Journal: EBioMedicine

Article Title: Intradermal vaccination prevents anti-MOG autoimmune encephalomyelitis in macaques

doi: 10.1016/j.ebiom.2019.08.052

Figure Lengend Snippet: Cytokine levels in plasma of treated and control animals. a) Heatmap showing cytokine levels in the plasma of all animals at several time points after sensitisation with rhMOG/IFA. Cytokine levels are represented by a colour gradient, ranging from yellow (no expression) to deep blue (highest concentration). Hierarchical clustering, represented by dendrograms, was performed at individual and cytokine levels. Four groups of animals were identified and are numbered from 1 to 4 in the text: animals treated with anti-DC-ASGPR-MOG are named T1, T2, and T3, controls treated with anti-DC-ASGPR-PSA, C1, C2, and C3, and untreated non-immunised naïve animals, 1, 2, 3, and 4; timepoints in dpi are numbered from D7 to D37; group I (red), group II (green), group III (purple), group IV (yellow); see also Supplemental table 4). b) Cytokine levels (expressed in log10 of pg/ml) in naïve animals relative to those measured in “EAE incubation” group II, in which there were significantly lower TGFβ1, TGFβ2, and IL-8 levels. c) Cytokine levels measured in “EAE resolution” group III, in which there were significantly higher TGFβ1, TGFβ2 and IL-8 levels in treated animals than naïve macaques. d) Varying cytokine levels between the two groups of treated and control animals and the various timepoints. In green, animals treated with anti-DC-ASGPR-MOG (T); in blue, control animals treated with anti-DC-ASGPR-PSA (C) and timepoints in day (d) post-sensitisation with rhMOG/IFA. The levels of the pro-inflammatory cytokines IL-1β, IFNγ, and TNFα were elevated at 35 dpi in controls but not treated animals. The levels of IL-8, TGFβ1, and TGFβ2 were elevated in treated animals at the last timepoint of 35 dpi, but not in controls. Statistics: exploratory analysis, with no multiple test correction, using the two-tailed unpaired t-test. (ns) p > .05; (*) p ≤ .050; (**) p ≤ .010; (***) p ≤ .0010. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Intracellular staining with anti-IL10-PE (JES3-9D7, BD) and anti-TGFβ1-AF488 (Mouse 9016, R&D system) and extracellular staining with anti-LAP-FITC (TGFβ1) (CH6-17E5.1, Miltenyi Biotec) of macaque PBMCs were also tested but did not permit detection of their targets.

Techniques: Clinical Proteomics, Control, Expressing, Concentration Assay, Incubation, Two Tailed Test

Journal: Molecular Metabolism

Article Title: Beta-hydroxybutyrate dampens adipose progenitors’ profibrotic activation through canonical Tgfβ signaling and non-canonical ZFP36 -dependent mechanisms

doi: 10.1016/j.molmet.2022.101512

Figure Lengend Snippet: βOHB counteracts Tgfβ-mediated stimulation of ECM genes . Combinatorial treatment with browning agents and Tgfb of visceral (A) or subcutaneous progenitors (B) on the expression of browning genes. βOHB (50 mM) or Bmp4 (10 ng/ml) was added 24 h after plating and Tgfβ (10 ng/ml) was added the following day, until day 5. Bars are mean+/- sem of three independent cell preparations. The same experimental scheme was used in (C–H) , to assess Ctgf , Loxl2 and Fn1 mRNA responses. Each point is an individual well from triplicates in at least 4 independent cell preparations. I: Dose-dependent response of Ctgf , Loxl2 and Fn1 gene expression to βOHB in the presence of Tgfβ in primary subcutaneous progenitors. Parentheses indicate significant differences between conditions, by Student's t test. J: Western blot analysis of Collagen1 protein expression in Tgfβ-stimulated visceral progenitors in the presence of βOHB or Acetoacetate. βOHB or Acetoacetate was added 1 day post plating and was maintained until cell harvest (Day 5). Chronic Tgfβ stimulation started from Day 2 to Day 5. Collagen1 antibody was from Proteintech, ref14695-1. Representative blot is shown, with quantification from 2 to 4 independent cell preparations.

Article Snippet: Tgfβ1 (human recombinant, ref 130-126-724) was from Myltenyi Biotech, Bmp4 (Human recombinant, ref PHC9534) from Invitrogen.

Techniques: Expressing, Gene Expression, Western Blot

Journal: Molecular Metabolism

Article Title: Beta-hydroxybutyrate dampens adipose progenitors’ profibrotic activation through canonical Tgfβ signaling and non-canonical ZFP36 -dependent mechanisms

doi: 10.1016/j.molmet.2022.101512

Figure Lengend Snippet: Mechanisms of fibrotic attenuation by βOHB . A : Smads mRNA expression in the presence of βOHB or chronic Tgfβ. Bars are mean values ± sem from 4 independent cell preparations. B: A representative western blot probed with antibodies against Smad2-3 (Cell signalling, ref 8685), Phospho Smad2 (Ser465/467)/Smad3 (Ser423/425) (Cell signalling, ref 8828 and Caveolin 1 (BD Transduction Laboratory, ref 610,060). βOHB concentration range is as in Figure 1 , in the presence or absence of a chronic Tgfβ stimulation. C-D : Quantitative analysis of protein signal intensity after normalization with Caveolin-1 as a loading control. Bars are mean values from 3 independent cell preparations. Parentheses indicate significant differences between groups by t test. E: ZFP36 mRNA upregulation by βOHB. F: Expression of ZFP36 and related transcripts by βOHB and Tgfβ in subcutaneous and visceral progenitors. Bars are mean values ± sem from 3 to 5 independent cell preparations. ∗ indicate significant differences compared to basal by Student t test. G–H: Gene expression in fibroblast cell lines from Wild Type (WT) or ZFP36 KO mice. Note that in KO mice, ZFP36 mRNA is transcribed but contains an insertion in exon 2 which prevents protein production. I-J: Knock-down of ZFP36 with siRNA in primary progenitors maintained with Tgfβ with or without βOHB. Bars are mean values from 3 independent experiments. Parentheses indicate significant differences between groups by t test.

Article Snippet: Tgfβ1 (human recombinant, ref 130-126-724) was from Myltenyi Biotech, Bmp4 (Human recombinant, ref PHC9534) from Invitrogen.

Techniques: Expressing, Western Blot, Transduction, Concentration Assay, Control, Gene Expression, Knockdown

Journal: Cell Reports Medicine

Article Title: Human astrocytes and microglia show augmented ingestion of synapses in Alzheimer’s disease via MFG-E8

doi: 10.1016/j.xcrm.2023.101175

Figure Lengend Snippet:

Article Snippet: Recombinant human TGFβ-1 , Miltenyi , 170-076-166.

Techniques: Labeling, Blocking Assay, Cell Isolation, Recombinant, Plasmid Preparation, Protease Inhibitor, Western Blot, Molecular Weight, Marker, Software

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: A CD8 T Cell/Indoleamine 2,3-Dioxygenase Axis Is Required for Mesenchymal Stem Cell Suppression of Human Systemic Lupus Erythematosus

doi: 10.1002/art.38674

Figure Lengend Snippet: Lupus CD8+ T cell–derived interferon-γ (IFNγ) promotes indoleamine 2,3-dioxygenase (IDO) production. A and B, Levels of IFNγ in supernatant are increased in the presence of UC-MSCs and SLE CD8+ T cells (A), while no change in levels of transforming growth factor β (TGFβ) is observed (B). Symbols represent individual subjects; horizontal lines show the mean (n = 7 per group). C, CD8+ T cells derived from patients with SLE produce much higher levels of intracellular IFNγ as compared to other cell subsets. D, Recombinant anti-human IFNγ antibody significantly abrogates SLE CD+ T cell–mediated IDO mRNA expression in UC-MSCs. E, Kynurenine levels in supernatant decrease in the presence of anti-human IFNγ antibody. F–H, In cocultured lupus peripheral blood mononuclear cells (PBMCs) and UC-MSCs, anti-human IFNγ antibodies (10 μg/ml) significantly inhibit kynurenine levels in supernatant (F), while the level of tryptophan is increased (G), and the ratio of kynurenine to tryptophan is decreased (H) (effects similar to those observed when 1-methyl- dl -tryptophan [1-DL-MT] is added). I, In the coculture system described above, UC-MSC–mediated inhibition of CD4+ T cell proliferation is abrogated by the addition of anti-human IFNγ antibody. Bars in C–I show the mean ± SEM (n = 7 per group in C, 8 per group in D, 8 per group in E, 5 per group in F–H, and 5 per group in I). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by one-way ANOVA followed by Bonferroni test. PE = phycoerythrin; FITC = fluorescein isothiocyanate; APC = allophycocyanin (see for other definitions). Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38674/abstract .

Article Snippet: The human TGFβ1 ELISA kit was from BioLegend.

Techniques: Derivative Assay, Recombinant, Expressing, Inhibition

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: A CD8 T Cell/Indoleamine 2,3-Dioxygenase Axis Is Required for Mesenchymal Stem Cell Suppression of Human Systemic Lupus Erythematosus

doi: 10.1002/art.38674

Figure Lengend Snippet: Defective indoleamine 2,3-dioxygenase (IDO) production in lupus bone marrow–derived MSCs (BM-MSCs). A–C, Expression of IFNGR1 (A), JAK-2 (B), and STAT-1 (C) was examined by real-time quantitative polymerase chain reaction. D, STAT-1, STAT-3, STAT-5, and their phosphorylated forms were assessed by Western blot analysis after treatment with UC-MSCs alone, UC-MSCs with different cell subsets (CD4+, CD8+, and non-CD4/CD8 T cells), or recombinant human interferon-γ (IFNγ). E, IDO mRNA expression in UC-MSCs, BM-MSCs from healthy controls, and BM-MSCs from SLE patients was examined after stimulation with different cytokines for 48 hours. F and G, Peripheral blood CD8+ T cells derived from patients with lupus were purified and used to stimulate UC-MSCs and BM-MSCs from healthy controls or lupus patients. Forty-eight hours later, IDO gene expression (F) and kynurenine enzyme activity (G) were determined. H, The ability of BM-MSCs from healthy controls and lupus patients to inhibit CD4+ T cell proliferation was compared. Bars in A–C and E–H show the mean ± SEM (n = 7 per group in A–D, 6 per group in E, 3 per group in F and G, and 4 per group in H). ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by one-way ANOVA. TGFβ = transforming growth factor β; IL-6 = interleukin-6; PBMC = peripheral blood mononuclear cell; NS = not significant (see for other definitions).

Article Snippet: The human TGFβ1 ELISA kit was from BioLegend.

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Recombinant, Purification, Gene Expression, Activity Assay