bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Genetic Screen for Zebrafish Mutants Affecting Early Hematopoiesis.
Authors: English Milton A. , Lei Lin , Giannetti Margot , Lyons Susan E. , Liu Paul P. 
Published: October 2019

Abstract
Hematopoiesis, or blood cell formation, starts from hematopoietic stem cells and goes through many rounds of cell proliferation and a number of commitment steps that progressively restrict cells to a particular lineage. The generation and maintenance of stem cell pool and the regulation of lineage commitment are poorly understood. Right now there is only one reported zebrafish mutant, cloche, that has defects in early hematopoiesis. We have undertaken a chemical mutagenesis screen using ENU in the zebrafish to uncover mutants in early hematopoiesis. Haploid embryos from F1 females were screened by RNA in situ hybridization using a stem cell marker, core binding factor ? (cbfb), and a myeloid-specific marker, leukocyte-specific plastin (l-plastin). We have screened 510 F1 females and 47 putative mutants have been identified. Of these, 22 showed decreased expression of l-plastin, 3 for cbfb, 2 for both l-plastin and cbfb and the remainder were mutated for l-plastin and other non-hematopoietic markers. Right now, we have focused our efforts on two mutants.
The first mutant line, L32, displayed a blood-less phenotype and showed a significant decrease in the expression of the hemangioblast marker scl. In addition to the marked reduction in the expression of the myeloid specific markers l-plastin, c/ebp1 and mpo, these embryos also showed decreased expression of the erythroid specific marker gata-1. Linkage analysis has placed this mutant to the proximal region of linkage group 12. Currently, we are in the process of testing candidate genes to identify the mutated gene. The second mutant line, L63 also has a blood less phenotype and showed decreased expression of both l-plastin and mpo. Like L32, L63 homozygous embryos have significant developmental defects and die 3 dpf. Low resolution mapping has placed this mutant to LG 8. Through this approach, we hope to identify novel genes that play critical roles in the developmental process regulating early hematopoiesis.
Fibroblast Growth Factors Regulate Stem Cell Functioning In Vitro and in Vivo.
Authors: Yeoh Joyce S.G , van Os Ronald , Weersing Ellen , Dontje Bert , Vellenga Edo , Bystrykh Leonid , de Haan Gerald 
Published: October 2019

Abstract
Fibroblast Growth Factors (FGF) are a large family of signaling molecules widely involved in tissue development, maintenance and repair. Little is known about the role of FGF/FGF-receptor signaling in the regulation of adult hematopoietic stem cells (HSC). In this study, we assessed the potential of exogenously added FGF-1/2, or retrovirally overexpressed FGF-1 to preserve HSC function in vitro and in vivo.
First, we demonstrate that in vitro culture of unfractionated mouse bone marrow cells, in serum-free medium, supplemented with FGF-1 or FGF-2 or FGF-1 + 2 resulted in the robust generation of long-term repopulating (LTR) HSCs. Cultures were maintained for 12 weeks and during that time in vivo competitive reconstitution assays were performed. Stem cell activity was detectable at 3, 5, and 8 weeks after initiation of culture, but lost after 12 weeks. However, whereas 3 and 5 week cultured cells provided radioprotection in non-competitive assays, animals transplanted with 8 or 12 week cultured cells succumbed due to bone marrow failure. So far, we have been unable to expand single, highly purified Lin−Sca-1+c-Kit+ using FGF-1 + 2. Consequently, we speculated that essential intermediate cell populations or signals are required for FGF-induced stem cell conservation. To test this we cultured highly purified CD45.1 Lin−Sca-1+c-Kit+ cells in a co-culture with CD45.2 unfractionated BM. Co-cultured cells were transplanted after 5 weeks in lethally irradiated recipients, and CD45.1 chimerism levels were assessed. High levels of CD45.1 chimerism confirmed that Lin−Sca-1+c-Kit+ cells require an accessory signal in addition to FGF to induced stem cell activity in vitro. We subsequently tested stem cell potential of cells cultured in FGF-1 + 2 for 5 weeks, with the addition of SCF + IL-11 + Flt3L for the last 2, 4 or 7 days. Cell numbers increased with increasing time of growth factor presence. However, only when growth factors were present for 2 days engraftment of cultured cells in a competitive repopulation assay was increased 3.5-fold.
Finally, we show by immunohistochemistry that ~10% of freshly isolated Lin−Sca-1+c-Kit+ expresses high levels of FGF-1. Retroviral overexpression of FGF-1 in stem cells resulted in increased growth potential and sustained clonogenic activity in vitro. Upon transplantation of transduced stem cells, FGF-1 overexpression resulted in increased white blood cell counts 4 weeks post-transplant compared to control animals. Most notable was a marked granulocytosis in FGF-1 overexpressing recipients Our results reveal FGF as an important regulator of HSC signaling and homeostasis. Importantly, in the presence of FGF stem cells can be maintained in vitro for 2 months. These findings open novel avenues for in vitro manipulation of stem cells for future clinical therapies.
T Cells Genetically Modified to Be CD19-Specific with Improved Potential for Trafficking and Activation.
Authors: Al-Kadhimi Zaid , Serrano Lisa Marie , Olivares Simon , Gonzalez Sergio , Pfeiffer Timothy , Vishwanath Reena , Jensen Michael C. , Forman Steven J. , Cooper Laurence J.N. 
Published: October 2019

Abstract
The safety and feasibility of adoptive immunotherapy using ex vivo-expanded differentiated human effector T cells that express tumor-specific chimeric receptors are being evaluated in clinical trials. Typically, these T cells are CCR7neg and bear a T-cell receptor of unknown specificity. To improve the therapeutic potential of genetically engineered T cells in general, and CD19-specific T cells in particular, strategies are needed to improve their ability traffic to sites of residual/macroscopic disease where infused T cells can be specifically activated for proliferation, cytokine secretion, and tumor-lysis. To accomplish these goals we have generated a selection process that uses genetically modified T cells, expressing influenza A matrix protein 1 (MP1) or CMV pp65, to act as antigen presenting cells (T-APC) in order to expand autologous viral-specific T cells in vitro and in vivo. The viral-specific effector T cells can then be genetically modified with a CD19-specific chimeric immunoreceptor (CD19R), which recognizes CD19 on malignant B cells, independent of MHC. By using these viral-specific T cells as a platform for the introduction of CD19R, we now demonstrate that bi-specific T cells express the chemokine receptor CCR7, which is implicated in the trafficking of T cells to lymph nodes. We demonstrate that this chemokine receptor functions to directionally chemotax the genetically modified bi-specific T-cells along concentration gradients of CCL19 or CCL21. We further demonstrate that both the endogenous and introduced chimeric immunoreceptor continue to function in CCR7+ bi-specific T cells. Indeed, the bi-specific T cells are capable of augmented cytokine production and proliferation upon docking with both CD19 and MP1 antigens, compared with these same T cells interacting with either CD19 or MP1 alone. This enhanced activation is an explanation for the enhanced in vivo anti-tumor activity demonstrated by bi-specific T-cells when stimulated with MP1+ T-APC in treating CD19+ lymphoma in NOD/scid mice. An advantage of this methodology is that the CCR7+ bi-specific T cells and T-APC can be genetically modified and expanded in compliance with current good manufacturing practice (cGMP) for 2nd generation Phase I/II clinical trials to test their ability to traffic to sites of lymphoma providing potent regional/local T-cell activation.
Legend: (A) CCR7+ viral- and CD19-bi-specific T cells migrate along recombinant CCL19 and CCL21 concentration gradients, whereas CCR7neg CD19-specific T cells do not. (B) Stimulation of both introduced chimeric immunoreceptor and endogenous T-cell receptor on CD19- and MP1- bi-specific T-cells, using artificial APC, results in augmented cytokine production.
Figure Figure
Analysis of Vector Integration with LM-PCR after Retroviral HSV-Tk/ΔLNGFR Gene Transfer.
Authors: Wehnert Stefanie , Krüger Tillmann , von Neuhoff Nils , Hertenstein Bernd , Ganser Arnold , Weissinger Eva-Maria 
Published: October 2019

Abstract
The development of secondary leukemia as an unexpected complication after retroviral transfer of potentially therapeutic genes was recently reported. In the pathogenesis of this complication, the upregulation of proto-oncogenes due to random vector integration played a crucial role. Therefore, the analysis of vector integration after retroviral gene transfer could become an interesting tool for risk assessment and safety measurement. In this study, 4 patients diagnosed with chronic myelogenous leukemia (CML) and 2 patients suffering from acute myelogenous leukemia (AML) were transplanted with T-cell-depleted CD34+ enriched stem cells (HSCT) from their HLA-identical siblings without further immunosuppression. After day +60 following HSCT, they received gene-modified donor T-cells infected with the replication-deficient retrovirus SFCMM-3, expressing the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene, and the truncated low affinity nerve growth factor (ΔLNGFR) to control graft versus leukemia (GvL) effects and graft versus host disease (GvHD). In order to assess the viral integration sites in the transfused gene-modified donor T-cells, we performed a ligation-mediated PCR (LM-PCR) to amplify the vector flanking sequences. Final PCR products were visualized by gel electrophoresis and silver staining. Afterwards, sequence analysis (CEQ 8000, Beckman Coulter GmbH, Krefeld, Germany) of the clonal products was carried out. Standard National Center for Biotechnology Information (NCBI) blast searches were done to identify the flanking sequences (http://www.ncbi.nlm.nih.gov/BLAST). Within the investigated gene-modified T-cells of our study population, we found several integration sites. Up to now there has been no evidence for retroviral integration in the vicinity of proto-oncogene promoters after sequence analysis and database search. Furthermore, we were able to monitor the presence of the transduced T-cells at several time points after HSCT by detecting the internal retroviral band. In conclusion, the LM-PCR is a specific method to amplify and identify the flanking sequences after retroviral gene transfer, contributing significantly to the safety of gene therapy.
Increased Incidence of Major Bleeding in Children Receiving Unfractionated Heparin for Clinical Management: A Prospective Cohort Study.
Authors: Mitchell Lesley G. , Kuhle Stefan , Massicotte Patricia M. , Vegh Patricia 
Published: October 2019

Abstract
BACKGROUND: Unfractionated heparin (UFH) is one of the most frequently prescribed drugs in paediatric tertiary care centres and is used in a diverse group of disorders including cardiopulmonary bypass, extra corporeal membrane oxygenation, dialyses and maintenance of both venous and arterial catheter patency. Dosing of UFH in children is extrapolated from adults and is assessed by either a chromogenic Anti-Xa assay or a clot-based activated partial thromboplastin time (aPTT). The overall objective of the study was to assess safety of current standard of practice in the use of therapeutic UFH in children.
Objective #1: The primary objective was to determine the incidence of bleeding and the incidence of recurrent thrombosis in children receiving UFH.
Objective #2: To assess the monitoring UFH by assessing the relationship of the aPTT and Anti-Xa heparin levels to heparin dose.
STUDY DESIGN: A prospective cohort study in nonselected children in a intensive care setting. The primary outcomes were major bleeding events and recurrent thrombosis. The secondary outcomes were assessing the APTT and Anti-Xa levels. Inclusion Criteria: Patients 〉 36 weeks gestation and 〈18 years of age requiring therapeutic doses of UFH. Exclusion Criteria: patients who received UFH for less than 1 day. Major bleeding was defined aprior as any of the following: CNS bleeding, retroperitoneal bleeding, and/or bleeding that results in stopping UFH infusion.
RESULTS: Patient Population 39 patients were enrolled, 22 (56%) male, 32 (82%) < 1 year of age and 90% of which where cardiac patients. Major Bleeding events: 11/39 patients had a major bleeding event 28.2% (95% CI 15.0–44.9%). No patient had recurrent thrombosis. Relationship of aPPT and Anti-Xa to heparin dose; A total of 188 paired aPTTs and anti-Xa levels were performed. There was little correlation between aPTT and anti-Xa levels (r2=0.205) and APTT and UFH dose (r2=0.054). There was no relationship between anti-Xa levels and UFH dose (r2=0.0089). (Figure 1 and 2)
Figure Figure
CONCLUSIONS:. There is an unacceptably high rate of bleeding in children receiving UFH for clinical care. There is little or no relationship of aPPT and Anti-Xa to heparin dose. Clinical trials are needed to assess the appropriate use of UFH therapy in children.
Prevention of Venous Thromboembolism and Pregnancy Complications using Low-Molecular-Weight Heparin during Pregnancy: A Systematic Review of Safety and Thromboprophylactic Efficacy.
Authors: Greer Ian A. , Nelson-Piercy Catherine 
Published: October 2019

Abstract
Introduction Low-molecular-weight heparin (LMWH) is used in pregnancy for venous thromboembolism (VTE) prophylaxis and for prevention of pregnancy complications, because of efficacy greater than or equal to unfractionated heparin, and a lower rate of side effects observed in non-pregnant patients. Due to the lack of data from large randomized controlled trials to guide physician’s practices, there is a need for increased data on safety and efficacy of LMWH for these indications. Our aim was to evaluate the safety and efficacy of using LMWH during pregnancy for preventing VTE and pregnancy complications by performing a systematic review of data from published literature.
Methods Data from published studies on the use of LMWH during pregnancy as VTE prophylaxis or LMWH for prevention of pregnancy complications were identified by searching MEDLINE and EMBASE databases up to the end of 2003. The reference lists from identified articles were also hand searched. Data on the LMWH regime, incidence of VTE, pregnancy complications, clinical outcomes and side effects were extracted and entered into pre-piloted forms.
Results Fifty studies reporting 2,322 pregnancies were included in this analysis. LMWH was received antenatally in 1883 (81%) of cases, and only peri or postpartum in 389 (17%) of cases. Dalteparin and enoxaparin were the most commonly used LMWH, but certoparin, nadroparin, rivaparin, and tinzaparin were also used. There were no maternal deaths. VTE was reported in 26 (1.1%) pregnancies. Severe maternal bleeding occurred in 46 (2%) pregnancies and was generally associated with obstetric causes. Thrombocytopenia occurred in 10 (0.4%) pregnancies and was not associated with thrombosis. Minor allergic skin reactions occurred in 23 (1%) pregnancies, and osteoporosis in two (0.09%) pregnancies.
Conclusion Data from this systematic review of the literature suggest that LMWH is both safe and effective for use as VTE prophylaxis during pregnancy.
Effect of Covalently-Bound Heparin on Insertion of the Reactive Centre Loop in Antithrombin.
Authors: Mewhort Tracy Anne , Berry Leslie R. , Chan Anthony K.C. 
Published: October 2019

Abstract
Heparin exerts its anticoagulant effects through its interaction with antithrombin (AT), leading to the inhibition of thrombin, a major player in the coagulation cascade. AT exists in equilibrium between a latent and an active state. In latent AT, the reactive centre loop (RCL) of AT is inserted into the central b-sheet (A-Sheet), forming a six-stranded A-Sheet. In active AT, residues P10-P14 of the RCL of AT are inserted into the A-sheet, retaining inhibitory activity. We have produced a molecule of covalently linked AT and heparin (ATH). Intrinsic fluorescence studies of ATH have shown that the AT is permanently activated in the covalent molecule. This study was designed to examine degree of the insertion of the RCL into the A-sheet of ATH. The tetradecapeptide P1-P14 was used to compete with the RCL of AT for insertion into the A-sheet. Peptides corresponding to the RCL residues P7-14, P3-P14 and P1-P14 of human AT were radiolabelled using reductive alkylation with formaldehyde + [3H]-NaBH4. Varying concentrations of radiolabelled peptide (0-3000 nM) were incubated with 1000 nM AT in the presence or absence of unfractionated heparin (UFH), and with ATH in 0.05 M Tris HCl buffer (pH 7.3) at 37°C. A 24-hour equilibrium dialysis against 0.05 M Tris HCl buffer followed. The amount of peptide bound to the AT or ATH was quantified following dialysis using liquid scintillation counting of samples taken from inside and outside the dialysis tubing. Results were expressed as bound vs. free peptide in moles. No binding to AT in the presence or absence of UFH, nor to ATH was seen using the P7-P14 peptide, while some binding to AT was seen for both the P3-P14 and P1-P14 peptides. There was a trend towards increased binding with AT in the presence of UFH over AT alone or ATH with the P3-P14 peptide. Binding to AT was lost in the presence of UFH, and no binding was seen with ATH in the P1-P14 condition, consistent with the literature. Furthermore, binding to AT was greatly reduced compared to that seen with the P3-P14 peptide. These data suggest that binding of heparin creates a conformational change in AT preventing the insertion of the RCL peptide into the molecule. Furthermore, results with the P3-P14 peptide may infer that it is the P1-P2 region that is most affected by the conformational change. This is consistent with the AT being in a partially activated conformation in the covalently linked ATH molecule.
Supression of FLT3-Expressing Leukemia by a Monoclonal Antibody-Auristatin Conjugate.
Authors: Li Yiwen , Li Hongli , Wang Mei-Nai , Bassi Rajiv , Ludwig Dale , Witte Larry , Zhu Zhenping , Bohlen Peter , Meyer Damon , Larkin Angelyn , Senter Peter , Hicklin Daniel 
Published: October 2019

Abstract
The receptor tyrosine kinase FLT3 is overexpressed in blasts of ~90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ~37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human monoclonal antibody (IMC-EB10) which binds with high affinity to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody raito = 8) was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH , EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro. In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 and BaF3-ITD leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias.
Assessment of Heparins, Heparinoids and Direct Thrombin Inhibitors Using a New FXa-and RVV-V-Based Assay.
Authors: Calatzis Andreas , Reininger Armin J. , Spannagl Michael 
Published: October 2019

Abstract
UFH and direct thrombin inhibitors are usually monitored using the activated partial thromboplastin time (aPTT), which has many known limitations. These include non-linear-dose response-relationships as well as a high variability among different reagents. In addition the aPTT has a low sensitivity towards low molecular weight heparin (LMWH). Newer methods such as chromogenic substrate analysis, ecarin clotting time or the Heptest assay have provided improved sensitivity and specificity, but still have not reached broad application in routine or emergency laboratories. The performance of a new universal monitoring method (Pefakit® PiCT®, Pentapharm, Basel) in detecting anticoagulant effects of anti-Xa and / or arti-IIa agents was evaluated.
Methods: Measurements were performed by addition of platelet poor plasma to a reagent containing a combination of FXa, RVV-V (FV activating snake venom) and phospholipids. After 180 s the samples were recalcified and the clotting time was recorded. Plasma samples from 30 healthy blood donors and 50 patients receiving low molecular weight heparin were analyzed. To evaluate the assay precision 30 samples were analyzed in triplicate. Plasma pools from healthy volunteers and patients under oral anticoagulant treatment were spiked with unfractionated heparin, low molecular weight heparin, argatroban and pentasaccharide.
Results: PiCT® clotting times of the healthy volunteers were 20.6 ± 1.2 sec. Clotting times of the patient samples ranged 29–195 sec. Correlation of anti-Xa activity to PiCT® clotting times was good (r=0.94). The mean variation coefficient of the repeated determinations was 2.6%. Addition of LMWH, UFH or Argatroban to normal plasma showed a steep increase and almost linear dose-response of the PiCT clotting times. Addition of pentasaccharide showed a non-linear dose-response with lesser increase of clotting times at concentrations above 0.75 μg/ml plasma. The addition of LMWH or UFH to plasma of patients under warfarin therapy showed moderate additive effects when compared to normal plasma, while stronger additive effects of warfarin and heparin therapy were found for argatroban.
Conclusion: The prothrombinase induced clotting test (PiCT ®) is a new clotting assay, which sensitively assesses LMWH, UFH and direct thrombin inhibitors with almost linear dose-response-relationships. The results revealed a wide measuring range which allows for assessment of prophylactic, therapeutic and supratherapeutic doses of these compounds. As a clotting test with pipetting volumes and an incubation regimen identical with the aPTT, the test can be used in routine or emergency laboratories, thus facilitating 24-hour availability of the method with short turn-around times. Allowing the assessment of heparins, heparinoids as well as direct thrombin inhibitor therapy with one method may simplify their management in clinical practice.