bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transfection with mRNA for CD19 Specific Chimeric Antigen Receptor Restores Natural Killer Cell Mediated Killing of CLL Cells.
Authors: Boissel Laurent , Betancur Monica , Tuncer Hande , Weitzman James , Klingemann Hans 
Published: October 2019

Abstract
B-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world and is characterized by monoclonal proliferation of functionally deficient lymphocytes, with relative decrease in T and Natural Killer (NK) cells. Available monoclonal antibody (mAb) therapies elicit a response in almost half of the patients, but durable remissions are rare. B-CLL cells are virtually impervious to T and NK cell-mediated cytotoxicity. However, a feasible approach to overcome this resistance is to redirect the specificity of T or NK cells by making them express chimeric antigen receptors (CAR). Such a method has been successfully tested in NK cells against a number of CD19-expressing cells; however the loading of cells with CD19-CAR relied on retrovirus-based transfection methods. We describe here a non-retroviral method for obtaining NK cells expressing high levels of CD19-CAR, based on electroporation of messenger RNA (mRNA). We used a CAR that is comprised of the single chain Fv portion of a mouse mAb against CD19 fused to the signaling intracellular domain of CD3ζ subunit. The corresponding cDNA was subcloned by PCR into a plasmid allowing T7-dependant mRNA synthesis. In vitro synthesis of the CD19-CAR mRNA was followed by a poly-adenylated tail elongation step in order to improve in vivo stability and translation of the messenger. Electroporation of NK-92 cells with this construct, using a Biorad GenePulser II apparatus, yields up to 78% expression efficiency and as high as 90% cell survival. Cytotoxicity of transfected NK-92 cells was tested against 3 target cell lines (NK sensitive, CD19 negative K562; CD19 expressing, NK resistant REH and SUPB15) as well as primary CLL cells from 6 untreated patients. A non-radioactive fluorochrome-based flow cytometric assay was used to determine NK-mediated cytotoxicity, at effector to target (E:T) ratios ranging from 1:1 to 10:1. Expression of the CD19-CAR on the surface of NK-92 cells do not change their cytolytic properties against K562: 70% ±5% killing compared to 74% ±2% killing for non-transfected NK-92, (E:T ratio of 10:1). Expression of CD19-CAR in NK-92 cells completely overcomes the resistance of REH and SUPB15 cell lines: 76% ±7% and 73% ±5% killing respectively, compared to 21% ±10% and 18% ±7% killing respectively with non-transfected NK-92 (E:T ratio of 10:1). Moreover, previously resistant primary CLL cells became highly sensitive to lysis by NK-92 expressing CD19-CAR: 46–82% killing, as opposed to 7–22% killing for non-transfected NK-92 (E:T ratio of 10:1). These results show that NK cells can be engineered using mRNA transfection by electroporation to target and effectively kill resistant primary CLL cells.
Valproic Acid Induces Apoptosis in Chronic Lymphocytic Leukemia Cells by Targeting Several Pro- and Anti-Apoptotic Genes.
Authors: Stamatopoulos Basile , Meuleman Nathalie , Kentos Alain , Hermans Philippe , Martiat Philippe , Bron Dominique , Lagneaux Laurence 
Published: October 2019

Abstract
Histone deacetylase inhibitors have been shown to modulate the cell cycle, to induce apoptosis and to sensitize cancer cells to other chemotherapeutics. Among these inhibitors, valproic acid (VPA), an antiepileptic drug, is being discussed as a promising novel anti-cancer drug. Chronic Lymphocytic Leukemia (CLL) is a clinically heterogeneous disease remaining incurable despite introducing new promising treatments. The effects of VPA and its mechanism of action were evaluated on mononuclear cells isolated from 40 CLL patients. Exposure of CLL cells to increased doses of VPA (0.5–5mM) leads to a dose-dependent cytotoxicity and apoptosis in all CLL patients. VPA treatment induced apoptotic changes in CLL cells including phosphatidylserine externalization and DNA fragmentation. The mean apoptotic rates were similar between IGHV mutated and unmutated patients, the latter presenting a more aggressive clinical course. VPA induced apoptosis via the extrinsic pathway involving engagement of the caspase-8-dependent cascade. Interestingly, VPA increased the sensitivity of leukemic cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) even among resistant patients. Moreover, VPA at physiological concentration of 1mM can significantly increase the in vitro cytotoxic effects of fludarabine, bortezomib and the natural product honokiol allowing the reduction of effective concentration 50% (EC50). In order to understand the early mechanism of action of VPA, we investigated gene expression profiles of 14 CLL-patient samples (7 with a good prognosis and 7 with a bad prognosis regarding IGHV mutational status and Zap-70 expression) treated in vitro during 4 hours with a physiological dose (1mM) of VPA and compared with their untreated counterpart using Affymetrix technology. No difference in gene modulation was observed between poor and good prognosis patients after VPA treatment. Modulation of several pro- and anti-apoptotic mRNA expression was confirmed by a real-time reverse transcription-PCR. The molecular analysis of the apoptotic machinery involved in VPA response revealed the up-regulation of APAF1 (5.5 fold, P<0.0001), BNIP3 (2.2 fold, P=0.0006), PTEN (1.9 fold, P=0.0002), CASP6 (2.5 fold, P<0.0001) and the down-regulation of CFLAR/FLIP (2.0 fold, P<0.0001), BCL2 (1.6 fold, P=0.0222), AVEN (1.9 fold, P<0.0001), BIRC4/XIAP (1.7 fold, P<0.0001) and BIRC1/NAIP (1.6 fold, P=0.0007). In conclusion, VPA induced apoptosis of CLL cells at clinically relevant concentration by selective activation of the caspase-8 (extrinsic) pathway and by targeting several pro- and anti-apoptotic genes. Therefore, the combined application of VPA with other drugs might be considered as a potential strategy for CLL treatment.
Tissue Factor and Cancer Procoagulant as Predictors of Thrombosis or Progression in Cancer Patients.
Authors: Molnar Soledad , Guglielmone Hugo , Minoldo Salvador , Rizzi Maria L. , Lavarda Marcelo , Jarchum Gustavo 
Published: October 2019

Abstract
Background: Two procoagulant factors are associated with the tumor cell: tissue factor (TF) and cancer procoagulant (CP). The activation of the coagulation system can cause thrombosis and it has also been involved in the development of metastasis.
Aims: 1- to establish the relationship between the presence and the levels of both procoagulants and the development of thrombosis or disease progression. 2- to analyse if the treatment with chemotherapy increases the levels of TF or CP.
Methods: Patients with recent diagnosis of cancer were included in this prospective study. TF and CP determinations were done at diagnosis, at three months after chemotherapy, and at either event of progression or thrombosis. TF evaluation was done through ELISA test (normal value 159 pg/ml) and CP evaluation by a coagulometric method (Gordon et al, Thromb Res1989;56:431–40). In the last test negative values indicate high CP activity. Statistical analysis of the data was performed by two-tailed Fisher exact test and Mann-Withney/Wilcoxon test.
Results: 31 patients, 61% female sex, age median 54 years. Digestive cancer 49%, breast 32%, genitourinary 13%, lung 2%. Stages of the disease were: early (I and II) 48% and advanced (III and IV) 52%. Median follow-up was 11.6 months. High levels of TF and CP at diagnosis were seen in 48% and 84% respectively. No patients developed thrombosis. Nine patients had disease progression (29%) and 4 (13%) died. Eigthy eight percent patients of this last group had advanced stages of the disease. The type of tumor more frequently associated with the event was digestive cancer (45%) and genitourinary cancer (33%). TF and CP values were increased by 40% and 50% respectively during chemotherapy treatment. Level of CP at diagnosis predicted progression (-26 sec versus -8 sec in patients with and without progression p 0.006) and also mortality (-34 sec versus -10 sec p0.003). TF at diagnosis predicted neither progression nor mortality. All patients who had progressive disease showed increased levels of TF (388 pg/ml vs 217 pg/ml p0.002) and CP (-63 sec vs -23 sec p.006) with respect to the values at diagnosis.
Conclusions: Higher levels of CP at diagnosis are associated with a greater frequency of progression and mortality. No patients developed thrombosis. TF and CP levels were increased at the time of progression with respect to the values obtained at diagnosis. Chemotherapy increases the values of both procoagulants. A larger follow-up and a large number of patients could clarify the role of these procoagulants in the development of disease progression (metastasis).
Enhanced Efficacy of Recombinant Factor VIII in Non-Covalent Complex with PEGylated Liposome in Hemophilia A Mice.
Authors: Pan Junliang , Liu Tongyao , Kim Ji-Yun , Patel Chandra , Newgren Jim , Sommer Jurg , Pierce Glenn , Jiang Haiyan 
Published: October 2019

Abstract
BAY 79-4980, a non-covalent complex of rFVIII (Kogenate® FS) reconstituted in PEGylated Liposome (PEG-Lip) diluent, was reported to double the bleed-free days in severe hemophilia A patients from 7 to 13 days (35 IU/kg) and 6 to 11 days (25 IU/kg), as compared to respectively corresponding doses of rFVIII1. However, in a more recent phase I trial, the FVIII pharmacokinetics (PK) in plasma was virtually identical in patients who received either rFVIII or BAY 79-4980. To understand the mechanism of action of BAY 79-4980, we compared the pharmacological efficacy and PK of BAY 79-4980 to rFVIII in Hemophilia A (HemA) mice. A tail vein transection model was established, in which the survival of animals following the injury correlated to the rFVIII dose administered at 24 hrs prior to the injury, and a 2.5 to 3-fold difference in rFVIII dose led to significantly different survival rates (p<0.05). In this bleeding model, BAY 79-4980 (13 IU/kg) demonstrated significantly enhanced (p=0.018) survival compared to rFVIII at 24 hrs post dosing. The formation of a non-covalent complex of rFVIII with PEG-Liposome prior to the administration is required for the improved efficacy of BAY 79-4980, as sequential infusion of rFVIII and PEG-Liposome was no longer different from the identical dose of rFVIII alone. The plasma PK of FVIII in HemA mice treated with either BAY 79-4980 or rFVIII displayed two-compartmental decay with comparable incremental recovery, clearance and volume of distribution. However, BAY 79-4980 showed a modest (13%) but significant (p=0.024) increase in terminal half-life, and a noticeable (42%) but not statistically significant (p>0.05) increase in AUC, compared to rFVIII. Similar results were also observed in a whole blood PK study in HemA mice that received 125I-labeled rFVIII reconstituted in PEG-liposome versus buffer. While the difference in plasma FVIII levels resulting from different terminal half-lives of BAY 79-4980 and rFVIII may account for the improved efficacy of BAY 79-4980 in HemA mice, other mechanisms, such as the association of PEG-liposome with platelets and monocytes, thereby altering their pro-hemostatic functions, are under investigation.
The Use of Ideal Amphipathic Helical Peptides To Reduce Hemorrhage In Vivo.
Authors: Charbonneau Sophie , Ganopolsky Jorge G. , Pang Henry T. , Shek Pang N. , Blostein Mark D. 
Published: October 2019

Abstract
We have previously demonstrated that a 22 amino acid ideal amphipathic peptide (IAP) of K7L15 composition dramatically accelerates both factor IXa and factor Xa activity. In the present work, we investigate the activity of IAP attached to a surface in view of designing a procoagulant surface to reduce hemorrhage. Our results show that IAP maintains its catalytic enhancing properties for factor IXa and factor Xa when attached to a surface. This enhancement is dependent on the presence of the gamma-carboxyglutamic acid domain of factor X, consistent with the hypothesis that IAP behaves as a phospholipid membrane, providing a surface for the assembly of procoagulant enzymes and substrates. To further confirm this hypothesis, we demonstrate direct binding between surface-bound IAP and the Gla domain of factor X using an ELISA-based binding assay. Based on the aforementioned evidence that immobilized IAP enhances procoagulant activity, we conducted in vivo experiments using an ear-bleeding model in rabbits. We incorporated IAP into DuraSeal, a commercially available sealing agent, and found that the addition of IAP decreases the bleeding time in rabbits by 25% (p=0.0065). In conclusion, the above data provide a rationale for designing procoagulant surfaces in vivo. Further evaluation in larger animal models is warranted.
Effects of Direct Factor Xa Inhibitor, YM150, on Clot Formation and Clot Lysis In Vitro Compared with Other Anticoagulants.
Authors: Kaku Seiji , Suzuki Ken-ichi , Funatsu Toshiyuki , Saitoh Minori , Koshio Hiroyuki , Ishihara Tsukasa , Hirayama Fukushi , Kawasaki Tomihisa 
Published: October 2019

Abstract
The objective of this study was to evaluate the effects of direct factor Xa inhibitor, YM150 and its major in vivo metabolite, YM-222714, on clot formation and clot lysis compared with other anticoagulants, such as a direct thrombin inhibitor (melagatran), a pentasaccharide (fondaparinux), low molecular weight heparin (enoxaparin) and unfractionated heparin. To assess clot lysis, the tissue plasminogen activator (tPA)-induced clot lysis assay was used with human plasma triggered by low and high levels of tissue factor (TF). Under low TF conditions, clot formation was completely prevented by melagatran at 1 μmol/L, by fondaparinux at all concentrations examined (0.1 to 1 μg/mL), by enoxaparin at 0.3 and 1 IU/mL and by heparin at 0.1 and 0.3 U/mL. Even under high TF conditions, 0.3 U/mL heparin prevented any clot formation. Although melagatran, fondaparinux, enoxaparin, and heparin potently prevented plasma clot formation under low TF conditions, under high TF conditions they were less effective at prolonging the clotting time. Under both low and high TF conditions, YM150 and YM-222714 prolonged the clotting time in a concentration dependent manner at concentrations between 0.3 and 3 μmol/L. YM150 and YM-222714 significantly accelerated clot lysis under both low and high TF conditions, but their effects were most evident under high TF conditions. Lower concentrations of melagatran (0.1 and 0.3 μmol/L) enhanced clot lysis under low TF conditions, but under high TF conditions, enhancement of clot lysis required higher melagatran concentrations (0.3 μmol/L or more). Under high TF conditions, fondaparinux enhanced clot lysis only at the highest concentration tested (1 μg/mL). Enoxaparin and heparin enhanced clot lysis under low TF conditions at the lowest test concentrations (0.1 IU/mL and 0.03 U/mL, respectively). Both also enhanced clot lysis under high TF conditions, but their effect reached statistical significance only at higher concentrations (1 IU/mL and 0.1 U/mL, respectively). These results suggested that direct factor Xa inhibitors, YM150 and YM-222714, exert stable anticoagulant effects independently of TF concentration. Both inhibitors enhanced tPA-induced fibrinolysis in human plasma clotted via the extrinsic coagulation pathway. Useful characteristics of YM150 and YM-222714, such as a linear dose response and reliable anticoagulation independent of TF concentration, may lead to the creation of an anticoagulant that is easier to use in the clinical setting than existing products. Potentially beneficial antithrombotic effects, which can be promoted by accelerating endogenous fibrinolytic pathways, may further aid in the prevention or treatment of thrombosis.
Comprehensive Molecular Characterization of Malignant and Microenvironmental Cells in Waldenstrom’s Macroglobulinemia by Gene Expression Profiling.
Authors: Hatjiharissi Evdoxia , Mitsiades Constantine S. , Bryan Ciccarelli T. , Lian Xu , Yang Cao , Adamia Sophia , Leleu Xavier , Roccaro Aldo , Ioakimidis Leukothea , Hunter Zachary R. , Patterson Christopher J. , Ghobrial Irene , Treon Steven P. 
Published: October 2019

Abstract
Waldenstrom’s Macroglobulinemia (WM) is an incurable B-cell malignancy characterized by bone marrow (BM) infiltration with a spectrum of clonally related cells, including small lymphocytes and lymphoplasmacytic cells (CD19+) as well as mature plasma cells (CD138+). The molecular pathogenesis of the disease remains to be defined. We therefore analyzed the gene expression profiles of CD19+ and CD138+ BM mononuclear cells from 30 untreated patients with WM and compared their gene expression profile to their normal counterparts from 10 healthy donors using Affymetrix microarrays (U133 plus 2.0). Since the microenvironment plays an important role in the pathogenesis of WM, we also profiled and compared gene expression profiling for CD19 and CD138 depleted BM mononuclear cells from the same patients and healthy donors. Gene expression analysis was performed using dChip software. Unsupervised hierarchical cluster analysis demonstrated distinct gene expression patterns between WM cells versus their normal counterparts. In supervised hierarchical cluster analysis selecting for genes with > 2 fold change in expression and a False discovery Rate (FDR) < 2%, a set of 1171, 582 and 360 genes were found to be differentially expressed between WM patient and healthy donor CD19+, CD138+, as well as CD19/CD138 depleted (microenvironmental) cells, respectively. Among the most significantly over-expressed genes in the CD19+ compartment in WM patients were: BCL2, TNFRSF13B, TNFRSF17, IGLL1, CCR2, CLLU1, whilst the AP1 family genes JUND and FOSB were among the most significantly down-regulated genes in both CD19+ and CD138+ cells in WM patients. Other interesting transcripts which were over-expressed in CD138+ cells from WM patients included those from genes involved in transcription regulation (ZKSCAN1, ZMYM1, ZNF189, ZNF19, and ZNF559) and interferon response (IFI16 and IFIH1). Of considerable interest was our observation that microenvironmental cells in WM patients demonstrated an overactive transcriptional profile composed of genes which are associated with immune and inflammatory responses including the Toll like receptors (TLR 1,5,7,8), interferon and cytokines (IFI16, IFNAR1, IL-10R, IL-8R), genes encoding extracellular matrix components (Fibronectin and Hepatocyte Growth Factor) as well as genes involved in apoptotic signaling (TNFSF10, TRAF4). These studies provide the first comprehensive molecular characterization of WM, dissecting the molecular features of the two immunophenotypically distinct populations of malignant cells, and providing for the first time evidence for a distinct molecular profile in BM microenviromental cells.
p53 Signaling in Response to Increased DNA Damage Sensitizes AML1-ETO Cells to Stress-Induced Death.
Authors: Krejci Ondrej , Wunderlich Mark , Geiger Hartmut , Chou Fu-Sheng , Schleimer David , Jansen Michael , Andreassen Paul R. , Mulloy James C. 
Published: October 2019

Abstract
Chromosomal translocation 8;21 is present in 10–15% of patients with acute myeloid leukemia and results in the expression of the AML1-ETO (AE) fusion protein. AE expression by itself is not sufficient for malignant transformation of bone marrow progenitor/stem cells, but the nature of the cooperating genetic alterations are unknown. It is unclear whether AE facilitates acquisition of these synergizing events. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic expansion of CD34+ progenitor cells that retain the ability to differentiate into myeloid, lymphoid, and erythroid lineages and engraft NOD/SCID mice. Consistent with mouse studies, these transgenic human cells fail to cause disease in immunodeficient mice or become transformed in vitro. These cells serve as a highly relevant pre-leukemia model for t(8;21) leukemogenesis. Here we demonstrate that AE downregulates genes involved in multiple DNA repair pathways, an effect that is evident as early as two days after retroviral transduction of human CD34+ cells. The human AE cells show delayed repair of induced DNA damage as shown by kinetics of a comet assay. During in vitro culture, AE cells show spontaneous accumulation of DNA damage, as demonstrated by γH2AX staining. Using a transgenic mutation frequency assay based on the lacZ reporter gene, we show that the in vivo mutation frequency of murine BM cells was increased approximately two fold by AE expression. Possibly as a result of this chronic DNA damage, AE cells have increased p53 protein expression and an upregulation of multiple p53 response genes. This results in increased basal apoptosis and enhanced sensitivity to DNA damaging agents compared to control cells. Intriguingly, microarray data indicate that t(8;21) patient samples also exhibit increased expression of p53 response genes when compared to other AML patient samples. Inhibition of the p53 pathway by shRNA interferes with the G1 cell cycle checkpoint and increases the resistance of AE cells to DNA damage. We thus speculate that AML1-ETO may indeed facilitate accumulation of additional genetic alterations by suppressing endogenous DNA repair, but that this activity also renders the cells more susceptible to stress. It is possible that the superior outcome of t(8;21) patients is partly due to an activated p53 pathway, and that loss of the p53 response pathway in disease progression will consequently negatively impact overall outcome. We are currently investigating whether a “p53 activation” profile can be used as a prognostic tool in AML.
Influence of Protein Kinase C and Calcium in the Course of Thrombin Receptors Activation.
Authors: Xie Ying , Han Yue , Wu De Pei , Sun Aining , Zhang Wei 
Published: October 2019

Abstract
Object In order to compare the functions of protein kinase C (PKC) and calcium (Ca2+) in platelet aggregation and platelet membrane surface glycoproteins GPIb expression after thrombin receptors activation, then to investigate the role of Gq signal transmission pathway in the course of thrombin receptors activation.
Methods Peptide SFLLRN (PAR1-AP) and AYPGKF (PAR4-AP) were used for stimulating platelet at different time point (0, 1, 2, 5, 10, 30min), then the alterations of platelet aggregation and GPIb were analyzed in the involvement of Ro-31-2220 (inhibitor of PKC) and BAPTA/AM (calcium chelator).
Results Either PAR1 or PAR4 peptide can induce absolute platelet aggregation, together with a reversible internalization of GPIb. Platelet aggregation was inhibited by Ro-31-2220 or BAPTA/AM while the shape change curve still occurred upon PARs activation. In addition, Ro-31-2220 decreases GPIb centralisation upon PAR1 stimulation (P <0.05 at 1, 2 min), though it blocks the pool of GPIb inside platelet in PAR4 activation (P <0.05 at 10, 30 min). Meanwhile, GPIb internalization was blocked by BAPTA for both peptides (P <0.05 at 1∼10 min).
Conclusion All the results confirm a critical role of Gq pathway in thrombin signal transmission through the involvement of protein kinase C and calcium. Calcium is closely correlated with the thrombin receptors activation, seemed to be similar for two PARs signal pathways. Protein kinase C urges GPIb centralisation in PAR1 pathway and accelerates GPIbα return in PAR4 pathway.