Key Points CD10 as a marker discriminating mature from immature neutrophils within heterogeneous neutrophil populations in pathological settings. Immunosuppressive mature CD66b+CD10+ and immunostimulatory immature CD66b+CD10− neutrophils coexist in G-CSF–treated donors.

© F e r r a t a S t o r t i F o u n d a t i o ncules might represent complementary therapeutic strategies to counteract the toxic effects of ROS in β-thalassemia. However, few of them have been shown to beneficially affect in vivo β-thalassemic red cell features and/or thalassemic ineffective erythropoiesis in vivo. 16,21 MethodsIn vitro erythropoiesis from CD34 + cells from peripheral circulation of normal and β-thalassemia-intermedia subjects Regarding cell culture, phenotypic analysis and cell sorting strategy, peripheral blood from adult normal volunteers and from transfusion independent β-thalassemia patients (β-thalassemia intermedia) was collected, after obtaining informed consent according to the guidelines established by the Ethic Committee for human subject studies of the University of Milan and the principles of the Declaration of Helsinki. Approval by the Ethic Committee of the University of Milan for human erythroid precursors studies was obtained. We analyzed 20 erythroid cultures from the peripheral blood of different normal subjects and 20 erythroid cultures from 10 homozygous β-thalassemic intermedia patients (β 0cod39 ). 11,17 Details on cell cultures are reported in the Online Supplementary Appendix. The Resveratrol concentration (5 mM; Sigma Aldrich, St. Louis, MO, USA) used in this study was selected from dose-response studies (Online Supplementary Figure 1SA) and a review of the literature. 3,5,6,[8][9][10]22 The erythroid cell antigen profile and the sorting of erythroid precursors were carried out as reported by Merry-Weather Clarke et al. 23 Details are reported in the Online Supplementary Appendix and Figure S1B).Quantitative real-time PCR was carried out as previously described. 24 Details are reported in the Online Supplementary Appendix. The primers used are listed in Online Supplementary Tables S1 and S2.For immunoblot-analysis of sorted human erythroid precursors and immunofluorescence assay for FoxO3a, 1 x 10 6 sorted CFU-E cells from both normal and β-thalassemic were solubilized as previously described. 11,20 Details on immunoblot and immunofluorescence analysis are reported in the Online Supplementary Appendix. Whenever indicated, sorted CFU-Es were separated into cytosol and nuclear fractions as previously reported. 25 The study was carried out in accordance with the Scientific Committee for Animal Experimentation (CIRSAL, University of Verona, Italy). C57B6/2J mice, wild-type controls (WT) and Hbb th3/+ mice were used as β-thalassemia models. Age-and sexmatched 2-month old mice (weight 20 g) were studied. The female/male ratio in the different groups was 1:1. Based on previous studies on resveratrol bioavailability in vivo, [26][27][28] the mice were placed either on resveratrol (2.5 mg/kg incorporated into AIN-93G diet) or standard diet (AIN-93G diet). The mice were fed with resveratrol diet for six months. Hematologic parameters and red cell indices were determined as previously reported. 16,[29][30][31][32] For cytofluorimetric analysis of mouse bone-marrow p...

To assess whether NLR pyrin domain-containing protein 3 (NLRP3) inflammasome, a multiprotein complex that mediates the activation of caspase-1 (CASP-1) and pro-inflammatory cytokines IL-18 and IL-1β, could be involved in the chronic inflammatory state observed in chronic kidney disease patients undergoing hemodialysis treatment (CKD-HD), we employed several biomolecular techniques including RT-PCR, western blot, FACS analysis, confocal microscopy and microarray. Interestingly, peripheral blood mononuclear cells from 15 CKD-HD patients showed higher mRNA levels of NLRP3, CASP-1, ASC, IL-1β, IL-18 and P2X7receptor compared to 15 healthy subjects. Western blotting analysis confirmed the above results. In particular, active forms of CASP-1, IL1-β and IL-18 resulted significantly up-regulated in CKD-HD versus controls. Additionally, elevated mitochondrial ROS level, colocalization of NLRP3/ASC/mitochondria in peripheral blood mononuclear cells from CKD-HD patients and down-regulation of CASP-1, IL1-β and IL-18 protein levels in immune-cells of CKD-HD patients stimulated with LPS/ATP in presence of mitoTEMPO, inhibitor of mitochondrial ROS production, suggested a possible role of this organelle in the aforementioned CKD-associated inflammasome activation. Then, microarray analysis confirmed, in an independent microarray study cohort, that NLRP3 and CASP-1, along with other inflammasome-related genes, were up-regulated in 17 CKD-HD patients and they were able to clearly discriminate these patients from 5 healthy subjects. All together these data showed, for the first time, that NLRP3 inflammasome was activated in uremic patients undergoing dialysis treatment and they suggested that this unphysiological condition could be possibly induced by mitochondrial dysfunction.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bi‑directional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphere‑forming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of E‑cadherin, the main marker of the epithelial state. PDAC CSCs were able to re‑differentiate into parental cells once cultured in parental growth condition, as demonstrated by re‑acquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds.

Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.