Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.

<p>Genes involved with a hyperproliferative response are shown.</p><p>Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced collagen expression.

<p>A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-collagen type I and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 3 separate occasions. (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) mRNA analysis Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1 (90 pM)). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown; * = p<0.05, One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 mRNA expression in human gingival fibroblasts.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. Values are expressed relative to untreated control. (N = 3; averages+/-SEM are shown; **** = p<0.0001, * = p<0.05 One-Way ANOVA).</p

(5Z)-7-oxozeaenol inhibits TGFβ1-induced mRNA expression in human gingival fibroblasts.

<p>(A) Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml^-1 (90 pM)) ligand or left untreated. Total RNA was harvested six hours later and subjected to gene expression profiling using GeneChip Human Gene 1.0 ST arrays (N = 2) as described in Methods. 147 genes were up-regulated in response to TGFβ1 (1.7 fold induction compared to DMSO control group) and 139 genes of the latter group were found to be (5<i>Z</i>)-7-Oxozeaenol sensitive. (B) Human gingival fibroblasts were treated as in (A) and subject to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown. * = p<0.05; ** = p<0.01; *** = p<0.001, One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 protein expression in human gingival fibroblasts.

<p>(A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-CCN2 and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 4 separate occasions and relative CCN2 expression in response to TGFβ1 was calculated using densitometry (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) Indirect immunofluorescence analysis. Human gingival fibroblasts cultured on glass coverslips as treated as in (A). Cells were fixed and stained with an anti-CCN2 antibody and DyLight 594 conjugated secondary antibody. Cells were counterstained with DAPI to detect nuclei. Representative photographs are shown. Experiments were conducted four times, and relative fluoresce intensity ratio was calculated as described in methods (N = 4, averages+/-SEM are shown. * = p<0.05, One-Way ANOVA). (C) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-phospho-TAK1 and anti-beta actin antibodies, as indicated.</p

(5Z)-7-oxozeaenol reduces TGFβ1 induced gingival fibroblast proliferation.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol ((5Z)-7-oxo; 400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml-1 (90 pM)) ligand or left untreated. Cultures were grown in the presence of BrdU for up to 72 hours as described in methods. One of three representative experiments is shown; (N = 4; averages+/-SEM are shown * p<0.05 for: DMSO vs TGFβ1, (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1; ** p<0.05 for: DMSO vs TGFβ1; (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1. Two-Way ANOVA followed by Tukey's Post Hoc analysis).</p

An investigation of the inter-molecular interaction, solid-state properties and dissolution properties of mixed copovidone hot-melt extruded solid dispersions

Previous research has focused on spray dried quaternary mixtures which, due to the addition of a surfactant, affected the physical stability and amorphous stability of selected model drugs. Very little research has focused on how inter-molecular interactions play a role in the successful formulation of hot-melt extruded quaternary amorphous blends and how they affect physical stability and solubility of amorphous solid dispersions (ASDs). Therefore the aim of this study was to investigate the role of inter-molecular interactions and their effect on the solid-state and dissolution properties of mixed copovidone amorphous solid dispersions (ASDs). The polymeric copovidone carriers used in this study were Poly (vinylpyrrolidone-vinyl acetate copolymer) and Plasdone S-630 (PL-S630) which in terms of monographs are the same, however they have different solid-state and dissolution properties. The ASDs showed a significantly higher dissolution rate compared to amorphous and pure INM in pH buffer 1.2 with a kinetic solubility of 24 μg/ml. The stability data showed that INM remained amorphous in solid solutions with PVP VA64 and Plasdone S-630, except for the higher drug loads. It was concluded that % drug loading did have a significant effect on the solubility of INM due to recrystallization at higher drug loads

Macrophage chitinase is required to lyse cysts in vitro.

<p>(A–E) Cysts purified from the brains of Me49 infected mice were cultured with or without BMDM in a 96 well plate. Cultures were imaged using an HT pathway microscope for 14 hours. Images were collected every 10 minutes and cyst survival time was calculated (A–D) Time-lapse images were taken from (A) cysts alone without macrophages, (B) cysts treated with 10 µg/ml trichoderma chitinase, (C) cysts with macrophages, and (D) cysts with macrophages treated with 100 µM allosamidin. (E) Graph of allosamidin concentration and cyst survival time. Data are representative of at least 3 individual experiments with a minimum of n = 6 and are represented as mean ± SEM, * p<0.05.</p

AMCase−/− mice have a higher parasite burden in the brain and succumb to infection during the chronic stage.

<p>(A–F) C57Bl/6 (WT) and AMCase−/− mice were infected with the Me49 strain of <i>T. gondii</i>. Serum was isolated from whole blood samples at days 3, 7 and 14 post infection and analyzed for (A) IFN-γ, (B) IL-6, (C) MCP-1, (D) TNF-α, (E) IL-12p70 (F) At day 7 DNA was isolated from the lungs and analyzed for parasite burden using qPCR. Results are displayed as parasites per mg tissue. (G–K) C57Bl/6 (WT) and AMCase−/− mice were infected with the Me49 strain of <i>T. gondii</i> and sacrificed at 5 weeks following infection. Brains were harvested and analyzed for cyst burden, cellular composition and histology. (G) Cyst counts were obtained from homogenized whole brain samples. (H) Cyst area, 20 cysts from each mouse were photographed microscopically and cyst area was determined using ImageJ software. (I) DNA from brains of WT and AMCase−/− was isolated and analyzed for parasite burden using qPCR. (J) Whole brains were fixed, frozen and stained for H&E to examine cyst burden and pathology. (K) BMNCs were isolated and analyzed for expression of CD4+ T cells, CD8+ T cells, macrophages (CD45^hi/CD11b+) and microglia (CD45^hi/CD11b+) by flow cytometry. Significance was determined using log rank test with p = 0.0177. Data are representative of at least 2 individual experiments with a minimum of n = 4 and are represented as mean ± SEM, ns = not significant, * p<0.05, ** p<0.01. (L) Survival data from C57Bl/6 (squares, n = 7) and AMCase−/− (triangles, n = 7). Data are representative of 4 individual experiments with C57Bl/6 (n>40) and AMCase−/− (n = 40) and significance tested using Log-rank (Mantel-Cox) and Gehan-Breslow Wilcoxan test * p<0.05.</p

Acidic mammalian chitinase is required to lyse cysts in vitro.

<p>(A) BMDM from WT and AMCase−/− mice were analyzed fluorometrically for chitinase activity. Chitinase activity from WT and AMCase−/− macrophages cultured with bradyzoites, tachyzoites, whole cysts, IL-4 or media alone. (B–D) Me49-RFP cysts purified from the brains of infected mice were cultured with or without CellTracker green labeled BMDM from either WT or AMCase null mice. Cultures were imaged using an HT pathway microscope for 16 hours. Images were collected every 10 minutes and cyst survival time was calculated. (B–C) Time-lapse images were taken from cyst cultures (B) WT macrophages or (C) AMCase−/− macrophages. (D) Graphical representation of cyst survival time over 16 hours. (E) Chitinase activity from WT bone marrow-derived macrophages cultured with whole cysts, whole cysts+LPS/IFN-γ, or media alone. Data are representative of at least 3 individual experiments with a minimum of n = 6 and are represented as mean ± SEM, * p<0.05, ** p<0.01 *** p<0.001.</p