bluuebunny/crossref_metadata_2025_split huggingface dataset:
mTOR Inhibitors Induce Apoptosis and Inhibit Growth of Primary Adult Human ALL in Xenograft and Tissue Culture Models.
Authors: Teachey David T. , Brown Valerie I. , Hall Junior , Cooperman Jonathan , Obzut Dana , Carroll Martin , Choi John K. , Grupp Stephan A. 
Published: April 2020

Abstract
While children with precursor B cell acute lymphoblastic leukemia (ALL) are often cured, adults with ALL usually succumb to their disease. Thus, the development of novel therapeutic agents is paramount. mTOR inhibitors (MTI) are a class of signal transduction inhibitors developed as immunosuppressive agents. We have previously shown the ability of MTI to inhibit growth and induce apoptosis in precursor B ALL cell lines and primary murine pre-B leukemia. To determine if this finding could be translated to clinical therapy, we explored if MTIs would be similarly effective in three primary human adult ALL samples. We have tested the MTIs rapamycin and CCI-779 in these models with similar results, but the results utilizing CCI-779 are presented below.
Stromal culture. ALL blasts were maintained in vitro on irradiated bone marrow stromal cells. Cells normally can be maintained for several weeks in these conditions. Cells were either untreated or treated with CCI-779 at 100ng/ml. Long-term cultures were assessed for the effect of MTI on cell proliferation and short term (48hr) cultures were assessed for induction of apoptosis. Treated cells showed a dramatic decrease in cell proliferation (6–24 fold compared to untreated) and a 4–5 fold increase in apoptotic cells as detected by Annexin-V compared to untreated cells.
NOD/SCID xenografts. To further evaluate the effect of MTI on ALL cells, patient samples were engrafted into NOD/SCID animals for analysis. Robust engraftment, expansion and repopulation in secondary hosts of ALL cells was seen in 78% of tested samples, demonstrating greater than 70% ALL in peripheral blood, bone marrow, and spleen in the majority of the mice. There was greater than 10 fold expansion of disease within the mouse. Engraftment was detected by flow cytometry for human CD19+/CD45+ cells. Engrafted animals with established disease (&gt;5% peripheral blasts) were either not treated or treated with the MTI CCI-779. Currently we have treated 45 mice engrafted from three separate patient samples. Untreated animals continued to show expansion of human ALL cells. In dramatic contrast, animals treated with CCI-779 as a single agent showed a 4–30 fold decrease in peripheral blood blasts and a decrease in splenomegaly (p&lt;.02).
Our data show that both in vitro and in an in vivo model of established ALL, MTIs decrease proliferation of lymphoblasts and promote apoptosis of ALL cells. These results suggest that the mTOR signaling pathway is necessary for the survival of ALL cells and that MTIs should be analyzed as therapeutic agents for the therapy of ALL.
Mutations in the VKORC1 Gene Cause Warfarin Resistance, Warfarin Sensitivity and Combined Deficiency of Vitamin K Dependent Coagulation Factors.
Authors: Oldenburg Johannes , Rost Simone , Fregin Andreas , Geisen Christof , Ivaskevicius Vytautas , Seifried Erhard , Scharrer Inge , Heistinger Maximilian , Tuddenham Edward , Muller-Reible Clemens , Zieger Barbara 
Published: April 2020

Abstract
Coumarins target blood coagulation via inhibition of the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, an essential cofactor for the post-translational gamma-carboxylation of several blood coagulation factors. Recently, two groups including ours identified a key component of the VKOR which we named Vitamin K Epoxid Reductase Subunit 1 (VKORC1). The corresponding gene comprises a 5 kb genomic region and consist of three exons encoding a small 163 aa transmembrane protein. Since VKOR was hypothesized to be involved in two heritable diseases, combined deficiency of vitamin K dependent clotting factors type 2 (VKCFD2) and resistance to coumarin-type drugs (warfarin/phenprocoumon resistance, WR) we sequenced the VKORC1 gene in patients with these phenotypes. The same homozygous missense mutation (R98W) was found in two families from Germany and Lebanon with a mild VKCFD2 phenotype. In 7 patients from 5 families with various degrees of WR (weekly warfarin dose ranged from 115 mg to 280 mg, two patients exhibited complete WR) different heterozygous missense mutations distributed throughout the gene (V29L, V45A, R58G, V66M, L128R) were identified. Furthermore, we proved a heterozygous nonsense mutation (W5Stop) in a young female patient with highly increased phenprocoumon sensitivity. Over-expression of the wild-type protein in HEK 293 cells leads to a striking increase in VKOR activity which is sensitive to warfarin inhibition. In contrast to the wild-type protein, the VKCFD2 variant exhibited a dramatically decrease of VKOR activity. The WR variants showed a varying reduction of the VKOR activity, but unexpectedly, were sensitive to warfarin in the HEK cell expression system The reason for the differing WR phenotypes in human and in the HEK cell expression system are still to be elucidated. In conclusion, VKORC1 protein most likely represents the molecular target of coumarins, which have prescribed for oral anticoagulant therapy since more than 60 years. Mutations in the VKORC1 gene are causative for the hereditary conditions of VKCFD2, warfarin resistance and also warfarin sensitivity. Our findings may provide a basis for a rational design of novel anticoagulants targeting VKOR in the future.
Optimal TBI Dose to Improve the Survival in Unrelated Matched Marrow Transplantation for Adult Patients with Severe Aplastic Anemia: Prospective De-Escalation Study of TBI Dose.
Authors: Lee Jong-Wook , Eom Ki Seong , Park Yoon Hee , Kim Yoo Jin , Lee Seok , Min Chang Ki , Kim Hee Je , Kim Dong Wook , Jin Jong Youl , Min Woo Sung , Park Chong Won , Kim Chun Choo 
Published: April 2020

Abstract
Background and Objectives: Patients with severe aplastic anemia (SAA) who do not have suitably HLA-matched related donor generally receive immunosuppressive therapy (IST) as first-line treatment and are considered for transplantation from matched unrelated donor (MUD) if they fail to respond to IST. To determine the optimal total body irradiation (TBI) dose as conditioning regimen in unrelated marrow transplantation for adult SAA patients, we conducted a prospective study to evaluate minimal dose of TBI sufficient to achieve sustained engraftment when it is used in combination with fixed dose of cyclophosphamide (CY).
Methods: Between May 1998 and December 2003 twenty-eight patients were enrolled with a median age of 23 years old (20–44). All patients were multiply transfused and had received 1 or 2 course of intensive IST. Conditioning regimen consisted of CY (120mg/kg) plus TBI and they were divided into 3 group according to the fractionated TBI dose: group 1 (1200 cGy, n=5), group 2 (1000 cGy, n=9), and group 3 (800 cGy, n=14). Donor/recipient pairs were matched for HLA-A, -B by serology and -DRB1 by low-resolution DNA typing (n=15) or HLA-A, -B, and -DRB1 by high-resolution typing (n=13). Patients received CsA+MTx (n=4) or FK506+MTx (n=24) as GVHD prophylaxis.
Results: All patients except one (group 2) achieved sustained engraftment. The incidence of acute GVHD more than grade II was not significantly different (60% in group 1, 38% in group 2, and 43% in group 3), but chronic GVHD developed more frequently in group 1 and 2 (60% & 80%, respectively) compared to group 3 (15%). Patients who received 800 cGy (group 3) had significantly better survival than those 1000 and 1200 cGy (93% in group 3, 44% in group 2, and 40% in group 1; group 3 vs group 1 and 2, p&lt;0.02).
Conclusions: Thus, a TBI dose of 800 cGy in combination with CY (120mg/kg) was sufficient to allow for engraftment in adult patients with SAA who received MUD BMT. Outcome in patients who received 800 cGy of TBI was superior to survival that among patients 1000 and 1200 cGy.
Conversion of Erythropoiesis to Myelopoiesis in Gata1-Deficient Zebrafish Embryos.
Authors: Galloway Jenna L. , Thisse Christine , Zhou Yi , Beltre Rosanna , Thisse Bernard , Zon Leonard I. 
Published: April 2020

Abstract
Primitive hematopoiesis in vertebrates initiates in the embryonic yolk sac and yields nucleated erythrocytes and macrophages that later enter circulation. In zebrafish, a group of blood cells contained within the axial vein make up the intermediate cell mass (ICM), the teleost equivalent of the mammalian yolk sac. To identify novel genes involved in hematopoiesis, a high-throughput whole embryo in situ hybridization screen was performed. Examination of the expression pattern of 3700 clones from an adult zebrafish hematopoietic cDNA library discovered 24 genes with expression in the blood during development. Each stage of hematopoiesis was defined by a subset of genes, providing a molecular signature of blood cell maturation, from hematopoietic progenitors to terminally differentiated erythrocytes. By using antisense morpholinos to the transcription factors gata1 and gata2, we were able to dissect the regulation of these 24 genes. Examination of gene expression in Gata1, Gata2, and Gata1/Gata2-deficient animals revealed that most erythroid genes are dependent upon Gata factors for expression. Surprisingly, three novel genes, expressed in hematopoietic progenitors, do not require Gata factors for their expression demonstrating that some erythroid genes are regulated in a Gata-independent manner. During our analysis, we also found persistent ectopic expression of the myeloid transcription factor, PU.1, in the ICM cells and a subsequent expansion of mpo expressing granulocytes and L-plastin expressing macrophages. By utilizing gata1-GFP transgenic zebrafish, we were able to isolate blood cells by flow cytometry and examine their morphology. We discovered that blood cells from the Gata1-deficient animals exhibited features characteristic of myeloid cells when compared to wild-type blood cells. By confocal microscopy, we detected some blood cells in the ICM of Gata1-deficient embryos that co-express globin and PU.1, while blood cells of wild-type embryos never co-express these markers at this stage. These observations demonstrate that in the absence of Gata1 the presumptive erythroid progenitors have transformed into the myeloid lineage, and that a major cell fate alteration has occurred. Ultimately, our studies have molecularly defined blood development by gene expression, and illustrated that Gata1 governs lineage fate decisions of hematopoietic progenitors in the developing embryo.
Expression of Erythropoietin Receptor-Like Molecule in Xenopus laevis (xeEPOR) and the Development of Anemia by the Administration of Its Recombinant Soluble Form.
Authors: Aizawa Youichi , Nogawa Nami , Kosaka Nobuyoshi , Maeda Yasutaka , Watanabe Takafumi , Miyazaki Hiroshi , Ochiya Takahiro , Kato Takashi 
Published: April 2020

Abstract
The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key ingredient of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. In this study, a cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, named xeEPOR tentatively, was cloned from cDNA library of Xenopus immature erythrocytes. The identities of the deduced entire amino acid sequence to human and murine EPO receptors were 24% and 25%, respectively; whereas transmembrane region and motifs of WSXWS and Box1/2 domains were found in the molecule. The northern analysis revealed that two types of xeEPOR RNAs were expressed in normal peripheral blood cells. In addition, by in situ hybridization and immunostaining with monoclonal antibodies raised against the extracellular domain of xeEPOR (soluble xeEPOR), immature basophilic erythrocytes expressing xeEPOR appeared in peripheral blood of phenylhydrazine-treated adult Xenopus. The fulllength xeEPOR cDNA was introduced into murine FDC/P2 cells and the signaling for the cellular proliferation and differentiation was examined in the presence of serum derived from anemic Xenopus as a stimulator. To further understanding the contribution of the xeEPOR gene expression to primitive and definitive hematopoiesis on Xenopus development, whole mount in situ hybridization was performed. As the binding motif of GATA-1, the hematopoietic specific transcription factor, was located at −24 to −15 base upstream of the translation initiation sequence, the correlated expressions of xeEPOR and Xenopus GATA-1 on developing embryo were evaluated with RT-PCR. The xeEPOR RNA was abundantly expressed at Nieuwkoop stage 30 (blood island formation) and thereafter, and temporally followed the expression of GATA-1, suggesting that the functional expression of xeEPOR was upregulated by GATA-1 in Xenopus as reported in studies on mammalian erythropoiesis. To confirm biological functions of the molecule, soluble xeEPOR was administered into adult Xenopus by intracardiac consecutive injections. The peripheral erythrocyte counts were gradually decreased; meanwhile immature erythrocytes were emerged in the circulation, demonstrating that this molecule plays a significant physiological role in erythropoiesis in Xenopus.
Generation of Human Regulatory T Cells Using NOD/SCID/γ CNULL Mice Model.
Authors: Hiramatsu Hidefumi , Fujino Hisanori , Heike Toshio , Ito Mamoru , Nakahata Tatsutoshi 
Published: April 2020

Abstract
We have reported a NOD/SCID/γcnull (NOG) mice model, which enable efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells. Using this model, we investigated whether various subpopulations of T cells were generated in this unique murine microenvironment.
Freshly collected cord blood was depleted of phagocytes with Silica ® followed by CD34 positive selection using auto MACS ®. The purity of CD34 positive cells always exceeded 98%. These cells were transplanted into irradiated NOG mice intravenously. About 3 months after the transplantation, human T cells in peripheral blood, bone marrow and spleen were analyzed by flow cytometry. As we have reported previously, more than half of the human cells seen in the spleen were human CD3+ T cells and as many as 30% of them expressed CD4 and CD25 without activation markers such as CD69. To examine if these CD4+ CD25+ cells have regulatory activity, CD4+ CD25− cells were stimulated with anti-human CD3 antibody along with irradiated autologous antigen presenting cells in the presence of limiting dose of CD4+ CD25+ cells. The inhibition of proliferation by CD4+ CD25+ cells was analyzed by 3H-thymidine uptake. CD4+ CD25+ cells successfully suppressed the CD4+ CD25− T cell proliferation and RT-PCR analysis revealed the expression of Foxp3, a marker for regulatory T cells, specifically in the CD4+ CD25+ cell population. These results suggest that regulatory T cells can develop from hematopoietic stem cells in our NOG mice model. As human T cells appear first in the thymus of NOG mice, these regulatory T cells are considered to arise in the murine thymus. Our model provides a new and versatile tool to investigate development and function of human regulatory T cells, which are often difficult to study because of complicated history of infection or genetic differences among individuals.
A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential.
Authors: Yang Liping , Aolfsson Jörgen , Månsson Robert , Bryder David , Borge Ole-Johan , Thoren Lina , Sitnicka Ewa , Sasaki Yutaka , Sigvardsson Mikael , Jacobsen Sten Eirik W. 
Published: April 2020

Abstract
The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.
Fludarabine Treated CLL Patients Showed an Increased Chemoresistance Ex-Vivo.
Authors: Schinkothe Timo , Hallek Michael J. , Staib Peter 
Published: April 2020

Abstract
Fludarabine is the most successful drug in patients with relapsed, progradient or refractory chronic lymphocytic leukemia (CLL). But also the use of fludarabine in primary therapy as an alternative to chlorambucil is in trial. Fludarabine induces higher response rates, but showed no improvement in overall survival. The reason for this clinical observation is unclear. In the present study we wanted to answer the question, if the induction of secondary resistance might be the cause for this contradiction.
METHODS. Peripheral blood samples from 42 patients with B-CLL were tested in the in-vitro chemosensitivity-test DiSC-assay with 11 drugs. 10 patients had received no therapy before the time of carrying out the In vitro test. 23 patients were pre-treated with prednisone, 8 patient with fludarabine and 5 patients with vincristine. Most of the patients received more then one drug before testing. The chemosensitivity were determined using the LC90.
RESULTS. The patients were divided into those who were pre-treated vs. not pre-treated with the fludarabine, prednisone or vincristine. The influences of pre-treatment to the sensitivity were analysed using the Mann-Whitney-U test for two independent groups. With fludarabine pre-treated patients had an significantly increased chemoresistance against fludarabine (2.4-fold; p=.004). The increase was also significant for bendamustin (4.4-fold), 2-CdA (3.4-fold), Gemcitabine (2.7-fold) and daunorubicin (1.5-fold). There was no significant influence observed onto the other drugs. Pre-treatment with vincristine or prednisone had no impact onto the chemosensitivity of any tested drug.
INTERPRETATION AND CONCLUSIONS. The results of this study demonstrate the capability of fludarabine to increase the chemoresistance against various drugs in CLL. The use of fludarabine in primary therapy should be discussable. The fact that fludarabine induces higher response rates, without an improvement in overall survival, might be understandable if the fludarabine treatment induces a negative long-term effect. The widely increase of the chemoresistance is such a negative long-term effect. Based on the presented data, we postulate that fludarabine induces a so called secondary resistance by selecting leukemic cells with a lower chemosensitivity under treatment.
Inhibition of Protein Kinase C (PKC) delta with Rottlerin Induces Apoptosis in B-CLL Cells, Augments Chemotherapeutic Cytotoxicity and Inhibits Antiapoptotic Survival Signals.
Authors: Ringshausen Ingo , Weick Kathrin , Oelsner Madlene , Peschel Christian , Decker Thomas 
Published: April 2020

Abstract
Purpose: We have previously shown that PKC delta is constitutively activated in B-CLL cells. In the present study, we have analyzed the mechanism of apotosis induction as well as the effets of PKC delta in the presence of survival signal and chemotherapeutic agents.
Methods: The mitochondrial membrane potential, presence of active caspase 3, conformational status of Bax and apoptosis assays (Annexin V stain, Tunel assay) were performed using flow cytometric analysis. Expression of BCL-2 family proteins and XIAP as well as processing of caspase 8 and 9 was revealed in western blot experiments.
Results: Rottlerin at 5μM (which is not toxic for normal lymphocytes) induced apoptosis in a large amount of B-CLL samples. Rottlerin was equally effective in zap70 positive- and negative samples. Rottlerin induced apoptosis was in part caspase dependent and involved processing of caspase 3, 8 and 9. Expression of antiapoptotic proteins MCL-1 and XIAP was reduced in Rottlerin treated cells and BAX expression increased and Bax conformation changed to its proapoptotic form.IN contrast, expression of bcl-2 was not changed.
B-CL cells are likely to receive survival signals from the microenvironment and might be rescued from cell death induced by chemotherapeutic agents. Rottlerin was very effective in inhibiting survival signals like IL-4 or stromal cell contact. In addition, treatment with Rottlerin enhanced the cytotoxicity of the chemotherapeutic agents Fludarabine, Vncristine and Daunorubicine.
Conclusion: Inhibition of PKC delta might be a powerful treatment option for B-CLL given its potential to induce apoptosis, inhibit antiapoptotic survival signals and augment the cytotoxicity of chemotherapeutic agents.
Chronic Lymphocytic Leukemia Cells Are Dependent on Autocrine VEGF and α4β1 Integrin for Chemokine-Induced Motility.
Authors: Till Kathleen J. , Spiller David G. , Harris Robert J. , Chen Haijuan , Zuzel Mirko , Cawley John C. 
Published: April 2020

Abstract
VEGF is a multifunctional cytokine involved in tumourigenesis. In chronic lymphocytic leukaemia (CLL), it is known that the malignant cells secrete VEGF and possess VEGF receptors. This suggests that an autocrine loop might be important in the pathogenesis of CLL. Here we show that, in patients with lymphadenopathy, autocrine VEGF and α4β1-integrin are involved in the chemokine-dependent motility of CLL cells on and through endothelium - processes important for the invasion of lymphoreticular tissues. In contrast, normal lymphocytes were not dependent on autocrine VEGF or α4β1 for either type of cell movement. Moreover, in contrast to normal B lymphocytes, CLL cells failed to cluster and activate αLβ2 in response to chemokines, unless VEGF receptor(s) and α4β1 were also engaged by their respective ligands. This is the first demonstration that autocrine VEGF is involved in CLL-cell motility and that the αLβ2 on the malignant cells is functionally altered in the disease. Taken together, the results suggest that VEGF and α4 blockade may be therapeutically useful in CLL patients with tissue disease.