Journal: bioRxiv

Article Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier inflammation of the vasculature, meninges and choroid plexus

doi: 10.1101/2020.04.15.043588

Figure Lengend Snippet: (A-B) Pericytes were treated with IL-1β for 24 hours and immunostained for CCL2 and ICAM-1 protein expression. Integrated intensity per cell was calculated using total cells (Hoechst), n = 3, significance determined using one-way ANOVA with Dunnett’s correction for multiple comparisons, **** p <0.0001. (C-D) Pericytes were screened for compounds that can modify IL-1β-induced CCL2 or ICAM-1 expression using the Prestwick chemical library. Integrated intensity per cell was calculated using total cell counts and normalized to IL-1β + vehicle condition (arrow)(blue arrow-digoxin, red arrow-lanatoside C, black arrows-hit compounds tested in (G)). Internal controls were present on each drug plate (TGFβ 1 , 1 or 10 ng/mL). E-F). Hits identified using cut-off criteria from primary screen that modified CCL2 or ICAM-1 expression with known therapeutic uses. (G) Conditioned media from pericytes pre-treated with hit compounds for 24 hours, then stimulated with IL-1β (0.05 ng/mL) for 24 hours was analysed by CBA, data was normalized to cell number and presented as the logged value of cytokine secretion in pg/mL/10,000 cells. Control (vehicle for IL-1β (0.01% BSA in PBS), IL-1β (0.05 ng/mL + dimethylsulfoxide (DMSO))(n = 2, media pooled from triplicate wells for analysis). (H) Drug screening pipeline in primary human brain pericytes. Initial screen in pericytes completed in duplicate wells and secondary screen in 83 compounds that met cut-off criteria was completed in triplicate wells. Effects that were reproduced were examined using a 3 point concentration test, then tested for effects on cell secretion (data in fig. S1).

Article Snippet: Secretome analysis of the clarified media was performed using the Proteome ProfilerTM Human XL Cytokine Array Kit (R & D Systems), as per manufacturer’s instructions.

Techniques: Expressing, Modification, Control, Drug discovery, Concentration Assay

Journal: bioRxiv

Article Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier inflammation of the vasculature, meninges and choroid plexus

doi: 10.1101/2020.04.15.043588

Figure Lengend Snippet: Endothelial cells derived from human brain tissue express endothelial markers, Claudin-5 (A), ZO-1, (B), ERG (C), and CD31 (D). (E-G) Human endothelial cells were pretreated with digoxin (100 nM) or lanatoside C (1 µM) for 24 hours, followed by 24 hours of IL-1β (0.05 ng/mL) treatment. Staining quantification of CCL2 (E), ICAM-1 (F), and nuclear CEBPδ (G), in endothelial cells. Representative images of (n = 3) (H, I). (J-R) Conditioned media was analysed by CBA in endothelial cells treated as above (n = 3). Cytokine/chemokine concentration was normalized to total cell counts (Hoechst)). Two-way ANOVA with Tukey’s multiple comparison test *** p <0.001, ** p <0.01, * p <0.05.

Article Snippet: Secretome analysis of the clarified media was performed using the Proteome ProfilerTM Human XL Cytokine Array Kit (R & D Systems), as per manufacturer’s instructions.

Techniques: Derivative Assay, Staining, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier inflammation of the vasculature, meninges and choroid plexus

doi: 10.1101/2020.04.15.043588

Figure Lengend Snippet: A) Immunohistochemical staining of LME for vascular cells types including pericytes (PDGFRβ) endothelial cells (CD31), fibroblasts (PDLIM3), smooth muscle cells (αSMA) and extracellular matrix protein deposition (COL1A1). (B-G) Secretions from meningeal explants following inflammatory stimulation were investigated using cytometric bead arrays (n = 2 cases, 3-4 explants per case), values normalized to vehicle condition, raw values Fig S7, 8. (H) Proteome profilers were used to detect secretions from human brain meningeal explants (pooled from 5 explants per condition, one case) after 24 hours of inflammatory stimulation with vehicle, IFNγ, IL-1β, or LPS (10 ng/mL), raw intensities were normalized to reference spots.

Article Snippet: Secretome analysis of the clarified media was performed using the Proteome ProfilerTM Human XL Cytokine Array Kit (R & D Systems), as per manufacturer’s instructions.

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier inflammation of the vasculature, meninges and choroid plexus

doi: 10.1101/2020.04.15.043588

Figure Lengend Snippet: A-I) meningeal explant secretions were measured using CBA (n = 3-5 explants per condition, 5 cases). Secretions in pg/mL were normalized to vehicle for each case-arbitrary units (A.U.) except for G-CSF, GM-CSF and RANTES where cytokines were below the detection level in vehicle conditions. Two-way ANOVA with Tukey’s multiple comparison test **** p <0.0001, *** p < 0.001, ** p < 0.01, * p <0.05. J) Proteome profilers were used to characterise LME secretions in response to vehicle or IL-1β (10 ng/mL) with either digoxin or lanatoside C pre-treatment (10 µM). Average intensity values from proteome profilers were normalised to the reference spots on each blot (heatmap is average of n=3 cases, each pooled from 3-4 explants per case).

Article Snippet: Secretome analysis of the clarified media was performed using the Proteome ProfilerTM Human XL Cytokine Array Kit (R & D Systems), as per manufacturer’s instructions.

Techniques: Comparison

Journal: bioRxiv

Article Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier inflammation of the vasculature, meninges and choroid plexus

doi: 10.1101/2020.04.15.043588

Figure Lengend Snippet: (A, B) Immunohistochemical staining of CPE for vascular cells types including pericytes (PDGFRβ) endothelial cells (CD31), fibroblasts (PDLIM3), smooth muscle cells (αSMA) and epithelial cells (transthyretin (TTR)). (C-K) Secretions from CPE following inflammatory stimulation were investigated using cytometric bead arrays (n = 3 cases, 3 explants per case), values normalized to vehicle condition, raw values Fig S8, 9. One way ANOVA, Dunnett’s multiple comparisons test, *** p < 0.001, ** p < 0.01, * p <0.05. (L) Proteome profilers were used to detect secretions from human brain choroid plexus explants (average of n = 2 cases each pooled from 3 explants per condition) after 24 hours of inflammatory stimulation with vehicle, IFNγ, IL-1β, or LPS (10 ng/mL), raw intensities were normalized to reference spots.

Article Snippet: Secretome analysis of the clarified media was performed using the Proteome ProfilerTM Human XL Cytokine Array Kit (R & D Systems), as per manufacturer’s instructions.

Techniques: Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier inflammation of the vasculature, meninges and choroid plexus

doi: 10.1101/2020.04.15.043588

Figure Lengend Snippet: A-I) Choroid plexus explants were treated with vehicle, digoxin or lanatoside C (both 10 µM) for 24 hours followed by IL1β (10 ng/mL) for 24 hours. Secretions were measured using CBA (n=2 cases, 3 explants per case). Values normalized to vehicle for each case, except for G-CSF, GM-CSF and RANTES as vehicle condition was under the level of detection, (raw data provided in supplementary, Fig S9). J) Proteome profiler analysis of pooled choroid plexus explant secretions (3 pooled explants per case). Data are presented from the average of each case normalised to reference point.

Article Snippet: Secretome analysis of the clarified media was performed using the Proteome ProfilerTM Human XL Cytokine Array Kit (R & D Systems), as per manufacturer’s instructions.

Techniques: