bluuebunny/crossref_metadata_2025_split huggingface dataset:
Divergent Point of Developmental Cell Fate of Common Precursor for Cardiomyocyte and Hemangioblast Is Accompanied by the Presence or Absence of Expression of PDGFRalpha and Flk-1.
Authors: Hirata Hirokazu , Murakami Yoshinobu , Tanaka Yosuke , Inoue Kayoko , Ishikawa Masakazu , Sasada Masataka , Asahara Takayuki , Kawamata Shin 
Published: April 2020

Abstract
Pluripotency of embryonic stem (ES) cells provides a powerful tool for dissecting the cellular and molecular mechanisms regulating germ layer fate determination and tissue formation. Both of hemangioblasts and cardiomyocytes are thought to be derived from lateral plate mesoderm of early embryo, but little is known about the phenotypic specification in these two lines of differentiation at the divergent point through development. Utilizing the in vitro differentiation system of murine ES cells in the presence or absence of stromal cell layer, and gene expression data in the microarray analysis, we determined a set of cell-surface molecules as specific markers, which enabled efficient enrichment of hemangioblasts or cardiac myocytes. Furthermore, we represent the evidences that the sorted fraction with hemangioblast-specific markers markedly produced both of blood island and endotherial colonies in stromal-free culture system.
Materials and Methods
Murine embryonic stem cell line, EB5 was cultured on gelatin-coated plates for mesodermal differentiation after withdrawal of LIF and Blastcidin. The expression of surface molecules related to mesodermal differentiation was evaluated with flowcytometry or quantitative RT-PCR techniques. The cell population defined by combination of the candidate surface molecules was sorted at day 5 and seeded on PA-6 or OP-9 stromal cell layers, or in conditioned medium of PA-6 cells for stroma-free assay until day 12 of differentiation. Differentiational direction of each culture sample was evaluated by the expression of genes or membrane molecules specific for tissue lineages with quantitative RT-PCR or flowcytometric techniques. The cardiogenic potential was also assessed by scoring beating colonies co-cultured on stromal cells.
Results and Discussion
In combination with cell surface expression of Flk-1 (VEGFR2), which is known to be a restricted marker for mesodermal tissues during early embryonic development, upregulated expression of Platelet-Derived Growth Factor (PDGF) receptor alpha on cell surface was detected more frequently in the fraction containing pre-cardiomyocytic cells. In contrast, negative fraction for PDGFRalpha contributed to efficient formation of hematopoietic burst colonies growing together with endotherial cells engulfing DiI- acetylated LDL. We also cofirmed that these suspended hematopoietic cells were spurting out of hemangioblast-like compartment in time-lapse imaging analysis. Our findings may provide a new experimantal window to analyze the common mesodermal precursors for hemangioblasts and cardiomyocytes in vitro. We will search for tissue stem cells bearing the corresponding surface markers in human cell sources, and evaluate their differentiation potentials and therapeutic effect on ischemic diseases.
Specificity of Smad Signaling during Primitive Erythropoiesis.
Authors: Zafonte Brian T. , Huber Tara L. , Keller Gordon , Evans Todd 
Published: April 2020

Abstract
Bone morphogenetic proteins (BMPs) comprise a sub-family of TGF-beta-like molecules that exert a wide range of biological activities during development, and are essential for normal hematopoiesis. However, the precise stage in development that BMP signaling regulates hematopoiesis is not defined. Three proteins, Smad1, Smad5, and Smad8 transmit BMP signals to the nucleus to activate the expression of hematopoietic-specific transcription factors. These Smads are homologous in their sequences, and appear to be regulated similarly, however their specificity in regulating hematopoiesis remains undefined. Although Smad proteins are regulated post-translationally, their expression is also under transcriptional control during development. We examined the specificity of Smad1/5/8 activity in the context of primitive erythropoiesis, using the mouse embryonic stem cell /embryoid body (ES/EB) system. We exploited ES cells with GFP targeted to the brachyury locus, in order to identify specific sub-sets of progenitors. Smad1 transcript levels are initially upregulated as ES cells become fated to mesoderm and hematopoietic progenitors, but the levels are significantly decreased in cells derived from differentiating primitive erythroid colonies. In contrast, Smad5 transcript levels show the opposite profile, being more correlated with erythroid differentiation. To directly assess the role of these Smads during erythropoiesis, their activity is being manipulated in ES cells during the commitment phases of embryonic hematopoiesis. For this purpose, inducible ES cell lines were generated capable of forcing the expression of wildtype Smad1 or Smad5, or a dominant-negative isoform of Smad5, at any stage of ES/EB development. Colony assays were used to analyze quantitatively the hematopoietic potential of these cells. Forced expression of Smad1 results in a marked increase in primitive red blood cell colony formation as compared to control ES cells. Maintenance of Smad1 expression does not appear to inhibit terminal differentiation. Based on a time-study of the induction, the effect on erythoid colonies could be due to expansion of earlier progenitors. Current experiments using the in vitro blast assay are examining the direct effect of Smad1 expression on earlier (hemangioblast) development. This data, and analogous analyses of cells induced to express Smad5 or the dominant-negative Smad isoform are in progress and will be presented. These studies should facilitate our understanding of the specificity of BMP-regulated Smads during commitment and differentiation of embryonic stem cells and hematopoietic progenitors.
HLA-G Antigen Expression Is Associated with a More Aggressive, Unfavorable Outcome and Immunosuppression in Chronic Lymphocytic Leukemia.
Authors: Nueckel Holger , Rebmann Vera , Jan Duerig , Ulrich Duehrsen , Hans Grosse-Wilde 
Published: April 2020

Abstract
Background: The human leukocyte antigen (HLA)-G molecule exhibits limited tissue distribution and exerts multiple immunoregulatory functions. Recent studies indicate an ectopic up-regulation in tumor cells which may favor their escape from antitumor immune responses. The role of HLA-G in B cell chronic lymphocytic leukemia (B-CLL) has not been defined.
Experimental design: HLA-G expression was studied retrospectively in circulating B-CLL cells from 47 patients by flow cytometry using the MEM/G9 monoclonal antibody.
Results: The proportion of leukemic cells expressing HLA-G varied from 1% to 54%. Patients with &lt; 23% HLA-G positive cells (according to ROC analysis; designated as the HLA-G negative group) had a significantly longer progression-free survival (PFS) time than patients with &gt; 23% of positive cells (median PFS 120 versus 23 months, p=0.0001). Multivariate analysis revealed that HLA-G expression (hazard ratio 4.81; p=0.002) was next to the initial staging according to Binet (hazard ratio 8.6; p=0.0001) the best independent prognostic factor compared to other known prognostic factors like ZAP-70 status (hazard ratio 3.6; p=0.029) or CD38 status (hazard ratio 1.83; p=0.37). Humoral and cellular immunosuppression was significantly more prominent in HLA-G positive as compared to HLA-G negative patients group (median immunglobuline G (g/l) 4.3 versus 7.3; median total T-cells (per μl) 824 versus 2540).
Conclusions: In B-CLL the level of HLA-G expression is correlated with the degree of immunosuppression and prognosis. To our knowledge this is the first report that describes an association of HLA-G antigen expression and the course of the disease in B-CLL patients. Thus, HLA-G may contribute to the impairment of immune responses against tumor cells and infections. These findings need to be confirmed in a prospective study.
Potential Value of N-Desulfated Heparin without Anticoagulant Activity in Treatment of Complement-Mediated Lysis of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes.
Authors: Ninomiya Haruhiko , Nagasawa Toshiro 
Published: April 2020

Abstract
Paroxysmal nocturnal hemoglobinuria (PNH) is clinically characterized by the complement (C’)-mediated intravascular lysis of PNH-affected erythrocytes and thrombotic diathesis. Treatment methods of intravascular hemolysis, especially on the hemolytic crisis, have not been established yet. Although several reagents have been used for the treatment of PNH hemolysis, the value of prednisone/prednisolone in treating the hemolysis of PNH is controversial; anti-C5 antibody is under clinical study. We previously demonstrated that heparin and low-molecular weight heparin (LMWH) inhibit C’-mediated hemolysis of PNH erythrocytes in vitro, by inhibiting insertion of C5b-7 into the erythrocyte membranes (Ninomiya et al: Inhibition of complement-mediated haemolysis in paroxysmal nocturnal haemoglobinuria by heparin or low-molecular weight heparon. Brit J Haematol109,875–881,2000). However, that the concentration of heparin (or even that of LMWH) required to inhibit C’-mediated lysis of PNH erythrocytes efficiently induces a profound prolongation of APTT (activated partial thromboplastin time) of plasma unables their clinical application. In the current study, we examined the inhibitory effects of heparin, LMWH (dalteparin), and N-desulfated heparin on C’-mediated lysis of PNH erythrocytes (sucrose hemolytic assay) and their anticoagulant effects on normal plasma coagulation system (APTT, TT (thrombin time), and AT-III (antithrombin)-enhancing activity). N-desulfated heparin inhibited C’-mediated lysis of PNH erythrocytes, equivalently to heparin or LMWH, on the basis of their uronic acid (UA) contents; IC50 values of heparin, LMWH, and N-desulfated heparin, was 0.8 U/ml (UA 2.9 μg/ml), 0.5 IU/ml (UA 2.3μg/ml), and 3.0μg/ml (UA 0.9μg/ml), respectively. Whereas the concentrations of heparin and LMWH, that exerted their anti-C’ activity efficiently, induced prolongations of APTT profoundly to clinically unapplicable extents, N-desulfated heparin (up to 50 μg/ml) did not show any anticoagulant activity. These data show a potential value of N-desulfated heparin for the treatment of C’-mediated intravascular lysis of PNH erythrocytes without bleeding complications due to its administration. Because anticoagulant therapy may be beneficial and indicated for the treatment of hemolysis in PNH, especially in hemolytic crisis increasing the risk of thrombotic complications, cocktail therapy of heparin (or LMWH) with N-desulfated heparin may be proposed.
Functional Characterization of Telomerase RNA Variants Found in Patients with Hematological Disorders.
Authors: Ly Hinh , Calado Rodrigo T. , Allard Paulette A. , Baerlocher Gabriela M. , Lansdorp Peter M. , Young Neal S. , Parslow Tristram G. 
Published: April 2020

Abstract
Human telomerase uses a portion of its integral RNA component (hTER) as the template to synthesize telomeres at chromosome ends. hTER sequence polymorphisms have been observed in some patients with bone marrow failure syndromes such as aplastic anemia, but the functional significance of most such variants is unknown. Here, we report the functional characteristics of ten previously-described and two newly discovered hTER disease-associated polymorphisms. Most of these hTER variants adversely affected telomerase enzymatic function as measured in the telomerase reconstituted human cells. Similar loss-of-function effects were also seen directly in primary lymphocytes collected from two of the patients. The majority of the functional deficits stemmed from perturbations of the predicted hTER RNA secondary structure, and corresponded well with the degrees of telomere shortening observed in patients. In contrast, hTER variants anticipated to be inconsequential polymorphisms, which were also found in healthy subjects, did not interfere with telomerase function. Loss of telomerase activity and of telomere maintenance resulting from inherited hTER mutations may predispose some patients to aplastic anemia and other marrow failure disorders.
FLT3 Is Fused to ETV6 in a Myeloproliferative Disorder with a t(12;13)(p13;q12) Translocation.
Authors: Vu Hoang Anh , Xinh Phan Thi , Shimizu Seiko , Masuda Michihiko , Motoji Toshiko , Tokunaga Katsushi , Sato Yuko 
Published: April 2020

Abstract
The FLT3, located at band 13q12 and encoding a receptor tyrosine kinase (RTK), is one of the most frequently mutated genes in hematologic malignancies including ALL, MDS, and AML. The most common mutation of the FLT3 is an internal tandem duplication in exons 14 and 15, whereas other mutations have also been found at and around codon 835 of exon 20. These activating mutations promote constitutive RTK activity in the absence of ligand, proposing FLT3 as an attractive therapeutic target for directed inhibition. However, many questions with regards to the biology of FLT3 and its role in leukemogenesis remain to be clarified. Despite its highly frequent mutations, FLT3 has never been reported to fuse to any other genes, a phenomenon usually observed in other RTKs. Here, we report a case of a novel fusion gene between FLT3 and ETV6 at 12p13, a well-known target for a number of translocations. The patient, a 68-year-old female, was diagnosed as myeloproliferative disorder with hypereosinophilia in May 2002. Peripheral blood showed WBC 33.6x106/L (3% myelocytes, 33.5% neutrophils, 54% eosinophils, 1.5% basophils, 1.0% monocytes and 7% lymphocytes), Hb 119g/L and platelet counts 5,450x106/L. The bone marrow (BM) was marked hypercellular with 0.9% blasts, 6.0% promyelocytes, 15.6% myelocytes, 8.1% immature eosinophils and 19.2% mature eosinophils. Karyotype of BM cells was 46, XX, t(12;13)(p13.1;q12.3–13)[28]/46, XX[2]. Under the suspicion of Ph-negative CML, she was treated with IFNα with no response. Then, HU was started and her WBC decreased to 30x106/L. FISH analysis showed that the breakpoint at 12p13 occurred within ETV6, while the breakpoints at 13q12 occurred at two locations, within FLT3 or CDX2. To identify the fusion partner of ETV6, 3′-RACE PCR was performed. Sequence analysis of PCR-products revealed 4 types of ETV6/FLT3 transcripts. These fusion transcripts were confirmed by Northern blot analysis. Each ETV6/FLT3 transcript contained the entire helix-loop-helix domain of ETV6 (exons 1 to 4 or 5) and almost all of the functional domains of FLT3 including the tyrosine kinase domain (from exons 14, 16 or 17), suggesting that the resultant chimeric protein would be constitutively activated FLT3 kinase. Of them, three are in-frame fusion, presumably encoding for the approximately 58, 62, and 83 kD fusion proteins. However, Western blot analysis showed only expression of the 58 and 83 kD proteins. RT-PCR detected the reciprocal FLT3/ETV6 transcript, comprising the FLT3 exons 1 to 13 frameshifly fused to the ETV6 exons 6 to 8, within which a stop codon appeared at codon 33- downstream from the fusion point. Functional studies to assess the oncogenic properties of these fusion proteins are now in progress. Our findings provide an evidence that FLT3 is also involved in hematologic malignancies as a fusion gene.
A “Single” Dose of G-CSF after Autologous Stem Cell Transplantation in Patients with Malignant Lymphoma - Third Interim Analysis of Multicentre Randomized Trial Comparing Standard Schedule with Delayed Application and Placebo.
Authors: Faber Edgar , Pytlik Robert , Trneny Marek , Slaby Jiri , Kozak Tomas , Raida Ludek , Papajik Tomas , Zikesova Eva , Zapletalova Jana , Maresova Ivana , Hamouzova Michaela , Indrak Karel 
Published: April 2020

Abstract
The optimal schedule of G-CSF given after autologous peripheral stem cell transplantation (ASCT) has not been defined yet. Here we present results of the third interim analysis of randomized multicentre trial comparing standard application of G-CSF from day +5 (arm A) with delayed dosing (G-CSF was started when WBC reached 0,5x109/l and neutrophil count 0,1x109/l; arm B) and placebo (arm C). In order to eliminate the influence of diagnosis and conditioning and to minimize the impact of graft quality only patients with malignant lymphoma conditioned by BEAM and with graft harvested in maximum 3 aphereses containing at least 5,0x106 CD 34+cells/kg were included. Patients signed informed consent before registration into the study that had been approved by local ethical and national health care authorities. 115 patients at median age 45 (range 21 – 64) were randomized by July 15th, 2004. The data of 97 patients were analysed. There was no difference in age, lymphoma bone marrow involvement at diagnosis, number of chemotherapy cycles and the use of radiotherapy before transplantation and CD 34+ cells content in graft between any two of the three arms. Duration of neutropenia below 0,5x109/l and 1,0x109/l and number of days to neutrophil engraftment over 0,5x109/l and 1,0x109/l are listed in the table. A significant difference in duration of neutropenia and time to neutrophil engraftment was found between the arms A and B (p=0,002 – 0,005), the arms A and C (p&lt;0,0001) and also between the arms B and C (p=0,04–0,0001). A trend for delayed platelet engraftment was observed in both arms with G-CSF (A and B). Duration of hospitalisation was significantly longer in placebo group (arm C), however, there was no prolongation of fever or antibiotic use. The delayed „single” application of G-CSF (usually with one or two doses sufficient for full neutrophil recovery) appears to be safe and cost effective. Comparing with standard G-CSF schedule only one day delay in median neutrophil engraftment was observed. We hope to finish the study within the year 2004. Supported by the grant MSN 15110004.
Arm A / G-CSF from day +5 (n=33) Arm B / Tailored initiation of G-CSF (n=32) Arm C / Placebo (n=32) * median(range) Days with neutropenia &lt;0,5x109/l (days) 7 (4–10)* 8 (6–11)* 10 (6–30)* Days with neutropenia &lt;1,0x109/l (days) 8 (5–15)* 9 (7–11)* 11,5 (6–30)* Neutrophil engraftment &gt;0,5x109/l (day +) 10 (9–12)* 11 (9–15)* 14 (9–30)* Neutrophil engraftment &gt;1,0x109/l (day +) 11 (9–22)* 12 (9–15)* 15 (10–30)* Platelet engraftment &gt;20x109/l (day +) 12 (2–33)* 12 (7–30)* 11 (7–24)* Platelet engraftment &gt;50x109/l (day +) 17,5 (8–100)* 16,5 (9–115)* 13 (8–32)*
Insertion Sites of Retroviral Gene-Marking Vectors Influence the Contribution of Individual, Non-Malignant Cell Clones to Long-Term Hematopoiesis.
Authors: Kustikova Olga , Li Zhixiong , Baum Christopher , Fehse Boris 
Published: April 2020

Abstract
Replication-defective gammaretroviral vectors have successfully been used for gene marking and therapy studies. Recently, we and others have shown that proto-oncogene upregulation due to retroviral vector insertion may trigger malignant transformation of engineered cells. We have now investigated whether insertional mutagenesis may also influence the contribution of individual cell clones to normal long-term hematopoiesis. Therefore, using LM-PCR we retrieved retroviral insertion sites from hematopoietic cells of mice exhibiting progression to oligoclonal or monoclonal hematopoiesis upon serial bone marrow transplantation. From 28 mice analyzed (12 primary, 16 secondary) we obtained a total of 67 insertions that matched the murine genome. In samples harvested from primary recipients seven months after transplantation of gene-marked cells a number of intriguing hits (HoxB4, Evi1, Ly78, Ccnd3, Pip5k2a) was found obviously already reflecting selection for long-term repopulating ability. Strikingly, almost all clones analyzed five months after serial transplantation revealed hits in genes with an established or potential role in the self-renewal process of hematopoietic stem cells (e.g., Vegfa, HoxB4, CyclinD3, Evi1) or other genes involved in the regulation of cell survival (Ly78, Pip5k2a, Irf2bp, Cflar). Two (Evi1, Ccnd3) among the 13 insertions associated with serial repopulation activity were already established common integration sites (CIS) in the context of mouse leukemias elicited by replication-competent retroviruses (RTCGD database). Using RT-PCR transcriptional activation involving fusion transcripts originating from the retroviral LTR into exons could be detected for the two genes (HoxB4, Vegfa) tested. Interestingly, Evi1 was the only gene targeted several times - with distinct insertions in 5 different clones. However, despite the fact that Evi1 was also the only established proto-oncogene found to be marked, four of the clones detected in primary recipients were lost upon serial transplantation. This indicates that insertion into the Evi1 locus, although having the potential to promote engraftment and/or survival of a given clone, was not sufficient for malignant transformation. Thus our data demonstrate a delicate regulatory balance following insertional mutagenesis by replication-deficient retroviral vectors. These findings have major implications for interpreting results from diagnostic gene marking experiments, for gene therapy, and the discovery of genes regulating stem cell turnover.
(The first two authors contributed equally to this work)
Breakpoint-Specific Targeting of p210BCR-ABL mRNA by siRNA Alters Progenitor Cell Proliferative Rate in CML.
Authors: Marley Stephen B. , Withey Jane , Harvey Amanda , Crompton Mark , Gordon Myrtle 
Published: April 2020

Abstract
Abrogating expression of p210BCR-ABL or of associated cell-signalling proteins in CML through siRNA-targeted destruction of mRNA has enormous potential in CML, both as a research tool and as a potential therapy, especially for imatinib resistant disease. We have investigated a p210BCR-ABL junction-specific (b3a2) siRNA. We used an Oligofectamine transfection protocol which gives high transfection rates with minimal toxicity. In the Bcr-Abl-positive cell line, K562, this siRNA reduced p210BCR-ABL expression by up to 80% compared with actin controls and induced significant levels of apoptosis. Silencing of p210BCR-ABL has been reported by Scherr et al (Blood 2003, 101:1566–69) not to induce apoptosis in primary Ph+ cells. Similarly, we showed that the initial CFU-GM plating efficiency from b3a2 CML peripheral bloods treated with breakpoint-specific siRNA was not significantly reduced (100+/−5% of untreated controls). Replating large numbers of individual CFU-GM and analysing the distribution of secondary colonies can quantify the proliferative capacity of the myeloid progenitor pool. Using this assay, we showed that siRNA transfection significantly reduced the proliferative capacity of CML CFU-GM in a breakpoint-specific fashion (56+/−18% of untreated controls). This reduction in proliferative capacity is very similar to the CML-specific reduction in proliferative capacity induced by Interferon-alpha (50+/−40% of untreated controls) or by imatinib mesylate (50+/−30% of untreated controls). To our knowledge, this is the first demonstration of a functional consequence of siRNA treatment of primary CML haemopoietic cells. Our findings support the view that siRNA-based therapies may ultimately be of benefit, particularly in the management of CML