Journal: PLoS ONE

Article Title: A GPBAR1 (TGR5) Small Molecule Agonist Shows Specific Inhibitory Effects on Myeloid Cell Activation In Vitro and Reduces Experimental Autoimmune Encephalitis (EAE) In Vivo

doi: 10.1371/journal.pone.0100883

Figure Lengend Snippet: (A) Immune cell populations (CD3 + T cells, CD19 + B cells, CD56 + NK cells, Neutrophils and CD14 + monocytes) were enriched from blood and Gpbar1 mRNA levels were assessed by microarray using Affymetrix chip technology. Absolute expression is normalized to relative expression of 62 control probes across the entire chip. CHO cells expressing human GPBAR1 were generated and stimulated with GPBAR1 agonists Lithocholic acid (B) or EX00000246 and BIX02694 (C) and cAMP production was assessed. The chemical structures of the molecules are shown in each case. Human monocytes (4 donors, assayed in triplicate) were isolated by negative selection and pre-treated with increasing concentrations of Lithocholic acid (LCA) or EX00000246 or BIX02694 at 1 µM for 30 minutes before stimulation with IFNγ + LPS and data is presented as percent of control (POC) (D–G) or pg/ml (H, I). Controls with no ligand were all treated with 0.1% DMSO in media. Supernatants were collected after 24 hours and assessed for IL-12p40 (D, F, H) and TNFα (E, G, I) by a sandwich immunoassay. Production of IL-12p40 (J) and TNFα (K) by LPS-stimulated monocytes was assessed in the presence of histamine and isoprenaline at 1 µM. Protein levels of IL-12p40 and TNFα were assessed after 24 hours.

Article Snippet: Levels of cytokine in the supernatants were determined by sandwich immunoassay (TNFα and IL-12p40, MesoScale Discovery, as individual analytes or in a multiplex between IL-12p40, IL-6, IL-1β and TNFα proinflammatory 39-plex, MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel, HCYTOMAG-60K proinflammatory, Millipore for BioRad FlexMap 3D.

Techniques: Microarray, Expressing, Control, Generated, Isolation, Selection

Journal: PLoS ONE

Article Title: A GPBAR1 (TGR5) Small Molecule Agonist Shows Specific Inhibitory Effects on Myeloid Cell Activation In Vitro and Reduces Experimental Autoimmune Encephalitis (EAE) In Vivo

doi: 10.1371/journal.pone.0100883

Figure Lengend Snippet: (A) Production of cytokines at 24 hrs by LPS-stimulated monocytes (1 donor, quadruplicate) was assessed in the presence of EX00000246 (500 nM) or Lithocholic acid (10 µM) by BioRad FlexMap 3D technology using a multi-plex kit purchased from Millipore.Human monocytes (B) and monocyte-derived macrophages (C) (4 donors, assayed in triplicate) were stimulated with various TLR ligands in the presence of EX00000246 and production of IL-12p40 as measured by MSD was assessed at 24 hrs. Untreated negative controls reported cytokine levels at or below the detection limit and therefore are not plotted in these graphs.

Article Snippet: Levels of cytokine in the supernatants were determined by sandwich immunoassay (TNFα and IL-12p40, MesoScale Discovery, as individual analytes or in a multiplex between IL-12p40, IL-6, IL-1β and TNFα proinflammatory 39-plex, MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel, HCYTOMAG-60K proinflammatory, Millipore for BioRad FlexMap 3D.

Techniques: Derivative Assay

Journal: PLoS ONE

Article Title: A GPBAR1 (TGR5) Small Molecule Agonist Shows Specific Inhibitory Effects on Myeloid Cell Activation In Vitro and Reduces Experimental Autoimmune Encephalitis (EAE) In Vivo

doi: 10.1371/journal.pone.0100883

Figure Lengend Snippet: (A) Gpbar1 and 18s mRNA levels were assessed by Taqman in C57BL/6 mouse whole blood and isolated leukocytes (n = 2 for leukocytes, n = 5 for blood). (B–C) GPBAR1 protein levels were assessed on mouse blood cells by flow cytometry using CD115 to stain monocytes. (B) Gating scheme and representative histograms of lymphocytes, granulocytes and monocytes are shown (n = 1 is shown, representative of 3 mice). (C) Median fluorescence intensity of GPBAR1 on monocytes, granulocytes, and lymphocytes is shown (n = 3, averaged). Monocytes stained with an isotype control are the control sample (B) and (C). (D–F) Impact of GPBAR1 agonism on LPS induced cytokines. Blood from C57BL/6 mice (n = 5, averaged) was stimulated ex vivo with 100 ng LPS in the presence of BIX02694 and intracellular TNFα in monocytes was measured by flow cytometry (D). Balb/c mice were challenged with 2 µg LPS, in the presence of vehicle, Prednisolone (3 mg/kg), or BIX02694 (0.3, 3, 10 mg/kg) and serum cytokine levels were measured; TNFα at 1 hour (E) IL-12p40 (F) and CCL2 (G) both at 4 hours post LPS administration.

Article Snippet: Levels of cytokine in the supernatants were determined by sandwich immunoassay (TNFα and IL-12p40, MesoScale Discovery, as individual analytes or in a multiplex between IL-12p40, IL-6, IL-1β and TNFα proinflammatory 39-plex, MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel, HCYTOMAG-60K proinflammatory, Millipore for BioRad FlexMap 3D.

Techniques: Isolation, Flow Cytometry, Staining, Fluorescence, Control, Ex Vivo

Journal: Biomedicines

Article Title: Cow Milk Extracellular Vesicle Effects on an In Vitro Model of Intestinal Inflammation

doi: 10.3390/biomedicines10030570

Figure Lengend Snippet: The 10 10 mEV impact on cytokine production by Caco-2. Caco-2 and THP-1 cells were left untreated (basal condition, C0) or stimulated with IFN-γ and LPS (inflamed co-culture, C1). At 24 h post stimulation, 10 10 mEV suspension was added to Caco-2 cells in inflamed co-culture (C2a) or to both Caco-2 and THP-1 cells in inflamed co-culture (C2b). A total of 24 h later, culture supernatants were collected, and levels of cytokines were determined through ELISA. Data are presented as box-and-whisker plots displaying median and interquartile range (boxes) and minimum and maximum values (whiskers). Values of C0, C2a, C2b were compared to C1, using an ANOVA followed by Dunnett’s multiple comparison test or a Kruskal–Wallis test followed by Dunn’s multiple comparison test; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12p70, IL-12p40, IL17, TNF-α were determined using MILLIPLEX ® Premixed Human Cytokine Panel A (Merck Millipore, Darmstadt, Germany) and a Bioplex MAGPIX Multiplex Reader (Bio-Rad, Hercules, CA, USA), according to the manufacturers’ instructions, as previously described [ ].

Techniques: Co-Culture Assay, Suspension, Enzyme-linked Immunosorbent Assay, Whisker Assay, Comparison

Journal: Biomedicines

Article Title: Cow Milk Extracellular Vesicle Effects on an In Vitro Model of Intestinal Inflammation

doi: 10.3390/biomedicines10030570

Figure Lengend Snippet: The 10 10 mEV impact on cytokine production by THP-1. Co-culture of Caco-2 and THP-1 cells were left untreated (basal condition, C0) or stimulated with IFN-γ and LPS (inflamed co-culture, C1). At 24 h post stimulation, 10 10 mEV suspension was added to both Caco-2 and THP-1 cells in inflamed co-culture (C2b). A total of 24 h later, culture supernatants were collected, and levels of cytokines were determined through ELISA. Data are presented as box-and-whisker plots displaying median and interquartile range (boxes) and minimum and maximum values (whiskers). Values of C0, C2a, C2b were compared to C1, using an ANOVA followed by Dunnett’s multiple comparison test or a Kruskal–Wallis test followed by Dunn’s multiple comparison test; * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Levels of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12p70, IL-12p40, IL17, TNF-α were determined using MILLIPLEX ® Premixed Human Cytokine Panel A (Merck Millipore, Darmstadt, Germany) and a Bioplex MAGPIX Multiplex Reader (Bio-Rad, Hercules, CA, USA), according to the manufacturers’ instructions, as previously described [ ].

Techniques: Co-Culture Assay, Suspension, Enzyme-linked Immunosorbent Assay, Whisker Assay, Comparison