bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neuronal/Mitochondrial Nitric Oxide Synthase Homologous Deletion Recombinant Negative Mice (NOS1 −/−) Long-Term Bone Marrow Cultures (LTBMCs) Demonstrate Increased Longevity and Radioresistance of Derived Cell Lines.
Authors: Epperly Michael W. , Greenberger Joel S. , Cao Shaonan , Franicola Darcy , Shen Hongmei , Greenberger Emily E. , Epperly Laura D. , Zhang Xicheng 
Published: April 2020

Abstract
Neuronal NOS (NOS1) is localized to mitochondria. Ionizing irradiation results in influx of calcium into mitochondria stimulating production of nitric oxide (NO), and also increases production of superoxide which reacts with NO to produce peroxynitrite. Peroxidation of mitochondrial lipids, release of cytochrome C and apoptosis is directly related to mitochondrial peroxynitrite. We hypothesized that reduction of mitochondrial NO production should provide radioresistance. In addition, since ROS production is associated with aging NOS1−/− mouse LTBMCs should demonstrate greater hematopoietic longevity. LTBMCs established from NOS1 −/− mice demonstrated increased cumulative adherent cobblestone islands (adherent stem cell containing islands), production of total nonadherent cells, and cumulative day 7 and day 14 CFU-GEMM hematopoietic multi-lineage colony forming cells (over 65 weeks) compared to NOS1 +/+ controls (22 weeks) (p &lt; 0.0001). Seven and 14 day CFU-GEMM production in nonadherent cell harvests from NOS1 −/− LTBMCs continued for 65 weeks compared to 15 weeks for NOS1 +/+ LTBMCs (p &lt; 0.0001). Marrow stromal cell lines derived from NOS1 −/− and NOS1 +/+ culture were irradiated to doses from 0 to 800 cGy, plated in 4 well tissue culture plates, stained with crystal violet 7 days later and colonies of greater than 50 cells were counted. NOS1 −/− stromal cell lines had an increased shoulder on the survival curve compared to the NOS1 +/+ cells (n = 32.15 ± 1.21 and 10.47 ± 3.20, respectively, p=0.0026). Cell cycle analysis of NOS1 −/− and NOS1 +/+ cell lines following 10 Gy irradiation demonstrated a G1 arrest 6 hr after irradiation, in both; however, by 24 hr, NOS1 +/+ but not NOS1−/− cells resumed normal cycling. To determine whether the radioresistance of NOS1−/− cells was attributable to expected higher levels of antioxidants, cells were analyzed for glutathione (GSH) and glutathione peroxidase (GPX). NOS1 −/− cells demonstrated increased GSH compared to NOS1 +/+ cells at 0, 30 min and 24 hr following irradiation (p &lt; 0.0001) with no significant difference in GPX before or after irradiation. NOS1 −/− compared to NOS1+/+. IL-3 dependent hematopoietic cells from NOS1 −/− LTBMCs had significantly decreased apoptosis, 24 hrs following 10 Gy irradiation (5.3 ± 2.4 vs. 14.8 ± 3.3 %, respectively, p = 0.049). Therefore, reduction of NOS1 in bone marrow increases hematopoietic longevity in LTBMCs and radioresistance of derived cell lines.
Bis(1H-indol-2yl)methanones Are Effective Inhibitors of Mutated FLT3 Tyrosine Kinase, Overcome Resistance to PKC412A In Vitro and Show Synergy with Chemotherapy.
Authors: Heidel Florian , Mirea Fian K. , Breitenbucher Frank , Dove Stefan , Huber Christoph , Bohmer Frank D. , Fischer Thomas 
Published: April 2020

Abstract
Inhibition of mutated FLT3 receptor tyrosine kinase has become a promising therapeutic strategy in AML. Currently, a phase III trial testing the tyrosine kinase inhibitor (TKI) PKC412A in combination with induction chemotherapy is under way. Development of resistance to PKC412A mediated by secondary FLT3 mutations has been described recently (Heidel et al., Blood 2006). This observation may have an increasing impact on duration of response in future clinical trials. Herein we investigated two novel TKIs (methanones) to determine their efficacy on FLT3-ITD transfected 32D cells as well as on primary ITD positive AML blasts.
The bis(1H-indol-2-yl)methanone compounds Cpd.98 and Cpd.102 (Mahboobi et al. J. Med. Chem. 2006) that have recently shown efficacy in proliferation assays using FLT3-ITD transfected cells were characterized. Western Blotting experiments of murine 32D-FLT3-ITD cells confirmed dose dependent dephosphorylation of FLT3-ITD and its downstream target STAT5 upon incubation with Cpd.98 and Cpd.102, respectively. To define a non-myelotoxic range of these inhibitors, bone-marrow (BM) cells of healthy donors were tested with different methanone concentrations to determine the rate of apoptosis (sub G1 fraction in cell cycle analysis). No significant increase in apoptosis was seen up to 3μM of inhibitor concentration for either compound.
Additionally, colony assays of healthy BM-cells also showed no significant reduction of colony formation up to 3μM. To determine the efficacy of these compounds in PKC412A-resistant cells, transfected 32D cells containing the previously described resistance mediating mutation (32D-FLT3-ITD-N676K) were also tested at different concentrations for induction of apoptosis. Resistance in these cells could be overcome by 300nM (Cpd.102) and 1μM (Cpd.98), respectively.
As clinical studies in AML will evaluate FLT3 inhibitors in combination with chemotherapy, we tested both compounds in vitro in combination with cytosine arabinosid (Ara-C) and daunorubicine (DNR). Both compounds showed synergistic effects in combination with chemotherapy in 32D-FLT3-ITD cells as revealed by apoptosis assays.
Primary AML blasts harbouring the FLT3-ITD length mutation were also incubated with both compounds to determine the efficacy in patient material. These experiments revealed apoptosis rates of up to 30% upon incubation for 72 hours. In conclusion bis(1H-indol-2-yl)methanone compounds overcome resistance mediated by the previously described FLT3-ITD mutation N676K in vitro and show efficacy on FLT3-ITD-positive cells alone as well as in combination with chemotherapy. Experiments evaluating toxicity in murine models are currently planned to explore a possible clinical application.
Prolonged Exposure to FLT3 Inhibitors Leads to Resistance Via Activation of Parallel Signaling Pathways.
Authors: Piloto Obdulio , Wright Melissa , Brown Patrick , Kim Kyu-Tae , Levis Mark , Small Donald 
Published: April 2020

Abstract
A number of tyrosine kinase inhibitors (TKI) have been developed to treat a variety of malignancies. However, continuous treatment with TKIs may select for resistant clones as has been seen with Gleevec treatment of CML. To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in AML, we developed resistant human cell lines through prolonged co-culture with FLT3 TKIs. Both FLT3 TKI sensitive and resistant cell lines exhibit inhibition of FLT3 phosphorylation upon FLT3 TKI treatment. However, FLT3 TKI resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI resistant cell lines and primary samples failed to reveal any mutations in FLT3 or in 100 kinases/phosphatases tested but did reveal activating N-Ras mutations that were not present in the parental FLT3 TKI sensitive cell line. Taken together, these data indicate that FLT3 TKI resistant cells most frequently become FLT3 independent due to activation of parallel signaling pathways that provide compensatory survival / proliferation signals when FLT3 is inhibited. IMC-EB10, an unconjugated monoclonal antibody against FLT3, is still cytotoxic to FLT3 TKI resistant clones in vivo. An approach combining FLT3 TKIs with anti-FLT3 antibodies may prove superior and result in reduced chances of developing resistance.
Erythropoietin Modulation of Podocalyxin within an Erythroblast Niche.
Authors: Sathyanarayana Pradeep , Menon Madhu P. , Bogacheva Olga , Bogachev Oleg , Kapelle William S. , Houde Estelle , Wojchowski Don M. 
Published: April 2020

Abstract
Epo’s capacity to promote red cell production is ascribed largely to anti-apoptotic effects on erythroid progenitor pools. Via gene profiling of bone marrow- derived erythroblasts, Epo is now revealed to down-modulate chemokine receptor-4 (Cxcr4) and integrin alpha-4 (Itga4), and to selectively up-modulate the expression of growth differentiation factor 3 (Gdf3), oncostatin-M (Onco-M), and the sialomucin podocalyxin like-1 (PODXL). Epo dose-dependent induction of PODXL was approximately 20 fold; was discovered in KitposCD71high erythroblasts; and peaked at subsequent KitnegCD71high and Ter119pos stages. As studied using minimal knocked-in Epo receptor (EpoR) alleles, efficient PODXL expression depended upon a proximal EpoR PY343 Stat5 binding site. In mice expressing a phosphotyrosine-null EpoR-HM allele, compromised PODXL expression further correlated with an abnormal representation of anucleated red cells in marrow (and with defective Epo-induced production of PODXLpos reticulocytes). By selectively modulating Gdf3, Onco-M, Cxcr4, Itga-4 and PODXL expression, Epo is proposed to modify a unique erythroid niche, mobilize early-stage erythroblasts, and promote reticulocyte emigration to blood.
The Role of Gja1 Gap Junctions in the Bone Marrow Niche.
Authors: Anderson Nicole M. , Zirngibi Ralph , Chen Frieda , Owen Celeste , Zitano Lia , Lau Esther , Aubin Jane , Stanford William 
Published: April 2020

Abstract
The interplay between the stroma and hematopoietic progenitors within the bone marrow niche is critical for the homeostatic regulation of both mesenchymal and blood lineages. Gap junctions play an important role in the communication between hematopoietic supportive cells of the fetal liver and bone marrow stroma. Targeted deletion of the gap junction protein Gja1 (connexin 43) demonstrated a requirement of Gja1 for placental and cardiac function leading to neonatal lethality. Surprisingly, Gja-1 heterozygous animals demonstrated no steady state hematopoietic defects, suggesting that either Gja-1 is not critical for hematopoietic stromal cell communication or that Gja-1 hemizygosity does not functionally alter gap junctions. We have identified an N-ethyl-N-nitrosourea (ENU)-induced strain (Gja1JRT) with a dominant negative mutation in Gja1 which phenocopies the human autosomal dominant disorder oculodentodigital dysplasia (ODDD). ODDD is characterized by a variety of developmental abnormalities including syndactyly, enamel hypoplasia, craniofacial abnormalities and cardiac dysfunction. In addition to these phenotypes, Gja-1JRT mice exhibit decreased bone mass and mechanical strength as well as alterations in hematopoietic progenitor frequencies, abnormalities not previously reported in human ODDD patients (Development132: 4375–4386, 2005). We are currently performing a longitudinal analysis of 2, 4, 8 and 12 month old Gja-1JRT animals, examining hematopoietic including osteoclastic and mesenchymal/osteoblast progenitors to evaluate the dynamic interplay between these two tissues. To date, we have observed alterations in hematopoietic parameters including increased peripheral blood cell number and increased myeloid colony number at 2 months of age. At 4 months of age, Gja-1JRT mice demonstrated a significant decrease in overall clonogenic (CFU-C) cells, while some peripheral blood cell parameters remained elevated. Despite the increases in differentiated myeloid cells and their precursors at 2 months of age, analysis of hematopoietic-derived bone resorbing osteoclasts showed a reduction in osteoclasts at 2 months of age but not at later time points. Analysis of the mesenchymal compartment of the bone marrow demonstrated increased total mesenchymal progenitors (CFU-F) and osteoprogenitors (CFU-O) at 8 and 12 months of age. Thus, alterations in hematopoietic and osteoclastic cell frequencies preceded alterations in the bone marrow mesenchymal compartment in the Gja-1JRT mice. In addition to a complete longitudinal analysis, we will report on the alterations in trabecular bone, which create the niche microenvironment, and the impact of these changes on the hematopoietic stem cell frequency.
Adhesion of Hematopoietic Progenitor Cells to Human Mesenchymal Stromal Cells as a Model for Interaction between Stem Cells and Their Niche.
Authors: Wagner Wolfgang , Wein Frederik , Roderburg Christoph , Benes Vladimir , Diehlmann Anke , Krause Ulf , Frankhauser Maria , Wuchter Patrick , Volker Eckstein , Ho Anthony D. 
Published: April 2020

Abstract
Objective: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining “stemness” has been demonstrated. Human mesenchymal stromal cell (MSC) feeder layers represent a surrogate model for this interaction. The molecular composition of this heterotypic cell-cell contact is yet unknown.
Methods: To define this cell-cell contact between HPC and MSC, we have studied adhesion of various fractions of HPC with different preparations of MSC by using a novel assay based on gravitational force upon inversion. Adherent and non-adherent cells were then separated. Gene expression analysis by microarray (GeneChip Human Genome U133_Plus_2.0, Affymetrix) of the two populations was performed and the relationship to long-term hematopoietic culture initiating cell (LTC-IC) frequency examined.
Results: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to MSC from human bone marrow (CD34+vs. CD34−, CD34+/CD38−vs. CD34+/CD38+, slow dividing fraction vs. fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the non-adherent CD34+ cells, thus providing evidence for specific adhesive interaction of primitive HPC with MSC. Genes coding for adhesion proteins and extracellular matrix were highly expressed in the adherent fraction compared to non-adherent CD34+ cells. These genes included fibronectin1 (FN1), cadherin11, VCAM1, connexin43 and ITGBL1. Furthermore, affinity of CD34+ cells was analyzed on human MSC isolated from bone marrow (BM), adipose tissue (AT) and cord blood (CB). Affinity to BM-MSC was significantly higher compared to AT-MSC and CB-MSC. Gene expression in different MSC preparations (BM-MSC, AT-MSC and CB-MSC) correlated in various adhesion proteins with the differences observed in affinity of HPC (including cadherin11, VCAM1, N-cadherin, ITGB1, ITGA1, ITGA5, SDF-1 and osteopontin). Western blot analysis also confirmed higher protein expression of FN1, cadherin11, N-cadherin and ITGB1 in BM-MSC compared to AT-MSC and CB-MSC.
Conclusion: MSC represent a model for the human hematopoietic niche. Primitive subsets of HPC have significantly higher affinity to BM-MSC. The essential role of specific junction proteins (cadherin11, VCAM1, N-cadherin) for stabilization of cell-cell contact is indicated by their significant higher expression on both sides of the heterotypic interaction.
Bmi1- Is a SALL4 Target Gene in Hematopoeitic and Leukemic Cells.
Authors: Ma Yupo , Yang Jianchang , Chai Li , Liu Fang , Amin Hesham M. 
Published: April 2020

Abstract
Bmi-1 and SALL4 are putative oncogenes modulating stem cell pluripotency and playing a role in leukemogenesis. Previously, we report that SALL4, a zinc finger transcription factor, is constitutively expressed in human acute myeloid leukemia (AML) and induces AML in transgenic mice. Here we demonstrate that transcription from the Bmi-1 promoter is strikingly activated by SALL4. The SALL4 responsiveness of the Bmi-1 promoter exhibits dose-dependent activation in a luciferase reporter gene assay. We mapped the N-terminal domain of SALL4 responsible for activation of the Bmi-1 promoter. Chromatin immunoprecipitation from a myeloid stem cell line, 32D demonstrated that SALL4 bound directly to the Bmi-1 promoter in a region involving SALL4 stimulation. The over-expression of SALL4 in the 32D cell line up-regulated Bmi-1expression and was associated with increased proliferation and inhibition of apoptosis. Deletion of one copy of SALL4 by the gene target in the mouse marrow significantly reduced Bmi-1 expression. SALL4 up-regulated Bmi-1 expression in SALL4 transgenic mice and the up-regulated levels of Bmi-1 in SALL4 transgenic mice correlated to that of disease progression from normal, to preleukemic and leukemic stages. Similar to Bmi-1, SALL4 was expressed highly in hematopoietic cells (HSCs) and was down-regulated as differentiation proceeds. These findings, when taken together, reveal a novel link between SALL4 and Bmi-1 in regulating self-renewal of normal and leukemic stem cells.
Different Kinetics and Function of Vascular Endothelial Growth Factor Recepotor-1 and −2 during Hemangioblast Development from Primate Embryonic Stem Cells.
Authors: Umeda Katsutsugu , Heike Toshio , Shinoda Gen , Niwa Akira , Arai Masato , Matsubara Hiroshi , Ma Feng , Adachi Souichi , Suemori Hirofumi , Torii Ryuzo , Shibuya Masabumi , Nakatsuji Norio , Nakahata Tatsutoshi 
Published: April 2020

Abstract
The close developmental association between hematopoietic and endothelial cells suggests that both lineage cells share a common precursor, the hemangioblast. The vascular endothelial growth factor (VEGF)-A system has been proven to have roles in the embryonic development of hemangioblast in mouse by the use of embryos or embryonic stem (ES) cells deprived of the genes encoding its ligand or receptors (VEGFR-1 and VEGFR-2). On the other hand, there have been only a few reports on the hemangioblast development during primate (human and monkey) embryogenesis.
We have previously demonstrated that the hemangioblast is highly enriched in the VEGFR-2high CD34+ cell fraction, differentiated from cynomolgus monkey ES cells by 6-day coculture with OP9 stromal cells. In the current study, we examined whether the VEGF-A system was involved in the development of hemangioblast induced from primate ES cells by using the coculture system. VEGFR-1 and −2 were expressed by undifferentiated monkey ES cells at low levels. The VEGFR-2low cells gradually decreased while VEGFR-2high cells could be detected on day 6. On the other hand, VEGFR-1 was constantly expressed at low levels on day 6 and thereafter. Exogenous VEGF-A165, the action of which is mediated VEGFR-1 and VEGFR-2, increased the proportion of VEGFR-2high CD34+ cells in a dose-dependent manner. Addition of VEGF-E (VEGFR-2-specific agonist) and VEGFR-1 blocking antibody resulted in the increase of VEGFR-2high CD34+ cells in a dose-dependent manner, while VEGFR-2 blocking antibody suppressed their development. Thus, VEGF-A/VEGFR-2 interaction promoted the development of hemangioblast, while VEGF-A/VEGFR-1 interaction acted as a negative regulator.
We then examined when VEGF-related signals work on VEGFR-2high cell development by changing factors on day 4. The proportion of VEGFR-2high CD34+ cells with the last 2-day VEGF-A165 and -E treatment were almost equivalent to that with continuous exposure to the VEGF. On the contrary, initial 4-day VEGF-A165 and -E treatment did not any stimulatory effects on VEGFR-2high CD34+ cell development. Our previous results show that during differentiation, Brachyury (early mesodermal marker) is first expressed on day 4, followed by the up-regulation of genes, such as SCL, LMO2, and MYB, representing hematopoietic and/or endothelial potentials on day 6. Collectively, VEGFR-2-mediated signals might contribute to the commitment and/or expansion of the hemangioblast rather than their development.
This coculture system provides an opportunity to better understand the regulative mechanisms on early hematopoietic and endothelial cell development, which remains unresolved by experiments using human embryos.
Performance of Murine Soluble Thrombomodulin (rmsTM) in a Murine Model of Endotoxin-Induced Sepsis.
Authors: Joyce David E. , Xu Haifeng , Mathur Sumeet , Castellino Francis J. 
Published: April 2020

Abstract
The endothelial surface protein thrombomodulin (TM) generates activated protein C (aPC) that protects from sepsis-related DIC, vascular inflammation, and apoptosis. The purpose of this study is to evaluate a novel recombinant mouse soluble TM (Eli Lilly, Indianapolis, IN) in a murine lethal endotoxin (LPS) sepsis model to determine both kinetic and survival endpoints. A dose effective in rodent reperfusion studies was employed in this sepsis model (D. Berg, Eli Lilly). C57BL6 mice were injected concomitantly with rmsTM 5 mg/kg (s.c.) and LPS 14 ug/g (i.p.) at time zero. Plasma concentrations were determined from citrated plasma at 3, 12, 24, and 48 hr, and plasma rmsTM concentrations were determined by ELISA. In vitro determinations of mouse aPC amidolytic activity and rmsTM dependent fibrinogen clotting time assays utilized rmsTM, murine thrombin, fibrinogen, and Protein C (Enzyme Research Labs). The in-vivo time-dependent mouse plasma kinetic studies confirmed plasma concentrations comparable to that achieved in human sTM antithrombotic studies. Thus, an rmsTM dose of 5 mg/kg was chosen for the 7-day LPS survival study (20% survival model). Twenty age-and weight-matched male C57BL6 mice, age 8–14 weeks, were treated with LPS 14 ug/g or LPS and rmsTM, 5 mg/kg, s.c. Survival curves were analyzed by Wilcoxon rank sum test (power 80%, alpha 2.5, p&lt;0.05). An initial time course after rmsTM s.c. injection yielded a peak plasma rmsTM level at 3 hr (5,000 ng/mL) which persisted up to 48 hr (2,000 ng/mL). In vitro activation of aPC was saturating (2,000 ng/mL aPC with &lt;20 ng/mL rmsTM). Prolongation of the thrombin clot time occurred at approximately 10 ug/ml of rmsTM. The LPS survival study in C57BL6 administered LPS (14 ug/g, i.p.) or LPS and rmsTM 5mg/kg s.c. demonstrated no survival difference (P=0.63). In vitro evaluation of aPC generation by rmsTM yielded ample amounts of aPC. Similarly, reduction of thrombin activity by rmsTM occurred at higher rmsTM concentrations. Thus, the lack of survival difference seen between endotoxemic mice treated with saline or adjunctive rmsTM in this model may be from the lack of thrombin inhibition, the lack of persistent (rmsTM:Thrombin) PC activation, or by more efficient clearance of aPC. Species specificity of TM is less likely a contributing factor. Further elucidation of the murine thrombin/thrombomodulin regulatory mechanism in vivo may be necessary to help explain our unexpected sTM survival results.