Project description:Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic and refractory disease, characterized by necrotizing small to medium vessel vasculitides. AAV constitutes three distinct disorders: microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis), and eosinophilic granulomatosis with polyangiitis (EGPA) (formerly known as Churg-Strauss syndrome). ANCA, a characteristic autoantibody for AAV consists of two major subtypes: one against myeloperoxidase (MPO-ANCA) and the other against leukocyte proteinase 3 (PR3-ANCA). MPO-ANCA is mainly detected in patients with MPA (55-90%) and in those with EGPA (20-40%) and less frequently detected in patients with GPA (20-30%). We hypothesized that an aberrant PTM occurred in neutrophil MPO in patients with MPO-ANCA-positive AAV (MPO-AAV) is involved in the production of MPO-ANCA. To test the hypothesis, SWATH-MS was used to comprehensively quantify PTMs of human MPO purified from neutrophils of MPO-AAV and healthy controls, and PTMs of mouse MPO treated with hydrogen peroxide in vitro.
Project description:To study monocyte and macrophage activation in ANCA-associtated vasculitis (AAV), we performed bulk RNA sequencing of bead-selected monocytes and in vitro cultured monocyte-derived macrophages from AAV patients and healthy controls. Overview patients included for sequencing monocytes: - AAV active disease, n=4, MPO-AAV=4 - AAV remission, n=10, PR3-AAV=5, MPO-AAV=5 - Healthy controls, n=6 Overview patients included for sequencing monocyte-derived macrophages: - AAV active, n=1, PR3-AAV=1 - AAV remission, n=3, PR3-AAV=3 - Healthy controls, n=3
Project description:These data are part of a body of work exploring the effect of IgG from patients with ANCA vasculitis on human monocytes in vitro. Please see the relevant publication for a full description.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data
