Between 1 June and 31 December 2016, 13,023 blood donations from the University Hospital Aachen in Germany were routinely screened for West Nile virus (WNV) RNA using the cobas TaqScreen WNV Test. On 28 September 2016, one blood donor was tested positive. Subsequent analysis revealed an acute Usutu virus (USUV) infection. During the ongoing USUV epizootics in Germany, blood transfusion services, public health authorities and clinicians should be aware of increased human USUV infections.
Background Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type–specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated. Methods We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR). Results Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number. Conclusions Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.
we did have some individuals falling within that range (Table 1). Sacerdoti et al. (7 ) studied eight healthy individuals with 20-HETE expressed as the excretion rate (1.6 ng/min). It is difficult to compare these data with our own because these individuals were receiving an infusion of aminohippurate in 50 g/L dextrose in water at 1.5 mL/min for 5 h to determine renal plasma flow.Information on the physiologic effects of 20-HETE in humans is limited. Sacerdoti et al. (7 ) showed that the rate of 20-HETE excretion was increased in individuals with hepatic cirrhosis. Laffer et al. (5 ) recently showed a positive correlation between diastolic BP and 20-HETE excretion rate in a group of 13 salt-sensitive hypertensive individuals. In some rat models of hypertension, high BP has been associated with increased 20-HETE production (2 ), but this is yet to be confirmed in human studies.In conclusion, the sample preparation procedure using solid-phase cartridge extraction and HPLC purification would lend itself to automation and enable the convenient analysis of 20-HETE excretion in large human studies. Such studies could examine in more detail the potential role of this vasoactive CYP450 metabolite in vascular function and human hypertension.This study was funded in part by grants from the National Health and Medical Research Council of Australia (NHMRC; Grant 139067) and NIH Grant GM31278 (to J.R.F.). 9. Lin F, Rios A, Falck JR, Belosludtsev Y, Schwartzman ML. 20-Hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule. Am
Lipocalin 2 (LCN2), a proinflammatory mediator, is involved in the pathogenesis of myeloproliferative neoplasms (MPN). Here, we investigated the molecular mechanisms of LCN2 overexpression in MPN. LCN2 mRNA expression was 20-fold upregulated in peripheral blood (PB) mononuclear cells of chronic myeloid leukemia (CML) and myelofibrosis (MF) patients vs. healthy controls. In addition, LCN2 serum levels were significantly increased in polycythemia vera (PV) and MF and positively correlated with JAK2V617F and mutated CALR allele burden and neutrophil counts. Mechanistically, we identified endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) as a main driver of LCN2 expression in BCR-ABL- and JAK2V617F-positive 32D cells. The UPR inducer thapsigargin increased LCN2 expression >100-fold, and this was not affected by kinase inhibition of BCR-ABL or JAK2V617F. Interestingly, inhibition of the UPR regulators inositol-requiring enzyme 1 (IRE1) and c-Jun N-terminal kinase (JNK) significantly reduced thapsigargin-induced LCN2 RNA and protein expression, and luciferase promoter assays identified nuclear factor kappa B (NF-κB) and CCAAT binding protein (C/EBP) as critical regulators of mLCN2 transcription. In conclusion, the IRE1–JNK-NF-κB–C/EBP axis is a major driver of LCN2 expression in MPN, and targeting UPR and LCN2 may represent a promising novel therapeutic approach in MPN.
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