Subcloning potential of EPC/ECFC generated from the PBMC of ACS patients.

<p>After <i>ex-vivo</i> expansion, primary EPC/ECFC colonies were trypsinized and assessed for clonogenic potential capacity by single cells replating assay. In <b>A</b>, single cells derived from EPC/ECPF colonies were seeded in collagen I coated wells and monitored day by day (<b>a</b>: day 1; <b>b</b>: day 2; <b>c</b>: day 3; <b>e–f</b>: day 4; <b>a–e</b>: original magnification 25X; <b>f</b>: original magnification 40X). One representative experiment is shown. In <b>B</b>, secondary clones were classified on the basis of their proliferation properties. Data are mean±SD derived from six independent experiments.</p

Phalaena sp.

<div><h3>Background</h3><p>The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the <em>in vitro</em> clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin.</p> <h3>Methodology/Principal Findings</h3><p>By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7–14 days after the acute cardiovascular event. Notably, <em>ex-vivo</em> generation of EPC/ECFC was correlated to a higher <em>in vitro</em> release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed <em>in vitro</em> expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: <em>i</em>) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, <em>i</em>) variable expression of the CD34, CD106 and CD184 markers, and <em>iii</em>) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities.</p> <h3>Conclusion/Significance</h3><p>Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.</p> </div

Identification of optimal culture conditions for the <i>ex-vivo</i> expansion of ACS PB-derived EPC/ECFC.

<p> Primary EPC/ECFC colonies were generated by plating patient PBMC in M⁵¹⁰⁰ medium, as detailed in the Method section. In <b>A</b>, after the colony identification (at day 5 after plating), medium was change (arrow) and replaced either with fresh M⁵¹⁰⁰, or M^EGM or M¹⁹⁹ and the development of the colonies was monitored over the time. The growth kinetics of a representative experiment out of five independent experiments is shown. At each indicated time point, the mean cell number/ECFC was determined by two independent operators; standard deviations were below 10% and are not shown. Asterisk, p<0.05. In <b>B</b>, immunocytochemical analysis of <i>in vitro</i> expanded EPC/ECFC documenting positivity for CD105 antigen (original magnification: 20X) and for the specific endothelial marker Factor VIII (original magnification: 40X). In <b>C</b>, FISH analysis performed on <i>in vitro</i> expanded EPC/ECFC by using the centromeric enumeration probe CEP9 (white arrows) documenting a normal diploid chromosomal pattern (original magnification: 40X).</p

Correlation analysis between INR and warfarin / 3’-hydroxywarfarin in the two cohorts of patients investigated.

<p>Correlation analysis between INR and warfarin / 3’-hydroxywarfarin in the two cohorts of patients investigated.</p

Time-frame findings in patients starting OAT (n = 52).

<p>Time-frame findings in patients starting OAT (n = 52).</p

Univariate and multivariate analyses to estimate the contribution of different variables on INR.

<p>Univariate and multivariate analyses to estimate the contribution of different variables on INR.</p

Correlation analyses between: INR, serum warfarin (ng/mL) and 3’-hydroxywarfarin (ng/mL), and the amount of drug (warfarin week) taken by the whole cohort of patients on oral anticoagulant therapy.

<p>Correlation analyses between: INR, serum warfarin (ng/mL) and 3’-hydroxywarfarin (ng/mL), and the amount of drug (warfarin week) taken by the whole cohort of patients on oral anticoagulant therapy.</p

MOESM2 of Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients

Additional file 2: Table S1. List of genes involved in innate and adaptive immunity

HPLC calibration curves.

<p>Warfarin (1A) and 3’-hydroxywarfarin (1B) calibration curves; R expresses the ratio between the area under the analyte peak (warfarin or 3’-hydroxywarfarin respectively) and the area of the internal standard.</p

Warfarin daily dose for different WRI.

<p>Mean and median warfarin dose increased as WRI increased. WR 0, WRI 1 and WRI 2 classes are as specified in text. Continuous line indicates the median; dashed line indicates the mean, vertical bars indicate the 1^st and 99^th percentile of warfarin day (mg).</p