia.

  14. 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

  15. 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.

  16. 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.

  17. Human smooth muscle autoantibody. Its identification as antiactin antibody and a study of its binding to "nonmuscular" cells.

    PubMed

    Gabbiani, G; Ryan, G B; Lamelin, J P; Vassalli, P; Majno, G; Bouvier, C A; Cruchaud, A; Lüscher, E F

    1973-09-01

    When human serum containing smooth muscle autoantibodies (SMA) is incubated with extracts containing thrombosthenin (the contractile material of platelets) or thrombosthenin-A (the actin-like moiety of thrombosthenin), it loses its ability to bind to smooth muscle. Such binding is also diminished when SMA serum is incubated with lysed platelets; this effect is not seen if the SMA serum is incubated with intact platelets. The incubation of other autoantibodies (such as antimitochondrial or antinuclear antibodies) with thrombosthenin does not affect their binding to the specific antigens. It appears that SMA is directed against the actin fraction of thrombosthenin-ie, SMA is an antiactin antibody. Hence the name of antiactin autoantibody (AAA) seems more appropriate than smooth muscle autoantibody (SMA). A study of the distribution of antiactin autoantibody binding in rat, rabbit and man shows that several "nonmuscular" structures contain actin under normal conditions; these include megakaryocytes and platelets, normal rat hepatocytes, the brush borders of renal tubules, the periphery of epithelial cells of the intestine, polymorphs and lymphocytes in lymph nodes (but not thymic cortical lymphocytes). In addition, certain cell types (such as granulation tissue fibroblasts, cultivated fibroblasts, hepatocytes or regenerating liver and epidermal cells growing over a skin wound) can reversibly acquire a massive network of actin-containing microfilaments resembling those in smooth muscle.

  18. 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.

  19. 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.

  20. 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.

  1. Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

    PubMed

    Mallows, R S; Bolton, T B

    1987-06-15

    Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.

  2. 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

  3. 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.

  4. Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells.

    PubMed

    Brun, Paola; Gobbo, Serena; Caputi, Valentina; Spagnol, Lisa; Schirato, Giulia; Pasqualin, Matteo; Levorato, Elia; Palù, Giorgio; Giron, Maria Cecilia; Castagliuolo, Ignazio

    2015-09-01

    Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2(-/-)) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2(-/-) mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2(-/-) mice exhibited smaller ganglia, fewer HuC/D(+ve) and nNOS(+ve) neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with

  5. Effects of epithelium removal on relaxation of airway smooth muscle induced by vasoactive intestinal peptide and electrical field stimulation.

    PubMed Central

    Farmer, S. G.; Togo, J.

    1990-01-01

    1. We have studied the effect of epithelium removal on relaxation of guinea-pig isolated tracheal smooth muscle induced by vasoactive intestinal peptide (VIP) or stimulation of non-adrenergic, non-cholinergic (NANC) inhibitory nerves. Also examined were the effects of inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE). 2. Epithelium removal produced a 3.6 +/- 0.4 fold leftward shift in the VIP concentration-response curve. The supersensitivity to VIP, following epithelium removal was abolished by phosphoramidon or thiorphan (NEP inhibitors), but unaffected by captopril (an ACE inhibitor). In intact trachea, the NEP inhibitors produced leftward shifts in the VIP curves similar to those produced by epithelium removal. 3. In contrast to responses to exogenous VIP, neurogenic NANC inhibitory responses to electrical field stimulation were affected neither by epithelial denudation nor by the peptidase inhibitors. 4. As in previous studies, epithelium removal increased tracheal sensitivity to isoprenaline. This was not altered by pretreatment with a cocktail of peptidase inhibitors. Thus, the effect of the NEP inhibitors on responses to VIP appears to be relatively specific. 5. These data indicate that exogenous VIP is a substrate for airway NEP, since inhibition of the enzyme potentiates the peptide. This is further evidence that the airway epithelium provides a source for the metabolism of mediators. 6. In guinea-pig trachea the NEP responsible for cleaving VIP may be located largely in the epithelial layer, since NEP inhibition was without effect on sensitivity to VIP in epithelium-denuded preparations. If VIP is a NANC inhibitory neurotransmitter in this tissue its degradation endogenously does not appear to involve epithelial NEP. PMID:2196967

  6. 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.

  7. 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

  8. Experimental evidence and mathematical modeling of thermal effects on human colonic smooth muscle contractility.

    PubMed

    Altomare, A; Gizzi, A; Guarino, M P L; Loppini, A; Cocca, S; Dipaola, M; Alloni, R; Cicala, M; Filippi, S

    2014-07-01

    It has been shown, in animal models, that gastrointestinal tract (GIT) motility is influenced by temperature; nevertheless, the basic mechanism governing thermal GIT smooth muscle responses has not been fully investigated. Studies based on physiologically tuned mathematical models have predicted that thermal inhomogeneity may induce an electrochemical destabilization of peristaltic activity. In the present study, the effect of thermal cooling on human colonic muscle strip (HCMS) contractility was studied. HCMSs were obtained from disease-free margins of resected segments for cancer. After removal of the mucosa and serosa layers, strips were mounted in separate chambers. After 30 min, spontaneous contractions developed, which were measured using force displacement transducers. Temperature was changed every hour (37, 34, and 31°C). The effect of cooling was analyzed on mean contractile activity, oscillation amplitude, frequency, and contraction to ACh (10(-5) M). At 37°C, HCMSs developed a stable phasic contraction (~0.02 Hz) with a significant ACh-elicited mean contractile response (31% and 22% compared with baseline in the circular and longitudinal axis, respectively). At a lower bath temperature, higher mean contractile amplitude was observed, and it increased in the presence of ACh (78% and 43% higher than the basal tone in the circular and longitudinal axis, respectively, at 31°C). A simplified thermochemomechanical model was tuned on experimental data characterizing the stress state coupling the intracellular Ca(2+) concentration to tissue temperature. In conclusion, acute thermal cooling affects colonic muscular function. Further studies are needed to establish the exact mechanisms involved to better understand clinical consequences of hypothermia on intestinal contractile activity.

  9. 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.

  10. SREBP inhibits VEGF expression in human smooth muscle cells

    SciTech Connect

    Motoyama, Koka; Fukumoto, Shinya . E-mail: sfukumoto@med.osaka-cu.ac.jp; Koyama, Hidenori; Emoto, Masanori; Shimano, Hitoshi; Maemura, Koji; Nishizawa, Yoshiki

    2006-03-31

    Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate expression of genes encoding enzymes for lipid biosynthesis. SREBPs are activated by HMG-CoA reductase inhibitors (statins). Statins have been also reported to suppress vascular endothelial growth factor (VEGF) expression in vascular smooth muscle cells (VSMCs). Therefore, we hypothesized that SREBPs are involved in statin-mediated regulation of VEGF production in VSMCs. SREBP1 was robustly expressed, and was activated by atorvastatin in VSMCs, as demonstrated by increased levels of the mature nuclear form of SREBP1, and increased promoter activities of a reporter containing sterol regulatory elements by atorvastatin. Moreover, overexpression of SREBP1a dose-dependently suppressed VEGF promoter activity. Site-specific mutation or deletion of the proximal Sp1 sites reduced the inhibitory effects of SREBP1a on VEGF promoter activity. These data demonstrated that SREBP1, activated by atorvastatin, suppressed VEGF expression through the indirect interaction with the proximal tandem Sp1 sites in VSMCs.

  11. The Effects of Lactobacillus acidophilus on the Intestinal Smooth Muscle Contraction through PKC/MLCK/MLC Signaling Pathway in TBI Mouse Model.

    PubMed

    Sun, Bo; Hu, Chen; Fang, Huan; Zhu, Lina; Gao, Ning; Zhu, Jingci

    2015-01-01

    Clinical studies have shown that probiotics influence gastrointestinal motility. However, the molecular mechanisms by which probiotic Lactobacillus modulates intestinal motility in traumatic brain injury (TBI) mouse model have not been explored. In the present study, we provided evidence showing that treatment of TBI mice with Lactobacillus acidophilus significantly improved the terminal ileum villus morphology, restored the impaired interstitial cells of Cajal (ICC) and the disrupted ICC networks after TBI, and prevented TBI-mediated inhibition of contractile activity in intestinal smooth muscle. Mechanistically, the decreased concentration of MLCK, phospho-MLC20 and phospho-MYPT1 and increased concentration of MLCP and PKC were observed after TBI, and these events mediated by TBI were efficiently prevented by Lactobacillus acidophilus application. These findings may provide a novel mechanistic basis for the application of Lactobacillus acidophilus in the treatment of TBI.

  12. Ranolazine inhibits voltage-gated mechanosensitive sodium channels in human colon circular smooth muscle cells.

    PubMed

    Neshatian, Leila; Strege, Peter R; Rhee, Poong-Lyul; Kraichely, Robert E; Mazzone, Amelia; Bernard, Cheryl E; Cima, Robert R; Larson, David W; Dozois, Eric J; Kline, Crystal F; Mohler, Peter J; Beyder, Arthur; Farrugia, Gianrico

    2015-09-15

    Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na(+) current in the human colon smooth muscle and to examine the effects of ranolazine on Na(+) current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na(+) currents (-1.36 ± 0.36 pA/pF), shear stress increased Na(+) peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 μM) blocked peak Na(+) current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na(+) current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine.

  13. 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.

  14. Smooth muscle in the wall of the developing human urinary bladder and urethra.

    PubMed Central

    Gilpin, S A; Gosling, J A

    1983-01-01

    A series of human fetal and neonatal specimens ranging in age from the second month of intrauterine development to 4 1/2 years after birth has been examined using histological and histochemical techniques. In both sexes histologically differentiated smooth muscle cells were evident in the bladder wall from the 52 mm crown-rump length stage onwards--urethral smooth muscle was not distinguishable until 119 mm crown-rump length. In addition to relatively late differentiation, urethral smooth muscle was histochemically distinct from the urinary bladder detrusor muscle. Sex differences in the arrangement and innervation of smooth muscle in the proximal urethra have also been observed, and these findings lend support to the presence of a pre-prostatic urethra sphincter. It seems likely that this sphincter acts principally to prevent reflux of ejaculate into the bladder during seminal emission. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:6654742

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. [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.

  20. 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.

  1. 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

  2. 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.

  3. [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.

  4. 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).

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. Model emulates human smooth pursuit system producing zero-latency target tracking.

    PubMed

    Bahill, A T; McDonald, J D

    1983-01-01

    Humans can overcome the 150 ms time delay of the smooth pursuit eye movement system and track smoothly moving visual targets with zero-latency. Our target-selective adaptive control model can also overcome an inherent time delay and produce zero-latency tracking. No other model or man-made system can do this. Our model is physically realizable and physiologically realistic. The technique used in our model should be useful for analyzing other time-delay systems, such as man-machine systems and robots.

  10. [Vascular smooth muscle cells from human umbilical artery undergo osteoblast differentiation and calcification in vitro].

    PubMed

    Guo, Yong Ping; Sun, Ming Shu; Qian, Jia Qi; Ni, Zhao Hui

    2008-04-01

    To research if the vascular smooth muscle cells (VSMCs) from human umbilical artery undergo osteoblast differentiation spontaneously in vitro. The growth curve of vascular smooth muscle cells from human umbilical artery was obtained by MTT method. The course of multicell nodule formation spontaneously by VSMCs was observed morphologically. The apoptosis of VSMCs in the nodules was detected by Hoechst 33258 and TUNEL methods respectively. The expression of alkaline phosphotase in the nodules was detected by immunohistochemical method. And the calcification was studied with transmission electron microscope and by alizarin red S respectively. We found that the umbilical artery smooth muscle cells confluenced after 7 days of passage and exhibited typical "hill and valley" pattern under light microscope. The cells grew into aggregation and formed nodules at the "hill" region with culture-time prolongation. After 4-5 weeks culture, these nodules built up and calcified spontaneously. We also found alkaline phosphotase expression and apoptosis of VSMCs in these nodules at the same time. We conclude that the vascular smooth muscle cells from human umbilical artery just like from aortic artery can undergo osteoblast differentiation spontaneously in vitro, and apoptosis participate this procedure probably.

  11. 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.

  12. Smooth versus Textured Surfaces: Feature-Based Category Selectivity in Human Visual Cortex

    PubMed Central

    Tootell, Roger

    2016-01-01

    Abstract In fMRI studies, human lateral occipital (LO) cortex is thought to respond selectively to images of objects, compared with nonobjects. However, it remains unresolved whether all objects evoke equivalent levels of activity in LO, and, if not, which image features produce stronger activation. Here, we used an unbiased parametric texture model to predict preferred versus nonpreferred stimuli in LO. Observation and psychophysical results showed that predicted preferred stimuli (both objects and nonobjects) had smooth (rather than textured) surfaces. These predictions were confirmed using fMRI, for objects and nonobjects. Similar preferences were also found in the fusiform face area (FFA). Consistent with this: (1) FFA and LO responded more strongly to nonfreckled (smooth) faces, compared with otherwise identical freckled (textured) faces; and (2) strong functional connections were found between LO and FFA. Thus, LO and FFA may be part of an information-processing stream distinguished by feature-based category selectivity (smooth > textured). PMID:27699206

  13. Influence of micropattern width on differentiation of human mesenchymal stem cells to vascular smooth muscle cells.

    PubMed

    Nakamoto, Tomoko; Wang, Xinlong; Kawazoe, Naoki; Chen, Guoping

    2014-10-01

    In recent years, various approaches have been taken to generate functional muscle tissue by tissue engineering. However, in vitro methods to generate smooth muscle with physiologically aligned structure remains limited. In order to mimic the in vivo highly organized structure of smooth muscle cells, we used micropatterning technology for engineering parallel aligned cells. In this study, a gradient micropattern of different width of cell-adhesive polystyrene stripes (5, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800 and 1000μm) was prepared and the effects of micropattern width on human mesenchymal stem cells (hMSCs) orientation, morphology and smooth muscle cell differentiation were investigated. The width of micropattern stripes showed obvious effect on cell orientation, morphology and smooth muscle cell differentiation. The cells showed higher degree of orientation when the micropattern stripes became narrower. Higher expression of calponin and smooth muscle actin was observed among the narrow micropatterns ranging from 200μm to 20μm, compared to the non-patterned area and wide micropattern areas which showed similar levels of expression.

  14. Smooth muscle in the annulus fibrosus of the tympanic membrane in bats, rodents, insectivores, and humans.

    PubMed

    Henson, M M; Madden, V J; Rask-Andersen, H; Henson, O W

    2005-02-01

    The annulus fibrosus and its attachment to the bony tympanic ring were studied in a series of mammals. In the pallid bat, Antrozous pallidus, there is an extensive plexus of large interconnected blood sinuses in the part of the annulus that borders the tympanic bone. The spaces between the sinuses are packed with smooth muscle cells. Most of the cells have a predominately radial orientation; they extend from the bony tympanic sulcus to a dense collagenous matrix (apical zone) where radially oriented fibers of the pars tensa are confluent with the annulus. The muscles and vessels constitute a myovascular zone. A structurally similar myovascular zone is also present in the European hedgehog. In rodents, the annulus lacks the large interconnected blood sinuses but many small vessels are present. Smooth muscle is concentrated in the broad area of attachment of the annulus to the tympanic bone. In the gerbil, smooth muscle seems to be concentrated in the central part of the width of the annulus where it is attached to bone and radiates toward the tympanic membrane. In humans collections of radially oriented smooth muscle cells were found in several locations. The smooth muscle in all species studied appears to form a rim of contractile elements for the pars tensa. This arrangement suggests a role in controlling blood flow and/or creating and maintaining tension on the tympanic membrane.

  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. 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.

  18. 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.

  19. 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

  20. 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.

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

    PubMed

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

    2012-10-01

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

  2. Tachyphylaxis to beta-adrenoceptor agonists in human bronchial smooth muscle: studies in vitro.

    PubMed Central

    Davis, C; Conolly, M E

    1980-01-01

    In studies on human isolated peripheral airway smooth muscle; 1 A concentration dependent beta-adrenoceptor tachyphylaxis was observed to isoprenaline. 2 Cross desensitization to other beta-adrenoceptor agonists was demonstrated. 3 The desensitization was reversible with time. Hydrocortisone appeared to accelerate the recovery from the desensitized state. Low concentration isoprenaline (10(-9) mol l-1) prevented recovery whereas cyclohexamide 1.8 x 10(-4) mol l-1 had no noticeable effect on recovery. Continued occupancy of the receptor appears to prevent recovery. The recovery from the desensitized state does not apparently require synthesis of new proteins. 4 Bronchial wall cyclic AMP response to isoprenaline was attenuated after isoprenaline induced desensitization whereas total phosphodiesterase activity of bronchial wall was not altered by desensitization. Thus by exclusion the adenylate cyclase receptor complex may be altered in human peripheral airway smooth muscle beta-adrenoceptor tachyphylaxis. PMID:6108126

  3. Assays for in vitro monitoring of human airway smooth muscle (ASM) and human pulmonary arterial vascular smooth muscle (VSM) cell migration.

    PubMed

    Goncharova, Elena A; Goncharov, Dmitry A; Krymskaya, Vera P

    2006-01-01

    Migration of human pulmonary vascular smooth muscle (VSM) cells contributes to vascular remodeling in pulmonary arterial hypertension and atherosclerosis. Evidence also indicates that, in part, migration of airway smooth muscle (ASM) cells may contribute to airway remodeling associated with asthma. Here we describe migration of VSM and ASM cells in vitro using Transwell or Boyden chamber assays. Because dissecting signaling mechanisms regulating cell migration requires molecular approaches, our protocol also describes how to assess migration of transfected VSM and ASM cells. Transwell or Boyden chamber assays can be completed in approximately 8 h and include plating of serum-deprived VSM or ASM cell suspension on membrane precoated with collagen, migration of cells toward chemotactic gradient and visual (Transwell) or digital (Boyden chamber) analysis of membrane. Although the Transwell assay is easy, the Boyden chamber assay requires hands-on experience; however, both assays are reliable cell-based approaches providing valuable information on how chemotactic and inflammatory factors modulate VSM and ASM migration.

  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. Macrophages and smooth muscle cells express lipoprotein lipase in human and rabbit atherosclerotic lesions.

    PubMed Central

    Ylä-Herttuala, S; Lipton, B A; Rosenfeld, M E; Goldberg, I J; Steinberg, D; Witztum, J L

    1991-01-01

    Lipoprotein lipase (LPL; EC 3.1.1.34) may promote atherogenesis by producing remnant lipoproteins on the endothelial surface and by acting on lipoproteins in the artery wall. In vitro, smooth muscle cells and macrophages synthesize LPL, but in human carotid lesions only a few smooth muscle cells were reported to contain LPL protein. Endothelial cells do not synthesize LPL in vitro, but in normal arteries intense immunostaining for LPL is present on the endothelium. We used Northern blot analysis, in situ hybridization, and immunocytochemistry of human and rabbit arteries to determine cellular distribution and the site of the synthesis of LPL in atherosclerotic lesions. Northern blot analysis showed that LPL mRNA was detectable in macrophage-derived foam cells isolated from arterial lesions of "ballooned" cholesterol-fed rabbits. In situ hybridization studies of atherosclerotic lesions with an antisense riboprobe showed a strong hybridization signal for LPL mRNA in some, but not all, lesion macrophages, which were mostly located in the subendothelial and edge areas of the lesions. Also, some smooth muscle cells in lesion areas also expressed LPL mRNA. Immunocytochemistry of frozen sections of rabbit lesions with a monoclonal antibody to human milk LPL showed intense staining for LPL protein in macrophage-rich intimal lesions. The results suggest that lesion macrophages and macrophage-derived foam cells express LPL mRNA and protein. Some smooth muscle cells in the lesion areas also synthesize LPL. These data are consistent with an important role for LPL in atherogenesis. Images PMID:1719546

  9. Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: A model for gene therapy

    SciTech Connect

    Lynch, C.M.; Miller, A.D. ); Clowes, M.M.; Osborne, W.R.A.; Clowes, A.W. )

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli {beta}-galactosidase gene or a human adenosine deaminase gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  10. Comparison of human bronchiolar smooth muscle responsiveness in vitro with histological signs of inflammation.

    PubMed Central

    de Jongste, J C; Mons, H; Van Strik, R; Bonta, I L; Kerrebijn, K F

    1987-01-01

    A study was carried out to test the hypothesis that chronic inflammation is associated with increased sensitivity or contractility of human airway smooth muscle. Bronchiolar strips from 30 patients, 12 of whom had chronic bronchitis, were examined in the organ bath for their responses to histamine, methacholine, and leukotriene (LT) C4. The same airways were also studied histologically and small airway disease was quantified by subjective grading of the degree of inflammatory cell infiltration, smooth muscle hypertrophy, fibrosis, and goblet cell hyperplasia. The degree of small airway disease varied widely among patients both with and without chronic bronchitis. Multiple regression analysis failed to show increased sensitivity (-log EC50) to histamine, methacholine, or LTC4 in relation to small airway disease. In contrast, the only significant correlations found were between a decreased -log EC50 to histamine and methacholine and an increased small airway disease score. Contractile responses (Tmax) to histamine and methacholine in peripheral airways tended to be higher in patients with chronic bronchitis than in those without. Tmax was not related to small airway disease scores. These results suggest that chronic airway inflammation does not cause in vitro hyperresponsiveness of human small airway smooth muscle. Images PMID:3321543

  11. 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

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. Altered Expression of Human Smooth Muscle Myosin Phosphatase Targeting (MYPT) Isovariants with Pregnancy and Labor

    PubMed Central

    Taggart, Julie; Robson, Stephen; Taggart, Michael

    2016-01-01

    Background Myosin light-chain phosphatase is a trimeric protein that hydrolyses phosphorylated myosin II light chains (MYLII) to cause relaxation in smooth muscle cells including those of the uterus. A major component of the phosphatase is the myosin targeting subunit (MYPT), which directs a catalytic subunit to dephosphorylate MYLII. There are 5 main MYPT family members (MYPT1 (PPP1R12A), MYPT2 (PPP1R12B), MYPT3 (PPP1R16A), myosin binding subunit 85 MBS85 (PPP1R12C) and TIMAP (TGF-beta-inhibited membrane-associated protein (PPP1R16B)). Nitric oxide (NO)-mediated smooth muscle relaxation has in part been attributed to activation of the phosphatase by PKG binding to a leucine zipper (LZ) dimerization domain located at the carboxyl-terminus of PPP1R12A. In animal studies, alternative splicing of PPP1R12A can lead to the inclusion of a 31-nucleotide exonic segment that generates a LZ negative (LZ-) isovariant rendering the phosphatase less sensitive to NO vasodilators and alterations in PPP1R12ALZ- and LZ+ expression have been linked to phenotypic changes in smooth muscle function. Moreover, PPP1R12B and PPP1R12C, but not PPP1R16A or PPP1R16B, have the potential for LZ+/LZ- alternative splicing. Yet, by comparison to animal studies, the information on human MYPT genomic sequences/mRNA expressions is scant. As uterine smooth muscle undergoes substantial remodeling during pregnancy we were interested in establishing the patterns of expression of human MYPT isovariants during this process and also following labor onset as this could have important implications for determining successful pregnancy outcome. Objectives We used cross-species genome alignment, to infer putative human sequences not available in the public domain, and isovariant-specific quantitative PCR, to analyse the expression of mRNA encoding putative LZ+ and LZ- forms of PPP1R12A, PPP1R12B and PPP1R12C as well as canonical PPP1R16A and PPP1R16B genes in human uterine smooth muscle from non

  17. Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells.

    PubMed

    Iyer, Dharini; Gambardella, Laure; Bernard, William G; Serrano, Felipe; Mascetti, Victoria L; Pedersen, Roger A; Talasila, Amarnath; Sinha, Sanjay

    2015-04-15

    The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening.

  18. Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells

    PubMed Central

    Iyer, Dharini; Gambardella, Laure; Bernard, William G.; Serrano, Felipe; Mascetti, Victoria L.; Pedersen, Roger A.; Talasila, Amarnath; Sinha, Sanjay

    2015-01-01

    The epicardium has emerged as a multipotent cardiovascular progenitor source with therapeutic potential for coronary smooth muscle cell, cardiac fibroblast (CF) and cardiomyocyte regeneration, owing to its fundamental role in heart development and its potential ability to initiate myocardial repair in injured adult tissues. Here, we describe a chemically defined method for generating epicardium and epicardium-derived smooth muscle cells (EPI-SMCs) and CFs from human pluripotent stem cells (HPSCs) through an intermediate lateral plate mesoderm (LM) stage. HPSCs were initially differentiated to LM in the presence of FGF2 and high levels of BMP4. The LM was robustly differentiated to an epicardial lineage by activation of WNT, BMP and retinoic acid signalling pathways. HPSC-derived epicardium displayed enhanced expression of epithelial- and epicardium-specific markers, exhibited morphological features comparable with human foetal epicardial explants and engrafted in the subepicardial space in vivo. The in vitro-derived epicardial cells underwent an epithelial-to-mesenchymal transition when treated with PDGF-BB and TGFβ1, resulting in vascular SMCs that displayed contractile ability in response to vasoconstrictors. Furthermore, the EPI-SMCs displayed low density lipoprotein uptake and effective lowering of lipoprotein levels upon treatment with statins, similar to primary human coronary artery SMCs. Cumulatively, these findings suggest that HPSC-derived epicardium and EPI-SMCs could serve as important tools for studying human cardiogenesis, and as a platform for vascular disease modelling and drug screening. PMID:25813541

  19. 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.

  20. 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  

  1. 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.

  2. 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.

  3. Heparin fragments inhibit human vascular smooth muscle cell proliferation in vitro

    SciTech Connect

    Selden, S.C.; Johnson, W.V.; Maciag, T.

    1986-03-01

    The authors have examined the effect of heparin on human abdominal aortic smooth muscle cell growth. Cell proliferation was inhibited by more than 90% at a concentration of 20 ..mu..g/ml in a 12 day growth assay using heparin from Sigma, Upjohn or Calbiochem. Additionally, 200 ..mu..g/ml Upjohn heparin inhibits /sup 3/H-thymidine incorporation by 50% in short term assays using serum or purified platelet-derived growth factor (25-100ng/ml) to initiate the cell cycle. Homogeneous size classes of heparin fragments were prepared by nitrous acid cleavage and BioGel P-10 filtration chromatography. Deca-, octa-, hexa-, tetra-, and di-saccharides inhibited proliferation by 90% at concentrations of 280, 320, 260, 180 and 100 ..mu..g/ml, respectively, in a 12 day growth assay. These data confirm the work of Castellot et.al. and extend the range of inhibitory fragments down to the tetra- and di-saccharide size. These data suggest, therefore, that di-saccharide subunit of heparin is sufficient to inhibit vascular smooth muscle cell proliferation. The authors are now examining the role of the anhydromannose moiety on the reducing end of the nitrous acid generated fragments as a possible mediator of heparin-induced inhibition of vascular smooth muscle cell proliferation.

  4. Matrix metalloproteinase expression and activity in human airway smooth muscle cells

    PubMed Central

    Elshaw, Shona R; Henderson, Neil; Knox, Alan J; Watson, Susan A; Buttle, David J; Johnson, Simon R

    2004-01-01

    Airway remodelling is a feature of chronic asthma comprising smooth muscle hypertrophy and deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinases (MMPs) breakdown ECM, are involved in tissue remodelling and have been implicated in airway remodelling. Although mesenchymal cells are an important source of MMPs, little data are available on airway smooth muscle (ASM) derived MMPs. We therefore investigated MMP and tissue inhibitor of metalloproteinase (TIMP) production and activity in human ASM cells.MMPs and TIMPs were examined using quantitative real-time RT–PCR, Western blotting, zymography and a quench fluorescence (QF) assay of total MMP activity.The most abundant MMPs were pro-MMP-2, pro- MMP-3, active MMP-3 and MT1-MMP. TIMP-1 and TIMP-2 expression was low in cell lysates but high in conditioned medium. High TIMP secretion was confirmed by the ability of ASM-conditioned medium to inhibit recombinant MMP-2 in a QF assay. Thrombin increased MMP activity by activation of pro-MMP-2 independent of the conventional smooth muscle thrombin receptors PAR 1 and 4.In conclusion, ASM cells express pro-MMP-2, pro and active MMP-3, MMP-9 and MT1-MMP. Unstimulated cells secrete excess TIMP 1 and 2, preventing proteolytic activity. MMP-2 can be activated by thrombin which may contribute to airway remodelling. PMID:15265805

  5. Inhibitory effects of SKF96365 on the activities of K(+) channels in mouse small intestinal smooth muscle cells.

    PubMed

    Tanahashi, Yasuyuki; Wang, Ban; Murakami, Yuri; Unno, Toshihiro; Matsuyama, Hayato; Nagano, Hiroshi; Komori, Seiichi

    2016-02-01

    In order to investigate the effects of SKF96365 (SKF), which is a non-selective cationic channel blocker, on K(+) channel currents, we recorded currents through ATP sensitive K(+) (IKATP), voltage-gated K(+) (IKv) and Ca(2+) activated K(+) channels (IBK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished IKATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K(+) channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC50) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC50 value in IKATP suppression) reversibly inhibited the IKv that was induced by repetitive depolarizing pulses from -80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient IBK and caffeine-induced IBK. These results indicated that SKF inhibited ATP sensitive K(+) channels and voltage-gated K(+) channels, with the ATP sensitive K(+) channels being more sensitive than the voltage-gated K(+) channels. These inhibitory effects on K(+) channels should be considered when SKF is used as a cationic channel blocker.

  6. Inhibitory effects of SKF96365 on the activities of K+ channels in mouse small intestinal smooth muscle cells

    PubMed Central

    TANAHASHI, Yasuyuki; WANG, Ban; MURAKAMI, Yuri; UNNO, Toshihiro; MATSUYAMA, Hayato; NAGANO, Hiroshi; KOMORI, Seiichi

    2015-01-01

    In order to investigate the effects of SKF96365 (SKF), which is a non-selective cationic channel blocker, on K+ channel currents, we recorded currents through ATP sensitive K+ (IKATP), voltage-gated K+ (IKv) and Ca2+ activated K+ channels (IBK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished IKATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K+ channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC50) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC50 value in IKATP suppression) reversibly inhibited the IKv that was induced by repetitive depolarizing pulses from −80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient IBK and caffeine-induced IBK. These results indicated that SKF inhibited ATP sensitive K+ channels and voltage-gated K+ channels, with the ATP sensitive K+ channels being more sensitive than the voltage-gated K+ channels. These inhibitory effects on K+ channels should be considered when SKF is used as a cationic channel blocker. PMID:26498720

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Fluid flow releases fibroblast growth factor-2 from human aortic smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Rhoads, D. N.; Eskin, S. G.; McIntire, L. V.

    2000-01-01

    This study tested the hypothesis that fluid shear stress regulates the release of fibroblast growth factor (FGF)-2 from human aortic smooth muscle cells. FGF-2 is a potent mitogen that is involved in the response to vascular injury and is expressed in a wide variety of cell types. FGF-2 is found in the cytoplasm of cells and outside cells, where it associates with extracellular proteoglycans. To test the hypothesis that shear stress regulates FGF-2 release, cells were exposed to flow, and FGF-2 amounts were measured from the conditioned medium, pericellular fraction (extracted by heparin treatment), and cell lysate. Results from the present study show that after 15 minutes of shear stress at 25 dyne/cm(2) in a parallel-plate flow system, a small but significant fraction (17%) of the total FGF-2 was released from human aortic smooth muscle cells. FGF-2 levels in the circulating medium increased 10-fold over medium from static controls (P<0.01). A 50% increase in FGF-2 content versus control (P<0.01) was found in the pericellular fraction (extracted by heparin treatment). Furthermore, a significant decrease in FGF-2 was detected in the cell lysate, indicating that FGF-2 was released from inside the cell. Cell permeability studies with fluorescent dextran were performed to examine whether transient membrane disruption caused FGF-2 release. Flow cytometry detected a 50% increase in mean fluorescence of cells exposed to 25 dyne/cm(2) versus control cells. This indicates that the observed FGF-2 release from human aortic smooth muscle cells is likely due to transient membrane disruption on initiation of flow.

  13. An electrophysiological study of the smooth muscle of the human colon.

    PubMed Central

    Kirk, D.

    1981-01-01

    Electrical recordings were made in vitro from preparations of human colonic smooth muscle from surgically resected specimens. The behaviour of the taenia consisted of regular spike action potentials based on a slow wave rhythm (22 +/- 5 c.p.m.), with tetanic contractions of the muscle. The actions of cholinergic drugs were studied and experiments performed to investigate the mechanism of the action potentials. The circular muscle produced clusters of spikes with solitary contractions. The differences between the two muscle layers may be of relevance to understanding the colonic electromyogram as recorded in vivo. PMID:7294682

  14. Remeshed smoothed particle hydrodynamics simulation of the mechanical behavior of human organs.

    PubMed

    Hieber, Simone E; Walther, Jens H; Koumoutsakos, Petros

    2004-01-01

    In computer aided surgery the accurate simulation of the mechanical behavior of human organs is essential for the development of surgical simulators. In this paper we introduce particle based simulations of two different human organ materials modeled as linear viscoelastic solids. The constitutive equations for the material behavior are discretized using a particle approach based on the Smoothed Particle Hydrodynamics (SPH) method while the body surface is tracked using level sets. A key aspect of this approach is its flexibility which allows the simulation of complex time varying topologies with large deformations. The accuracy of the original formulation is significantly enhanced by using a particle reinitialization technique resulting in remeshed Smoothed Particle Hydrodynamics (rSPH). The mechanical parameters of the systems used in the simulations are derived from experimental measurements on human cadaver organs. We compare the mechanical behavior of liver- and kidney-like materials based on the dynamic simulations of a tensile test case. Moreover, we present a particle based reconstruction of the liver topology and its strain distribution under a small local load. Finally, we demonstrate a unified formulation of fluid structure interaction based on particle methods.

  15. 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

  16. 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.

  17. 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.

  18. Prostanoid receptors mediating contraction in rat, macaque and human bladder smooth muscle in vitro.

    PubMed

    Root, James A; Davey, Dorren A; Af Forselles, Kerry J

    2015-12-15

    Selective prostaglandin EP1 antagonists have been suggested for the treatment of bladder dysfunction. This study assessed the contractile prostanoid receptor subtypes in human and non-human bladder in vitro. Classical tissue bath studies were conducted using bladder strips exposed to prostanoid agonists and antagonists. Prostaglandin E2 (PGE2) contracted rat, macaque and human bladder smooth muscle strips (pEC50 7.91±0.06 (n=7), 6.40±0.13 (n=7), and 6.07±0.11 (n=5), respectively). The EP1 receptor antagonist, PF2907617 (300nM), caused a rightward shift of the PGE2 concentration-response curve in the rat bladder only (pKB 8.40±0.15, n=3). PGE2 responses in rat and macaque bladders, but not human, were antagonised by the EP3 antagonist CJ24979 (1µM). Sulprostone, a mixed EP1/EP3/FP receptor agonist, induced potent contractions of rat bladder muscle (pEC50 7.94±0.31, n=6). The FP receptor agonist, prostaglandin F2α (PGF2α), induced bladder contraction in all species tested, but with a lower potency in rat. The selective FP receptor agonist latanoprost caused potent contractions of macaque and human bladder strips only. SQ29548, a selective TP antagonist, and GW848687X, a mixed EP1/TP antagonist caused rightward shifts of the concentration-response curves to the selective TP agonist, U46619 (pKB estimates 8.53±0.07 and 7.56±0.06, n=3, respectively). Responses to U46619 were absent in rat preparations. These data suggest significant species differences exist in bladder contractile prostanoid receptor subtypes. We conclude that the EP1 subtype does not represent the best approach to the clinical treatment of bladder disorders targeting inhibition of smooth muscle contraction.

  19. Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue.

    PubMed Central

    Lazard, D; Sastre, X; Frid, M G; Glukhova, M A; Thiery, J P; Koteliansky, V E

    1993-01-01

    The expression of several differentiation markers in normal human mammary gland myoepithelium and in certain stromal fibroblasts ("myofibroblasts") associated with breast carcinomas was studied by immunofluorescence microscopy of frozen sections. Several antibodies to smooth muscle-specific proteins (smooth muscle alpha-actin, smooth muscle myosin heavy chains, calponin, alpha 1-integrin, and high molecular weight caldesmon) and to epithelial-specific proteins (cytokeratins, E-cadherin, and desmoplakin) were used to show that myoepithelial cells concomitantly express epithelial and smooth muscle markers whereas adjacent luminal cells express only epithelial markers. The same antibodies were used to establish that stromal myofibroblasts exhibit smooth muscle phenotypic properties characterized by the expression of all the smooth muscle markers examined except for high molecular weight caldesmon. In addition, both myoepithelium and myofibroblasts show a significant degree of heterogeneity in smooth muscle protein expression. Thus, myoepithelial cells and stromal myofibroblasts are epithelial and mesenchymal cells, respectively, which coordinately express a set of smooth muscle markers while maintaining their specific original features. The dual nature of myoepithelial cells and the phenotypic transition of fibroblasts to myofibroblasts are examples of the plasticity of the differentiated cell phenotype. Images PMID:8430113

  20. 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.

  1. Regulatory mechanism of human vascular smooth muscle cell phenotypic transformation induced by NELIN

    PubMed Central

    PEI, CHANGAN; QIN, SHIYONG; WANG, MINGHAI; ZHANG, SHUGUANG

    2015-01-01

    Vascular disorders, including hypertension, atherosclerosis and restenosis, arise from dysregulation of vascular smooth muscle cell (VSMC) differentiation, which can be controlled by regulatory factors. The present study investigated the regulatory mechanism of the phenotypic transformation of human VSMCs by NELIN in order to evaluate its potential as a preventive and therapeutic of vascular disorders. An in vitro model of NELIN-overexpressing VSMCs was prepared by transfection with a lentiviral (LV) vector (NELIN-VSMCs) and NELIN was slienced using an a lentiviral vector with small interfering (si)RNA in another group (LV-NELIN-siRNA-VSMCs). The effects of NELIN overexpression or knockdown on the phenotypic transformation of human VSMCs were observed, and its regulatory mechanism was studied. Compared with the control group, cells in the NELIN-VSMCs group presented a contractile phenotype with a significant increase of NELIN mRNA, NELIN protein, smooth muscle (SM)α-actin and total Ras homolog gene family member A (RhoA) protein expression. The intra-nuclear translocation of SMα-actin-serum response factor (SMα-actin-SRF) occurred in these cells simultaneously. Following exposure to Rho kinsase inhibitor Y-27632, SRF and SMα-actin expression decreased. However, cells in the LV-NELIN-siRNA-VSMCs group presented a synthetic phenotype, and the expression of NELIN mRNA, NELIN protein, SMα-actin protein and total RhoA protein was decreased. The occurrence of SRF extra-nuclear translocation was observed. In conclusion, the present study suggested that NELIN was able to activate regulatory factors of SMα-actin, RhoA and SRF successively in human VSMCs cultured in vitro. Furthermore, NELIN-induced phenotypic transformation of human VSMCs was regulated via the RhoA/SRF signaling pathway. The results of the present study provide a foundation for the use of NELIN in preventive and therapeutic treatment of vascular remodeling diseases, including varicosity and

  2. 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.

  3. 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.

  4. The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

    PubMed Central

    Köhler, Eleonore S; Sankaranarayanan, Selvakumari; van Ginneken, Christa J; van Dijk, Paul; Vermeulen, Jacqueline LM; Ruijter, Jan M; Lamers, Wouter H; Bruder, Elisabeth

    2008-01-01

    Background Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens. Results Between 23 weeks of gestation and 3 years after birth, CPS- and ASS-protein content in enterocytes was high and then declined to reach adult levels at 5 years. OAT levels declined more gradually, whereas ARG-1 was not expressed. ARG-2 expression increased neonatally to adult levels. Neurons in the enteric plexus strongly expressed ASS, OAT, NOS1 and ARG2, while varicose nerve fibers in the circular layer of the muscularis propria stained for ASS and NOS1 only. The endothelium of small arterioles expressed ASS and NOS3, while their smooth-muscle layer expressed OAT and ARG2. Conclusion The human small intestine acquires the potential to produce arginine well before fetuses become viable outside the uterus. The perinatal human intestine therefore resembles that of rodents and pigs. Enteral ASS behaves as a typical suckling enzyme because its expression all but disappears in the putative weaning period of human infants. PMID:19000307

  5. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration

    SciTech Connect

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation and migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that Ipt

  6. 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

  7. 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.

  8. 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.

  9. Estrogen effects on human airway smooth muscle involve cAMP and protein kinase A.

    PubMed

    Townsend, Elizabeth A; Sathish, Venkatachalem; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S

    2012-11-15

    Clinically observed differences in airway reactivity and asthma exacerbations in women at different life stages suggest a role for sex steroids in modulating airway function although their targets and mechanisms of action are still being explored. We have previously shown that clinically relevant concentrations of exogenous estrogen acutely decrease intracellular calcium ([Ca(2+)](i)) in human airway smooth muscle (ASM), thereby facilitating bronchodilation. In this study, we hypothesized that estrogens modulate cyclic nucleotide regulation, resulting in decreased [Ca(2+)](i) in human ASM. In Fura-2-loaded human ASM cells, 1 nM 17β-estradiol (E(2)) potentiated the inhibitory effect of the β-adrenoceptor (β-AR) agonist isoproterenol (ISO; 100 nM) on histamine-mediated Ca(2+) entry. Inhibition of protein kinase A (PKA) activity (KT5720; 100 nM) attenuated E(2) effects on [Ca(2+)](i). Acute treatment with E(2) increased cAMP levels in ASM cells comparable to that of ISO (100 pM). In acetylcholine-contracted airways from female guinea pigs or female humans, E(2) potentiated ISO-induced relaxation. These novel data suggest that, in human ASM, physiologically relevant concentrations of estrogens act via estrogen receptors (ERs) and the cAMP pathway to nongenomically reduce [Ca(2+)](i), thus promoting bronchodilation. Activation of ERs may be a novel adjunct therapeutic avenue in reactive airway diseases in combination with established cAMP-activating therapies such as β(2)-agonists.

  10. Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration.

    PubMed

    Liu, Wenrui; Kong, Hui; Zeng, Xiaoning; Wang, Jingjing; Wang, Zailiang; Yan, Xiaopei; Wang, Yanli; Xie, Weiping; Wang, Hong

    2015-08-15

    Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (KATP) channels have been identified in ASMCs. Mount evidence has suggested that KATP channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K(+) channels triggers K(+) efflux, which leading to membrane hyperpolarization, preventing Ca(2+)entry through closing voltage-operated Ca(2+) channels. Intracellular Ca(2+) is the most important regulator of muscle contraction, cell proliferation and migration. K(+) efflux decreases Ca(2+) influx, which consequently influences ASMCs proliferation and migration. As a KATP channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2'-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca(2+)/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective KATP channel antagonist. These findings provide a strong evidence to support that Ipt antagonize the proliferating and migrating effects of PDGF-BB on

  11. Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

    PubMed Central

    Yang, Libang; Geng, Zhaohui; Nickel, Thomas; Johnson, Caitlin; Gao, Lin; Dutton, James; Hou, Cody; Zhang, Jianyi

    2016-01-01

    Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs. PMID:26771193

  12. Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells.

    PubMed

    Hung, Shih-Chieh; Kuo, Pei-Yin; Chang, Ching-Fang; Chen, Tain-Hsiung; Ho, Larry Low-Tone

    2006-06-01

    The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.

  13. Receptor-based differences in human aortic smooth muscle cell membrane stiffness

    NASA Technical Reports Server (NTRS)

    Huang, H.; Kamm, R. D.; So, P. T.; Lee, R. T.

    2001-01-01

    Cells respond to mechanical stimuli with diverse molecular responses. The nature of the sensory mechanism involved in mechanotransduction is not known, but integrins may play an important role. The integrins are linked to both the cytoskeleton and extracellular matrix, suggesting that probing cells via integrins should yield different mechanical properties than probing cells via non-cytoskeleton-associated receptors. To test the hypothesis that the mechanical properties of a cell are dependent on the receptor on which the stress is applied, human aortic smooth muscle cells were plated, and magnetic beads, targeted either to the integrins via fibronectin or to the transferrin receptor by use of an IgG antibody, were attached to the cell surface. The resistance of the cell to deformation ("stiffness") was estimated by oscillating the magnetic beads at 1 Hz by use of single-pole magnetic tweezers at 2 different magnitudes. The ratio of bead displacements at different magnitudes was used to explore the mechanical properties of the cells. Cells stressed via the integrins required approximately 10-fold more force to obtain the same bead displacements as the cells stressed via the transferrin receptors. Cells stressed via integrins showed stiffening behavior as the force was increased, whereas this stiffening was significantly less for cells stressed via the transferrin receptor (P<0.001). Mechanical characteristics of vascular smooth muscle cells depend on the receptor by which the stress is applied, with integrin-based linkages demonstrating cell-stiffening behavior.

  14. Intracellular Ca(2+) remodeling during the phenotypic journey of human coronary smooth muscle cells.

    PubMed

    Muñoz, Eva; Hernández-Morales, Miriam; Sobradillo, Diego; Rocher, Asunción; Núñez, Lucía; Villalobos, Carlos

    2013-11-01

    Vascular smooth muscle cells undergo phenotypic switches after damage which may contribute to proliferative disorders of the vessel wall. This process has been related to remodeling of Ca(2+) channels. We have tested the ability of cultured human coronary artery smooth muscle cells (hCASMCs) to return from a proliferative to a quiescent behavior and the contribution of intracellular Ca(2+) remodeling to the process. We found that cultured, early passage hCASMCs showed a high proliferation rate, sustained increases in cytosolic [Ca(2+)] in response to angiotensin II, residual voltage-operated Ca(2+) entry, increased Stim1 and enhanced store-operated currents. Non-steroidal anti-inflammatory drugs inhibited store-operated Ca(2+) entry and abolished cell proliferation in a mitochondria-dependent manner. After a few passages, hCASMCs turned to a quiescent phenotype characterized by lack of proliferation, oscillatory Ca(2+) response to angiotensin II, increased Ca(2+) store content, enhanced voltage-operated Ca(2+) entry and Cav1.2 expression, and decreases in Stim1, store-operated current and store-operated Ca(2+) entry. We conclude that proliferating hCASMCs return to quiescence and this switch is associated to a remodeling of Ca(2+) channels and their control by subcellular organelles, thus providing a window of opportunity for targeting phenotype-specific Ca(2+) channels involved in proliferation.

  15. Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate.

    PubMed

    Ždychová, J; Čejková, S; Králová Lesná, I; Králová, A; Malušková, J; Janoušek, L; Kazdová, L

    2014-01-01

    The abnormal proliferation of vascular smooth muscle cells (VSMC) is thought to play a role in the pathogenesis of atherosclerosis. Adipocytes produce several bioactive paracrine substances that can affect the growth and migration of VSMCs. Our study focuses on the direct effect of the bioactive substances in conditioned media (CM) that was obtained by incubation with primary adipocyte-derived cell lines, including cell lines derived from both preadipocytes and from more mature cells, on the proliferation rate of human aortic smooth muscle cells (HAoSMCs). We used a Luminex assay to measure the adipokine content of the CM and showed that there was a higher concentration of monocyte chemoattractant protein-1 in renal preadipocyte-CM compared with the HAoSMC control (p<0.5). The addition of both renal preadipocyte- and epicardial adipocyte- CM resulted in the elevated production of vascular endothelial growth factor compared with the control HASoSMC CM (p<0.001). The adiponectin content in renal adipocyte-CM was increased compared to all the remaining adipocyte-CM (p<0.01). Moreover, the results showed a higher proliferation rate of HAoSMCs after co-culture with epicardial adipocyte-CM compared to the HAoSMC control (p<0.05). These results suggest that bioactive substances produced by adipocytes have a stimulatory effect on the proliferation of VSMCs.

  16. Smooth enlargement of human standing sway by instability due to weak reaction floor and noise

    PubMed Central

    Funato, Tetsuro; Aoi, Shinya; Tomita, Nozomi; Tsuchiya, Kazuo

    2016-01-01

    Human quiet standing is accompanied by body sway. The amplitude of this body sway is known to be larger than would be predicted from simple noise effects, and sway characteristics are changed by neurological disorders. This large sway is thought to arise from nonlinear control with prolonged periods of no control (intermittent control), and a nonlinear control system of this kind has been predicted to exhibit bifurcation. The presence of stability-dependent transition enables dynamic reaction that depends on the stability of the environment, and can explain the change in sway characteristics that accompanies some neurological disorders. This research analyses the characteristics of a system model that induces transition, and discusses whether human standing reflects such a mechanism. In mathematical analysis of system models, (intermittent control-like) nonlinear control with integral control is shown to exhibit Hopf bifurcation. Moreover, from the analytical solution of the system model with noise, noise is shown to work to smooth the enlargement of sway around the bifurcation point. This solution is compared with measured human standing sway on floors with different stabilities. By quantitatively comparing the control parameters between human observation and model prediction, enlargement of sway is shown to appear as predicted by the model analysis. PMID:26909186

  17. 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.

  18. 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

  19. Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells

    PubMed Central

    Brun, Juliane; Lutz, Katrin A.; Neumayer, Katharina M. H.; Klein, Gerd; Seeger, Tanja; Uynuk-Ool, Tatiana; Wörgötter, Katharina; Schmid, Sandra; Kraushaar, Udo; Guenther, Elke; Rolauffs, Bernd; Aicher, Wilhelm K.; Hart, Melanie L.

    2015-01-01

    The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel

  20. Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells*

    PubMed Central

    Vigetti, Davide; Rizzi, Manuela; Moretto, Paola; Deleonibus, Sara; Dreyfuss, Jonathan M.; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Hascall, Vincent C.; Ramoni, Marco F.; De Luca, Giancarlo; Passi, Alberto

    2011-01-01

    Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2. PMID:21768115

  1. Glycosaminoglycans and glucose prevent apoptosis in 4-methylumbelliferone-treated human aortic smooth muscle cells.

    PubMed

    Vigetti, Davide; Rizzi, Manuela; Moretto, Paola; Deleonibus, Sara; Dreyfuss, Jonathan M; Karousou, Evgenia; Viola, Manuela; Clerici, Moira; Hascall, Vincent C; Ramoni, Marco F; De Luca, Giancarlo; Passi, Alberto

    2011-10-07

    Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2.

  2. Smooth-muscle-like cells derived from human embryonic stem cells support and augment cord-like structures in vitro.

    PubMed

    Vo, Elaine; Hanjaya-Putra, Donny; Zha, Yuanting; Kusuma, Sravanti; Gerecht, Sharon

    2010-06-01

    Engineering vascularized tissue is crucial for its successful implantation, survival, and integration with the host tissue. Vascular smooth muscle cells (v-SMCs) provide physical support to the vasculature and aid in maintaining endothelial viability. In this study, we show an efficient derivation of v-SMCs from human embryonic stem cells (hESCs), and demonstrate their functionality and ability to support the vasculature in vitro. Human ESCs were differentiated in monolayers and supplemented with platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta 1 (TGF-beta1). Human ESC-derived smooth-muscle-like cells (SMLCs) were found to highly express specific smooth muscle cell (SMC) markers--including alpha-smooth muscle actin, calponin, SM22, and smooth muscle myosin heavy chain--to produce and secrete fibronectin and collagen, and to contract in response to carbachol. In vitro tubulogenesis assays revealed that these hESC-derived SMLCs interacted with human endothelial progenitor cell (EPCs) to form longer and thicker cord-like structures in vitro. We have demonstrated a simple protocol for the efficient derivation of highly purified SMLCs from hESCs. These in vitro functional SMLCs interacted with EPCs to support and augment capillary-like structures (CLSs), demonstrating the potential of hESCs as a cell source for therapeutic vascular tissue engineering.

  3. Effect of dexamethasone on voltage-gated Na+ channel in cultured human bronchial smooth muscle cells.

    PubMed

    Nakajima, Toshiaki; Jo, Taisuke; Meguro, Kentaro; Oonuma, Hitoshi; Ma, Ji; Kubota, Nami; Imuta, Hiroyuki; Takano, Haruhito; Iida, Haruko; Nagase, Takahide; Nagata, Taiji

    2008-06-06

    Voltage-gated Na(+) channel (I(Na)) encoded by SCN9A mRNA is expressed in cultured human bronchial smooth muscle cells. We investigated the effects of dexamethasone on I(Na), by using whole-cell voltage clamp techniques, reverse transcriptase/polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Acute application of dexamethasone (10(-6) M) did not affect I(Na). However, the percentage of the cells with I(Na) was significantly less in cells pretreated with dexamethasone for 48 h, and the current-density of I(Na) adjusted by cell capacitance in cells with I(Na) was also decreased in cells treated with dexamethasone. RT-PCR analysis showed that alpha and beta subunits mRNA of I(Na) mainly consisted of SCN9A and SCN1beta, respectively. Treatment with dexamethasone for 24-48 h inhibited the expression of SCN9A mRNA. The inhibitory effect of dexamethasone was concentration-dependent, and was observed at a concentration higher than 0.1 nM. The effect of dexamethasone on SCN9A mRNA was not blocked by spironolactone, but inhibited by mifepristone. The inhibitory effects of dexamethasone on SCN9A mRNA could not be explained by the changes of the stabilization of mRNA measured by using actinomycin D. These results suggest that dexamethasone inhibited I(Na) encoded by SCN9A mRNA in cultured human bronchial smooth muscle cells by inhibiting the transcription via the glucocorticoid receptor.

  4. Oxidized low density lipoprotein (LDL) affects hyaluronan synthesis in human aortic smooth muscle cells.

    PubMed

    Viola, Manuela; Bartolini, Barbara; Vigetti, Davide; Karousou, Evgenia; Moretto, Paola; Deleonibus, Sara; Sawamura, Tatsuya; Wight, Thomas N; Hascall, Vincent C; De Luca, Giancarlo; Passi, Alberto

    2013-10-11

    Thickening of the vessel in response to high low density lipoprotein(s) (LDL) levels is a hallmark of atherosclerosis, characterized by increased hyaluronan (HA) deposition in the neointima. Human native LDL trapped within the arterial wall undergoes modifications such as oxidation (oxLDL). The aim of our study is to elucidate the link between internalization of oxLDL and HA production in vitro, using human aortic smooth muscle cells. LDL were used at an effective protein concentration of 20-50 μg/ml, which allowed 80% cell viability. HA content in the medium of untreated cells was 28.9 ± 3.7 nmol HA-disaccharide/cell and increased after oxLDL treatment to 53.9 ± 5.6. OxLDL treatments doubled the transcripts of HA synthase HAS2 and HAS3. Accumulated HA stimulated migration of aortic smooth muscle cells and monocyte adhesiveness to extracellular matrix. The effects induced by oxLDL were inhibited by blocking LOX-1 scavenger receptor with a specific antibody (10 μg/ml). The cholesterol moiety of LDL has an important role in HA accumulation because cholesterol-free oxLDL failed to induce HA synthesis. Nevertheless, cholesterol-free oxLDL and unmodified cholesterol (20 μg/ml) induce only HAS3 transcription, whereas 22,oxysterol affects both HAS2 and HAS3. Moreover, HA deposition was associated with higher expression of endoplasmic reticulum stress markers (CHOP and GRP78). Our data suggest that HA synthesis can be induced in response to specific oxidized sterol-related species delivered through oxLDL.

  5. Effects of menthol on circular smooth muscle of human colon: analysis of the mechanism of action.

    PubMed

    Amato, Antonella; Liotta, Rosa; Mulè, Flavia

    2014-10-05

    Menthol is the major constituent of peppermint oil, an herbal preparation commonly used to treat nausea, spasms during colonoscopy and irritable bowel disease. The mechanism responsible for its spasmolytic action remains unclear. The aims of this study were to investigate the effects induced by menthol on the human distal colon mechanical activity in vitro and to analyze the mechanism of action. The spontaneous or evoked-contractions of the circular smooth muscle were recorded using vertical organ bath. Menthol (0.1 mM-30 mM) reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. The inhibitory effect was not affected by 5-benzyloxytryptamine (1 μM), a transient receptor potential-melastatin8 channel antagonist, or tetrodotoxin (1 μM), a neural blocker, or 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (10 µM), inhibitor of nitric oxide (NO)-sensitive soluble guanylyl cyclase, or tetraethylammonium (10 mM), a blocker of potassium (K+)-channels. On the contrary, nifedipine (3 nM), a voltage-activated L-type Ca2+ channel blocker, significantly reduced the inhibitory menthol actions. Menthol also reduced in a concentration-dependent manner the contractile responses caused by exogenous application of Ca2+ (75-375 μM) in a Ca2+-free solution, or induced by potassium chloride (KCl; 40 mM). Moreover menthol (1-3 mM) strongly reduced the electrical field stimulation (EFS)-evoked atropine-sensitive contractions and the carbachol-contractile responses. The present results suggest that menthol induces spasmolytic effects in human colon circular muscle inhibiting directly the gastrointestinal smooth muscle contractility, through the block of Ca2+ influx through sarcolemma L-type Ca2+ channels.

  6. Biomechanical effects of environmental and engineered particles on human airway smooth muscle cells.

    PubMed

    Berntsen, P; Park, C Y; Rothen-Rutishauser, B; Tsuda, A; Sager, T M; Molina, R M; Donaghey, T C; Alencar, A M; Kasahara, D I; Ericsson, T; Millet, E J; Swenson, J; Tschumperlin, D J; Butler, J P; Brain, J D; Fredberg, J J; Gehr, P; Zhou, E H

    2010-06-06

    The past decade has seen significant increases in combustion-generated ambient particles, which contain a nanosized fraction (less than 100 nm), and even greater increases have occurred in engineered nanoparticles (NPs) propelled by the booming nanotechnology industry. Although inhalation of these particulates has become a public health concern, human health effects and mechanisms of action for NPs are not well understood. Focusing on the human airway smooth muscle cell, here we show that the cellular mechanical function is altered by particulate exposure in a manner that is dependent upon particle material, size and dose. We used Alamar Blue assay to measure cell viability and optical magnetic twisting cytometry to measure cell stiffness and agonist-induced contractility. The eight particle species fell into four categories, based on their respective effect on cell viability and on mechanical function. Cell viability was impaired and cell contractility was decreased by (i) zinc oxide (40-100 nm and less than 44 microm) and copper(II) oxide (less than 50 nm); cell contractility was decreased by (ii) fluorescent polystyrene spheres (40 nm), increased by (iii) welding fumes and unchanged by (iv) diesel exhaust particles, titanium dioxide (25 nm) and copper(II) oxide (less than 5 microm), although in none of these cases was cell viability impaired. Treatment with hydrogen peroxide up to 500 microM did not alter viability or cell mechanics, suggesting that the particle effects are unlikely to be mediated by particle-generated reactive oxygen species. Our results highlight the susceptibility of cellular mechanical function to particulate exposures and suggest that direct exposure of the airway smooth muscle cells to particulates may initiate or aggravate respiratory diseases.

  7. Inhibition of delayed rectifier K(+)-current by levcromakalim in single intestinal smooth muscle cells: effects of cations and dependence on K(+)-flux.

    PubMed Central

    McHugh, D; Beech, D J

    1995-01-01

    1. Whole-cell voltage-clamp recordings were made from single smooth muscle cells isolated from the longitudinal layer of the guinea-pig small intestine. 2. Levcromakalim ((-)Ckm) inhibited delayed rectifier K-current (IK(DR)) and induced a voltage-independent K-current (IK(-Ckm)). Both effects were inhibited similarly by glibenclamide. In some cells, however, IK(-Ckm) could be induced without any effect on IK(DR). 3. Ba2+ caused a voltage-dependent block of IK(-Ckm). The IC50 was 0.2 mM at -40 mV (6 cells), but at 0 mV 2 mM Ba2+ caused only a 26 +/- 7% inhibition (n = 5). Ba2+ had much less effect on IK(DR), 2 mM Ba2+ having no inhibitory effect on current elicited by depolarization to -30 mV (n = 6) or 0 mV (n = 5). 4. Low concentrations of Zn2+ blocked IK(-Ckm) while having little effect on IK(DR). Zn2+ (40 microM) caused a 77 +/- 1% reduction of IK(-Ckm) at -30 mV (n = 4) but IK(DR) was inhibited by only 10 +/- 3% at the same voltage (n = 4). 5. Inward current amplitudes were compared in 135 mM Rb+ and 135 mM K+ bath solutions. (-)Ckm-activated Rb(+)-current was only 4% of the K(+)-current, whereas delayed rectifier Rb(+)-current was larger than K(+)-current. 6. (-)Ckm did not inhibit IK(DR) if IK(-Ckm) was blocked. In the presence of 2 mM Ba2+ or 135 mM Rb+, (-)Ckm did not induce current nor did it inhibit the delayed rectifier.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7881739

  8. 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.

  9. 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

  10. SIRT1 is required for mitochondrial biogenesis reprogramming in hypoxic human pulmonary arteriolar smooth muscle cells.

    PubMed

    Li, Pengyun; Liu, Yan; Burns, Nana; Zhao, Ke-Seng; Song, Rui

    2017-03-22

    Although recent studies have reported that mitochondria are putative oxygen sensors underlying hypoxic pulmonary vasoconstriction, little is known concerning the sirtuin 1 (SIRT1)-mediated mitochondrial biogenesis regulatory program in pulmonary arteriolar smooth muscle cells (PASMCs) during hypoxia/reoxygenation (H/R). We investigated the epigenetic regulatory mechanism of mitochondrial biogenesis and function in human PASMCs during H/R. Human PASMCs were exposed to hypoxia of 24-48 h and reoxygenation of 24-48 h. The expression of SIRT1 was reduced in a time-dependent manner. Mitochondrial transcription factor A (TFAM) expression was increased during hypoxia and decreased during reoxygenation, while the release of TFAM was increased in a time-dependent manner. Lentiviral overexpression of SIRT1 preserved SIRT3 deacetylase activity in human PASMCs exposed to H/R. Knockdown of PGC-1α suppressed the effect of SIRT1 on SIRT3 activity. Knockdown of SIRT3 abrogated SIRT1-mediated deacetylation of cyclophilin D (CyPD). Notably, knockdown of SIRT3 or PGC-1α suppressed the incremental effect of SIRT1 on mitochondrial TFAM, mitochondrial DNA (mtDNA) content and cellular ATP levels. Importantly, polydatin restored SIRT1 levels in human PASMCs exposed to H/R. Knockdown of SIRT1 suppressed the effect of polydatin on mitochondrial TFAM, mtDNA content and cellular ATP levels. In conclusion, SIRT1 expression is decreased in human PASMCs during H/R. TFAM expression in mitochondria is reduced and the release of TFAM is increased by H/R. PGC-1α/SIRT3/CyPD mediates the protective effect of SIRT1 on expression and release of TFAM and mitochondrial biogenesis and function. Polydatin improves mitochondrial biogenesis and function by enhancing SIRT1 expression in hypoxic human PASMCs.

  11. 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.

  12. 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.

  13. Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

    PubMed

    Eoh, Joon H; Shen, Nian; Burke, Jacqueline A; Hinderer, Svenja; Xia, Zhiyong; Schenke-Layland, Katja; Gerecht, Sharon

    2017-04-01

    Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications.

  14. Expression and function of K(V)2-containing channels in human urinary bladder smooth muscle.

    PubMed

    Hristov, Kiril L; Chen, Muyan; Afeli, Serge A Y; Cheng, Qiuping; Rovner, Eric S; Petkov, Georgi V

    2012-06-01

    The functional role of the voltage-gated K(+) (K(V)) channels in human detrusor smooth muscle (DSM) is largely unexplored. Here, we provide molecular, electrophysiological, and functional evidence for the expression of K(V)2.1, K(V)2.2, and the electrically silent K(V)9.3 subunits in human DSM. Stromatoxin-1 (ScTx1), a selective inhibitor of K(V)2.1, K(V)2.2, and K(V)4.2 homotetrameric channels and of K(V)2.1/9.3 heterotetrameric channels, was used to examine the role of these channels in human DSM function. Human DSM tissues were obtained during open bladder surgeries from patients without a history of overactive bladder. Freshly isolated human DSM cells were studied using RT-PCR, immunocytochemistry, live-cell Ca(2+) imaging, and the perforated whole cell patch-clamp technique. Isometric DSM tension recordings of human DSM isolated strips were conducted using tissue baths. RT-PCR experiments showed mRNA expression of K(V)2.1, K(V)2.2, and K(V)9.3 (but not K(V)4.2) channel subunits in human isolated DSM cells. K(V)2.1 and K(V)2.2 protein expression was confirmed by Western blot analysis and immunocytochemistry. Perforated whole cell patch-clamp experiments revealed that ScTx1 (100 nM) inhibited the amplitude of the voltage step-induced K(V) current in freshly isolated human DSM cells. ScTx1 (100 nM) significantly increased the intracellular Ca(2+) level in DSM cells. In human DSM isolated strips, ScTx1 (100 nM) increased the spontaneous phasic contraction amplitude and muscle force, and enhanced the amplitude of the electrical field stimulation-induced contractions within the range of 3.5-30 Hz stimulation frequencies. These findings reveal that ScTx1-sensitive K(V)2-containing channels are key regulators of human DSM excitability and contractility and may represent new targets for pharmacological or genetic intervention for bladder dysfunction.

  15. 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.

  16. Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells.

    PubMed Central

    Chen, J K; Hoshi, H; McKeehan, W L

    1987-01-01

    Myo-intimal proteoglycan metabolism is thought to be important in blood vessel homeostasis, blood clotting, atherogenesis, and atherosclerosis. Human platelet-derived transforming growth factor type beta (TGF-beta) specifically stimulated synthesis of at least two types of chondroitin sulfate proteoglycans in nonproliferating human adult arterial smooth muscle cells in culture. Stimulation of smooth muscle cell proteoglycan synthesis by smooth muscle cell growth promoters (epidermal growth factor, platelet-derived growth factor, and heparin-binding growth factors) was less than 20% of that elicited by TGF-beta. TGF-beta neither significantly stimulated proliferation of quiescent smooth muscle cells nor inhibited proliferating cells. The extent of TGF-beta stimulation of smooth muscle cell proteoglycan synthesis was similar in both nonproliferating and growth-stimulated cells. TGF-beta, which is a reversible inhibitor of endothelial cell proliferation, had no comparable effect on endothelial cell proteoglycan synthesis. These results are consistent with the hypothesis that TGF-beta is a cell-type-specific regulator of proteoglycan synthesis in human blood vessels and may contribute to the myo-intimal accumulation of proteoglycan in atherosclerotic lesions. Images PMID:3474655

  17. 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.

  18. Smoking and Female Sex: Independent Predictors of Human Vascular Smooth Muscle Cells Stiffening

    PubMed Central

    Dinardo, Carla Luana; Santos, Hadassa Campos; Vaquero, André Ramos; Martelini, André Ricardo; Dallan, Luis Alberto Oliveira; Alencar, Adriano Mesquita; Krieger, José Eduardo; Pereira, Alexandre Costa

    2015-01-01

    Aims Recent evidence shows the rigidity of vascular smooth muscle cells (VSMC) contributes to vascular mechanics. Arterial rigidity is an independent cardiovascular risk factor whose associated modifications in VSMC viscoelasticity have never been investigated. This study’s objective was to evaluate if the arterial rigidity risk factors aging, African ancestry, female sex, smoking and diabetes mellitus are associated with VMSC stiffening in an experimental model using a human derived vascular smooth muscle primary cell line repository. Methods Eighty patients subjected to coronary artery bypass surgery were enrolled. VSMCs were extracted from internal thoracic artery fragments and mechanically evaluated using Optical Magnetic Twisting Cytometry assay. The obtained mechanical variables were correlated with the clinical variables: age, gender, African ancestry, smoking and diabetes mellitus. Results The mechanical variables Gr, G’r and G”r had a normal distribution, demonstrating an inter-individual variability of VSMC viscoelasticity, which has never been reported before. Female sex and smoking were independently associated with VSMC stiffening: Gr (apparent cell stiffness) p = 0.022 and p = 0.018, R2 0.164; G’r (elastic modulus) p = 0.019 and p = 0.009, R2 0.184 and G”r (dissipative modulus) p = 0.011 and p = 0.66, R2 0.141. Conclusion Female sex and smoking are independent predictors of VSMC stiffening. This pro-rigidity effect represents an important element for understanding the vascular rigidity observed in post-menopausal females and smokers, as well as a potential therapeutic target to be explored in the future. There is a significant inter-individual variation of VSMC viscoelasticity, which is slightly modulated by clinical variables and probably relies on molecular factors. PMID:26661469

  19. 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

  20. 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.

  1. Nitric oxide production by cultured human aortic smooth muscle cells: stimulation by fluid flow

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Tilton, R. G.; Eskin, S. G.; McIntire, L. V.

    1998-01-01

    This study demonstrated that exposure of cultured human aortic smooth muscle cells (SMC) to fluid flow resulted in nitric oxide (NO) production, monitored by nitrite and guanosine 3',5'-cyclic monophosphate production. A rapid burst in nitrite production rate was followed by a more gradual increase throughout the period of flow exposure. Neither the initial burst nor the prolonged nitrite production was dependent on the level of shear stress in the range of 1.1-25 dyn/cm2. Repeated exposure to shear stress after a 30-min static period restimulated nitrite production similar to the initial burst. Ca(2+)-calmodulin antagonists blocked the initial burst in nitrite release. An inhibitor of nitric oxide synthase (NOS) blocked nitrite production, indicating that changes in nitrite reflect NO production. Treatment with dexamethasone or cycloheximide had no effect on nitrite production. Monoclonal antibodies directed against the inducible and endothelial NOS isoforms showed no immunoreactivity on Western blots, whereas monoclonal antibodies directed against the neuronal NOS gave specific products. These findings suggest that human aortic SMC express a constitutive neuronal NOS isoform, the enzymatic activity of which is modulated by flow.

  2. Dihydrotestosterone alters cyclooxygenase-2 levels in human coronary artery smooth muscle cells

    PubMed Central

    Osterlund, Kristen L.; Handa, Robert J.

    2010-01-01

    Both protective and nonprotective effects of androgens on the cardiovascular system have been reported. Our previous studies show that the potent androgen receptor (AR) agonist dihydrotestosterone (DHT) increases levels of the vascular inflammatory mediator cyclooxygenase (COX)-2 in rodent cerebral arteries independent of an inflammatory stimulus. Little is known about the effects of androgens on inflammation in human vascular tissues. Therefore, we tested the hypothesis that DHT alters COX-2 levels in the absence and presence of induced inflammation in primary human coronary artery smooth muscle cells (HCASMC). Furthermore, we tested the ancillary hypothesis that DHT's effects on COX-2 levels are AR-dependent. Cells were treated with DHT (10 nM) or vehicle for 6 h in the presence or absence of LPS or IL-1β. Similar to previous observations in rodent arteries, in HCASMC, DHT alone increased COX-2 levels compared with vehicle. This effect of DHT was attenuated in the presence of the AR antagonist bicalutamide. Conversely, in the presence of LPS or IL-1β, increases in COX-2 were attenuated by cotreatment with DHT. Bicalutamide did not affect this response, suggesting that DHT-induced decreases in COX-2 levels occur independent of AR stimulation. Thus we conclude that DHT differentially influences COX-2 levels under physiological and pathophysiological conditions in HCASMC. This effect of DHT on COX-2 involves AR-dependent and- independent mechanisms, depending on the physiological state of the cell. PMID:20103743

  3. Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model.

    PubMed

    Kusch, Angelika; Tkachuk, Sergey; Lutter, Steffen; Haller, Hermann; Dietz, Rainer; Lipp, Martin; Dumler, Inna

    2002-01-01

    Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinase-type plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral blood-derived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPA-induced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture five-fold, whereas VSMC display an increased expression of cell surface-associated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.

  4. Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression.

    PubMed

    Hojo, Y; Ikeda, U; Maeda, Y; Takahashi, M; Takizawa, T; Okada, M; Funayama, H; Shimada, K

    2000-05-01

    The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured. VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay. Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe. Immunohistochemistry was performed to determine which types of cell produce VEGF. Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively. Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h. Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF. Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production. Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs. A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell. An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures. Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.

  5. Monocyte prostaglandins inhibit procollagen secretion by human vascular smooth muscle cells: implications for plaque stability.

    PubMed

    Fitzsimmons, C; Proudfoot, D; Bowyer, D E

    1999-02-01

    Extracellular matrix remodelling occurs during atherosclerosis dictating the structure of the plaque and thus the resistance to rupture. Monocytes and macrophages are believed to play a role in this remodelling. In the present study, filter-separated co-culture has been used to study the effect of monocytes on procollagen turnover by human vascular smooth muscle cells (VSMC). In this system, freshly isolated human peripheral blood monocytes inhibited procollagen secretion from VSMC without affecting either degradation of procollagen, or DNA synthesis by the VSMC. Insertion of a 12 kDa dialysis membrane between the two cell types and treatment with indomethacin showed that the inhibitory factor was of low molecular weight and was cyclooxygenase-dependent. Pre-incubation of each cell type with indomethacin demonstrated that monocyte, but not VSMC cyclooxygenase was required. Thus, the inhibitory effect on procollagen secretion was due, most likely, to monocyte prostaglandins. Neither inhibition of thromboxane synthetase, nor blocking IL-1 activity, reduced the inhibitory activity. Addition of prostaglandins PGE1, PGE2 and PGF2alpha to VSMC cultures caused a reduction in procollagen secretion which was equivalent to, but was not additive with, the maximal effect achieved by monocytes. Monocytes and macrophages are a major source of prostaglandins and these molecules are likely to play an important role in collagen turnover within lesions.

  6. Olfactory Receptors Modulate Physiological Processes in Human Airway Smooth Muscle Cells

    PubMed Central

    Kalbe, Benjamin; Knobloch, Jürgen; Schulz, Viola M.; Wecker, Christine; Schlimm, Marian; Scholz, Paul; Jansen, Fabian; Stoelben, Erich; Philippou, Stathis; Hecker, Erich; Lübbert, Hermann; Koch, Andrea; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    Pathophysiological mechanisms in human airway smooth muscle cells (HASMCs) significantly contribute to the progression of chronic inflammatory airway diseases with limited therapeutic options, such as severe asthma and COPD. These abnormalities include the contractility and hyperproduction of inflammatory proteins. To develop therapeutic strategies, key pathological mechanisms, and putative clinical targets need to be identified. In the present study, we demonstrated that the human olfactory receptors (ORs) OR1D2 and OR2AG1 are expressed at the RNA and protein levels in HASMCs. Using fluorometric calcium imaging, specific agonists for OR2AG1 and OR1D2 were identified to trigger transient Ca2+ increases in HASMCs via a cAMP-dependent signal transduction cascade. Furthermore, the activation of OR2AG1 via amyl butyrate inhibited the histamine-induced contraction of HASMCs, whereas the stimulation of OR1D2 with bourgeonal led to an increase in cell contractility. In addition, OR1D2 activation induced the secretion of IL-8 and GM-CSF. Both effects were inhibited by the specific OR1D2 antagonist undecanal. We herein provide the first evidence to show that ORs are functionally expressed in HASMCs and regulate pathophysiological processes. Therefore, ORs might be new therapeutic targets for these diseases, and blocking ORs could be an auspicious strategy for the treatment of early-stage chronic inflammatory lung diseases. PMID:27540365

  7. Glycosylated human oxyhaemoglobin activates nuclear factor-κB and activator protein-1 in cultured human aortic smooth muscle

    PubMed Central

    Peiró, Concepción; Matesanz, Nuria; Nevado, Julián; Lafuente, Nuria; Cercas, Elena; Azcutia, Verónica; Vallejo, Susana; Rodríguez-Mañas, Leocadio; Sánchez-Ferrer, Carlos F

    2003-01-01

    Diabetic vessels undergo structural changes that are linked to a high incidence of cardiovascular diseases. Reactive oxygen species (ROS) mediate cell signalling in the vasculature, where they can promote cell growth and activate redox-regulated transcription factors, like activator protein-1 (AP-1) or nuclear factor-κB (NF-κB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes. In this study, we analysed whether Amadori-modified human oxyhaemoglobin, glycosylated at either normal (N-Hb) or elevated (E-Hb) levels, can induce cell growth and activate AP-1 and NF-κB in cultured human aortic smooth muscle cells (HASMC). E-Hb (1 nM–1 μM), but not N-Hb, promoted a concentration-dependent increase in cell size from nanomolar concentrations, although it failed to stimulate HASMC proliferation. At 10 nM, E-Hb stimulated both AP-1 and NF-κB activity, as assessed by transient transfection, electromobility shift assays or immunofluorescence staining. The effects of E-Hb resembled those of the proinflammatory cytokine tumour necrosis factor-α (TNF-α). E-Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers. In conclusion, E-Hb stimulates growth and activates AP-1 and NF-κB in human vascular smooth muscle by redox-sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy. PMID:14504138

  8. TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle.

    PubMed

    Vohra, Pawan K; Thompson, Michael A; Sathish, Venkatachalem; Kiel, Alexander; Jerde, Calvin; Pabelick, Christina M; Singh, Brij B; Prakash, Y S

    2013-12-01

    Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca(2+) signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca(2+) entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP-BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca(2+) (EGTA; 1mM) or intracellular Ca(2+) (BAPTA; 5μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca(2+) influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca(2+) and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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.

  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. Tumor necrosis factor gene expression in human vascular intimal smooth muscle cells detected by in situ hybridization.

    PubMed Central

    Barath, P.; Fishbein, M. C.; Cao, J.; Berenson, J.; Helfant, R. H.; Forrester, J. S.

    1990-01-01

    We used immunohistochemistry to detect tumor necrosis factor (TNF) and in situ hybridization to detect TNF messenger RNA (mRNA) in the intimal mesenchymal-appearing cells and in the medial smooth muscle cells of human atherosclerotic arteries. Medial smooth muscle cells showed localization of immunoreactive TNF on the cell surface and did not express TNF mRNA. Conversely, in intimal mesenchymal-appearing cells, TNF was localized in the cytoplasm and TNF mRNA was expressed by in situ hybridization. Thus 89% of intimal cells were immunohistochemically positive for TNF, 96% of them were positive by in situ hybridization, and 76% were positive for the smooth muscle cell marker, HHF35. Our results suggest that intimal mesenchymal-appearing cells are mostly, but not exclusively, derived from smooth muscle cells. These cells express TNF, whereas the medial smooth muscle cells in the atherosclerotic human arteries do not. The expression of TNF by these mesenchymal-appearing cells may have implications regarding the evolution of the atherosclerotic plaque. Images Figure 1 to Figure 4 Figure 3 PMID:1698022

  16. 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.

  17. 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

  18. 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).

  19. Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes.

    PubMed

    Kranzhöfer, R; Clinton, S K; Ishii, K; Coughlin, S R; Fenton, J W; Libby, P

    1996-08-01

    Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human alpha-thrombin (EC50, approximately equal to 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, alpha-thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only alpha-thrombin concentrations of > or = 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor-alpha (TNF alpha) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only alpha-thrombin concentrations of > or = 100 nmol/L increased levels of IL-6, IL-1 beta, MCP-1, or TNF alpha mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca2+]i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.

  20. Ca2+ inhibition of inositol trisphosphate-induced Ca2+ release in single smooth muscle cells of guinea-pig small intestine.

    PubMed Central

    Zholos, A V; Komori, S; Ohashi, H; Bolton, T B

    1994-01-01

    1. Single smooth muscle cells from the longitudinal muscle layer of guinea-pig small intestine were voltage clamped using patch pipettes in the whole-cell mode. 2. When D-myo-inositol 1,4,5-trisphosphate (InsP3) was released at intervals, by photolysis of 'caged' InsP3 within the cell, increases in [Ca2+]i in many cells, as judged from Ca(2+)-activated K(+)-current, were all-or-none; release of InsP3 before a critical interval had elapsed, which was quite stable for an individual cell, resulted in no response. After Ca(2+)-induced Ca2+ release had been evoked by depolarization, the InsP3 response was inhibited. Oscillations in [Ca2+]i evoked by muscarinic receptor activation were unaffected by Ruthenium Red; during these oscillations exogenous InsP3 was not effective close to, or shortly after, peak [Ca2+]i but was effective at other times. 3. Reproducible release of Ca2+ and elevation of [Ca2+]i could be produced by brief (up to 0.5 s) pressure applications of 10 mM caffeine at intervals of 10 s or greater but caffeine itself rarely evoked oscillations in [Ca2+]i. Responses to flash release of InsP3 were reduced after caffeine-induced responses and recovery of caffeine-induced Ca2+ release was faster than recovery of InsP3-induced Ca2+ release. 4. The results support the idea that InsP3-induced Ca(2+)-store release can be inhibited by a certain level of [Ca2+]i at a time when Ca2+ stores have refilled and can be released by caffeine; they also support the suggestion that during oscillations of [Ca2+]i evoked by muscarinic receptor activation, Ca2+ inhibition of InsP3-induced Ca2+ release at some critical level of [Ca2+]i allows Ca2+ stores to refill and leads to a fall in [Ca2+]i so contributing to the oscillations which are observed. PMID:7531770

  1. 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.

  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. Spatial and temporal traction response in human airway smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Tolic-Norrelykke, Iva Marija; Butler, James P.; Chen, Jianxin; Wang, Ning

    2002-01-01

    Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.

  6. Vitamin D attenuates cytokine-induced remodeling in human fetal airway smooth muscle cells.

    PubMed

    Britt, Rodney D; Faksh, Arij; Vogel, Elizabeth R; Thompson, Michael A; Chu, Vivian; Pandya, Hitesh C; Amrani, Yassine; Martin, Richard J; Pabelick, Christina M; Prakash, Y S

    2015-06-01

    Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25-dihydroxyvitamin D3 ; 1,25(OH)2 D3 ), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro-inflammatory cytokines. Cells were pre-treated with calcitriol and then exposed to TNFα or TGFβ for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP-9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFβ-induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFβ in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood.

  7. On the Visual Input Driving Human Smooth-Pursuit Eye Movements

    NASA Technical Reports Server (NTRS)

    Stone, Leland S.; Beutter, Brent R.; Lorenceau, Jean

    1996-01-01

    Current computational models of smooth-pursuit eye movements assume that the primary visual input is local retinal-image motion (often referred to as retinal slip). However, we show that humans can pursue object motion with considerable accuracy, even in the presence of conflicting local image motion. This finding indicates that the visual cortical area(s) controlling pursuit must be able to perform a spatio-temporal integration of local image motion into a signal related to object motion. We also provide evidence that the object-motion signal that drives pursuit is related to the signal that supports perception. We conclude that current models of pursuit should be modified to include a visual input that encodes perceived object motion and not merely retinal image motion. Finally, our findings suggest that the measurement of eye movements can be used to monitor visual perception, with particular value in applied settings as this non-intrusive approach would not require interrupting ongoing work or training.

  8. Effects of cigarette smoke extract on human airway smooth muscle cells in COPD.

    PubMed

    Chen, Ling; Ge, Qi; Tjin, Gavin; Alkhouri, Hatem; Deng, Linghong; Brandsma, Corry-Anke; Adcock, Ian; Timens, Wim; Postma, Dirkje; Burgess, Janette K; Black, Judith L; Oliver, Brian G G

    2014-09-01

    We hypothesised that the response to cigarette smoke in airway smooth muscle (ASM) cells from smokers with chronic obstructive pulmonary disease (COPD) would be intrinsically different from smokers without COPD, producing greater pro-inflammatory mediators and factors relating to airway remodelling. ASM cells were obtained from smokers with or without COPD, and then stimulated with cigarette smoke extract (CSE) or transforming growth factor-β1. The production of chemokines and matrix metalloproteinases (MMPs) were measured by ELISA, and the deposition of collagens by extracellular matrix ELISA. The effects of CSE on cell attachment and wound healing were measured by toluidine blue attachment and cell tracker green wound healing assays. CSE increased the release of CXCL8 and CXCL1 from human ASM cells, and cells from smokers with COPD produced more CSE-induced CXCL1. The production of MMP-1, -3 and -10, and the deposition of collagen VIII alpha 1 (COL8A1) were increased by CSE, especially in the COPD group which had higher production of MMP-1 and deposition of COL8A1. CSE decreased ASM cell attachment and wound healing in the COPD group only. ASM cells from smokers with COPD were more sensitive to CSE stimulation, which may explain, in part, why some smokers develop COPD.

  9. Coherent anti-Stokes Raman scattering microscopy of human smooth muscle cells in bioengineered tissue scaffolds

    NASA Astrophysics Data System (ADS)

    Brackmann, Christian; Esguerra, Maricris; Olausson, Daniel; Delbro, Dick; Krettek, Alexandra; Gatenholm, Paul; Enejder, Annika

    2011-02-01

    The integration of living, human smooth muscle cells in biosynthesized cellulose scaffolds was monitored by nonlinear microscopy toward contractile artificial blood vessels. Combined coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy was applied for studies of the cell interaction with the biopolymer network. CARS microscopy probing CH2-groups at 2845 cm-1 permitted three-dimensional imaging of the cells with high contrast for lipid-rich intracellular structures. SHG microscopy visualized the fibers of the cellulose scaffold, together with a small signal obtained from the cytoplasmic myosin of the muscle cells. From the overlay images we conclude a close interaction between cells and cellulose fibers. We followed the cell migration into the three-dimensional structure, illustrating that while the cells submerge into the scaffold they extrude filopodia on top of the surface. A comparison between compact and porous scaffolds reveals a migration depth of <10 μm for the former, whereas the porous type shows cells further submerged into the cellulose. Thus, the scaffold architecture determines the degree of cell integration. We conclude that the unique ability of nonlinear microscopy to visualize the three-dimensional composition of living, soft matter makes it an ideal instrument within tissue engineering.

  10. Cigarette smoke-induced mitochondrial fragmentation and dysfunction in human airway smooth muscle

    PubMed Central

    Aravamudan, Bharathi; Kiel, Alexander; Freeman, Michelle; Delmotte, Philippe; Thompson, Michael; Vassallo, Robert; Sieck, Gary C.; Pabelick, Christina M.

    2014-01-01

    The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases. PMID:24610934

  11. Transcriptional profiling of human smooth muscle cells infected with gingipain and fimbriae mutants of Porphyromonas gingivalis

    PubMed Central

    Zhang, Boxi; Sirsjö, Allan; Khalaf, Hazem; Bengtsson, Torbjörn

    2016-01-01

    Porphyromonas gingivalis (P. gingivalis) is considered to be involved in the development of atherosclerosis. However, the role of different virulence factors produced by P. gingivalis in this process is still uncertain. The aim of this study was to investigate the transcriptional profiling of human aortic smooth muscle cells (AoSMCs) infected with wild type, gingipain mutants or fimbriae mutants of P. gingivalis. AoSMCs were exposed to wild type (W50 and 381), gingipain mutants (E8 and K1A), or fimbriae mutants (DPG-3 and KRX-178) of P. gingivalis. We observed that wild type P. gingivalis changes the expression of a considerable larger number of genes in AoSMCs compare to gingipain and fimbriae mutants, respectively. The results from pathway analysis revealed that the common differentially expressed genes for AoSMCs infected by 3 different wild type P. gingivalis strains were enriched in pathways of cancer, cytokine-cytokine receptor interaction, regulation of the actin cytoskeleton, focal adhesion, and MAPK signaling pathway. Disease ontology analysis showed that various strains of P. gingivalis were associated with different disease profilings. Our results suggest that gingipains and fimbriae, especially arginine-specific gingipain, produced by P. gingivalis play important roles in the association between periodontitis and other inflammatory diseases, including atherosclerosis. PMID:26907358

  12. Human discrimination of visual direction of motion with and without smooth pursuit eye movements

    NASA Technical Reports Server (NTRS)

    Krukowski, Anton E.; Pirog, Kathleen A.; Beutter, Brent R.; Brooks, Kevin R.; Stone, Leland S.

    2003-01-01

    It has long been known that ocular pursuit of a moving target has a major influence on its perceived speed (Aubert, 1886; Fleischl, 1882). However, little is known about the effect of smooth pursuit on the perception of target direction. Here we compare the precision of human visual-direction judgments under two oculomotor conditions (pursuit vs. fixation). We also examine the impact of stimulus duration (200 ms vs. 800 ms) and absolute direction (cardinal vs. oblique). Our main finding is that direction discrimination thresholds in the fixation and pursuit conditions are indistinguishable. Furthermore, the two oculomotor conditions showed oblique effects of similar magnitudes. These data suggest that the neural direction signals supporting perception are the same with or without pursuit, despite remarkably different retinal stimulation. During fixation, the stimulus information is restricted to large, purely peripheral retinal motion, while during steady-state pursuit, the stimulus information consists of small, unreliable foveal retinal motion and a large efference-copy signal. A parsimonious explanation of our findings is that the signal limiting the precision of direction judgments is a neural estimate of target motion in head-centered (or world-centered) coordinates (i.e., a combined retinal and eye motion signal) as found in the medial superior temporal area (MST), and not simply an estimate of retinal motion as found in the middle temporal area (MT).

  13. Inhibition of human arterial smooth muscle cell growth by human monocyte/macrophages: a co-culture study.

    PubMed

    Proudfoot, D; Fitzsimmons, C; Torzewski, J; Bowyer, D E

    1999-07-01

    Monocyte/macrophages produce a variety of substances which may influence the function of smooth muscle cells (SMC). During atherogenesis, macrophages are thought to modulate SMC migration, proliferation and synthesis of extracellular matrix. Such modulation is the balance between stimulatory and inhibitory influences. Thus, for example, our earlier studies have shown that macrophages not only secrete mitogens, but also produce small molecular weight inhibitors of SMC proliferation. In the present study, we have used a co-culture system in which human monocyte/macrophages were separated from human arterial SMC (hSMC) by a filter with the optional addition of a 12 kDa cut-off dialysis membrane, in order to assess their effect on hSMC growth. We have found that human peripheral blood-derived monocytes produced a substance of < 12 kDa that inhibited hSMC growth in the co-culture system. The monocyte-derived factor causing this effect was completely blocked by indomethacin, indicating that growth-inhibitory factors produced by the monocytes were cyclooxygenase products. We have shown that PGE1 and PGE2 inhibit hSMC growth, making them likely candidates for the effector molecules released from monocytes in our co-culture system.

  14. The human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor

    PubMed Central

    Ulrich, Craig; Quilici, David R.; Schlauch, Karen A.

    2013-01-01

    Molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor at term are incompletely known. More than 10% of babies born worldwide are premature and 1,000,000 die annually. Preterm labor results in preterm delivery in 50% of cases in the United States explaining 75% of fetal morbidity and mortality. There is no Food and Drug Administration-approved treatment to prevent preterm delivery. Nitric oxide-mediated relaxation of human uterine smooth muscle is independent of global elevation of cGMP following activation of soluble guanylyl cyclase. S-nitrosation is a likely mechanism to explain cGMP-independent relaxation to nitric oxide and may reveal S-nitrosated proteins as new therapeutic targets for the treatment of preterm labor. Employing S-nitrosoglutathione as an nitric oxide donor, we identified 110 proteins that are S-nitrosated in 1 or more states of human pregnancy. Using area under the curve of extracted ion chromatograms as well as normalized spectral counts to quantify relative expression levels for 62 of these proteins, we show that 26 proteins demonstrate statistically significant S-nitrosation differences in myometrium from spontaneously laboring preterm patients compared with nonlaboring patients. We identified proteins that were up-S-nitrosated as well as proteins that were down-S-nitrosated in preterm laboring tissues. Identification and relative quantification of the S-nitrosoproteome provide a fingerprint of proteins that can form the basis of hypothesis-directed efforts to understand the regulation of uterine contraction-relaxation and the development of new treatment for preterm labor. PMID:23948706

  15. BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells

    PubMed Central

    Wakle-Prabagaran, Monali; Lorca, Ramón A.; Ma, Xiaofeng; Stamnes, Susan J.; Amazu, Chinwendu; Hsiao, Jordy J.; Hyrc, Krzysztof L.; Wright, Michael E.; England, Sarah K.

    2016-01-01

    The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) plays an important role in regulating Ca2+ signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCa in myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCa and both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCa in an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca2+ to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca2+-free medium and inhibitors of various Ca2+ signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca2+ through store-operated Ca2+ channels. Finally, we found that the specific BKCa blocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCa channel in human myometrium and that BKCa and its immunomodulatory interacting partners regulate Ca2+ dynamics in hMSMCs during pregnancy. PMID:27044074

  16. Human Embryonic Stem Cell Derived Vascular Progenitor Cells Capable of Endothelial and Smooth Muscle Cell Function

    PubMed Central

    Hill, Katherine L; Obrtlikova, Petra; Alvarez, Diego F; King, Judy A; Keirstead, Susan A; Allred, Jeremy R; Kaufman, Dan S

    2010-01-01

    OBJECTIVE Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34+ cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization. MATERIALS AND METHODS hESCs are co-cultured on M2-10B4 stromal cells or Wnt1 expressing M2-10B4 for 13–15 days to generate a CD34+ cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24–48 hours. RESULTS CD34+ vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when co-cultured in Matrigel. Human umbilical vein endothelial cells (HUVEC) cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1 overexpressing stromal cells produced an increased number of progenitor cells. CONCLUSIONS The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The step-wise differentiation outlined here is an efficient, reproducible method with potential for large scale cultures suitable for clinical applications. PMID:20067819

  17. Heterogeneity of smooth muscle cells in atheromatous plaque of human aorta.

    PubMed Central

    Babaev, V. R.; Bobryshev, Y. V.; Stenina, O. V.; Tararak, E. M.; Gabbiani, G.

    1990-01-01

    This study was undertaken to investigate the expression of cytoskeletal proteins and the ultrastructure of cells in normal intima and atheromatous plaque of human aorta. It has been established, using double-labeling immunofluorescence, that smooth muscle cells (SMC) in normal aortic intima contain myosin, vimentin, and alpha-actin but do not react with antibodies against desmin. In contrast, 7 of 28 atherosclerotic plaques contained many cells expressing desmin in addition to the other cytoskeletal proteins characteristic of normal intima SMC. These cells were localized predominantly in the plaque cap and had the ultrastructural features of modulated SMC, ie, well-developed endoplasmic reticulum and Golgi apparatus. Besides, some cells in the 13 atherosclerotic plaques proved to be myosin, alpha actin, and desmin negative but contained vimentin and actin as revealed by fluorescent phalloidin. These cells were found in the immediate proximity of atheromatous material and reacted with a monoclonal antibody specific to SMC surface protein (11G10) but not with monoclonal anti-muscle actin (HHF35) and anti-macrophage (HAM56) antibodies. Electron microscopy of this plaque zone revealed that the cytoplasm of these cells was filled with rough endoplasmic reticulum and a developed Golgi complex. At the same time, a certain proportion of cells in this region retained morphologic features of differentiated SMC such as the presence of a basal lamina and myofilament bundles. The revealed peculiarities of cytoskeletal protein expression and the ultrastructure of cells in human aortic atherosclerotic plaques may be explained by a phenotypic modulation of vascular SMC. Images Figure 4 Figure 1 Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 PMID:2190471

  18. 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

  19. 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

  20. 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

  1. Lipoprotein(a) Promotes Smooth Muscle Cell Proliferation and Dedifferentiation in Atherosclerotic Lesions of Human Apo(a) Transgenic Rabbits

    PubMed Central

    Ichikawa, Tomonaga; Unoki, Hiroyuki; Sun, Huijun; Shimoyamada, Hiroaki; Marcovina, Santica; Shikama, Hisataka; Watanabe, Teruo; Fan, Jianglin

    2002-01-01

    Elevated plasma lipoprotein(a) [Lp(a)] levels constitute an independent risk factor for the development of atherosclerosis. However, the mechanism underlying Lp(a) atherogenicity is unclear. Recently, we demonstrated that Lp(a) may potentially be proatherogenic in transgenic rabbits expressing human apolipoprotein(a) [apo(a)]. In this study, we further investigated atherosclerotic lesions of transgenic rabbits by morphometry and immunohistochemistry. On a cholesterol diet, human apo(a) transgenic rabbits had more extensive atherosclerotic lesions of the aorta, carotid artery, iliac artery, and coronary artery than did nontransgenic littermate rabbits as defined by increased intimal lesion area. Enhanced lesion development in transgenic rabbits was characterized by increased accumulation of smooth muscle cells, that was often associated with the Lp(a) deposition. To explore the possibility that Lp(a) may be involved in the smooth-muscle cell phenotypic modulation, we stained the lesions using a panel of monoclonal antibodies against smooth-muscle myosin heavy-chain isoforms (SM1, SM2, and SMemb) and basic transcriptional element binding protein-2 (BTEB2). We found that a large number of smooth muscle cells located in the apo(a)-containing areas of transgenic rabbits were positive for SMemb and BTEB2, suggesting that these smooth muscle cells were either immature or in the state of activation. In addition, transgenic rabbits showed delayed fibrinolytic activity accompanied by increased plasma plasminogen activator inhibitor-1. We conclude that Lp(a) may enhance the lesion development by mediating smooth muscle cell proliferation and dedifferentiation possibly because of impaired fibrinolytic activity. PMID:11786416

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. TREK-1 currents in smooth muscle cells from pregnant human myometrium.

    PubMed

    Heyman, Nathanael S; Cowles, Chad L; Barnett, Scott D; Wu, Yi-Ying; Cullison, Charles; Singer, Cherie A; Leblanc, Normand; Buxton, Iain L O

    2013-09-15

    The mechanisms governing maintenance of quiescence during pregnancy remain largely unknown. The current study characterizes a stretch-activated, tetraethylammonium-insensitive K(+) current in smooth muscle cells isolated from pregnant human myometrium. This study hypothesizes that these K(+) currents can be attributed to TREK-1 and that upregulation of this channel during pregnancy assists with the maintenance of a negative cell membrane potential, conceivably contributing to uterine quiescence until full term. The results of this study demonstrate that, in pregnant human myometrial cells, outward currents at 80 mV increased from 4.8 ± 1.5 to 19.4 ± 7.5 pA/pF and from 3.0 ± 0.8 to 11.8 ± 2.7 pA/pF with application of arachidonic acid (AA) and NaHCO3, respectively, causing intracellular acidification. Similarly, outward currents were inhibited following application of 10 μM fluphenazine by 51.2 ± 9.8% after activation by AA and by 73.9 ± 4.2% after activation by NaHCO3. In human embryonic kidney (HEK-293) cells stably expressing TREK-1, outward currents at 80 mV increased from 91.0 ± 23.8 to 247.5 ± 73.3 pA/pF and from 34.8 ± 8.9 to 218.6 ± 45.0 pA/pF with application of AA and NaHCO3, respectively. Correspondingly, outward currents were inhibited 89.5 ± 2.3% by 10 μM fluphenazine following activation by AA and by 91.6 ± 3.4% following activation by NaHCO3. Moreover, currents in human myometrial cells were activated by stretch and were reduced by transfection with small interfering RNA or extracellular acidification. Understanding gestational regulation of expression and gating of TREK-1 channels could be important in determining appropriate maintenance of uterine quiescence during pregnancy.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells

    PubMed Central

    Damián-Zamacona, Salvador; Toledo-Ibelles, Paola; Ibarra-Abundis, Mabel Z.; Uribe-Figueroa, Laura; Hernández-Lemus, Enrique; Macedo-Alcibia, Karla Paola; Delgado–Coello, Blanca; Mas-Oliva, Jaime; Reyes-Grajeda, Juan Pablo

    2016-01-01

    Background Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved. Objective The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC) stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively), with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis. Approach and Results Total RNA was isolated with TRIzol reagent (Life Technologies) and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL) was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix) designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (P< 0.05), and 8 genes validated by qPCR using Taqman probes. Conclusions 10 molecular processes were significantly affected in hVSMC: Apoptosis and cell cycle, extracellular matrix remodeling, DNA repair, cholesterol efflux, cGMP biosynthesis, endocytic mechanisms, calcium homeostasis, redox balance, membrane trafficking and finally, the immune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and

  15. Pharmacological evidence for a novel cysteinyl-leukotriene receptor subtype in human pulmonary artery smooth muscle

    PubMed Central

    Walch, Laurence; Norel, Xavier; Bäck, Magnus; Gascard, Jean-Pierre; Dahlén, Sven-Erik; Brink, Charles

    2002-01-01

    To characterize the cysteinyl-leukotriene receptors (CysLT receptors) in isolated human pulmonary arteries, ring preparations were contracted with leukotriene C4 (LTC4) and leukotriene D4 (LTD4) in either the absence or presence of the selective CysLT1 receptor antagonists, ICI 198615, MK 571 or the dual CysLT1/CysLT2 receptor antagonist, BAY u9773. Since the contractions induced by the cysteinyl-leukotrienes (cysLTs) in intact preparations failed to attain a plateau response over the concentration range studied, the endothelium was removed and the tissue treated continuously with indomethacin (Rubbed+INDO). In these latter preparations, the pEC50 for LTC4 and LTD4 were not significantly different (7.61±0.07, n=20 and 7.96±0.09, n=22, respectively). However, the LTC4 and LTD4 contractions were markedly potentiated when compared with data from intact tissues. Leukotriene E4 (LTE4) did not contract human isolated pulmonary arterial preparations. In addition, treatment of preparations with LTE4 (1 μM; 30 min) did not modify either the LTC4 or LTD4 contractions. Treatment of preparations with the S-conjugated glutathione (S-hexyl-GSH; 100 μM, 30 min), an inhibitor of the metabolism of LTC4 to LTD4, did not modify LTC4 contractions. The pEC50 values for LTC4 were significantly reduced by treatment of the preparations with either ICI 198615, MK 571 or BAY u9773 and the pKB values were: 7.20, 7.02 and 6.26, respectively. In contrast, these antagonists did not modify the LTD4 pEC50 values. These findings suggest the presence of two CysLT receptors on human pulmonary arterial vascular smooth muscle. A CysLT1 receptor with a low affinity for CysLT1 antagonists and a novel CysLT receptor subtype, both responsible for vasoconstriction. Activation of this latter receptor by LTC4 and LTD4 induced a contractile response which was resistant to the selective CysLT1 antagonists (ICI 198615 and MK 571) as well as the non-selective (CysLT1/CysLT2) antagonist, BAY u9773. PMID

  16. Expression of myosin isoforms in the smooth muscle of human corpus cavernosum.

    PubMed

    Koi, P T; Milhoua, P M; Monrose, V; Melman, A; DiSanto, M E

    2007-01-01

    The molecular interaction between smooth muscle (SM) myosin and actin in the corpus cavernosum (CC) determines the erectile state of the penis. A key mechanism regulating this interaction and subsequent development and maintenance of force is alternative splicing of SM myosin heavy chain (MHC) and 17 kDa essential SM myosin light chain (MLC) pre-mRNAs. Our aim was to examine the relative SM myosin isoform composition in human CC. Tissue samples were obtained from 18 patients with erectile dysfunction (ED), Peyronie's disease, or both. One specimen was obtained during a transgender operation. Patients then were stratified according to presence of diabetes mellitus, hypertension, ED, or Peyronie's disease, as well as failure of phosphodiesterase-5 (PDE5) inhibitors and history of previous pelvic or penile surgeries, radiation, or both. Our results revealed that all human CC samples expressed only the SM-A isoform. There was a predominance of SM2 isoform mRNA relative to SM1 across all samples, with a mean of 63.8%, which correlated with protein analysis by gel electrophoresis. A statistically significant difference was found between patients who had undergone previous pelvic surgery, radiation, or both and those who did not. The ratio of LC(17b) to LC(17a) was approximately 1:1 for all patients, with a mean of 48.9% LC(17b). Statistical difference was seen in the relative ratio of LC(17b) to LC(17a) among the group who failed conservative therapy with PDE5 inhibitors compared with all others. In conclusion, we determined the SM myosin isoform composition of human CC and present for the first time differences in relative myosin isoform expression among patients with several risk factors contributing to their cause of ED. Our data reflect the fact that alternative splicing events in the MHC and 17 kDa MLC pre-mRNA may be a possible molecular mechanism involved in the altered contractility of the CCSM in patients with ED.

  17. 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.

  18. Calcium Phosphate Crystals from Uremic Serum Promote Osteogenic Differentiation in Human Aortic Smooth Muscle Cells.

    PubMed

    Liu, Yaorong; Zhang, Lin; Ni, Zhaohui; Qian, Jiaqi; Fang, Wei

    2016-11-01

    Recent study demonstrated that calcium phosphate (CaP) crystals isolated from high phosphate medium were a key contributor to arterial calcification. The present study further investigated the effects of CaP crystals induced by uremic serum on calcification of human aortic smooth muscle cells. This may provide a new insight for the development of uremic cardiovascular calcification. We tested the effects of uremic serum or normal serum on cell calcification. Calcification was visualized by staining and calcium deposition quantified. Expression of various bone-calcifying genes was detected by real-time PCR, and protein levels were quantified by western blotting or enzyme-linked immunosorbent assays. Pyrophosphate was used to investigate the effects of CaP crystals' inhibition. Finally, CaP crystals were separated from uremic serum to determine its specific pro-calcification effects. Uremic serum incubation resulted in progressively increased calcification staining and increased calcium deposition in HASMCs after 4, 8 and 12 days (P vs 0 day <0.001 for all). Compared to cells incubated in control serum, uremic serum significantly induced the mRNA expression of bone morphogenetic factor-2, osteopontin and RUNX2, and increased their protein levels as well (P < 0.05 for all). Inhibition of CaP crystals with pyrophosphate incubation prevented calcium deposition and bone-calcifying gene over-expression increased by uremic serum. CaP crystals, rather than the rest of uremic serum, were responsible for these effects. Uremic serum accelerates arterial calcification by mediating osteogenic differentiation. This effect might be mainly attributed to the CaP crystal content.

  19. Soluble guanylate cyclase modulators blunt hyperoxia effects on calcium responses of developing human airway smooth muscle.

    PubMed

    Britt, Rodney D; Thompson, Michael A; Kuipers, Ine; Stewart, Alecia; Vogel, Elizabeth R; Thu, James; Martin, Richard J; Pabelick, Christina M; Prakash, Y S

    2015-09-15

    Exposure to moderate hyperoxia in prematurity contributes to subsequent airway dysfunction and increases the risk of developing recurrent wheeze and asthma. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) axis modulates airway tone by regulating airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)]i) and contractility. However, the effects of hyperoxia on this axis in the context of Ca(2+)/contractility are not known. In developing human ASM, we explored the effects of novel drugs that activate sGC independent of NO on alleviating hyperoxia (50% oxygen)-induced enhancement of Ca(2+) responses to bronchoconstrictor agonists. Treatment with BAY 41-2272 (sGC stimulator) and BAY 60-2770 (sGC activator) increased cGMP levels during exposure to 50% O2. Although 50% O2 did not alter sGCα1 or sGCβ1 expression, BAY 60-2770 did increase sGCβ1 expression. BAY 41-2272 and BAY 60-2770 blunted Ca(2+) responses to histamine in cells exposed to 50% O2. The effects of BAY 41-2272 and BAY 60-2770 were reversed by protein kinase G inhibition. These novel data demonstrate that BAY 41-2272 and BAY 60-2770 stimulate production of cGMP and blunt hyperoxia-induced increases in Ca(2+) responses in developing ASM. Accordingly, sGC stimulators/activators may be a useful therapeutic strategy in improving bronchodilation in preterm infants.

  20. Keratose Hydrogels Promote Vascular Smooth Muscle Differentiation from C-kit Positive Human Cardiac Stem Cells.

    PubMed

    Ledford, Benjamin T; Simmons, Jamelle; Chen, Miao; Fan, Huimin; Barron, Catherine; Liu, Zhongmin; Van Dyke, Mark; He, Jia-Qiang

    2017-03-28

    Stem cell-based therapies have demonstrated great potential for the treatment of cardiac diseases, e.g., myocardial infarction; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation are still major limitations, which lead low therapeutic efficiency. Biomaterials provide a promising solution to overcome these issues due to their biocompatibility, biodegradability, low/non-immunogenicity, and low/non-cytotoxicity. The present study aims to investigate the impacts of Keratose (KOS) hydrogel biomaterial on cellular viability, proliferation, and differentiation of c-kit+ human cardiac stem cells (hCSCs). Briefly, hCSCs were cultured on both KOS hydrogel-coated dishes and regular tissue culture dishes (Blank control). Cell viability, stemness, proliferation, cellular morphology, and cardiac lineage differentiation were compared between KOS hydrogel and the Blank control at different time points. We found that KOS hydrogel is effective in maintaining hCSCs without any observable toxic effects, although cell size and proliferation rate appeared smaller on the KOS hydrogel compared to the Blank control. To our surprise, KOS hydrogel significantly promoted vascular smooth muscle cell (VSMC) differentiation (~72%), while on the Blank control dishes, most of the hCSCs (~78%) became cardiomyocytes. Further, we also observed "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. To the best of our knowledge, this is the first report to discover the preferred differentiation of hCSCs toward VSMCs on KOS hydrogel. The underlying mechanism remains unknown. This innovative methodology may offer a new approach to generate a robust number of VSMCs simply by culturing hCSCs on KOS hydrogel, and the resulting VSMCs may be used in animal studies and clinical trials in

  1. Sex-specific pharmacological modulation of autophagic process in human umbilical artery smooth muscle cells.

    PubMed

    Campesi, Ilaria; Occhioni, Stefano; Capobianco, Giampiero; Fois, Marco; Montella, Andrea; Dessole, Salvatore; Franconi, Flavia

    2016-11-01

    Sex has largely been neglected in cell studies. Therefore, we investigated the occurrence of sexual dimorphism in human umbilical artery smooth muscle cells (HUASMCs). In particular, we investigated the existence of sex differences in basal and in drug-induced autophagy, a process involved in cardiovascular diseases. HUASMCs were isolated from healthy and normal weight male and female newborns (MHUASMCs and FHUASMCs, respectively). Expression of the primary molecules involved in the autophagic process [beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)], and PmTOR were detected using western blotting in basal conditions, after serum starvation, rapamycin and verapamil treatments. The level of constitutive autophagy, measured as the LC3II/I ratio, was similar in male and female HUASMCs in the basal condition. Serum starvation promoted autophagy in both cell types, but the increase was more pronounced in FHUASMCs, while 250nM rapamycin induced autophagy only in female cells. Moreover, the level of verapamil-induced autophagy was not different between the two sexes. Notably, in the basal condition, Beclin-1 was more elevated in MHUASMCs than in FHUASMCs, and the difference disappeared after serum starvation and exposure to rapamycin. After exposure to verapamil, the differences in Beclin-1 increased, with more elevated expression levels in female cells. PmTor did not differ in basal conditions, but it was significantly down-regulated by starvation only in FHUASMCs and by rapamycin both in male and female cells. Finally, a strong negative correlation was observed between the newborn's weight and basal autophagy in female cells and between the newborn's weight and the LC3II/I ratio in male verapamil-treated cells. These results indicate that sex-differences begin in utero, are parameter-specific and drug specific suggesting that HUASMCs are a suitable model for the screening of drugs and to study the influence of sex. The sex differences in the autophagy

  2. Concerted upregulation of CLP36 and smooth muscle actin protein expression in human endometrium during decidualization.

    PubMed

    Miehe, Ulrich; Neumaier-Wagner, Peruka; Kadyrov, Mamed; Goyal, Pankaj; Alfer, Joachim; Rath, Werner; Huppertz, Berthold

    2005-01-01

    The human endometrium prepares for implantation of the blastocyst by reorganization of its whole cellular network. Endometrial stroma cells change their phenotype starting around the 23rd day of the menstrual cycle. These predecidual stroma cells first appear next to spiral arteries, and after implantation these cells further differentiate into decidual stroma cells. The phenotypical changes in these cells during decidualization are characterized by distinct changes in the actin filaments and filament-related proteins such as alpha-actinin. The carboxy-terminal LIM domain protein with a molecular weight of 36 kDa (CLP36) is a cytoskeletal component that has been shown to associate with contractile actin filaments and to bind to alpha-actinin supporting a role for CLP36 in cytoskeletal reorganization and signal transduction by binding to signaling proteins. The expression patterns of CLP36, alpha-actinin and actin were studied in endometrial stroma cells from different stages of the menstrual cycle and in decidual stroma cells from the 6th week of gestation until the end of pregnancy. During the menstrual cycle, CLP36 is only expressed in the luminal and glandular epithelium but not in endometrial stroma cells. During decidualization and throughout pregnancy, a parallel upregulation of CLP36 and smooth muscle actin, an early marker of decidualization in the baboon, was observed in endometrial decidual cells. Since both proteins maintain a high expression level throughout pregnancy, a role of both proteins is suggested in the stabilization of the cytoskeleton of these cells that come into close contact with invading trophoblast cells.

  3. 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.

  4. 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

  5. Regional differences of energetics, mechanics, and kinetics of myosin cross-bridge in human ureter smooth muscle.

    PubMed

    Vargiu, Romina; Perinu, Anna; Tintrup, Frank; Broccia, Francesca; Lisa, Antonello De

    2015-01-01

    This study provides information about baseline mechanical properties of the entire muscle and the molecular contractile mechanism in human ureter smooth muscle and proposed to investigate if changes in mechanical motor performance in different regions of isolated human ureter are attributable to differences in myosin crossbridge interactions. Classic mechanical, contraction and energetic parameters derived from the tension-velocity relationship were studied in ureteral smooth muscle strips oriented longitudinally and circularly from abdominal and pelvic human ureter parts. By applying of Huxley's mathematical model we calculated the total working crossbridge number per mm(2) (Ψ), elementary force per single crossbridge (Π0), duration of maximum rate constant of crossbridge attachment 1/f1 and detachment 1/g2 and peak mechanical efficiency (Eff.max). Abdominal longitudinal smooth muscle strips exhibited significantly higher maximum isometric tension and faster maximum unloaded shortening velocity compared to pelvic ones. Contractile differences were associated with significantly higher crossbridge number per mm(2). Abdominal longitudinal muscle strips showed a lower duration of maximum rate constant of crossbridge attachment and detachment and higher peak mechanical efficiency than pelvic ones. Such data suggest that the abdominal human ureter showed better mechanical motor performance mainly related to a higher crossbridge number and crossbridge kinetics differences. Such results were more evident in the longitudinal rather than in the circular layer.

  6. 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

  7. 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.

  8. Decreased vascular smooth muscle cell density in medial degeneration of human abdominal aortic aneurysms.

    PubMed Central

    López-Candales, A.; Holmes, D. R.; Liao, S.; Scott, M. J.; Wickline, S. A.; Thompson, R. W.

    1997-01-01

    Abdominal aortic aneurysms (AAAs) are characterized by structural deterioration of the aortic wall leading to progressive aortic dilatation and eventual rupture. The histopathological changes in AAAs are particularly evident within the elastic media, which is normally dominated by vascular smooth muscle cells (SMCs). To determine whether a decrease in vascular SMCs contributes to medial degeneration, we measured SMC density in 21 normal and pathological human abdominal aortic tissue specimens using immunohistochemistry for alpha-SMC actin and direct cell counts (medial SMCs per high-power field (HPF)). Medial SMC density was not significantly different between normal aorta (n = 5; 199.5 +/- 14.9 SMCs/HPF) and atherosclerotic occlusive disease (n = 6; 176.4 +/- 13.9 SMCs/HPF), but it was reduced by 74% in AAA (n = 10; 50.9 +/- 6.1 SMCs/HPF; P < 0.01 versus normal aorta). Light and electron microscopy revealed no evidence of overt cellular necrosis, but SMCs in AAAs exhibited ultrastructural changes consistent with apoptosis. Using in situ end-labeling (ISEL) of fragmented DNA to detect apoptotic cells, up to 30% of aortic wall cells were ISEL positive in AAAs. By double-labeling techniques, many of these cells were alpha-actin-positive SMCs distributed throughout the degenerative media. In contrast, ISEL-positive cells were observed only within the intimal plaque in atherosclerotic occlusive disease. The amount of p53 protein detected by immunoblotting was increased nearly fourfold in AAA compared with normal aorta and atherosclerotic occlusive disease (P < 0.01), and immunoreactive p53 was localized to lymphocytes and residual SMCs in the aneurysm wall. Using reverse transcription polymerase chain reaction assays a substantial amount of p53 mRNA expression was observed in AAAs. These results demonstrate that medial SMC density is significantly decreased in human AAA tissues associated with evidence of SMC apoptosis and increased production of p53, a potential

  9. 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.

  10. 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

  11. 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.

  12. Human motion perception and smooth eye movements show similar directional biases for elongated apertures

    NASA Technical Reports Server (NTRS)

    Beutter, B. R.; Stone, L. S.

    1998-01-01

    Although numerous studies have examined the relationship between smooth-pursuit eye movements and motion perception, it remains unresolved whether a common motion-processing system subserves both perception and pursuit. To address this question, we simultaneously recorded perceptual direction judgments and the concomitant smooth eye-movement response to a plaid stimulus that we have previously shown generates systematic perceptual errors. We measured the perceptual direction biases psychophysically and the smooth eye-movement direction biases using two methods (standard averaging and oculometric analysis). We found that the perceptual and oculomotor biases were nearly identical, suggesting that pursuit and perception share a critical motion processing stage, perhaps in area MT or MST of extrastriate visual cortex.

  13. Human Motion Perception and Smooth Eye Movements Show Similar Directional Biases for Elongated Apertures

    NASA Technical Reports Server (NTRS)

    Beutter, Brent R.; Stone, Leland S.

    1997-01-01

    Although numerous studies have examined the relationship between smooth-pursuit eye movements and motion perception, it remains unresolved whether a common motion-processing system subserves both perception and pursuit. To address this question, we simultaneously recorded perceptual direction judgments and the concomitant smooth eye movement response to a plaid stimulus that we have previously shown generates systematic perceptual errors. We measured the perceptual direction biases psychophysically and the smooth eye-movement direction biases using two methods (standard averaging and oculometric analysis). We found that the perceptual and oculomotor biases were nearly identical suggesting that pursuit and perception share a critical motion processing stage, perhaps in area MT or MST of extrastriate visual cortex.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Differential effects of blinks on horizontal saccade and smooth pursuit initiation in humans.

    PubMed

    Rambold, Holger; El Baz, Ieman; Helmchen, Christoph

    2004-06-01

    Blinks executed during eye movements affect kinetic eye movement parameters, e.g., peak velocity of saccades is decreased, their duration is increased, but their amplitude is not altered. This effect is mainly explained by the decreased activity of premotor neurons in the brainstem: omni-pause neurons (OPN) in the nucleus raphe interpositus. Previous studies examined the immediate effect of blinks directly on eye movements but not their effect when they are elicited several hundred milliseconds before the eye movements. In order to address this question we tested blinks elicited before the target onset of saccades and pursuit and compared the results to the gap effect: if a fixation light is extinguished for several hundred milliseconds, the reaction time (latency) for subsequent saccades or smooth pursuit eye movements is decreased. Monocular eye and lid movements were recorded in nine healthy subjects using the scleral search-coil system. Laser stimuli were front-projected onto a tangent screen in front of the subjects. Horizontal step-ramp smooth pursuit of 20 deg/s was elicited in one session, or 5 deg horizontal visually guided saccades in another experimental session. In one-third of the trials (smooth pursuit or saccades) the fixation light was extinguished for 200 ms before stimulus onset (gap condition), and in another third of the trials reflexive blinks were elicited by a short airpuff before the stimulus onset (blink condition). The last third of the trials served as controls (control condition). Stimulus direction and the three conditions were randomized for saccades and smooth pursuit separately. The latency of the step-ramp smooth pursuit in the blink condition was found to be decreased by 10 ms, which was less than in the gap condition (38 ms). However, the initial acceleration and steady-state velocity of smooth pursuit did not differ in the three conditions. In contrast, the latency of the saccades in the gap condition was decreased by 39 ms, but

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Free Fatty Acid Palmitate Impairs the Vitality and Function of Cultured Human Bladder Smooth Muscle Cells

    PubMed Central

    Oberbach, Andreas; Schlichting, Nadine; Heinrich, Marco; Till, Holger; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

    2012-01-01

    Background Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways. Methodology/Principal Findings HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1. Conclusions/Significance Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain

  6. The purinergic component of human bladder smooth muscle cells’ proliferation and contraction under physiological stretch

    SciTech Connect

    Wazir, Romel; Luo, De-Yi; Tian, Ye; Yue, Xuan; Li, Hong; Wang, Kun-Jie

    2013-07-26

    Highlights: •Stretch induces proliferation and contraction. •Optimum applied stretch in vitro is 5% and 10% equibiaxial stretching respectively. •Expression of P2X1 and P2X2 is upregulated after application of stretch. •P2X2 is possibly more susceptible to stretch related changes. •Purinoceptors functioning may explain conditions with atropine resistance. -- Abstract: Objective: To investigate whether cyclic stretch induces proliferation and contraction of human smooth muscle cells (HBSMCs), mediated by P2X purinoceptor 1 and 2 and the signal transduction mechanisms of this process. Methods: HBSMCs were seeded on silicone membrane and stretched under varying parameters; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (Frequency: 0.05 Hz, 0.1 Hz, 0.2 Hz, 0.5 Hz, 1 Hz). 5-Bromo-2-deoxyuridine assay was employed for proliferative studies. Contractility of the cells was determined using collagen gel contraction assay. After optimal physiological stretch was established; P2X1 and P2X2 were analyzed by real time polymerase chain reaction and Western Blot. Specificity of purinoceptors was maintained by employing specific inhibitors; (NF023 for P2X1, and A317491for P2X2), in some experiments. Results: Optimum proliferation and contractility were observed at 5% and 10% equibiaxial stretching respectively, applied at a frequency of 0.1 Hz; At 5% stretch, proliferation increased from 0.837 ± 0.026 (control) to 1.462 ± 0.023%, p < 0.05. Mean contraction at 10% stretching increased from 31.7 ± 2.3%, (control) to 78.28 ±1.45%, p < 0.05. Expression of P2X1 and P2X2 was upregulated after application of stretch. Inhibition had effects on proliferation (1.232 ± 0.051, p < 0.05 NF023) and (1.302 ± 0.021, p < 0.05 A314791) while contractility was markedly reduced (68.24 ± 2.31, p < 0.05 NF023) and (73.2 ± 2.87, p < 0.05 A314791). These findings shows that mechanical stretch can promote magnitude-dependent proliferative and contractile modulation of HBSMCs in

  7. Iptakalim influences the proliferation and apoptosis of human pulmonary artery smooth muscle cells

    PubMed Central

    LI, QINGLIN; YAN, XIAOPEI; KONG, HUI; XIE, WEIPING; WANG, HONG

    2016-01-01

    The aim of the present study was to determine the effect of an ATP-sensitive K+ (KATP) channel opener iptakalim (IPT) on the proliferation and apoptosis of human pulmonary artery smooth muscle cells (HPASMCs), and examine the potential value of IPT to hypoxic pulmonary hypertension (HPH) at a cellular level. HPASMCs were divided into the control, ET-1, ET-1+IPT and ET-1+IPT+glibenclamide (GLI) groups. GLI was administered 30 min prior to ET-1 and IPT. The 4 groups were incubated with corresponding reagents for 24 h. Cell viability was evaluated using a CCK-8 assay, cell proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, and cell apoptosis via the expression of apoptosis-related proteins, i.e., Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) using western blotting. We incubated HPASMCs with varying concentrations of ET-1 for 24, 48 and 72 h, and found that cell survival rate was increased in a dose-dependent manner (P<0.05) rather than in a time-dependent manner (P>0.05). After co-incubation of HPASMCs with varying concentrations of IPT and ET-1 for 24 h, the cell survival rate was decreased in a dose-dependent manner. The cell survival rate in the IPT+ET-1 group was significantly lower than that in the ET-1 group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1 group were higher than those in the control group, and the expression of Bax/Bcl-2 was lower than the control group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1+IPT group were lower than those in the ET-1 group, and the expression of Bax/Bcl-2 was higher than that in the ET-1 group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1+IPT+GLI group were higher than those in the ET-1+IPT group, and the expression of Bax/Bcl-2 was lower than that in the ET-1+IPT group (P<0.05). In conclusion, IPT inhibited ET-1-induced HPASMC proliferation and promoted cell apoptosis. Thus, it may play an

  8. 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

  9. 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.

  10. Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells

    PubMed Central

    2014-01-01

    Background Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. Results Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. Conclusions These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of

  11. 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.

  12. 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.

  13. 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

  14. Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

    PubMed

    Yazawa, H; Hirasawa, A; Horie, K; Saita, Y; Iida, E; Honda, K; Tsujimoto, G

    1996-03-01

    1. In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]-arginine8-vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg-1 protein. 2. Nonlabelled compounds compete for [3H]-AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin > or = AVP > Thr4, Gly7-oxytocin > (beta-mercapto-beta-beta-cyclopentamethylenepropionyl-O-Me Tyr2, Arg8) vasopressin > desmopressin > OPC21268 > OPC31260. This order was markedly different from that observed in rat vascular smooth muscle cells (A10), a well-established V1A receptor system. 3. In HVSMC both oxytocin and AVP increased inositol 1,4,5-trisphosphate (IP3) production and [Ca2+]i response, but the efficacy of the responses was greater for oxytocin than AVP. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected only oxytocin receptor but not V1A or V2 receptors in HVSMC, whereas only V1A receptors were found in A10 cells. 5. In conclusion, in HVSMC only oxytocin receptors are expressed among the vasopressin receptor family, and they coupled to phosphatidyl inositol (PI) turnover/Ca2+ signalling. This unexpected observation should provide new insight into the functional role of the oxytocin receptor in a human vascular smooth muscle cell line.

  15. H2 Receptor-Mediated Relaxation of Circular Smooth Muscle in Human Gastric Corpus: the Role of Nitric Oxide (NO).

    PubMed

    Lee, Sang Eok; Kim, Dae Hoon; Kim, Young Chul; Han, Joung-Ho; Choi, Woong; Kim, Chan Hyung; Jeong, Hye Won; Park, Seon-Mee; Yun, Sei Jin; Choi, Song-Yi; Sung, Rohyun; Kim, Young Ho; Yoo, Ra Young; Sun, Park Hee; Kim, Heon; Song, Young-Jin; Xu, Wen-Xie; Yun, Hyo-Yung; Lee, Sang Jin

    2014-10-01

    This study was designed to examine the effects of histamine on gastric motility and its specific receptor in the circular smooth muscle of the human gastric corpus. Histamine mainly produced tonic relaxation in a concentration-dependent and reversible manner, although histamine enhanced contractility in a minor portion of tissues tested. Histamine-induced tonic relaxation was nerve-insensitive because pretreatment with nerve blockers cocktail (NBC) did not inhibit relaxation. Additionally, K(+) channel blockers, such as tetraethylammonium (TEA), apamin (APA), and glibenclamide (Glib), had no effect. However, N(G)-nitro-L-arginine methyl ester (L-NAME) and 1H-(1,2,4)oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), did inhibit histamine-induced tonic relaxation. In particular, histamine-induced tonic relaxation was converted to tonic contraction by pretreatment with L-NAME. Ranitidine, the H2 receptor blocker, inhibited histamine-induced tonic relaxation. These findings suggest that histamine produced relaxation in circular smooth muscle of human gastric smooth muscle through H2 receptor and NO/sGC pathways.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. Cell-to-cell contact of human monocytes with infected arterial smooth-muscle cells enhances growth of Chlamydia pneumoniae.

    PubMed

    Puolakkainen, Mirja; Campbell, Lee Ann; Lin, Tsun-Mei; Richards, Theresa; Patton, Dorothy L; Kuo, Cho-Chou

    2003-02-01

    Chlamydia pneumoniae can infect arterial cells. It has been shown that coculture of human monocytes (U937) and endothelial cells promotes infection of C. pneumoniae in endothelial cells and that the enhancement was mediated by a soluble factor (insulin-like growth factor 2) secreted by monocytes. In this study, it is shown that coculture of monocytes with C. pneumoniae enhances infection of C. pneumoniae in arterial smooth-muscle cells 5.3-fold at a monocyte-to-smooth-muscle cell ratio of 5. However, unlike endothelial cells, no enhancement was observed if monocytes were placed in cell culture inserts or if conditioned medium from monocyte cultures was used, which suggests that cell-to-cell contact is critical. The addition of mannose 6-phosphate or octyl glucoside, a nonionic detergent containing a sugar group, to cocultures inhibited the enhancement. These findings suggest that the monocyte-smooth-muscle cell interaction may be mediated by mannose 6-phosphate receptors present on monocytes.

  15. Human coronary artery smooth muscle cell response to a novel PLA textile/fibrin gel composite scaffold.

    PubMed

    Gundy, Sarah; Manning, Grainne; O'Connell, Enda; Ellä, Ville; Harwoko, Marvi Sri; Rochev, Yuri; Smith, Terry; Barron, Valerie

    2008-11-01

    Previous studies have demonstrated the potential of fibrin as a cell carrier for cardiovascular tissue engineering applications. Unfortunately, fibrin exhibits poor mechanical properties. One method of addressing this issue is to incorporate a textile in fibrin to provide structural support. However, it is first necessary to develop a deeper understanding of the effect of the textile on cell response. In this study, the cytotoxicity of a polylactic acid (PLA) warp-knit textile was assessed with human coronary artery smooth muscle cells (HCASMC). Subsequently, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was employed to examine the gene expression of HCASMC embedded in fibrin with and without the textile. Five genes were examined over a 3-week period: smooth muscle alpha-actin (SMalphaA), myosin heavy chain 11 smooth muscle (SM1/SM2), calponin, myosin heavy chain 10 non-muscle (SMemb) and collagen. Additionally, a microarray analysis was performed to examine a wider range of genes. The knitting process did not adversely affect the cell response; there was no dramatic change in cell number or metabolic rate compared to the negative control. After 3 weeks, there was no significant difference in gene expression, except for a slight decrease of 10% in SMemb in the fibrin with textile. After 3 weeks, there were no obvious cytotoxic effects observed as a result of the knitting process and the gene expression profile did not appear to be altered in the presence of the mesh in the fibrin gel.

  16. 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

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. The role of K⁺ conductances in regulating membrane excitability in human gastric corpus smooth muscle.

    PubMed

    Lee, Ji Yeon; Ko, Eun-Ju; Ahn, Ki Duck; Kim, Sung; Rhee, Poong-Lyul

    2015-04-01

    Changes in resting membrane potential (RMP) regulate membrane excitability. K(+) conductance(s) are one of the main factors in regulating RMP. The functional role of K(+) conductances has not been studied the in human gastric corpus smooth muscles (HGCS). To examine the role of K(+) channels in regulation of RMP in HGCS we employed microelectrode recordings, patch-clamp, and molecular approaches. Tetraethylammonium and charybdotoxin did not affect the RMP, suggesting that BK channels are not involved in regulating RMP. Apamin, a selective small conductance Ca(2+)-activated K(+) channel (SK) blocker, did not show a significant effect on the membrane excitability. 4-Aminopyridine, a Kv channel blocker, caused depolarization and increased the duration of slow wave potentials. 4-Aminopyridine also inhibited a delayed rectifying K(+) current in isolated smooth muscle cells. End-product RT-PCR gel detected Kv1.2 and Kv1.5 in human gastric corpus muscles. Glibenclamide, an ATP-sensitive K(+) channel (KATP) blocker, did not induce depolarization, but nicorandil, a KATP opener, hyperpolarized HGCS, suggesting that KATP are expressed but not basally activated. Kir6.2 transcript, a pore-forming subunit of KATP was expressed in HGCS. A low concentration of Ba(2+), a Kir blocker, induced strong depolarization. Interestingly, Ba(2+)-sensitive currents were minimally expressed in isolated smooth muscle cells under whole-cell patch configuration. KCNJ2 (Kir2.1) transcript was expressed in HGCS. Unique K(+) conductances regulate the RMP in HGCS. Delayed and inwardly rectifying K(+) channels are the main candidates in regulating membrane excitability in HGCS. With the development of cell dispersion techniques of interstitial cells, the cell-specific functional significance will require further analysis.

  9. PKC-DEPENDENT REGULATION OF Kv7.5 CHANNELS BY THE BRONCHOCONSTRICTOR HISTAMINE IN HUMAN AIRWAY SMOOTH MUSCLE CELLS.

    PubMed

    Haick, Jennifer M; Brueggemann, Lioubov I; Cribbs, Leanne L; Denning, Mitchell F; Schwartz, Jeffrey; Byron, Kenneth L

    2017-03-10

    Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, though the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells

    SciTech Connect

    Cushing, S.D.; Berliner, J.A.; Valente, A.J.; Territo, M.C.; Navab, M.; Parhami, F.; Gerrity, R.; Schwartz, C.J.; Fogelman, A.M.

    1990-07-01

    After exposure to low density lipoprotein (LDL) that had been minimally modified by oxidation (MM-LDL), human endothelial cells (EC) and smooth muscle cells (SMC) cultured separately or together produced 2- to 3-fold more monocyte chemotactic activity than did control cells or cells exposed to freshly isolated LDL. This increase in monocyte chemotactic activity was paralleled by increases in mRNA levels for a monocyte chemotactic protein 1 (MCP-1) that is constitutively produced by the human glioma U-105MG cell line. Antibody that had been prepared against cultured baboon smooth muscle cell chemotactic factor (anti-SMCF) did not inhibit monocyte migration induced by the potent bacterial chemotactic factor f-Met-Leu-Phe. However, anti-SMCF completely inhibited the monocyte chemotactic activity found in the media of U-105MG cells, EC, and SMC before and after exposure to MM-LDL. Moreover, monocyte migration into the subendothelial space of a coculture of EC and SMC that had been exposed to MM-LDL was completely inhibited by anti-SMCF. Anti-SMCF specifically immunoprecipitated 10-kDa and 12.5-kDa proteins from EC. Incorporation of (35S)methionine into the immunoprecipitated proteins paralleled the monocyte chemotactic activity found in the medium of MM-LDL stimulated EC and the levels of MCP-1 mRNA found in the EC. We conclude that SMCF is in fact MCP-1 and MCP-1 is induced by MM-LDL.

  18. The Impact of Vitamin D on Asthmatic Human Airway Smooth Muscle

    PubMed Central

    Hall, Sannette C.; Fischer, Kimberly D.; Agrawal, Devendra K.

    2016-01-01

    Asthma is a chronic heterogeneous disorder, which involves airway inflammation, airway hyperresponsiveness (AHR) and airway remodeling. The airway smooth muscle (ASM) bundle regulates the broncho-motor tone and plays a critical role in AHR as well as orchestrating inflammation. Vitamin D deficiency has been linked to increased severity and exacerbations of symptoms in asthmatic patients. It has been shown to modulate both immune and structural cells, including ASM cells, in inflammatory diseases. Given that current asthma therapies have not been successful in reversing airway remodeling, vitamin D supplementation as a potential therapeutic option has gained a great deal of attention. Here, we highlight the potential immunomodulatory properties of vitamin D in regulating ASM function and airway inflammation in bronchial asthma. PMID:26634624

  19. 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.

  20. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  1. 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.

  2. 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.

  3. 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.

  4. Human bronchial smooth muscle cells express adenylyl cyclase isoforms 2, 4, and 6 in distinct membrane microdomains.

    PubMed

    Bogard, Amy S; Xu, Congfeng; Ostrom, Rennolds S

    2011-04-01

    Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because β-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter. We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC). Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6. Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca(2+)-inhibited and G(βγ)-stimulated AC activity, consistent with expression of AC6, AC2, and AC4. Isoproterenol-stimulated AC activity was inhibited by Ca(2+) but unaltered by G(βγ), whereas butaprost-stimulated AC activity was stimulated by G(βγ) but unaffected by Ca(2+) addition. Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions. Immunoisolation of caveolae using caveolin-1 antibodies yielded Ca(2+)-inhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G(βγ)-stimulated AC activity (consistent with expression of AC2 and/or AC4). Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E(2) or butaprost. β(2)AR, but not prostanoid EP(2) or EP(4) receptors, colocalized with AC5/6 in lipid raft fractions. Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains. These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca(2+) and G(βγ).

  5. Novel effect of 2-aminoethoxydiphenylborate through inhibition of calcium sensitization induced by Rho kinase activation in human detrusor smooth muscle.

    PubMed

    Shahab, Nouval; Kajioka, Shunichi; Takahashi, Ryosuke; Hayashi, Maya; Nakayama, Shinsuke; Sakamoto, Kazuyuki; Takeda, Masahiro; Masuda, Noriyuki; Naito, Seiji

    2013-05-15

    Since the introduction of 2-aminoethoxydiphenylborate (2-APB) as a membrane permeable modulator of inositol (1,4,5)-trisphosphate receptors, subsequent studies have revealed additional actions of this chemical on multiple Ca(2+)-permeable ionic channels in the plasma membrane. However, no reports have yet examined 2-APB as a modulator targeting contractile machinery in smooth muscle, independent of Ca(2+) mobilization, namely Ca(2+) sensitization. Here, we assessed whether or not 2-APB affects intracellular signaling pathways of Ca(2+) sensitization for contraction using α-toxin permeabilized human detrusor smooth muscle. Although contractions were induced by application of Ca(2+)-containing bath solutions, 2-APB had little effect on contractions induced by 1 µM Ca(2+) alone but significantly reversed the carbachol-induced augmentation of Ca(2+)-induced contraction in the presence of guanosine triphosphate (carbachol-induced Ca(2+) sensitization). The rho kinase inhibitor Y-27632 and protein kinase C inhibitor GF-109203X also reversed the carbachol-mediated Ca(2+) sensitization. Additional application of 2-APB caused a small but significant further attenuation of the contraction in the presence of GF-109203X but not in the presence of Y-27632. Like carbachol, the rho kinase activator; sphingosylphosphorylcholine, protein kinase C activator; phorbol 12,13 dibutyrate, and myosin light chain phosphatase inhibitor; calyculin-A all induced Ca(2+) sensitization. However, the inhibitory activity of 2-APB was limited with sphingosylphosphorylcholine-induced Ca(2+) sensitization. This study revealed a novel inhibitory effect of 2-APB on smooth muscle contractility through inhibition of the rho kinase pathway.

  6. 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

  7. Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Wang, Guoliang; Liu, Shenghua; Wang, Li; Meng, Liukun; Cui, Chuanjue; Zhang, Hao; Hu, Shengshou; Ma, Ning; Wei, Yingjie

    2017-01-01

    Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH. PMID:28255266

  8. G-Protein-Coupled Receptor 35 Mediates Human Saphenous Vein Vascular Smooth Muscle Cell Migration and Endothelial Cell Proliferation

    PubMed Central

    McCallum, Jennifer E.; Mackenzie, Amanda E.; Divorty, Nina; Clarke, Carolyn; Delles, Christian; Milligan, Graeme; Nicklin, Stuart A.

    2016-01-01

    Vascular smooth muscle cell (VSMC) migration and proliferation is central to neointima formation in vein graft failure following coronary artery bypass. However, there are currently no pharmacological interventions that prevent vein graft failure through intimal occlusion. It is hence a therapeutic target. Here, we investigated the contribution of GPR35 to human VSMC and endothelial cell (EC) migration, using a scratch-wound assay, and also the contribution to proliferation, using MTS and BrdU assays, in in vitro models using recently characterized human GPR35 ortholog-selective small-molecule agonists and antagonists. Real-time PCR studies showed GPR35 to be robustly expressed in human VSMCs and ECs. Stimulation of GPR35, with either the human-selective agonist pamoic acid or the reference agonist zaprinast, promoted VSMC migration in the scratch-wound assay. These effects were blocked by coincubation with either of the human GPR35-specific antagonists, CID-2745687 or ML-145. These GPR35-mediated effects were produced by inducing alterations in the actin cytoskeleton via the Rho A/Rho kinase signaling axis. Additionally, the agonist ligands stimulated a proliferative response in ECs. These studies highlight the potential that small molecules that stimulate or block GPR35 activity can modulate vascular proliferation and migration. These data propose GPR35 as a translational therapeutic target in vascular remodeling. PMID:27064272

  9. 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

  10. 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.

  11. 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.

  12. WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway.

    PubMed

    Lu, Shun; Liu, Hao; Lu, Lihe; Wan, Heng; Lin, Zhiqi; Qian, Kai; Yao, Xingxing; Chen, Qing; Liu, Wenjun; Yan, Jianyun; Liu, Zhengjun

    2016-10-05

    Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6h (4.56±1.14% vs. 11.23±2.25%, P<0.05) to 48h (25.25±5.51% vs. 97.54±13.12%, P<0.01) after injury. Transwell Migration Assay confirmed that WISP1 overexpression significantly promoted human VSMC migration by 2.25-fold compared with EV. Furthermore, WISP1 overexpression stimulated Akt signaling activation in human VSMCs. Blockage of Akt signaling by Akt inhibitor AZD5363 or PI3K inhibitor LY294002, led to an inhibitory effect of WISP1-induced proliferation and migration in human VSMCs. Moreover, we found that WISP1 overexpression stimulated GSK3α/β phosphorylation, and increased expression of cyclin D1 and MMP9 in human VSMCs, and this effect was abolished by AZD5363. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMCs, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.

  13. Oncostatin M Promotes Osteoblastic Differentiation of Human Vascular Smooth Muscle Cells Through JAK3-STAT3 Pathway.

    PubMed

    Kakutani, Yoshinori; Shioi, Atsushi; Shoji, Tetsuo; Okazaki, Hirokazu; Koyama, Hidenori; Emoto, Masanori; Inaba, Masaaki

    2015-07-01

    Vascular calcification is a clinically significant component of atherosclerosis and arises from chronic vascular inflammation. Oncostatin M (OSM) derived from plaque macrophages may contribute to the development of atherosclerotic calcification. Here, we investigated the stimulatory effects of OSM on osteoblastic differentiation of human vascular smooth muscle cells (HVSMC) derived from various arteries including umbilical artery, aorta, and coronary artery and its signaling pathway. Osteoblastic differentiation was induced by exposure of HVSMC to osteogenic differentiation medium (ODM) (10% fetal bovine serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid 2-phosphate in Dulbecco's modified Eagle's medium [DMEM]). OSM significantly increased alkaline phosphate (ALP) activity and matrix mineralization in HVSMC from all sources. Osteoblast marker genes such as ALP and Runx2 were also up-regulated by OSM in these cells. OSM treatment induced activation of STAT3 in HVSMC from umbilical artery as evidenced by immunoblot. Moreover, not only a JAK3 inhibitor, WHI-P154, but also knockdown of JAK3 by siRNA prevented the OSM-induced ALP activity and matrix mineralization in umbilical artery HVSMC. On the other hand, silencing of STAT3 almost completely suppressed OSM-induced ALP expression and matrix mineralization in HVSMC from all sources. These data suggest that OSM promotes osteoblastic differentiation of vascular smooth muscle cells through JAK3/STAT3 pathway and may contribute to the development of atherosclerotic calcification.

  14. Chrysin inhibits human airway smooth muscle cells proliferation through the extracellular signal-regulated kinase 1/2 signaling pathway.

    PubMed

    Yao, Jing; Zhang, Yun-Shi; Feng, Gan-Zhu; Du, Qiang

    2015-11-01

    Asthma is a chronic airway inflammatory disease characterized by an increased mass of airway smooth muscle (ASM). Chrysin (5,7-dihydroxyflavone), a natural flavonoid, has been shown to exert multiple biological activities, including anti-inflammatory, anti-proliferative and anti-oxidant effects, as well as the potency to ameliorate asthma in animal models. The objective of the present study was to identify the underlying mechanism of the therapeutic effects of chrysin. The impact of chrysin on basal and platelet-derived growth factor (PDGF)-induced proliferation and apoptosis of human airway smooth muscle cells (HASMCs) was investigated. Furthermore, the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway was evaluated in HASMCs. The results revealed that chrysin significantly inhibited basal as well as PDGF-induced HASMC proliferation, most likely through the suppression of ERK1/2 phosphorylation. However, chrysin did not significantly reduce PDGF-induced apoptosis of HASMCs. The present study indicated that chrysin may be a promising medication for controlling airway remodeling and clinical manifestations of asthma.

  15. A novel inhibitory effect of oxazol-5-one compounds on ROCKII signaling in human coronary artery vascular smooth muscle cells

    PubMed Central

    Al-Ghabkari, Abdulhameed; Deng, Jing-Ti; McDonald, Paul C.; Dedhar, Shoukat; Alshehri, Mana; Walsh, Michael P.; MacDonald, Justin A.

    2016-01-01

    The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII). In vitro studies showed DI to be a competitive inhibitor of ROCKII (Ki, 132 nM with respect to ATP). This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction (e.g., LC20, Par-4, MYPT1) and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII. PMID:27573465

  16. The anti-ageing hormone klotho induces Nrf2-mediated antioxidant defences in human aortic smooth muscle cells.

    PubMed

    Maltese, Giuseppe; Psefteli, Paraskevi-Maria; Rizzo, Benedetta; Srivastava, Salil; Gnudi, Luigi; Mann, Giovanni E; Siow, Richard C M

    2017-03-01

    Vascular ageing in conditions such as atherosclerosis, diabetes and chronic kidney disease, is associated with the activation of the renin angiotensin system (RAS) and diminished expression of antioxidant defences mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). The anti-ageing hormone klotho promotes longevity and protects against cardiovascular and renal diseases. Klotho has been shown to activate Nrf2 and attenuate oxidative damage in neuronal cells, however, the mechanisms by which it protects against vascular smooth muscle cell VSMC dysfunction elicited by Angiotensin II (AngII) remain to be elucidated. AngII contributes to vascular ageing and atherogenesis by enhancing VSMC oxidative stress, senescence and apoptosis. This study demonstrates that soluble klotho (1 nM, 24 hrs) significantly induces expression of Nrf2 and the antioxidant enzymes haeme oxygenase (HO-1) and peroxiredoxin-1 (Prx-1) and enhances glutathione levels in human aortic smooth muscle cells (HASMC). Silencing of Nrf2 attenuated the induction of HO-1 and Prx-1 expression by soluble klotho. Furthermore, soluble klotho protected against AngII-mediated HASMC apoptosis and senescence via activation of Nrf2. Thus, our findings highlight a novel Nrf2-mediated mechanism underlying the protective actions of soluble klotho in HAMSC. Targeting klotho may thus represent a therapeutic strategy against VSMC dysfunction and cardiovascular ageing.

  17. Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth

    SciTech Connect

    Patecki, Margret; Schaewen, Markus von; Tkachuk, Sergey; Jerke, Uwe; Dietz, Rainer; Dumler, Inna; Kusch, Angelika . E-mail: angelika.kusch@charite.de

    2007-08-03

    The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.

  18. Biphasic responses of human vascular smooth muscle cells to magnesium ion

    PubMed Central

    Ma, Jun; Zhao, Nan; Zhu, Donghui

    2016-01-01

    Magnesium-based alloys are promising in biodegradable cardiovascular stent applications. The degradation products of magnesium stents may have significant impacts on the surrounding vascular cells. However, knowledge on the interactions between magnesium ion and vascular cells at the molecular and cellular levels is still largely missing. Vascular smooth muscle cell (SMC) plays an important role in the pathogenesis of restenosis and wound healing after stent implantation. This study evaluated the short-term effects of extracellular magnesium ion (Mg2+) on the cellular behaviors of SMCs. Cellular responses to Mg2+ were biphasic and in a concentration-dependent manner. Low concentrations (10 mM) of Mg2+ increased cell viability, cell proliferation rate, cell adhesion, cell spreading, cell migration rate, and actin expression. In contrast, higher concentrations (40–60 mM) of Mg2+ had deleterious effects on cells. Gene expression analysis revealed that Mg2+ altered the expressions of genes mostly related to cell adhesion, cell injury, angiogenesis, inflammation, coagulation, and cell growth. Finding from this study provides some valuable information on SMC responses toward magnesium ions at the cellular and molecular levels, and guidance for future controlled release of magnesium from the stent material. PMID:26402437

  19. Biphasic responses of human vascular smooth muscle cells to magnesium ion.

    PubMed

    Ma, Jun; Zhao, Nan; Zhu, Donghui

    2016-02-01

    Magnesium-based alloys are promising in biodegradable cardiovascular stent applications. The degradation products of magnesium stents may have significant impacts on the surrounding vascular cells. However, knowledge on the interactions between magnesium ion and vascular cells at the molecular and cellular levels is still largely missing. Vascular smooth muscle cell (SMC) plays an important role in the pathogenesis of restenosis and wound healing after stent implantation. This study evaluated the short-term effects of extracellular magnesium ion (Mg(2+)) on the cellular behaviors of SMCs. Cellular responses to Mg(2+) were biphasic and in a concentration-dependent manner. Low concentrations (10 mM) of Mg(2+) increased cell viability, cell proliferation rate, cell adhesion, cell spreading, cell migration rate, and actin expression. In contrast, higher concentrations (40-60 mM) of Mg(2+) had deleterious effects on cells. Gene expression analysis revealed that Mg(2+) altered the expressions of genes mostly related to cell adhesion, cell injury, angiogenesis, inflammation, coagulation, and cell growth. Finding from this study provides some valuable information on SMC responses toward magnesium ions at the cellular and molecular levels, and guidance for future controlled release of magnesium from the stent material.

  20. Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth.

    PubMed

    Patecki, Margret; von Schaewen, Markus; Tkachuk, Sergey; Jerke, Uwe; Dietz, Rainer; Dumler, Inna; Kusch, Angelika

    2007-08-03

    The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.

  1. 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.

  2. 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.

  3. 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

  4. TRPC1/TRPC3 channels mediate lysophosphatidylcholine-induced apoptosis in cultured human coronary artery smooth muscles cells

    PubMed Central

    Wang, Yuan; Wang, Yan; Li, Gui-Rong

    2016-01-01

    The earlier study showed that lysophosphatidylcholine (lysoPC) induced apoptosis in human coronary artery smooth muscle cells (SMCs); however, the related molecular mechanisms are not fully understood. The present study investigated how lysoPC induces apoptosis in cultured human coronary artery SMCs using cell viability assay, flow cytometry, confocal microscopy, and molecular biological approaches. We found that lysoPC reduced cell viability in human coronary artery SMCs by eliciting a remarkable Ca2+ influx. The effect was antagonized by La3+, SKF-96365, or Pyr3 as well as by silencing TRPC1 or TRPC3. Co-immunoprecipitation revealed that TRPC1 and TRPC3 had protein-protein interaction. Silencing TRPC1 or TRPC3 countered the lysoPC-induced increase of Ca2+ influx and apoptosis, and the pro-apoptotic proteins Bax and cleaved caspase-3 and decrease of the anti-apoptotic protein Bcl-2 and the survival kinase pAkt. These results demonstrate the novel information that TRPC1/TRPC3 channels mediate lysoPC-induced Ca2+ influx and apoptosis via activating the pro-apoptotic proteins Bax and cleaved caspase-3 and inhibiting the anti-apoptotic protein Bcl-2 and the survival kinase pAkt in human coronary artery SMCs, which implies that TRPC1/TRC3 channels may be the therapeutic target of lysoPC-induced disorders such as atherosclerosis. PMID:27472391

  5. NOX4 downregulation leads to senescence of human vascular smooth muscle cells

    PubMed Central

    Przybylska, Dorota; Janiszewska, Dorota; Goździk, Aleksandra; Bielak-Zmijewska, Anna; Sunderland, Piotr; Sikora, Ewa; Mosieniak, Grażyna

    2016-01-01

    Senescence is a stress response characterized by an irreversible growth arrest and alterations in certain cell functions. It is believed that both double-strand DNA breaks (DSB) and increased ROS level are the main culprit of senescence. Excessive ROS production is also particularly important in the development of a number of cardiovascular disorders. In this context the involvement of professional ROS-producing enzymes, NADPH oxidases (NOX), was postulated. In contrary to the common knowledge, we have shown that not only increased ROS production but also diminished ROS level could be involved in the induction of senescence. Accordingly, our studies revealed that stress-induced premature senescence (SIPS) of vascular smooth muscle cells (VSMCs) induced by doxorubicin or H2O2, correlates with increased level of DSB and ROS. On the other hand, both SIPS and replicative senescence were accompanied by diminished expression of NOX4. Moreover, inhibition of NOX activity or decrease of NOX4 expression led to permanent growth arrest of VSMCs and secretion of interleukins and VEGF. Interestingly, cells undergoing senescence due to NOX4 depletion neither acquired DSB nor activated DNA damage response. Instead, transient induction of the p27, upregulation of HIF-1alpha, decreased expression of cyclin D1 and hypophosphorylated Rb was observed. Our results showed that lowering the level of ROS-producing enzyme - NOX4 oxidase below physiological level leads to cellular senescence of VSMCs which is correlated with secretion of pro-inflammatory cytokines. Thus the use of specific NOX4 inhibitors for pharmacotherapy of vascular diseases should be carefully considered. PMID:27655718

  6. 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.

  7. Voltage-gated sodium channel expressed in cultured human smooth muscle cells: involvement of SCN9A.

    PubMed

    Jo, Taisuke; Nagata, Taiji; Iida, Haruko; Imuta, Hiroyuki; Iwasawa, Kuniaki; Ma, Ji; Hara, Kei; Omata, Masao; Nagai, Ryozo; Takizawa, Hajime; Nagase, Takahide; Nakajima, Toshiaki

    2004-06-04

    Voltage-gated Na(+) channel (I(Na)) is expressed under culture conditions in human smooth muscle cells (hSMCs) such as coronary myocytes. The aim of this study is to clarify the physiological, pharmacological and molecular characteristics of I(Na) expressed in cultured hSMCs obtained from bronchus, main pulmonary and coronary artery. I(Na), was recorded in these hSMCs and inhibited by tetrodotoxin (TTX) with an IC(50) value of approximately 10 nM. Reverse transcriptase/polymerase chain reaction (RT-PCR) analysis of mRNA showed the prominent expression of transcripts for SCN9A, which was consistent with the results of real-time quantitative RT-PCR. These results provide novel evidence that TTX-sensitive Na(+) channel expressed in cultured hSMCs is mainly composed of Na(v)1.7.

  8. Differential responsiveness to contractile agents of isolated smooth muscle cells from human colons as a function of age and inflammation.

    PubMed

    Boyer, J C; Guitton, C; Pignodel, C; Cuq, P; Moussu, P; Pouderoux, P; Christen, M O; Balmes, J L; Bali, J P

    1997-11-01

    To study the involvement of age and inflammation in motor colonic activity in man, contractile responses to CCK, carbachol, and KCl of isolated colonic smooth muscle cells (SMC) from normal and inflamed human colons were evaluated; the incidence of sex and smoking on contraction was also analyzed. Contractile responses to the three agonists were significantly lower in tissues with a low degree of inflammation than in tissues with high level of inflammation or normal tissues. This reduction in cell responsiveness appears to be nonspecific and nonreceptor mediated. A positive correlation of the contractile responses to the three stimulants with the age of patients was observed. In contrast, no association was found between sex, smoking, and cell contraction. In conclusion, contractions of SMC due to CCK, carbachol, and KCl were significantly modified during life; inflammation of the colon led to a loss of SMC responsiveness.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress

    NASA Technical Reports Server (NTRS)

    Stamatas, G. N.; Patrick, C. W. Jr; McIntire, L. V.

    1997-01-01

    The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.

  16. Human vascular smooth muscle cells have at least two distinct PDGF receptors and can secrete PDGF-AA

    SciTech Connect

    Hosang, M.; Rouge, M. )

    1989-01-01

    Platelet-derived growth factor (PDGF), a potent mitogen and chemoattractant for smooth muscle cells and fibroblasts in culture, is believed to play an important role in the formation of proliferative lesions of arterio-sclerosis. PDGF appears as three different dimeric isoforms: AA, AB, and BB. These were recently found to bind to two different receptors, the A/B receptor (which binds all three isoforms) and the B receptor (which binds only PDGF-BB). To find out whether these receptors exhibit functional differences, we have monitored the binding and mitogenic activities of PDGF-AA and -BB in human umbilical vein smooth muscle cells (HSMCs), human dermal fibroblasts (HFs), and Swiss mouse 3T3 cells. With each cell type, there was a good correlation between the maximal levels of DNA synthesis achieved by these isoforms and the numbers of the appropriate receptor present on the cell surface: HMSCs, which have at least 32,000 B receptors but only 8,000 A/B receptors, responded well to PDGF-BB but responded poorly to PDGF-AA; whereas Swiss 3T3 cells, which have about equal numbers of B and A/B receptors (70,000 and 90,000, respectively), responded equally well to both isoforms. PDGF-AB was a more efficacious mitogen of HSMCs and HFs than was PDGF-AA and inhibited (125I)-PDGF-BB binding to HSMCs more effectively than PDGF-AA. This indicates that there may exist a third PDGF receptor type to which PDGF-BB and -AB but not PDGF-AA can bind.

  17. Differential gene expression profiling of human adipose stem cells differentiating into smooth muscle-like cells by TGFβ1/BMP4.

    PubMed

    Elçin, Ayşe Eser; Parmaksiz, Mahmut; Dogan, Arin; Seker, Sukran; Durkut, Serap; Dalva, Klara; Elçin, Yaşar Murat

    2017-03-15

    Regenerative repair of the vascular system is challenging from the perspectives of translational medicine and tissue engineering. There are fundamental hurdles in front of creating bioartificial arteries, which involve recaputilation of the three-layered structure under laboratory settings. Obtaining and maintaining smooth muscle characteristics is an important limitation, as the transdifferentiated cells fail to display mature phenotype. This study aims to shed light on the smooth muscle differentiation of human adipose stem cells (hASCs). To this end, we first acquired hASCs from lipoaspirate samples. Upon characterization, the cells were induced to differentiate into smooth muscle (SM)-like cells using a variety of inducer combinations. Among all, TGFβ1/BMP4 combination had the highest differentiation efficiency, based on immunohistochemical analyses. hSM-like cell samples were compared to hASCs and to the positive control, human coronary artery-smooth muscle cells (hCA-SMCs) through gene transcription profiling. Microarray findings revealed the activation of gene groups that function in smooth muscle differentiation, signaling pathways, extracellular modeling and cell proliferation. Our results underline the effectiveness of the growth factors and suggest some potential variables for detecting the SM-like cell characteristics. Evidence in transcriptome level was used to evaluate the TGFβ1/BMP4 combination as a previously unexplored effector for the smooth muscle differentiation of adipose stem cells.

  18. Effects of ginsenoside on large-conductance K(Ca) channels in human corporal smooth muscle cells.

    PubMed

    Sung, H H; Chae, M R; So, I; Jeon, J-H; Park, J K; Lee, S W

    2011-01-01

    Ginseng was known to be an effective natural product that enhances penile erection. However, the precise biological function and mechanisms of action of ginseng with regard to erectile function remain unknown. The principal objective of this study was to identify ginsenoside (principal molecular ingredients of ginseng)-induced activation of large-conductance K(Ca) channel in human corporal smooth muscle cells, and to determine ginseng's mechanism of action on penile erection. Electrophysiological studies using cultured human corporal smooth muscle cells were conducted. We evaluated the effects of total ginsenosides (TGS) and ginsenoside Rg3 on large-conductance K(Ca) channel by determining whole-cell currents and single-channel activities. There was an increase in outward current dependent on TGS concentration (at +60 mV, 1 μg ml(-1); 168.3±59.3%, n=6, P<0.05, 10 μg ml(-1); 173.2±36.8%, n=4, P<0.05, 50 μg ml(-1); 295.3±62.3%, n=19, P<0.001, 100 μg ml(-1); and 462.3±97.1%, n=5, P<0.001) and Rg3 concentration (at +60 mV, 1 μM (0.78 μg ml(-1)); 222.8±64.8%, n=11, P<0.0001, 10 μM; 672.6±137.1%, n=10, P<0.0001, 50 μM; and 1713.3±234.7%, n=15, P<0.001) in the solution that was blocked completely by tetraethylammonium (TEA). Channel opening in cell-attached mode and channel activity in the inside-out membrane patches was also increased significantly by 50 μg of TGS or 10 μM of Rg3. The results of this study suggested that the activation of large-conductance K(Ca) channels by ginsenoside could be one mechanism of ginsenoside-induced relaxation in corporal smooth muscle.

  19. 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.

  20. 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

  1. Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6.

    PubMed Central

    Loppnow, H; Libby, P

    1990-01-01

    The cells that make up blood vessel walls appear to participate actively in local immune and inflammatory responses, as well as in certain vascular diseases. We tested here whether smooth muscle cells (SMC) can produce the important inflammatory mediator IL6. Unstimulated SMC in vitro elaborated 5 X 10(3) pg recIL6/24h (i.e., biological activity equivalent to 5 X 10(3) pg recombinant IL6 (recIL6), as determined in B9-assay with a recIL6 standard). Several pathophysiologically relevant factors augmented IL6 release from SMC including 10 micrograms LPS/ml (10(4) pg recIL6), 10 ng tumor necrosis factor/ml (4 X 10(4) pg recIL6), and most notably 10 ng IL1/ml (greater than or equal to 3.2 X 10(5) pg recIL6). Production of IL6 activity corresponded to IL6 mRNA accumulation and de novo synthesis. SMC released newly synthesized IL6 rapidly, as little metabolically labeled material remained cell-associated. In supernatants of IL1-stimulated SMC, IL6 accounted for as much as 4% of the secreted proteins. In normal vessels SMC seldom divide, but SMC proliferation can occur in hypertension or during atherogenesis. We therefore tested the relationship between IL6 production and SMC proliferation in response to platelet-derived growth factor (PDGF) in vitro. Quiescent SMC released scant IL6 activity, whereas PDGF (1-100 ng/ml) produced concentration-dependent and coordinate enhancement of SMC proliferation and IL6 release (linear regression of growth vs. IL6 release yielded r greater than 0.9). IL6 itself neither stimulated nor inhibited SMC growth or IL6 production. Intact medial strips studied in short-term organoid culture produced large quantities of IL6, similar to the results obtained with cultured SMC. These findings illustrate a new function of vascular SMC by which these cells might participate in local immunoregulation and in the pathogenesis of various important vascular diseases as well as in inflammatory responses generally. Images PMID:2312724

  2. 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.

  3. LC/MS/MS data analysis of the human uterine smooth muscle S-nitrosoproteome fingerprint in pregnancy, labor, and preterm labor

    PubMed Central

    Ulrich, Craig C.; Quilici, David R.; Schlauch, Karen A.; Burkin, Heather R.; Buxton, Iain L.O.

    2015-01-01

    The data described in this article is the subject of an article in the American Journal of Physiology: Cell Physiology, titled “The Human Uterine Smooth Muscle S-nitrosoproteome Fingerprint in Pregnancy, Labor, and Preterm Labor” (doi:10.1152/ajpcell.00198.2013) (Ulrich et al., 2013) [1]. The data described is a large scale mass spectrometry data set that defines the human uterine smooth muscle S-nitrosoproteome differences among laboring, non-laboring, preterm laboring tissue after treatment with S-nitrosoglutathione. PMID:26322325

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. Methionine enhances the contractile activity of human colon circular smooth muscle in vitro.

    PubMed

    Choe, Eun Kyung; Moon, Jung Sun; Park, Kyu Joo

    2012-07-01

    Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 µM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 µM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and N(w)-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.

  15. Engineering vascular tissue with functional smooth muscle cells derived from human iPS cells and nanofibrous scaffolds.

    PubMed

    Wang, Yongyu; Hu, Jiang; Jiao, Jiao; Liu, Zhongning; Zhou, Zhou; Zhao, Chao; Chang, Lung-Ji; Chen, Y Eugene; Ma, Peter X; Yang, Bo

    2014-10-01

    Tissue-engineered blood vessels (TEBVs) are promising in the replacement of diseased vascular tissues. However, it remains a great challenge to obtain a sufficient number of functional smooth muscle cells (SMCs) in a clinical setting to construct patient-specific TEBVs. In addition, it is critical to develop a scaffold to accommodate these cells and retain their functional phenotype for the regeneration of TEBVs. In this study, human induced pluripotent stem cells (iPSCs) were established from primary human aortic fibroblasts, and characterized with the pluripotency markers expression and cells' capabilities to differentiate into all three germ layer cells. A highly efficient method was then developed to induce these human iPSCs into proliferative SMCs. After multiple times of expansion, the expanded SMCs retained the potential to be induced into the functional contractile phenotype of mature SMCs, which was characterized by the contractile response to carbachol treatment, up-regulation of specific collagen genes under transforming growth factor β1 treatment, and up-regulation of specific matrix metalloproteinase genes under cytokine stimulation. We also developed an advanced macroporous and nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffold with suitable pore size and interpore connectivity to seed these human iPSC-derived SMCs and maintain their differentiated phenotype. Subcutaneous implantation of the SMC-scaffold construct in nude mice demonstrated vascular tissue formation, with robust collagenous matrix deposition inside the scaffold and the maintenance of differentiated SMC phenotype. Taken together, this study established an exciting approach towards the construction of patient-specific TEBVs. We established patient-specific human iPSCs, derived proliferative SMCs for expansion, turned on their mature contractile SMC phenotype, and developed an advanced scaffold for these cells to regenerate vascular tissue in vivo.

  16. H2S relaxes isolated human airway smooth muscle cells via the sarcolemmal K(ATP) channel.

    PubMed

    Fitzgerald, Robert; DeSantiago, Breann; Lee, Danielle Y; Yang, Guangdong; Kim, Jae Yeon; Foster, D Brian; Chan-Li, Yee; Horton, Maureen R; Panettieri, Reynold A; Wang, Rui; An, Steven S

    2014-03-28

    Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)-the end effector of acute airway narrowing in asthma. Using magnetic twisting cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1-10mM). GYY4137 slowly released appreciable levels of H2S in the range of 10-275 μM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

  17. Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro

    PubMed Central

    Svegliati, Silvia; Amico, Donatella; Spadoni, Tatiana; Fischetti, Colomba; Finke, Doreen; Moroncini, Gianluca; Paolini, Chiara; Tonnini, Cecilia; Grieco, Antonella; Rovinelli, Marina; Gabrielli, Armando

    2017-01-01

    One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia. PMID:28228756

  18. Agonistic Anti-PDGF Receptor Autoantibodies from Patients with Systemic Sclerosis Impact Human Pulmonary Artery Smooth Muscle Cells Function In Vitro.

    PubMed

    Svegliati, Silvia; Amico, Donatella; Spadoni, Tatiana; Fischetti, Colomba; Finke, Doreen; Moroncini, Gianluca; Paolini, Chiara; Tonnini, Cecilia; Grieco, Antonella; Rovinelli, Marina; Gabrielli, Armando

    2017-01-01

    One of the earliest events in the pathogenesis of systemic sclerosis (SSc) is microvasculature damage with intimal hyperplasia and accumulation of cells expressing PDGF receptor. Stimulatory autoantibodies targeting PDGF receptor have been detected in SSc patients and demonstrated to induce fibrosis in vivo and convert in vitro normal fibroblasts into SSc-like cells. Since there is no evidence of the role of anti-PDGF receptor autoantibodies in the pathogenesis of SSc vascular lesions, we investigated the biologic effect of agonistic anti-PDGF receptor autoantibodies from SSc patients on human pulmonary artery smooth muscle cells and the signaling pathways involved. The synthetic (proliferation, migration, and type I collagen gene α1 chain expression) and contractile (smooth muscle-myosin heavy chain and smooth muscle-calponin expression) profiles of human pulmonary artery smooth muscle cells were assessed in vitro after incubation with SSc anti-PDGF receptors stimulatory autoantibodies. The role of reactive oxygen species, NOX isoforms, and mammalian target of rapamycin (mTOR) was investigated. Human pulmonary artery smooth muscle cells acquired a synthetic phenotype characterized by higher growth rate, migratory activity, gene expression of type I collagen α1 chain, and less expression of markers characteristic of the contractile phenotype such as smooth muscle-myosin heavy chain and smooth muscle-calponin when stimulated with PDGF and autoantibodies against PDGF receptor, but not with normal IgG. This phenotypic profile is mediated by increased generation of reactive oxygen species and expression of NOX4 and mTORC1. Our data indicate that agonistic anti-PDGF receptor autoantibodies may contribute to the pathogenesis of SSc intimal hyperplasia.

  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. Building additional complexity to in vitro-derived intestinal tissues.

    PubMed

    Brugmann, Samantha A; Wells, James M

    2013-01-01

    Gastrointestinal (GI) disorders affect up to 25% of the US population. Common intestinal disorders include malabsorption, irritable bowel syndrome and fecal incontinence. Some GI disorders such as Hirschsprung's disease have a genetic basis and are associated with an absence or paucity of enteric nerves. Current treatment plans for GI disorders range from changes in diet to bowel resection, and there are very few drugs available that target the primary deficiencies in intestinal function such as controlled peristalsis. While animal models can recapitulate the broad range of intestinal pathologies of the GI tract, they are intrinsically complicated and of low throughput. Several in vitro systems have been established, and these range from epithelial enteroids to more complex organoids, which contain most intestinal cell types. One of the more complex organoid systems was derived from adult mouse intestines and contains functional enteric nerves and smooth muscle capable of peristalsis. Establishing an equivalent human intestinal system is challenging due to limited access and variable quality of human intestinal tissues. However, owing to recent advances, it is possible to differentiate human induced and embryonic pluripotent stem cells, collectively called pluripotent stem cells, into human intestinal organoids (HIOs) in vitro. Although HIOs contain a significant degree of epithelial and mesenchymal complexity, they lack enteric nerves and thus are unable to model the peristaltic movements of the gut. The goal of this review is to discuss approaches to generate complex in vitro systems that can be used to more comprehensively model common intestinal pathologies. New and more biologically complete human models of the intestine would allow for unprecedented studies of the cellular and molecular basis of normal and pathological gut function. Furthermore, fully functional HIOs could serve as a platform for preclinical drug studies to model absorption and efficacy.

  1. The antifungal antibiotic, clotrimazole, inhibits chloride secretion by human intestinal T84 cells via blockade of distinct basolateral K+ conductances. Demonstration of efficacy in intact rabbit colon and in an in vivo mouse model of cholera.

    PubMed Central

    Rufo, P A; Merlin, D; Riegler, M; Ferguson-Maltzman, M H; Dickinson, B L; Brugnara, C; Alper, S L; Lencer, W I

    1997-01-01

    The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies. PMID:9399958

  2. High glucose induces cell death of cultured human aortic smooth muscle cells through the formation of hydrogen peroxide

    PubMed Central

    Peiró, Concepción; Lafuente, Nuria; Matesanz, Nuria; Cercas, Elena; Llergo, José L; Vallejo, Susana; Rodríguez-Mañas, Leocadio; Sánchez-Ferrer, Carlos F

    2001-01-01

    Alterations of the vessel structure, which is mainly determined by smooth muscle cells through cell growth and/or cell death mechanisms, are characteristic of diabetes complications. We analysed the influence of high glucose (22 mM) on cultured human aortic smooth muscle cell growth and death, as hyperglycaemia is considered one of the main factors involved in diabetic vasculopathy. Growth curves were performed over 96 h in medium containing 0.5% foetal calf serum. Cell number increased by 2–4 fold over the culture period in the presence of 5.5 mM (low) glucose, while a 20% reduction in final cell number was observed with high glucose. Under serum-free conditions, cell number remained constant in low glucose cultures, but a 40% decrease was observed in high glucose cultures, suggesting that high glucose may induce increased cell death rather than reduced proliferation. Reduced final cell number induced by high glucose was also observed after stimulation with 5 or 10% foetal calf serum. The possible participation of oxidative stress was investigated by co-incubating high glucose with different reactive oxygen species scavengers. Only catalase reversed the effect of high glucose. Intracellular H2O2 content, visualized with 2′,7′-dichlorofluorescein and quantified by flow cytometry, was increased after high glucose treatment. To investigate the cell death mechanism induced by high glucose, apoptosis and necrosis were quantified. No differences were observed regarding the apoptotic index between low and high glucose cultures, but lactate dehydrogenase activity was increased in high glucose cultures. In conclusion, high glucose promotes necrotic cell death through H2O2 formation, which may participate in the development of diabetic vasculopathy. PMID:11487505

  3. 2-Methoxycinnamaldehyde inhibits the TNF-α-induced proliferation and migration of human aortic smooth muscle cells.

    PubMed

    Jin, Young-Hee; Kim, Soo-A

    2017-01-01

    The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) is a crucial event in the development of atherosclerosis, and tumor necrosis factor-α (TNF-α) is actively involved in this process by enhancing the proliferation and migration of VSMCs. 2-Methoxycinnamaldehyde (MCA) is a natural compound of Cinnamomum cassia. Although 2-hydroxycinnamaldehyde (HCA), another compound from Cinnamomum cassia, has been widely studied with regard to its antitumor activity, MCA has not attracted researchers' interest due to its mild toxic effects on cancer cells and its mechanisms of action remain unknown. In this study, we examined the effects of MCA on the TNF-α-induced proliferation and migration of human aortic smooth muscle cells (HASMCs). As shown by our results, MCA inhibited TNF-α-induced cell proliferation by reducing the levels of cyclin D1, cyclin D3, CDK4 and CDK6, and increasing the levels of the cyclin-dependent kinase inhibitors, p21 and p27, without resulting in cellular cytotoxicity. Furthermore, MCA decreased the level of secreted matrix metalloproteinase (MMP)-9 by inhibiting MMP-9 transcription. Unexpectedly, MCA did not affect the TNF-α-induced levels of mitogen-activated protein kinases (MAPKs). However, by showing that MCA potently inhibited the degradation of IκBα and the subsequent nuclear translocation of nuclear factor-κB (NF-κB), we demonstrated that MCA exerts its effects through the NF-κB signaling pathway. MCA also effectively inhibited platelet-derived growth factor (PDGF)-induced HASMC migration. Taken together, these observations suggest that MCA has the potential for use as an anti-atherosclerotic agent.

  4. Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.

    PubMed

    Willets, Jonathon M; Brighton, Paul J; Mistry, Rajendra; Morris, Gavin E; Konje, Justin C; Challiss, R A John

    2009-08-01

    Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. The tyrosine phosphatase SHP-2 controls urokinase-dependent signaling and functions in human vascular smooth muscle cells

    SciTech Connect

    Kiyan, Julia Haller, Hermann; Dumler, Inna

    2009-04-01

    The urokinase (uPA)/urokinase receptor (uPAR) multifunctional system is an important mediator of functional behaviour of human vascular smooth muscle cells (VSMC). uPAR associates with platelet-derived growth factor receptor {beta} (PDGFR-{beta}), which serves as a transmembrane adaptor for uPAR in VSMC, to transduce intracellular signaling and initiate functional changes. The precise and rapid propagation of these signaling cascades demands both strict and flexible regulatory mechanisms that remain unexplored. We provide evidence that the tyrosine phosphatase SHP-2 mediates these processes. uPA regulated SHP-2 phosphorylation, catalytic activity, and its co-localization and association with the PDGFR-{beta}. Active PDGFR-{beta} was required for the uPA-induced SHP-2 phosphorylation. uPAR-directed STAT1 pathway was disturbed in cells expressing SHP-2 inactive mutant. Both, cell proliferation and migration were impaired in VSMC with downregulated SHP-2. Elucidating the underlying mechanisms, we found that uPA induced SHP-2 recruitment to lipid rafts. Disruption of rafts abolished uPA-related control of SHP-2 phosphorylation, its association with PDGFR-{beta} and finally the VSMC functional responses. Our results demonstrate that SHP-2 plays an important role in uPA-directed signaling and functional control of human VSMC and suggest that this phosphatase might contribute to the pathogenesis of the uPA-related vascular remodeling.

  14. LOX-1, a bridge between GLP-1R and mitochondrial ROS generation in human vascular smooth muscle cells.

    PubMed

    Dai, Yao; Mercanti, Federico; Dai, Dongsheng; Wang, Xianwei; Ding, Zufeng; Pothineni, Naga Venkata; Mehta, Jawahar L

    2013-07-19

    A growing body of evidence indicates that glucagon-like peptide-1 (GLP-1) agonists or dipeptidyl peptidase-4 (DPP-4) inhibitors play an important role in modulating oxidant stress in vascular beds. However, the underlying mechanism of this process remains unclear. In recent studies, we observed an increase in GLP-1 receptor (GLP-1R) expression in the aorta of LOX-1 knock-out mice. Since LOX-1 is a pivotal regulator of reactive oxygen species (ROS), we conducted studies to identify relationship between LOX-1, ROS and GLP-1 agonism or DPP-4 antagonism. We observed a sustained decrease in GLP-1R expression in human vascular smooth muscle cells (VSMCs) treated with ox-LDL. When VSMCs were treated with different concentration of liraglutide (a GLP-1 agonist) or NVPDPP728 (a DPP-4 inhibitor), expression of ROS decreased compared with ox-LDL alone treatment. To further prove that LOX-1 plays a pivotal role in ROS and GLP-1R expression, we treated VSMCs with LOX-1 antibody or transfected cells with human LOX-1 cDNA. The inhibitory effect of ox-LDL on GLP-1R expression was reversed with anti-LOX-1 antibody treatment, while the inhibitory effect of liraglutide and NVPDPP728 on ROS generation was attenuated when cells were transfected with LOX-1 cDNA. Our results suggest that LOX-1 may play a bridging role in GLP-1 activation and ROS interaction.

  15. CD38 and airway hyper-responsiveness: studies on human airway smooth muscle cells and mouse models.

    PubMed

    Guedes, Alonso G P; Deshpande, Deepak A; Dileepan, Mythili; Walseth, Timothy F; Panettieri, Reynold A; Subramanian, Subbaya; Kannan, Mathur S

    2015-02-01

    Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.

  16. In vitro photodynamic therapy with chlorin e6 leads to apoptosis of human vascular smooth muscle cells.

    PubMed

    Wawrzyńska, Magdalena; Kałas, Wojciech; Biały, Dariusz; Zioło, Ewa; Arkowski, Jacek; Mazurek, Walentyna; Strzadała, Leon

    2010-02-01

    Percutaneous coronary intervention has become the most common and widely implemented method of heart revascularization. However, the development of restenosis remains the major limitation of this method. Photodynamic therapy (PDT) recently emerged as a new and promising method for the prevention of arterial restenosis. Here the efficacy of chlorin e6 in PDT was investigated in vitro using human vascular smooth muscle cells (TG/HA-VSMCs) as one of the cell types crucial in the development of restenosis. PDT-induced cell death was studied on many levels,including annexin V staining, measurement of the generation reactive oxygen species (ROS) and caspase-3 activity,and assessment of changes in mitochondrial membrane potential and fragmentation of DNA. Photosensitization of TG/HA-VSMCs with a 170 lM of chlorin e6 and subsequent illumination with the light of a 672-nm diode laser(2 J/cm2) resulted in the generation of ROS, a decrease in cell membrane polarization, caspase-3 activation, as well as DNA fragmentation. Interestingly, the latter two apoptotic events could not be observed in photosensitized and illuminated NIH3T3 fibroblasts, suggesting different outcomes of the model of PDT in various types of cells. The results obtained with human VSMCs show that chlorin e6 may be useful in the PDT of aerial restenosis, but its efficacy still needs to be established in an animal model.

  17. Location of smooth-muscle myosin and tropomyosin binding sites in the C-terminal 288 residues of human caldesmon.

    PubMed Central

    Huber, P A; Fraser, I D; Marston, S B

    1995-01-01

    We have produced nine recombinant fragments, H1 to H9, from a human cDNA that codes for the C-terminal 288 residues of caldesmon. The fragment H1, encompassing the 288 residues, is equivalent to domains 3 and 4 of caldesmon (amino acids 506-793 in human, 476-737 in the chicken gizzard sequence). It has been shown [Huber, Redwood, Avent, Tanner and Marston (1993) J. Muscle Res. Cell Motil. 14, 385-391] to bind to actin, Ca(2+)-calmodulin, tropomyosin and myosin. The fragments, H2 to H9, differ in length between 60 and 176 residues and cover the whole of domains 3 and 4 with many of the fragments overlapping. We have characterized the myosin and tropomyosin binding of these fragments. The binding of both tropomyosin and myosin is highly dependent on salt concentration, indicating the ionic nature of these interactions. The location of the myosin binding is an extended region encompassing the junction of domains 3/4 and domain 4a (residues 622-714, human; 566-657, chicken gizzard). Tropomyosin binds in a smaller region within domain 4a of caldesmon (residues 663-714, human; 606-657 chicken gizzard). We confirmed predictions based on sequence similarities of a tropomyosin binding site in domain 3 of caldesmon; however, this site bound to skeletal-muscle tropomyosin and had little affinity for the smooth-muscle tropomyosin isoform. None of the protein fragments H2-H9 retained the affinity of the parent fragment H1 for either myosin or tropomyosin. This indicates the need for several interaction sites scattered over an extended region to attain higher affinity. The regions interacting with caldesmon in both tropomyosin and myosin are coiled-coil structures. This is probably the reason for their shared interaction sites on caldesmon and their similar natures of binding. Images Figure 1 Figure 2 Figure 9 PMID:8526878

  18. 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.

  19. 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

  20. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    SciTech Connect

    Petri, Marcelo H.; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

  1. Expression and proliferation profiles of PKC, JNK and p38MAPK in physiologically stretched human bladder smooth muscle cells

    SciTech Connect

    Wazir, Romel; Luo, De-Yi; Dai, Yi; Yue, Xuan; Tian, Ye; Wang, Kun-Jie

    2013-08-30

    Highlights: •Stretch induces proliferation in human bladder smooth muscle cells (HBSMC). •5% Equibiaxial elongation produces maximum proliferation. •Physiologic stretch decreases apoptotic cell death. •PKC is involved in functional modulation of bladder. •JNK and p38 are not involved in proliferating HBSMC. -- Abstract: Objective: To determine protein kinase C (PKC), c-Jun NH2-Terminal Kinase (JNK) and P38 mitogen-activated protein kinases (p38MAPK) expression levels and effects of their respective inhibitors on proliferation of human bladder smooth muscle cells (HBSMCs) when physiologically stretched in vitro. Materials and methods: HBSMCs were grown on silicone membrane and stretch was applied under varying conditions; (equibiaxial elongation: 2.5%, 5%, 10%, 15%, 20%, 25%), (frequency: 0.05, 0.1, 0.2, 0.5, 1 Hz). Optimal physiological stretch was established by assessing proliferation with 5-Bromo-2-deoxyuridine (BrdU) assay and flow cytometry. PKC, JNK and p38 expression levels were analyzed by Western blot. Specificity was maintained by employing specific inhibitors; (GF109203X for PKC, SP600125 for JNK and SB203580 for p38MAPK), in some experiments. Results: Optimum proliferation was observed at 5% equibiaxial stretch (BrdU: 0.837 ± 0.026 (control) to 1.462 ± 0.023)%, (P < 0.05) and apoptotic cell death rate decreased from 16.4 ± 0.21% (control) to 4.5 ± 0.13% (P < 0.05) applied at 0.1 Hz. Expression of PKC was upregulated with slight increase in JNK and no change in p38MAPK after application of stretch. Inhibition had effects on proliferation (1.075 ± 0.024, P < 0.05 GF109203X); (1.418 ± 0.021, P > 0.05 SP600125) and (1.461 ± 0.01, P > 0.05 SB203580). These findings show that mechanical stretch can promote magnitude-dependent proliferative modulation through PKC and possibly JNK but not via p38MAPK in hBSMCs.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Smooth Sailing.

    ERIC Educational Resources Information Center

    Price, Beverley; Pincott, Maxine; Rebman, Ashley; Northcutt, Jen; Barsanti, Amy; Silkunas, Betty; Brighton, Susan K.; Reitz, David; Winkler, Maureen

    1999-01-01

    Presents discipline tips from several teachers to keep classrooms running smoothly all year. Some of the suggestions include the following: a bear-cave warning system, peer mediation, a motivational mystery, problem students acting as the teacher's assistant, a positive-behavior-reward chain, a hallway scavenger hunt (to ensure quiet passage…

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Effective combination of hydrostatic pressure and aligned nanofibrous scaffolds on human bladder smooth muscle cells: implication for bladder tissue engineering.

    PubMed

    Ahvaz, Hana Hanaee; Soleimani, Masoud; Mobasheri, Hamid; Bakhshandeh, Behnaz; Shakhssalim, Naser; Soudi, Sara; Hafizi, Maryam; Vasei, Mohammad; Dodel, Masumeh

    2012-09-01

    Bladder tissue engineering has been the focus of many studies due to its highly therapeutic potential. In this regard many aspects such as biochemical and biomechanical factors need to be studied extensively. Mechanical stimulations such as hydrostatic pressure and topology of the matrices are critical features which affect the normal functions of cells involved in bladder regeneration. In this study, hydrostatic pressure (10 cm H(2)O) and stretch forces were exerted on human bladder smooth muscle cells (hBSMCs) seeded on aligned nanofibrous polycaprolactone/PLLA scaffolds, and the alterations in gene and protein expressions were studied. The gene transcription patterns for collagen type I, III, IV, elastin, α-SMA, calponin and caldesmon were monitored on days 3 and 5 quantitatively. Changes in the expressions of α-SMA, desmin, collagen type I and III were quantified by Enzyme-linked immuno-sorbent assay. The scaffolds were characterized using scanning electron microscope, contact angle measurement and tensile testing. The positive effect of mechanical forces on the functional improvement of the engineered tissue was supported by translational down-regulation of α-SMA and VWF, up-regulation of desmin and improvement of collagen type III:I ratio. Altogether, our study reveals that proper hydrostatic pressure in combination with appropriate surface stimulation on hBSMCs causes a tissue-specific phenotype that needs to be considered in bladder tissue engineering.

  18. TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

    SciTech Connect

    Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani . E-mail: chandraseka@uthscsa.edu

    2006-09-08

    CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-{kappa}B (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis.

  19. Meclofenamic Acid for Inhibition of Human Vascular Smooth Muscle Cell Proliferation and Migration: An In Vitro Study

    SciTech Connect

    Schober, Wolfgang; Kehlbach, Rainer; Gebert, Regina; Wiskirchen, Jakub; Rodegerdts, Enno; Claussen, Claus D.; Duda, Stephan H.

    2002-01-15

    Purpose: The aim of the study was to examine the effects of meclofenamic acid on proliferation, clonogenic activity,migratory ability, cell cycle distribution and p44/42 MAPK (mitogen activated protein kinase) expression in serum-stimulated human aortic smooth muscle cells (haSMCs). Methods: haSMCs were treated with meclofenamic acid in three different concentrations (10mM, 100 mM, 200 mM) for 4 days. Then meclofenamic acid-free culture medium was supplemented until day 20. Growth kinetics were assessed. Cell cycle analysis was performed by flow cytometry.Clonogenic activity was evaluated with colony formation assays.Migratory ability was investigated by stimulation with platelet-derived growth factor (PDGF-BB) in 24-well plates with 8 mm pores membrane inserts. p44/42 MAPK was detected by Western blot technique. Results: Meclofenamic acid inhibited the proliferation,clonogenic activity and migratory ability of haSMCs in a dose-dependent manner. Cell cycle analysis revealed a G2/M-phase block. The p44/42MAPK was significantly reduced. Conclusion: Meclofenamic acid inhibits the proliferation and migration of haSMCs. If a sufficient dose of meclofenamic acid can be applied systemically or by local drug delivery it could be a valuable substance to prevent restenosis after angioplasty.

  20. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

    SciTech Connect

    Weisinger, Gary . E-mail: gary_w@tasmc.health.gov.il; Limor, Rona; Marcus-Perlman, Yonit; Knoll, Esther; Kohen, Fortune; Schinder, Vera; Firer, Michael; Stern, Naftali

    2007-05-11

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

  1. Regulation of proliferation and gene expression in cultured human aortic smooth muscle cells by resveratrol and standardized grape extracts

    SciTech Connect

    Wang Zhirong; Chen Yan; Labinskyy, Nazar; Hsieh Tzechen; Ungvari, Zoltan; Wu, Joseph M. . E-mail: Joseph_Wu@nymc.edu

    2006-07-21

    Epidemiologic studies suggest that low to moderate consumption of red wine is inversely associated with the risk of coronary heart disease; the protection is in part attributed to grape-derived polyphenols, notably trans-resveratrol, present in red wine. It is not clear whether the cardioprotective effects of resveratrol can be reproduced by standardized grape extracts (SGE). In the present studies, we determined, using cultured human aortic smooth muscle cells (HASMC), growth and specific gene responses to resveratrol and SGE provided by the California Table Grape Commission. Suppression of HASMC proliferation by resveratrol was accompanied by a dose-dependent increase in the expression of tumor suppressor gene p53 and heat shock protein HSP27. Using resveratrol affinity chromatography and biochemical fractionation procedures, we showed by immunoblot analysis that treatment of HASMC with resveratrol increased the expression of quinone reductase I and II, and also altered their subcellular distribution. Growth of HASMC was significantly inhibited by 70% ethanolic SGE; however, gene expression patterns in various cellular compartments elicited in response to SGE were substantially different from those observed in resveratrol-treated cells. Further, SGE also differed from resveratrol in not being able to induce relaxation of rat carotid arterial rings. These results indicate that distinct mechanisms are involved in the regulation of HASMC growth and gene expression by SGE and resveratrol.

  2. Cyclic mechanical strain-induced proliferation and migration of human airway smooth muscle cells: role of EMMPRIN and MMPs.

    PubMed

    Hasaneen, Nadia A; Zucker, Stanley; Cao, Jian; Chiarelli, Christian; Panettieri, Reynold A; Foda, Hussein D

    2005-09-01

    Airway smooth muscle (ASM) proliferation and migration are major components of airway remodeling in asthma. Asthmatic airways are exposed to mechanical strain, which contributes to their remodeling. Matrix metalloproteinase (MMP) plays an important role in remodeling. In the present study, we examined if the mechanical strain of human ASM (HASM) cells contributes to their proliferation and migration and the role of MMPs in this process. HASM were exposed to mechanical strain using the FlexCell system. HASM cell proliferation, migration and MMP release, activation, and expression were assessed. Our results show that cyclic strain increased the proliferation and migration of HASM; cyclic strain increased release and activation of MMP-1, -2, and -3 and membrane type 1-MMP; MMP release was preceded by an increase in extracellular MMP inducer; Prinomastat [a MMP inhibitor (MMPI)] significantly decreased cyclic strain-induced proliferation and migration of HASM; and the strain-induced increase in the release of MMPs was accompanied by an increase in tenascin-C release. In conclusion, cyclic mechanical strain plays an important role in HASM cell proliferation and migration. This increase in proliferation and migration is through an increase in MMP release and activation. Pharmacological MMPIs should be considered in the pursuit of therapeutic options for airway remodeling in asthma.

  3. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.

    PubMed Central

    Schmitt-Gräff, A.; Krüger, S.; Bochard, F.; Gabbiani, G.; Denk, H.

    1991-01-01

    It has been suggested that perisinusoidal liver cells (PSC) play a pivotal role in the pathogenesis of fibrocontractive changes. Using light and electron microscopic immunolocalization techniques, a series of 207 normal and pathologic human liver specimens were evaluated for the expression of alpha smooth muscle (SM) actin and desmin in this and other nonparenchymal cell types. In normal adult liver tissue, PSCs were practically devoid of desmin and exceptionally stained for alpha-SM actin, whereas this actin isoform frequently was encountered in PSCs from the embryonic to the adolescent period. A broad spectrum of pathologic conditions was accompanied by the presence of alpha-SM actin containing PSCs; these were detected preferentially in periportal or perivenular zones according to the predominant location of the underlying hepatocellular damage. The occurrence of this PSC phenotype generally was associated with fibrogenesis and was in some cases detected earlier than overt collagen accumulation. Fibrous bands subdividing liver tissue in cirrhosis and focal nodular hyperplasia, as well as desmoplastic reaction to malignant tumors, contained alpha-SM actin-rich cells admixed with variable proportions of cells coexpressing desmin. In end stages, this population was less numerous than in active fibrotic or cirrhotic processes. Using immunogold electron microscopy, alpha-SM actin was localized in microfilament bundles of typical PSCs. Our results are compatible with the assumption that the appearance of alpha-SM actin and desmin-expressing myofibroblasts results at least in part from a phenotypic modulation of PSCs. Images Figure 1 Figure 2 PMID:2024709

  4. Sensory versus motor loci for integration of multiple motion signals in smooth pursuit eye movements and human motion perception.

    PubMed

    Niu, Yu-Qiong; Lisberger, Stephen G

    2011-08-01

    We have investigated how visual motion signals are integrated for smooth pursuit eye movements by measuring the initiation of pursuit in monkeys for pairs of moving stimuli of the same or differing luminance. The initiation of pursuit for pairs of stimuli of the same luminance could be accounted for as a vector average of the responses to the two stimuli singly. When stimuli comprised two superimposed patches of moving dot textures, the brighter stimulus suppressed the inputs from the dimmer stimulus, so that the initiation of pursuit became winner-take-all when the luminance ratio of the two stimuli was 8 or greater. The dominance of the brighter stimulus could be not attributed to either the latency difference or the ratio of the eye accelerations for the bright and dim stimuli presented singly. When stimuli comprised either spot targets or two patches of dots moving across separate locations in the visual field, the brighter stimulus had a much weaker suppressive influence; the initiation of pursuit could be accounted for by nearly equal vector averaging of the responses to the two stimuli singly. The suppressive effects of the brighter stimulus also appeared in human perceptual judgments, but again only for superimposed stimuli. We conclude that one locus of the interaction of two moving visual stimuli is shared by perception and action and resides in local inhibitory connections in the visual cortex. A second locus resides deeper in sensory-motor processing and may be more closely related to action selection than to stimulus selection.

  5. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  6. Activation of the phosphatidylinositol 3-kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation.

    PubMed

    Wang, Guoliang; Ma, Ning; Meng, Liukun; Wei, Yingjie; Gui, Jingang

    2015-12-01

    Over-activated PI3K/Akt signaling, a pathway strongly related to cancer survival and proliferation, has been reported recently to be involved in pulmonary artery smooth muscle cell apoptosis and proliferation in pulmonary hypertension (PH). In this study, we observed greatly increased lipocalin-2 (Lcn2) expression accompanied with over-activated PI3K/Akt signaling in a standard rat model of PH induced by monocrotaline. In view of the close relationship between Lcn2 and PI3K/Akt pathway, we hypothesized that the up-regulated Lcn2 might be a trigger of over-activated PI3K/Akt signaling in PH. Our results showed that Lcn2 significantly activated the PI3K/Akt pathway (determined by augmented Akt phosphorylation and up-regulated Mdm2) and significantly promoted proliferation (assessed by Ki67 staining) in cultured human pulmonary artery smooth muscle cells. Furthermore, we demonstrated that inhibition of Akt phosphorylation (LY294002) abrogated the Lcn2-promoted proliferation in cultured human pulmonary artery smooth muscle cells. In conclusion, Lcn2 significantly promoted human pulmonary artery smooth muscle cell proliferation by activating PI3K/Akt pathway. Further study on the role and mechanism of Lcn2 will help explore novel therapeutic strategies based on attenuating over-activated PI3K/Akt signaling in PH.

  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 muc