SERT function in response to TGF-β1 treatment of native mouse ileum.

<p>The distal small intestine was stripped off of the muscle layer, mounted in the Ussing chamber to expose tissue on both sides to Kreb’s solution supplemented with L-ascorbic acid (100 μM) and the monoamine oxidase inhibitor pargyline (100 μM). Tissues were treated with TGF-β1 for 1h from basolateral side and then incubated with ³[H]-5-HT for 30 min. Fluoxetine (10 μM) was added 30 min prior to TGF-β1 treatment from the apical side. Values represent mean ± SEM of 3 different experiments. *<i>P</i><0.05 compared with untreated control.</p

TGF-β1 increases SERT levels on the luminal surface in 3D-culture of Caco-2 cells.

<p>Caco-2 cells in 3D culture system untreated or treated with TGF-β1 (10 ng/ml, 1h) were stained for SERT (green), Phalloidin (red) and DAPI (blue) and visualized by confocal microscopy. XY planar images and orthogonal XZ images Orthognal <i>xz</i> images were obtained with a Zeiss LSM 510 confocal microscope.</p

BL-7010 Demonstrates Specific Binding to Gliadin and Reduces Gluten-Associated Pathology in a Chronic Mouse Model of Gliadin Sensitivity

<div><p>Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-<i>co</i>-styrene sulfonate) (P(HEMA-<i>co</i>-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated <i>in vivo</i> (in rats) and <i>in vitro</i> (genetic toxicity studies). <i>In vitro</i> binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.</p></div

Administration of BL-7010 (Polymers A and B) decreased CD3⁺ intraepithelial cells in villi tips of gliadin-sensitized mice.

<p>Small intestinal CD3⁺ stained small intestinal tissues from (A) non-sensitized mice (control) (B) gliadin-sensitized mice (gluten) (C) gliadin-sensitized mice, receiving Polymer A (D) gliadin-sensitized mice, receiving Polymer B. (E) Quantification of CD3⁺ cells/100 enterocytes in villi tips. Stained sections were viewed via optical microscopy (20X magnification). Bars represent the mean +/− SEM, statistical analysis was completed via one-way ANOVA and Bonferroni's post-hoc test (***p<0.001, ****p<0.0001) (n = 11–13).</p

Administration of BL-7010 (Polymers A and B) decreased paracellular permeability and PAT-1 mRNA levels in gliadin-sensitized mice.

<p>(A) Gliadin-sensitized mice had higher ⁵¹Cr-EDTA flux in comparison to non-sensitized control mice, and administration of Polymers A or B decreased it to the same level as the non-sensitized control mice. (B) Gliadin-sensitized mice had higher mRNA levels of PAT-1 in comparison to non-sensitized control mice, and administration of Polymers A or B to sensitized mice decreased expression to the same level as non-sensitized control mice. Bars represent the mean +/− SEM, statistical analysis was completed via one-way ANOVA and Bonferroni's post-hoc test (**p<0.01, ***p<0.001, ****p<0.0001) (n = 12–13).</p

An overlay of sensograms showing Polymer A interaction with gliadin at a series of concentrations of 10-640 ng/mL.

<p>KD was determined to be 12.1 nM, with kon of 2.39×105 1/M·s and koff of 2.89×10-3 1/s. B. An overlay of sensograms showing Polymer B interacting with gliadin, with KD of 2.44 nM, kon of 6.11×105 1/M·s and koff of 1.49×10-3 1/s using Biacore Evaluation Software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109972#s2" target="_blank">Methods</a>). For both graphs, colored lines indicate experimental sensograms at various concentrations, while corresponding black lines denote fitting of experimental data using 1∶1 binding model with global Rmax.</p

Interaction of BL-7010 (Polymer A) with gliadin, BSA, proteolytic enzymes and vitamins.

1<p>“No Interaction” means that no interaction signal (no increase in resonance units) was detected while increasing the concentration of the tested material and therefore no kinetics parameters could be calculated.</p><p>Interaction of BL-7010 (Polymer A) with gliadin, BSA, proteolytic enzymes and vitamins.</p