IgM autoantibody profiles exhibited similar reactivity patterns in the mixed PTM peptide panel, including acetyl peptides of histones H2B, H3 and H4, again con sistent with prior studies. CP-690550 We also observed con siderable reactivity to multiple methyl histone H3 peptides. However, both histone reactive and healthy Inhibitors,Modulators,Libraries sera shared this reactivity pattern and thus were not deter mined to be significant by SAM. Given that IgM antibo dies tend to be lower affinity with broader cross reactivity and may occur naturally with potential regula tory and protective roles against autoimmunity, the significance of reactivity among these epitopes is less clear. Modest levels of IgG or IgM reactivity to citrulli nated H3 and H4 peptides were also observed, with no significant difference between SLE and healthy sample groups.

Efficient production and visualization of NETs in vitro We next devised methods for generating Inhibitors,Modulators,Libraries NETs from human and murine myeloid cell lines, to facilitate the broad and uniform characterization of PTMs on human and murine NETs and to provide a supply of NETs for testing their immunogenicity in vivo. We employed two sources of murine and two sources of human NETs, including two murine cell lines derived from cells arrested in early myeloid differentiation, the human promyelocytic leukemia cell line HL60 as well as mature human granulocytes from healthy adult donors. To culture and differentiate the three immature cell lines into Inhibitors,Modulators,Libraries neutrophils, we adapted previous methods then stimulated them as well as primary human neutrophils to produce NETs using hydrogen peroxide, TNF and LPS.

We then visualized the result ing NETs using fluorescence microscopy. The corresponding NET DNA was characterized by gel elec trophoresis and the NET DNA yield from these diverse sources was quantified. Inhibitors,Modulators,Libraries The DNA yield of purified NETs relative to unstimulated neutrophils varied depending Inhibitors,Modulators,Libraries on the preparation source and type of stimulus used for NETosis, ranging from 10% for MPRO, 15% to 50% for EPRO, Palbociclib 9% to 45% for HL 60 and 70% for primary human neutrophils. Importantly, we found that induction of NETosis in HL 60 cells and primary human neutrophils did not result in a significant degree of apoptosis. Post translational modification profiles of human and murine NETs To assess the immunogenicity of NETs in the context of autoantibody binding reactivity profiles observed, we biochemically characterized the PTMs present on NETs by employing a high throughput immunoblotting assay that allows such profiling in a parallel manner using a MiniBlotter apparatus dur ing primary antibody incubation. A set of 22 commer cially available PTM specific anti histone antibodies was used to probe each immunoblot membrane, with a total of 44 epitopes profiled in two separate panels.