bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Genetic Mutation on Murine Chromosome 7 Causes Thrombocytosis Associated with Thrombopoietin Excess - A Model of Jak2V617F Negative Idiopathic Thrombocytosis.
Authors: Stevenson William S. , Metcalf Donald , Alexander Warren S. , Hilton Douglas J. , Roberts Andrew W. 
Published: April 2020

Abstract
A substantial proportion of patients with Essential Thrombocythemia (ET) have evidence of an acquired Jak2V617F mutation that causes the myeloproliferative phenotype. Jak2V617F negative ET patients presumably have mutations in other related pathways or alternatively may have chronic thrombocytosis secondary to factors extrinsic to the hemopoietic system. This second hypothesis is supported by clonality studies that demonstrate at least 30% of women with ET have evidence of polyclonal granulocytes. As part of an ENU mutation screen to identify novel genes associated with thrombocytosis, we have studied a mutant mouse strain, called plt2, with autosomal recessive thrombocytosis associated with increased thrombopoietin production. This mutation is linked to mouse chromosome 7, and therefore is unrelated to mutations in Jak2, thrombopoietin or the thrombopoietin receptor, c-Mpl. plt2/plt2 mice have platelet counts of 2038±347x109/L (n=107) increased by 47% compared to wild-type mice (1386±223; n=107, p&lt;0.001). This thrombocytosis was associated with increased numbers of megakaryocyte progenitor cells (plt2/plt2 28.8±4.6; n=4, Meg-CFC/5x105 cells to maximal cytokine stimulation compared to wild-type 18.3±5.5; n=4, p&lt;0.05) and megakaryocytes (108±15; n=7 megakaryocytes per hpf compared to 70±13; n=7, p&lt;0.01) that demonstrated a normal maturation profile based on ploidy measurement. plt2/plt2 mutant mice exhibited mild hepatomegaly (liver weight 1.6±0.2g; n=34 compared to 1.3±0.2g; n=23, p&lt;0.001) and serum thrombopoietin levels were elevated by 76% (4930±1309pg/mL; n=18 compared to 2802±1031; n=15, p&lt;0.001). There was no difference in relative TPO transcription between plt2/plt2 mice and wild-type mice in the liver (p=0.23), however, the TPO content of whole liver lysates was increased by 50% in the mutant mice (150%±50; n=8 per 1g liver weight compared to 100±32; n=8, p=0.03). To examine how the plt2 mutation interacts with other components of the TPO pathway, plt2/plt2 mice were intercrossed with mice lacking the TPO receptor (Mpl−/−). Mpl−/−plt2/plt2 mice have thrombocytopenia with platelet counts that are indistinguishable from Mpl−/−/ mice confirming that the plt2 mutation causes thrombocytosis by acting through the Mpl receptor. Surprisingly, Mpl+/−plt2/plt2 mice, with only one copy of the Mpl gene, had marked thrombocytosis with platelet counts of 2656±459x109/L (n=25) increased by 92% over wild-type mice suggesting an additive effect between excess thrombopoietin and Mpl heterozygosity on platelet count. We have utilized ENU mutagenesis to identify a novel mouse pedigree with heritable thrombocytosis linked to chromosome 7 that demonstrates altered cytokine production can mimic the chronic thrombocytosis that is the cardinal diagnostic feature of ET.
Effects of AMN107, a Novel Aminopyrimidine Tyrosine Kinase Inhibitor, on Human Mast Cells Bearing Wild-Type or Mutated Codon 816 c-kit.
Authors: Verstovsek Srdan , Akin Cem , Francis Giles J. , Taghi Manshouri , Huynh Ly , Manley Paul W. , Alland Leila , Dugan Margaret , Cortes Jorge E. , Hagop Kantarjian M. 
Published: April 2020

Abstract
Background. Majority of adult patients with systemic mastocytosis (SM) have activating mutation in codon 816 of c-kit (CD117), a receptor on the surface of mast cells. This abnormality is responsible for the pathogenesis of the disease.
Methods. We investigated the effects of a newly designed tyrosine kinase inhibitor, AMN107, by comparing its in vitro inhibitory potency on c-kit mutated mast cell lines and patient samples with that of imatinib mesylate, another tyrosine kinase inhibitor, effective in some patients with SM. Two cell lines, subclones of HMC-1 cells, were used: HMC-1560 carrying juxtamembrane domain mutation in codon 560 of c-kit, and HMC-1560, 816 carrying both codon 560 mutation and tyrosine kinase domain mutation in codon 816 of c-kit.
Results. In HMC-1560 mast cell line carrying wild-type codon 816, AMN107 was as potent as imatinib in inhibiting cellular proliferation, with IC50 values of 108 and 74 nM respectively, while in HMC-1560, 816 cell line carrying 816 mutation, neither medication had an effect. AMN107 was also as effective as imatinib in inhibiting phosphorylation of c-kit tyrosine kinase in HMC-1560 cells. The inhibition of cellular proliferation was associated with induction of apoptosis in HMC-1560 cells. AMN107 in concentrations up to 1 uM had no effect on bone marrow mast cells carrying D816V c-kit mutation obtained from patients with mastocytosis.
Conclusions. Our results suggest similar potency of AMN107 and imatinib in mast cells that carry wild-type codon 816, but no activity against codon 816 mutation carrying cells.
The Presence of the V617F Mutation Is Associated with Higher Haemoglobin, Older Age and an Increased Risk of Thrombosis in Essential Thrombocythemia.
Authors: Cheung Betty Y.Y. , Radia Deepti H. , Pantelidis Panagiotis , Yadegarfar Ghasem , Harrison Claire N. 
Published: April 2020

Abstract
The acquired single nucleotide valine to phenylalanine mutation (V617F) in the JAK2 tyrosine kinase is reported at variable frequencies in patients with the Philadelphia negative myeloproliferative disorders. As yet it is unclear whether its presence has an impact upon the clinical phenotype, complications or prognosis of these conditions. We have screened 87 patients under our care with myeloproliferative diseases (28 Polcythemia Vera [PV], 50 Essential Thrombocythemia [ET] and 9 Idiopathic Myelofibrosis[IMF]) using a modification of previously reported allele specific PCR on whole blood DNA (Baxter,E.J et al 2005Lancet, 365, 1054–1061) and then correlated these results with clinical data. Overall there were 50 females and 37 males with a median age of 57.4 years (range 11–92) and median follow-up of 61.4 months (1–313). V617F was detected in 26 (93%) patients with PV, 26 (52%) ET and 2 (22%) IMF. Further analysis was performed to correlate V617F status with clinical features in the patients with ET. No survival data is presented for these patients due to the inherent bias in the analysis of fresh rather than archived samples. This will be performed in a prospective manner. However, one of the V617F positive ET patients subsequently died from AML, interestingly remaining heterozygous for this mutation. When comparing ET patients with detectable V617F against those without the mutation, the following significant differences were identified at the time of diagnosis: an older age (median 49.5 vs 39.3 years, p=0.04), higher haemoglobin (median 14 vs 13g/dl, p=0.03) and documented thrombotic events (11/26 vs 2/24, p=0.01). There were no significant differences in gender, platelet count at diagnosis or duration of follow-up. Overall thrombotic events were also more frequent in V617F positive patients (16/26 vs 7/24, p=0.02). There was no difference in thrombotic events following diagnosis (5/26 vs 5/24) or in the type of thromboses (microvasular, arterial or venous). Advanced age is a well established risk factor for thrombosis in ET, hence it is possible that V617F was associated with thrombosis as a consequence of the older age of our ET patients. Multivariate analysis was limited by small patient numbers and failed to demonstrate a significant predictive impact of either age or V617F mutation upon thrombosis. Ongoing studies will hopefully provide larger numbers for further analysis.
Death-Associated Protein Kinase-2 (DAPK2) Potentiates Erythroid Progenitor Cell Apoptosis in Response to Cisplatin, and Cytokine Withdrawal.
Authors: Fang Jing , Menon Madhu P. , Tschan Mario , Torbett Bruce , Wojchowski Don 
Published: April 2020

Abstract
DAPK2 is a Ca++/CAM-regulated S/T kinase which when ectopically over-expressed in 293 cells, can induce membrane blebbing and apoptosis. In contrast to DAPK1 (its tumor-suppressor orthologue), DAPK2’s expression is tissue- restricted, and presently is shown to be markedly up-regulated in developing primary erythroblasts. To assess DAPK2 function in erythroid progenitor cells, wild-type and kinase-inactive K52A forms were expressed in UT7epo cells, and an siRNA lentivirus also was developed. In this Epo-dependent human erythroid progenitor cell model, wt-DAPK2 did not significantly induce apoptosis. Sensitivity to cell death due to exposure to cisplatin, however, was markedly potentiated, and altered cell morphologies were observed. These effects depended upon DAPK2’s kinase activity, and were not mediated by DAPK2-K52A. Furthermore, siRNA-mediated knock-down of DAPK2 proved to protect UT7epo erythroid progenitor cells from apoptosis due to cytokine-withdrawal. Knock-downs were confirmed at the protein level, and no such effects were exerted by negative control siRNA constructs. Within erythroid progenitor cells, DAPK2’s pro-apoptotic effects appear to be regulated by two clinically relevant cues - cisplatin exposure, and Epo deprivation. Mechanisms that regulate DAPK2, and its proposed caspase-independent apoptotic actions, presently are under investigation in primary erythroblast systems.
Randomized, Double-Blind, Placebo-Controlled Study of Darbepoetin alfa Every 3 Weeks for the Treatment of Chemotherapy-Induced Anemia.
Authors: Taylor Kerry , Ganly Peter , Charu Veena , DiBenedetto Joseph , Kracht Karolyn , Rossi Greg , Hernandez Enrique 
Published: April 2020

Abstract
Background: Darbepoetin alfa (Aranesp®; DA) has been shown to be safe and effective for treating chemotherapy-induced anemia (CIA). The ability to administer darbepoetin alfa every 3 weeks (Q3W) (coincident with chemotherapy) would simplify the treatment of CIA. We report results from the first multicenter, randomized, double-blind, placebo-controlled, phase 3 clinical trial evaluating efficacy and safety of fixed Q3W administration of an erythropoietic agent.
Methods: This study enrolled subjects ≥18 years, diagnosed with anemia (hemoglobin [Hb]&lt;11g/dL) and a nonmyeloid malignancy with ≥12 weeks of planned chemotherapy. Patients (N=391) were randomized 1:1 to receive DA 300 μg or placebo Q3W for 15 weeks. Dose adjustment rules included: increase (to 500 μg Q3W) if Hb concentration was &lt;9 g/dL at week 4 or &lt;10 g/dL (and had &lt;1-g/dL increase) at week 7, or decrease (dependent on previous dose) if Hb concentration was ≥13 g/dL or had ≥1-g/dL increase in any 2-week period. Efficacy was assessed by incidence of red blood cell (RBC) transfusions and achievement of target Hb of ≥11 g/dL (not exceeding 13 g/dL), consistent with ASH/ASCO, NCCN, EORTC evidence-based practice guidelines.
Results: A total of 386 randomized patients were included in the analysis. Demographic characteristics were similar between the 2 groups. Mean (SD) Hb levels at baseline were 10.03 (0.86) and 10.05 (0.92) g/dL in the placebo and DA groups, respectively. The most common tumor types were breast (23%), colon (11%), nonsmall-cell-lung cancer (10%), and hematologic malignancies (11%; 8% Non-Hodgkin’s Lymphoma). The incidence of RBC transfusions from week 5 to the end of treatment phase (EOTP) (the primary endpoint) was significantly lower for the DA group than for the placebo group (P&lt;0.001) (see Table). Hb levels rose steadily in the DA group through approximately week 9, increasing by a mean (SD) of 1.08 (1.28) g/dL from baseline, and then remained relatively stable (see Figure). The proportion of patients achieving Hb target range from week 5 to EOTP was significantly higher for the DA group than for the placebo group (P&lt;0.001). Dose adjustment rules helped to maintain Hb levels within target range. The safety profile of DA was consistent with that observed in previous studies. Rapid increases in Hb concentration or increases to ≥13 g/dL were not associated with adverse events.
Conclusions: Fixed Q3W administration of DA is well tolerated and effective for the treatment of CIA.
Summary of Results Placebo Darbepoetin alfa KM = Kaplan-Meier estimate Week 5 to EOTP N=185 N=181 Transfusions, KM (95% CL) (primary endpoint) 41% (34, 49) 24% (18, 30) Achievement of target Hb, KM (95% CL) 48% (41, 56) 82% (76, 88) Week 1 to EOTP N=193 N=193 Transfusions, KM (95% CL) 47% (40, 54) 30% (23, 36) Median time to target Hb, weeks (95% CL) 12 (9, 16) 6 (3, 7) Figure Figure
Effects of Mitochondrial Ferritin Expression on Tumor Iron Metabolism and Tumor Growth in Nude Mice Xenografts.
Authors: Nie Guangjun , Chen Guohua , Sheftel Alex , Pantopoulos Kostas , Ponka Prem 
Published: April 2020

Abstract
Mitochondrial ferritin (MtFt) is a mitochondrial iron storage protein, whose function and regulation is largely unknown. Our previous results have shown that MtFt markedly affects intracellular iron distribution and homeostasis in mammalian cells (Blood105: 2161–2167, 2005). Using tumor xenografts, we examined the effects of expression MtFt on tumor iron metabolism and growth. H1299 parental or MtFt overexpressing cells were implanted into nude mice. As compared to control tumor xenografts, the expression of MtFt dramatically reduced the implanted tumor growth. A cytosolic iron starvation phenotype in MtFt expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) and, concomitantly, both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to a decrease in both total cellular heme content and heme oxygenase-1 levels. In addition, the expression of MtFt in tumors was associated with a decrease in aconitase activity and lower frataxin protein levels. Mitochondrial iron deposition in MtFt expressing tumors was directly observed by transmission electron microscopy. The pattern of iron accumulation in MtFt overexpressing tumor cells is remarkably similar to that observed in the mitochondria of sideroblastic anemia patients. In conclusion, our study shows that MtFt expression significantly affected tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partial defects in heme and iron-sulfur cluster syntheses. It is likely that deprivation of iron in the cytosol is the cause of the significant inhibition of xenograft tumor growth.
Nitric Oxide Upregulates Ferritin Synthesis Independently of Iron Regulatory Protein-Iron Responsive Element Binding Activity.
Authors: Mikhael Marc , Kim Sangwon , Schranzhofer Matthias , Mullner Ernest W. , Ponka Prem 
Published: April 2020

Abstract
The remarkable post-transcriptional regulation of intracellular iron homeostasis by iron regulatory proteins 1 and 2 (IRP1 and IRP2) is touted as a classic example of translational control. During low levels of intracellular iron, IRPs bind to specific nucleotide sequences known as iron responsive elements (IRE) present in the 5′-untranslated region (UTR) of ferritin (Ft) mRNA and the 3′UTR of transferrin receptor (TfR) mRNA. Such binding during conditions of low intracellular iron, blocks the translation of Ft, the ubiquitous and major iron storing protein, while TfR mRNA is stabilized, leading to increased TfR synthesis and iron uptake. High levels of intracellular iron will abrogate IRP-IRE binding activity, de-repressing Ft translation on the one hand, while causing TfR mRNA instability on the other. This system ensures that potentially hazardous iron is safely stored when it is in abundance and acquired when its levels are low. Nitric oxide (NO), a gaseous molecule involved in a myriad of functions, is known to affect the IRP system in many ways. The NO+ redox form of nitric oxide is involved in the reversible post-translational regulation of numerous proteins (including IRP2), a process called S-nitrosylation. We previously reported that NO+ causes upregulation of Ft synthesis along with IRP2 degradation. Here we report that sodium nitroprusside (SNP), an NO+ donor, causes a dramatic and rapid increase of ferritin synthesis that for a time occurs without interfering with IRP-IRE binding activities. We also show that NO+ increases the translational efficiency of ferritin mRNA as compared to treatment with ferric ammonium citrate, an iron donor. In addition, we also report that SNP does not function as an iron donor. These results indicate that NO+-mediated upregulation of ferritin synthesis is, in part, due to an IRP-independent post-transcriptional mechanism of regulation.
A New Perspective on the Natural History of Hereditary Hemochromatosis.
Authors: Waalen Jill , Beutler Ernest 
Published: April 2020

Abstract
A few homozygotes for the C282Y mutation of HFE manifest severe and even fatal iron-storage disease; most have only a modest increase in iron stores, show no clinical manifestations, and have no shortening of lifespan. Screening for hemochromatosis has been advocated so that therapeutic phlebotomy could be offered to the mildly affected subjects, often termed "pre-cirrhotic", presumably protecting them against progression of the disease. The rationale of this approach depends upon the implicit assumption that the natural history of the disease is one of progressive accumulation of iron and consequently ever-increasing tissue damage. In 1998 we began screening for the C282Y homozygous genotype among patients in the Kaiser Permanente Health Appraisal Clinic in San Diego. We have now followed 144 patients from this cohort of 156 C282Y homozygotes with medical records spanning up to 10 years, including 35 patients who declined phlebotomy. Data from this analysis and other sources now suggest that the natural history of the disease might be quite different than has heretofore been assumed. Our revised perspective is based upon both cross-sectional and longitudinal observations. Cross-sectional analysis of levels of serum ferritin, a surrogate for total body iron, shows little or no change with age in C282Y homozygotes. Neither are abnormal liver function tests, which occur in a small minority of homozygotes, age-related. In Denmark, longitudinal analyses of serum ferritin levels in untreated patients show no increase over as much as 20 years. Our own longitudinal data on serum ferritin levels in homozygotes who were not phlebotomized confirm this observation. Longitudinal analyses of abnormal ALT levels in homozygotes show no upward trend over a four-year period. Accordingly, it seems likely that in hemochromatosis the maximum extent of disease has been achieved in most patients by the time they reach adulthood, and that patients who are diagnosed with mild disease are unlikely to show progression.
Human Brain Endothelial Cells (HUBEC) Can Expand Both Human Bone Marrow and Cord Blood SCID-Repopulating Cells (SRC) through Cell Contact Rather Than Soluble Factors.
Authors: Takebe Naoko , Cheng Xiangfei , Farese Ann M. , Welty Emily , Meisenberg Barry , MacVittie Thomas 
Published: April 2020

Abstract
Human brain endothelial cells (HUBEC), a U.S. Navy proprietary cell line, was reported previously by Chute et al as a promising co-culture ex vivo expansion system for both adult bone marrow (ABM) and cord blood (CB) hematopoietic stem cells (HSC).a,b,c We report here our results of using HUBEC in ex vivo expansion and in vivo engraftment assay using NOD-SCID mice. CD34+ enriched fresh ABM was obtained using the method as described previously.a,b However, we used frozen CB and the same cytokines for both ABM and CB expansion whereas Chute et al used fresh CB and different cytokines. Ex vivo expansion studies for both ABM and CB were performed for 7 days in the HUBEC coated plates with previously reported cell density and cytokine cocktail containing GM-CSF, IL-3, IL-6, SCF, and flt-3 (GM36SF) in IMDM 10% FBS media.a HSC injections and BM harvesting of NOD-SCID mice as well as flow cytometric analysis were performed using the methods of Chute et al.a NOD-SCID mice were transplanted with limiting doses of either fresh ABM CD34+ cells or freshly thawed CB CD34+. The progeny of the identical doses of ABM CD34+ or the progeny of the identical doses of CB CD34+ cells was then transplanted. Culture with GM36SF alone resulted in a 15.5-fold and 70-fold increase in total cells, a 3.4-fold and 32-fold increase in CD34+ cells, and a 4.8-fold and 4.1-fold increase in CD34+/CD38- cells for ABM and CB, respectively. In contrast, HUBEC co-culture with GM36SF yielded a 25-fold and 48-fold increase in total cells, a 8.9-fold and 13-fold increase in CD34+ cells, and 114-fold and 106-fold increase in CD34+/CD38- cells for ABM and CB, respectively. HUBEC co-culture without GM36SF supported a 1.0-fold and 1.0-fold increase in total cells, a 0.06-fold and 0.1-fold increase in CD34+ cells, and 0.25-fold and 0.2-fold increase in CD34+/CD38- cells for ABM and CB. HUBEC co-culture with GM36SF and transwell (non-contact culture) resulted in a 20-fold and 48-fold increase in total cells, a 6-fold and 8-fold increase in CD34+ cells, and a 32-fold and 38-fold increase in CD34+/CD38- cells for ABM and CB. Overall, the transwell expansion of CD34+/CD38- population in both ABM and CB was reduced to 30% of that achieved in the contact culture. ABM CD34+ cells (5 x 105) engrafted 60% and the progeny of 5 x 105 cultured in the HUBEC monolayer with GM36SF engrafted in 90% of transplanted mice. CB CD34+ cells (1 x 104) engrafted 27% and the progeny 1 x 104 CB CD34+ cells cultured in the HUBEC monolayer with GM36SF engrafted 64% of NOD-SCID mice. SRC frequencies calculated as a 3.12-fold and 2.7-fold increase in CD34+ enriched ABM and CB, respectively, which was less than reported previously.a,b In summary, HUBEC supports and expands SRC mainly through cell-to-cell contact between HSC and endothelial cells, with HUBEC-secreted factors playing a minor role.