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# 0.05 versus no inhibitor. We investigated whether ROS production in leukemic cells may induce the release of the histoneCDNA complex. measured in 80 patients with hematologic diseases and 40 healthy controls. ET formation and ROS levels were investigated during leukemic cell proliferation ROS/RNS assay kit (Cell Biolabs, San Diego, CA). Confocal microscopy U937 cells (fresh or cultured for 5 days) were fixed with 4% paraformaldehyde, stained with SYTOX green (Thermo Fisher Scientific) and mounted with Fluoroshield made up of DAPI (ImmunoBioScience Corporation, Mukilteo, Boc-NH-PEG2-C2-amido-C4-acid WA). Images were acquired on an Olympus FluoView FV1000 confocal microscope (Olympus, Tokyo, Japan) with a 100 objective. Flow cytometry hEC or HUVEC were stained with PE-conjugated E-selectin, PE-conjugated ICAM-1, and APC-conjugated VCAM-1 (all from BD Biosciences, Franklin Lakes, NJ). U937 cells were suspended at a final concentration of 1106 cells/mL in media and plated on hEC layers pre-treated with 50 g/mL histone for 1 h. The co-cultured cells were treated with cytosine DCarabinofuranoside (Ara-C; Hospira Pty Ltd., Mulgrave, Australia) for 24 h or without Ara-C for 48 h. Adherent and non-adherent U937 cells were collected separately and stained with FITC-conjugated CD45 (BD Biosciences), PE-conjugated CD105 (BD Biosciences), 7-AAD (Beckman Coulter, Fullerton, CA). Adhesion assay hEC were incubated with or without 50 g/mL histone for 5 h. U937 cells (1106 cells/mL) were added onto the hEC layer for 30 min. The non-adherent cells were collected. The adherent round U937 cells were enumerated under a light microscope (Olympus). For neutralizing histone, histone was pre-mixed with 62.5 g/mL polysialic acid (Sigma-Aldrich) for 1 h, 100 U/mL heparin (Sigma-Aldrich) for 10 min, and 100 nM activated protein C (APC; Haematologic Technologies Inc., Essex Junction, VA) for 30 min. The mixtures were then added to hEC. Anti-E-selectin antibody (50 g/mL), anti-ICAM-1 antibody (10 g/mL), or anti-VCAM-1 antibody (30 g/mL) (all from R&D Systems, Minneapolis, MN) was incubated with histoneCtreated hEC for 10 min. Then U937 cells were added. Before stimulated with histone, hEC were pre-treated for 1 h with 50 g/mL isotype-IgG2a, anti-TLR2, or anti-TLR4 antibody (all from eBioscience), or 5 M TLR9 antagonist (ODN TTAGGG; InvivoGen, San Diego, CA). Results Circulating levels of ET markers in patients with hematologic diseases The baseline characteristics of the study population are shown in Table 1. Final diagnosis of patients was acute leukemia (n = 21), myeloproliferative neoplasms (MPN, n = 45), and aplastic anemia (n = 14). The acute leukemia group was composed of acute myeloid leukemia (n = 14), acute lymphoblastic leukemia (n = 6), and mixed phenotype acute leukemia (n = 1). MPN patients were subdivided into 2 groups based on absolute neutrophil count (ANC): MPN with neutrophilia (ANC 7.5109/L; n = 13) and MPN without neutrophilia (ANC 7.5109/L; n = 32). Three ET markers (histoneCDNA complex, cell-free dsDNA, and neutrophil elastase) were measured. The level of the histoneCDNA complex was significantly higher in the acute leukemia group (311402) than in the MPN groups either with or without neutrophilia (118117, = 0.049 and 5341, = 0.008, respectively). No significant increase in the histoneCDNA complex level Boc-NH-PEG2-C2-amido-C4-acid Rabbit Polyclonal to AIFM2 was observed in patients with aplastic anemia compared with normal control. The circulating levels of cell-free dsDNA and neutrophil elastase were also highest in the acute leukemia group (Table 1). Among patients with MPN, those with neutrophilia exhibited a higher level of neutrophil elastase than those without neutrophilia. Based on the cut-off values (95 percentile of normal control values), positivity for the histoneCDNA complex and cell free dsDNA Boc-NH-PEG2-C2-amido-C4-acid was highest in the acute leukemia group (81.0% and 71.4%, respectively). Table 1 The baseline characteristics and laboratory results of the study populations. 0.05 ** 0.001 vs normal control (test for comparisons of mean values and Chi-square test for comparisons of positivity). Abbreviations: MPN, myeloproliferative neoplasms; ANC; absolute neutrophil count; PT, prothrombin time; aPTT, activated partial prothrombin time; PB, peripheral blood. To investigate the factor(s) contributing to the circulating levels of the histoneCDNA complex, cell-free dsDNA, and neutrophil elastase, we performed multiple linear regression analysis (Table 2). Peripheral blast count.