Blockade of adenosine A2B receptors ameliorates murine colitis

Background and purpose: The adenosine 2B (A2B) receptor is the predominant adenosine receptor expressed in the colon. Acting through the A2B receptor, adenosine mediates chloride secretion, as well as fibronectin and interleukin (IL)-6 synthesis and secretion in intestinal epithelial cells. A2B receptor mRNA and protein expression are increased during human and murine colitis. However, the effect of the A2B receptor in the activation of the intestinal inflammatory response is not known. In this study, we examined the effect of A2B receptor antagonism on murine colitis. Experimental approach: Dextran sodium sulphate (DSS)-treated mice and piroxicam-treated IL-10/ mice were used as animal models of colitis. The A2B receptor-selective antagonist, ATL-801, was given in the diet. Key results: Mice fed ATL-801 along with DSS showed a significantly lower extent and severity of colitis than mice treated with DSS alone, as shown by reduced clinical symptoms, histological scores, IL-6 levels and proliferation indices. The administration of ATL-801 prevented weight loss, suppressed the inflammatory infiltrate into colonic mucosa and decreased epithelial hyperplasia in piroxicam-treated IL-10/ mice. IL-6 and keratinocyte-derived chemokine (KC) concentrations in the supernatants of colonic organ cultures from colitic mice were significantly reduced by ATL-801 administration. Conclusions and implications: Taken together, these data demonstrate that the intestinal epithelial A2B receptor is an important mediator of pro-inflammatory responses in the intestine and that A2B receptor blockade may be an effectiv

Effects of experience and body size on refuge choice in the crayfish Orconectes immunis

We investigated whether refuge size or experience with a refuge affected the refuge use of male Orconectes immunis crayfish. Individuals were given choices among seven refuges for 10 consecutive days. Refuges were formed from equal length but different diameter PVC pipe and placed in an array in a random sequence. Three treatments were used. In the Novel Refuge treatment, individuals were placed in a new test arena with a new arrangement of cleaned refuges every day. In the Nonremoval treatment, individuals were left in the same arena with the same set of refuges each day. In the Removal treatment, individuals were removed from the refuges each day but placed back in the same arena with the same set of refuges after the refuges had been cleaned. We found that refuge occupation was correlated with an individual\u27s size; smaller crayfish tended to use smaller refuges than larger crayfish, even though all crayfish could fit in all of the different sized refuges. When first tested, individuals initially chose larger refuges than they would subsequently settle in, suggesting that under duress, they were not as particular about refuge characteristics. Individuals in the Nonremoval and Removal treatments were significantly more consistent in their refuge use than those in the Novel Refuge treatment, suggesting that experience with a particular refuge increased use of that refuge. Individuals from the Novel Refuge treatment that were housed for a month with a single refuge did not increase their use of that sized refuge more than those that were housed without a refuge, indicating that simply occupying a refuge of a given size did not affect refuge preference

Exposure of Induced Pluripotent Stem Cell-Derived Vascular Endothelial and Smooth Muscle Cells in Coculture to Hemodynamics Induces Primary Vascular Cell-Like Phenotypes

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation. Here we tested the hypothesis that hemodynamic exposure of iECs in coculture with iSMCs induces an in vivo-like phenotype. iECs and iSMCs were cocultured under vascular region-specific blood flow hemodynamics, and compared to hemodynamic cocultures of blood vessel-derived endothelial (pEC) and smooth muscle (pSMC) cells. Hemodynamic flow-induced gene expression positively correlated between pECs and iECs as well as pSMCs and iSMCs. While endothelial nitric oxide synthase 3 protein was lower in iECs than pECs, iECs were functionally mature as seen by acetylated-low-density lipoprotein (LDL) uptake. SMC contractile protein markers were also positively correlated between pSMCs and iSMCs. Exposure of iECs and pECs to atheroprone hemodynamics with oxidized-LDL induced an inflammatory response in both. Dysfunction of the transforming growth factor β (TGFβ) pathway is seen in several vascular diseases, and iECs and iSMCs exhibited a transcriptomic prolife similar to pECs and pSMCs, respectively, in their responses to LY2109761-mediated transforming growth factor β receptor I/II (TGFβRI/II) inhibition. Although there are differences between ECs and SMCs derived from iPSCs versus blood vessels, hemodynamic coculture restores a high degree of similarity in their responses to pathological stimuli associated with vascular diseases. Thus, iPSC-derived vascular cells exposed to hemodynamics may provide a viable system for modeling rare vascular diseases and testing new therapeutic approaches.This work was supported by a grant from the British Heart Foun-dation (FS/13/29/30024) (to F.S. and S.S.). All other authors were supported by the SBIR Grant NCATS R43TR001206

Heterologous Overexpression and Mutagenesis of the Human Bile Salt Export Pump (ABCB11) Using DREAM (Directed REcombination-Assisted Mutagenesis)

Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines

Effects of adenosine A2A receptor activation and alanyl-glutamine in Clostridium difficile toxin-induced ileitis in rabbits and cecitis in mice

<p>Abstract</p> <p>Background</p> <p>Severe <it>Clostridium difficile </it>toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A_2Areceptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A_2Areceptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of <it>C. difficile </it>toxin A-induced epithelial injury.</p> <p>Methods</p> <p>Toxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10.</p> <p>Results</p> <p>ATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A_2Areceptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10.</p> <p>Conclusions</p> <p>Combination therapy with an adenosine A_2Areceptor agonist and alanyl-glutamine is effective in reversing <it>C. difficile </it>toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of <it>C. difficile </it>infection.</p

Confocal microscopy 3D imaging of diesel particulate matter

To date, diesel particulate matter (DPM) has been described as aggregates of spherule particles with a smooth appearing surface. We have used a new colour confocal microscope imaging method to study the 3D shape of diesel particulate matter (DPM); we observed that the particles can have sharp jagged appearing edges and consistent with these findings, 2D light microscopy demonstrated that DPM adheres to human lung epithelial cells. Importantly, the slide preparation and confocal microscopy method applied avoids possible alteration to the particles' surfaces and enables colour 3D visualisation of the particles. From twenty-one PM particles, the mean (standard deviation) major axis length was 5.6 (2.25) μm with corresponding values for the minor axis length of 3.8 (1.25) μm. These new findings may help explain why air pollution particulate matter (PM) has the ability to infiltrate human airway cells, potentially leading to respiratory tract, cardiovascular and neurological disease