In Vitro Megakaryocyte Differentiation and Proplatelet Formation in Ph-Negative Classical Myeloproliferative Neoplasms: Distinct Patterns in the Different Clinical Phenotypes

Background: Ph-negative myeloproliferative neoplasms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET). Although the pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature. Methodology/Principal Findings: We analyzed the in vitro megakaryocyte differentiation and proplatelet formation in 30 PMF, 8 ET, 8 PV patients, and 17 healthy controls (CTRL). Megakaryocytes were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of thrombopoietin. Megakaryocyte output was higher in MPN patients than in CTRL with no correlation with the JAK2 V617F mutation. PMF-derived megakaryocytes displayed nuclei with a bulbous appearance, were smaller than ET- or PV-derived megakaryocytes and formed proplatelets that presented several structural alterations. In contrast, ET- and PV-derived megakaryocytes produced more proplatelets with a striking increase in bifurcations and tips compared to both control and PMF. Proplatelets formation was correlated with platelet counts in patient peripheral blood. Patients with pre-fibrotic PMF had a pattern of megakaryocyte proliferation and proplatelet formation that was similar to that of fibrotic PMF and different from that of ET. Conclusions/Significance: In conclusion, MPNs are associated with high megakaryocyte proliferative potential. Profound differences in megakaryocyte morphology and proplatelet formation distinguish PMF, both fibrotic and prefibrotic, from ET and PV

Method of Forming Microchannels on the Surface of a Glass Substrate

A method of forming arbitrarily shaped microchannels on substrates of brittle dielectric materials, such as optical glasses and optical crystals, is considered. This method makes it possible to ensure the identity of microchannel edges over the entire perimeter</jats:p

Method of crack-free laser writing of microchannels on glass substrates

Abstract Approaches to the problem of the formation of microchannels of arbitrary shape on optical glass substrates and other fragile materials without microdefects (cracks and chips) both on the surface of the substrate and inside the substrates are considered. Different approaches to the formation of laser beam trajectories within microchannels are considered. It was found that the raster scanning method improves the performance of the laser micromachining process and provides the ability to write structures of any length without longitudinal cracks with the energy characteristics of the laser spot in the focus much higher than those in the vector scanning method.</jats:p

The BH3-mimetic ABT-737 effectively kills acute myeloid leukemia initiating cells

AbstractThe anti-apoptotic proteins Bcl-XL and Bcl-2 are abundantly expressed in hematopoietic stem cells and/or progenitor cells. Furthermore, leukemic cells expressing these proteins are enriched in minimal residual disease cell populations. This prompted us to test the BH3-mimetic compound ABT-737 for its ability to eradicate putative leukemic stem cells. ABT-737 demonstrated potent cytotoxic effects in all patient samples tested. The efficacy of ABT-737 against AML blasts and the primitive CD34+/CD38− population was equal and independent of sensitivity to cytarabine/daunorubicin. These results, together with previously reported synergistic effects of ABT-737 with chemotherapeutics make BH3-mimetics promising candidates for future AML treatment regimens

Ex Vivo Co-Culture of AML Blasts and Bone Marrow Mesenchymal Stromal Cells to Accurately Predict the Clinical Efficacy of Cytarabine-Daunorubicin Treatment

Abstract Introduction- The mainstream therapy for AML is cytarabine (AraC) and anthracycline-based intensive chemotherapy. Between 10-40% of patients however have a refractory disease. It is becoming increasingly accepted that the risk for refractory disease could be reduced by finding better ways of selecting the induction therapy. While multiple in vitro and in vivo experimental models exist for pre-clinical drug efficacy testing, they are either labour intensive, too time consuming or generate variable results, making them unsuitable for clinical application. Furthermore, very few, if any of these methods have been shown to replicate the clinical response of patients. The aim of this study was to develop a functional drug testing system that can rapidly and faithfully predict the clinical response of the patient and therefore be utilized to help the identification of patients suitable for AraC+daunorubicin (DnR) therapy. Methods- Primary samples were obtained through Blood Cancer Biobank Ireland. All patients were consented and the research project was approved by the local Research Ethics Committee of NUI, Galway. Mononuclear AML blasts were isolated from bone marrow (BM) aspirates of newly diagnosed patients by ficoll-paque gradient separation. HS-5 human bone marrow mesenchymal stromal cells (BMSC), healthy donor (HD)-derived BMSCs immortalised with hTERT (iMSC) and primary BMSCs (non-immortalised, pBMSC) from HDs and AML patients were used as feeder layers. Cytokine and chemokine secretion by BMSCs was determined using antibody arrays (R&amp;D systems). Extracellular matrix (ECM) scaffold formation was determined by immunocytochemical detection of collagen I, III, IV, V, VI, laminin and fibronectin. AML blasts were cultured in direct contact on established BMSC feeder layer. BMSCs were identifiable by either GFP expression or green cell tracker labelling (CFSE) and the percentage of dying AML cells (negative for GFP/CFSE and positive for the viability dye, ToPro-3) was determined with FACS. Results- In order to choose the most suitable BMSC feeder layer, three main characteristics were studied: cytokine/chemokine secretion, formation of ECM-BMSC proteocellular scaffold and ability to support ex vivo AML blast survival were determined. HS-5 cells, iMSCs and primary, HD pBMSC secreted 66, 59 and 51 paracrine factors, respectively including angiopoietin 1, fibroblast growth factor, interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte colony stimulating factor (G-CSF), C-X-C motif chemokine 12 (CXCL12), CXCL10 and vascular endothelial growth factor. 51% of cytokines/chemokines were common between iMSCs and HD pBMSC, while the HS-5 secretome shared 43% commonality with HD pBMSC. iMSCs also showed an equal ability to establish an ECM-BMSC scaffold with HD pBMSC and they had a superior ability to support the ex vivo survival of AML blasts over HS-5 cells. To determine how closely iMSCs can replicate the effects of BMSCs of AML patients, AML blasts were cultured on matching BMSCs (BMSCs isolated from the same patient). When these AML-BMSC co-cultures were exposed to the BH3 mimetics ABT-199 and ABT-737 or AraC+DnR, iMSCs closely replicated the protective effect of the patients' own BMSCs. Using this optimized ex vivo co-culture model, we tested 16 AML patient samples by exposing iMSC-AML blast co-cultures to a clinically-relevant dosage of AraC+DnR. To quantify drug efficacy, the area under the curve (AUC) for each treatment timepoint (24 h and 48 h) was calculated. By applying a cut-off value of 35% efficacy, we found that the ex vivo test could predict the clinical response of the patient in 81.2% of cases (p-value= 0.002, Figure). Conclusions- The developed drug efficacy test can recapitulate the key features of the BM microenvironment and could predict the clinical response of patients with high accuracy. The advantages of this model over more complex pre-clinical AML models is its suitability to be developed into a laboratory diagnostic tool which could greatly advance the clinical decision on treatment choice. Figure. Figure. Disclosures Quinn: Janssen: Honoraria. </jats:sec

Type I interferons induce peripheral T regulatory cell differentiation under tolerogenic conditions

Abstract The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/β is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/β play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/β on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.</jats:p