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Blood transfusion is an essential and irreplaceable part of modern medicine, as a therapeutic modality or additional support to other clinical therapies. Nevertheless, the entire procedure from blood collection to administration, absorbs a significant amount of resources and has a number of problems that need to be addressed. The paucity of donors, the transmission of pathogenic microorganisms and the overall costs of the process have switched the scientific interest to the quest of alternative transfusion methods. The industrial ex vivo production of transfusable red blood cells capable of replacing a unit of packed red blood cells is a very attractive prospect, let alone the idea of a massive production of such a biological material. Various scientific groups, by exploiting erythropoiesis, the stem cells' characteristics and the constantly renewed knowledge in the fields of collection, culture, preservation and expansion of stem cells, have made significant progress towards the realization of such an idea. All three major sources of stem cells, haematopoietic stem/progenitor cells, human embryonic stem cells and induced pluripotent stem cells are thought to be capable of generating adequate amounts of red blood cells. By further studying and refining the in vitro red cell production protocols, it is anticipated that the economic and biotechnological obstacles of the current methods will be overcome in the near future. This manuscript is a brief revisit of their current state of the art, potentials and obstacles that are associated with industrial and clinical application issues.

Aims Sudden cardiac death (SCD) annual incidence is 0.6–1% in post-myocardial infarction (MI) patients with left ventricular ejection fraction (LVEF)≥40%. No recommendations for implantable cardioverter-defibrillator (ICD) use exist in this population. Methods and results We introduced a combined non-invasive/invasive risk stratification approach in post-MI ischaemia-free patients, with LVEF ≥ 40%, in a multicentre, prospective, observational cohort study. Patients with at least one positive electrocardiographic non-invasive risk factor (NIRF): premature ventricular complexes, non-sustained ventricular tachycardia, late potentials, prolonged QTc, increased T-wave alternans, reduced heart rate variability, abnormal deceleration capacity with abnormal turbulence, were referred for programmed ventricular stimulation (PVS), with ICDs offered to those inducible. The primary endpoint was the occurrence of a major arrhythmic event (MAE), namely sustained ventricular tachycardia/fibrillation, appropriate ICD activation or SCD. We screened and included 575 consecutive patients (mean age 57 years, LVEF 50.8%). Of them, 204 (35.5%) had at least one positive NIRF. Forty-one of 152 patients undergoing PVS (27–7.1% of total sample) were inducible. Thirty-seven (90.2%) of them received an ICD. Mean follow-up was 32 months and no SCDs were observed, while 9 ICDs (1.57% of total screened population) were appropriately activated. None patient without NIRFs or with NIRFs but negative PVS met the primary endpoint. The algorithm yielded the following: sensitivity 100%, specificity 93.8%, positive predictive value 22%, and negative predictive value 100%. Conclusion The two-step approach of the PRESERVE EF study detects a subpopulation of post-MI patients with preserved LVEF at risk for MAEs that can be effectively addressed with an ICD. Clinicaltrials.gov identifier NCT02124018

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

AIMS: Country differences in management practices are evident, and the publication of management guidelines by the European Society of Cardiology (ESC) and other learned societies has tried to recommend a uniform evidence-based approach to management. Despite the availability of guidelines and efforts to improve implementation, differences in guideline adherence are evident, and differences between countries and regions within Europe are therefore likely.METHODS AND RESULTS: In this analysis from the baseline dataset of the EORP-AF Pilot survey, we examined regional differences in presentation and treatment of contemporary patients with atrial fibrillation (AF) in Europe, as managed by European cardiologists. We focused on a subgroup of 902 hospital admitted patients in whom no rhythm control was performed or planned. Chronic heart failure was more common in East countries (P < 0.0001) while hypertension and peripheral artery disease were more common in South countries (both P < 0.0001). Previous bleeding and chronic kidney disease were more common in South countries (both P < 0.0001). A CHA2DS2-VASc score of ≥2 was highest in East and South countries (93.0 and 95.3%, respectively) compared with 80.8% in West countries (P < 0.0001). A HAS-BLED score of ≥3 was also highest in East and South countries (18.0 and 29.2% respectively) compared with 4.8% in West countries (P < 0.0001). Oral anticoagulation (OAC) use (either as OAC or OAC plus antiplatelet therapy) in West, East, and South countries was 72.0, 74.7, and 76.2%, respectively. Only antiplatelet therapy was used in 13.6, 15.4, and 12.4%, respectively. An initial rate control strategy only was most common in South countries (77.8%) (P < 0.0001).CONCLUSION: From the systematic collection of contemporary data regarding the management and treatment of AF in nine participating member ESC countries, we provide hypothesis-generating insights into regional management practices in Europe with regard to patient characteristics and treatment options.

Abstract 1250 The CCAAT-binding transcription factor NF-Y, consists of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YA is characterized as the regulatory subunit of the complex because of its differential expression during cell cycle. NF-YA appears to have a complex role in hematopoietic stem cell (HSC) biology. So far, however, there are no available data on the role of NF-Y in mesenchymal stem cell (MSC) biology. The aim of this study is the evaluation of NF-YA expression levels during the ex vivo expansion of human bone marrow (BM)-MSCs and the assessment of its possible role for the BM-MSC differentiation process towards the osteoblastic and adipocytic lineages. BM-MSCs were isolated from posterior iliac crest aspirates of hematologically healthy individuals undergoing orthopedic surgery after written informed consent. BM-MSCs were ex vivo expanded until passage 10 (P10). Total RNA was isolated from BM-MSCs at P2 and P10 for the evaluation of NF-YA mRNA levels. P3 BM-MSCs' ex vivo differentiation into adipocytes and osteoblasts was induced using the appropriate culture media. Mineralization was evidenced via Alizarin Red and Von Kosa staining and lipid droplets were revealed with Oil Red O staining. Total RNA and proteins were isolated from undifferentiated and differentiated BM-MSCs at various time-points. Protein levels of NF-YA were immunodetected by Western blot. The relative gene expression of NF-YA, as well as that of lineage-specific markers was evaluated by real-time PCR. All PCR results are expressed as 2−ΔCt. Adipocytic and osteoblastic differentiation of BM-MSCs was also induced following knockdown of NF-YA expression after BM-MSC transduction with lentiviral particles carrying either shRNA against NF-YA or nonsense shRNA. Total RNA was isolated from transduced BM-MSCs at several time-points during differentiation. A statistically significant (p=0.04) reduction in the mRNA levels of NF-YA was found in P10, as compared to P2 BM-MSC cultures (n=12). During adipogenic differentiation, NF-YA significantly increased (p=0.0478) at day 16, as compared to the onset of differentiation induction (day 0), (n=14). Similarly, protein levels of NF-YA also increased during adipogenesis Furthermore, NF-YA expression levels strongly correlated with the mRNA levels of adipogenesis-associated genes PPARG (r=0.74, p In conclusion we have shown for the first time, that BM-MSCs display decreased NF-YA mRNA levels after prolonged ex vivo expansion, a finding that might be associated with MSCs' senescence and/or loss of stemness., We have also shown that NF-YA does not have any major effect on BM-MSCs' osteogenic differentiation but displays a significant role in their adipogenic differentiation as was shown by (a) the increased NF-YA mRNA and protein levels during the ex vivo adipogenesis, (b) the positive correlation between the NF-YA mRNA expression levels and the adipocyte-associated genes PPARG, C/EBPA and LPL and (c) the impaired adipogenic differentiation following NF-YA knock down. Collectively our results imply a novel role for NF-YA in BM-MSCs' biology Disclosures: No relevant conflicts of interest to declare.