Journal: Nature Communications

Article Title: NETosis and thrombosis in vaccine-induced immune thrombotic thrombocytopenia

doi: 10.1038/s41467-022-32946-1

Figure Lengend Snippet: a PF4 and PF4-heparin ELISA experiment of VITT serum and controls. The cut-off, 0.50 OD units (Vax, VTE n = 7; AB, VITT Pt and HIT n = 3). b 14 C-serotonin release assay for VITT samples with buffer alone, PF4 (10 μg/mL), 0.1 or 100 U/mL heparin or IV.3 antibody (50 μg/mL). Each dot represents the mean of assays done in triplicate. The cut-off was set at 20% CPM. c Platelet aggregation responses. Purified IgG from VITT patients induced aggregation in platelet-rich plasma (red, blue and black traces). Blockage of FcγRIIa with IV.3 inhibited aggregation (purple, green and light blue traces). d Nucleosomal CitH3 (H3R8Cit ELISA, * p = 0.03), e myeloperoxidase (ELISA, ** p = 0.01), and f cfDNA (PicoGreen fluorescence assay, **** p < 0.0001) levels in VITT patients’ plasma ( n = 7) relative to controls ( n = 7) were determined. g Representative side and forward scatter flow cytometry plot backgated for neutrophils (yellow) and monocytes (blue) from VITT patient’s and vaccine control blood. LDG are indicated. h LDG quantitated as number of LDG events relative to 200 monocytes ( n = 7, except VITT n = 3; * p = 0.02). i Representative plot of NPA from VITT and vaccine control blood. j Quantification of NPA in VITT ( n = 7, except VITT n = 3; ** p = 0.004). k Representative plot of NETs from VITT and vaccine control blood. Quantification of NETs in VITT in l whole blood ( n = 7, except VITT n = 3; ** p = 0.005) and in m LDG population ( n = 6, except VITT n = 3; * p = 0.02). MPO + , CitH3 + double positive cells within the CD15 + population were defined as neutrophils undergoing NETosis. The percentage of gated events is indicated in each quadrant. Statistics: Kruskal-–Wallis ANOVA with Dunn’s correction. Data are presented as a , j , l , m mean ± SD; h mean ± SEM. OD optical density units, CPM counts per minute, Vax ctrl healthy vaccinated subject IgG; VTE venous thromboembolism patient IgG, ICU intensive care unit patient IgG, NPA neutrophil-platelet aggregates, LDG low density granulocytes, cfDNA cell-free DNA, CitH3 citrullinated histone H3, Pt patient. Source data are provided in the Source Data file.

Article Snippet: Plasma levels of myeloperoxidase and citrullinated histone H3 were determined using the human myeloperoxidase ELISA kit (ab119605, Abcam) and H3R8Cit ELISA Capture and Detection kit (R&D143002, EpiCypher) , respectively, following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Release Assay, Purification, Fluorescence, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Interferon-α and Interleukin-12 Are Induced Differentially by Toll-like Receptor 7 Ligands in Human Blood Dendritic Cell Subsets

doi: 10.1084/jem.20020207

Figure Lengend Snippet: TLR-7 ligands induce the production of IL-12 and IFN-α from MDCs and PDCs, respectively. (A and B) After 24-h culture with various stimuli, concentration of IL-12 p40+p70 (A) and IFN-α (B) in the culture supernatants of MDCs, PDCs, and PBMCs were measured by ELISA. The data are shown by means ± SEM of five independent experiments. (C) After 5-h culture with R-848 (10 −6 M), intracellular staining of IL-12 and IFN-α in MDCs and PDCs, together with the staining of surface expression of CD40, was performed. Percentages of the respective cytokine producing DCs are indicated. This figure represents the results from one of three experiments.

Article Snippet: 10 μg/ml brefeldin A (Sigma-Aldrich) was added during the last 1 h. After the stimulation, MDCs and PDCs were stained with Cy-Chrome-labeled CD40 (5C3; BD PharMingen), and then fixed, permeabilized (FIX and PERM kit; Caltag Laboratories), and stained with FITC-labeled anti–IL-12 p40+p70 mAb (B-P24; DIACLONE Research) or unconjugated mouse anti–human IFN-α mAb (MC-16; Genzyme).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing

Journal: Pharmaceutical Biology

Article Title: Tanshinone IIA alleviates ovalbumin-induced allergic rhinitis symptoms by inhibiting Th2 cytokine production and mast cell histamine release in mice

doi: 10.1080/13880209.2022.2034894

Figure Lengend Snippet: TIIA at 5 and 10 μmol/L had no effect on the viability of human mast cells, and reversed the promotion of histamine release and mast cell degranulation induced by C48/80. (A) The viability of human mast cell line HMC-1 cells after being treated with TIIA was examined by MTT assay. (B) Histamine content of human mast cell line HMC-1 cells after treatment with C48/80 and TIIA in culture medium (cell-free supernatants) and total suspensions was determined by ELISA. (C) The degranulation of human mast cell line HMC-1 cells after being treated with C48/80 and TIIA was detected by toluidine blue staining. *** p < 0.005, ### p < 0.005, * vs. control; # vs. OVA. TIIA: tanshinone IIA; MTT: methyl tetrazolium; ELISA: enzyme-linked immunosorbent assay; C48/80: compound 48/80.

Article Snippet: Mice serum was collected, and the concentrations of OVA-IgE and OVA-immunoglobulin G1 (IgG1) in mice serum were measured according to the instructions of mice OVA-IgE enzyme-linked immunosorbent assay (ELISA) kit (F10731, Westang, Shanghai, China) and mice OVA-IgG1 ELISA kit (3013, Chondrex, Woodinville, WA), respectively.

Techniques: MTT Assay, Enzyme-linked Immunosorbent Assay, Staining