👤 Shareef Jarvi Antar

Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent infection with severe acute respiratory syndrome coronavirus 2, but the underlying immunological mechanisms driving this distinct syndrome are unknown. We utilized high-dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute coronavirus disease 2019 (SAC). Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen-presenting cells. Interleukin 27 (IL-27), a cytokine known to drive hematopoietic stem cells toward EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing toward MIS-C, offering potential diagnostic and therapeutic targets. Show less

High-throughput reliable data generation has become a substantial requirement in many "omics" investigations. In proteomics the sample preparation workflow consists of multiple steps adding more bias to the sample with each additional manual step. Especially for label-free quantification experiments, this drastically impedes reproducible quantification of proteins in replicates. Here, a positive pressure workstation was evaluated to increase automation of sample preparation and reduce workload as well as consumables. Digested peptide samples were purified utilizing a new semi-automated sample preparation device, the Resolvex A200, followed by nanospray liquid chromatography/electrospray ionization (nLC/ESI) Orbitrap tandem mass spectrometry (MS/MS) measurements. In addition, the sorbents Maestro and WWP2 (available in conventional cartridge and dual-chamber narrow-bore extraction columns) were compared with Sep-Pak C18 cartridges. Raw data was analyzed by MaxQuant and Perseus software. The semi-automated workflow with the Resolvex A200 workstation and both new sorbents produced highly reproducible results within 10-300 μg of peptide starting material. The new workflow performed equally as well as the routinely conducted manual workflow with similar technical variability in MS/MS-based identifications of peptides and proteins. A first application of the system to a biological question contributed to highly reliable results, where time-resolved proteomic data was separated by principal component analysis (PCA) and hierarchical clustering. The new workstation was successfully established for proteolytic peptide purification in our proteomic workflow without any drawbacks. Highly reproducible results were obtained in decreased time per sample, which will facilitate further large-scale proteomic investigations. Show less