bluuebunny/crossref_metadata_2025_split huggingface dataset:
Congenital Amegakaryocytic Thrombocytopenia in a Thrombocytopenic Child Presenting without Amegakaryocytosis.
Authors: Geddis Amy , Fox Norma , Nguyen Hung , Keates-Baleeiro Jennifer , Frangoul Haydar 
Published: April 2020

Abstract
Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited bone marrow syndrome due mutation of the thrombopoietin receptor c-Mpl. Affected children present with thrombocytopenia at birth and absence or severe reduction of megakaryocytes in the bone marrow and usually progress to complete bone marrow failure within the first decade of life. Mutations of c-Mpl have been classified as either type I mutations, in which the receptor has lost all activity, or type II mutations, in which the receptor retains some degree of function. Clinically, type II patients have a slightly delayed onset of bone marrow failure (mean age 48 months) compared to type I patients (22 months). Here we describe a girl with CAMT who had a clinical course similar to patients with type I mutations but without marked amegakaryocytosis at onset. The patient was born with intracranial hemorrhage due to severe thrombocytopenia (platelets 17K), which was initially thought to be due to alloimmune thrombocytopenia because of consistent platelet antigen typing studies and because the child responded to transfusion with her mother’s platelets and not to random donor platelets. However, thrombocytopenia persisted and she did not respond to immune directed therapies such as IVIG, prednisone or Rituximab, and after 14 months of age she began to develop neutropenia and anemia and by 24 months she had progressed to severe aplastic anemia. Bone marrow evaluation at 3 months showed trileage hematopoiesis with only mildly decreased megakaryocytes although they were noted to be small with hypolobulated nuclei. By 6 months of age, however, megakaryocytes appeared mildly decreased but were morphologically unremarkable and the marrow showed increased hematogones. At 24 months, marrow cellularity had decreased to 5% of normal with frank amegakaryocytosis. Sequencing of c-Mpl revealed two heterozygous mutations, one at Arg102Pro in the extracellular domain and the other one resulting in a stop codon at amino acid 541 in the intracellular domain. Arg102Pro is the most commonly occurring mutation reported in CAMT and is generally associated with type II disease. 541Stop is the first mutation reported in exon 11 and results in truncation of the receptor shortly after the box 1 homology domain. Previous in vitro studies involving the murine homolog of this mutation, which is missing all but the proximal 28 amino acids of the intracellular domain, demonstrated that it does not signal in response to thrombopoietin. This case illustrates the variation in clinical phenotype that can be seen in CAMT and the importance of gene sequencing for accurate diagnosis.
Thrombocytopoiesis Is Not Regulated by Thrombopoietin (TPO) but Proplatelet Formation (PPF) Stimulating Factor.
Authors: Murai Kazunori , Kowata Shugo , Tsukushi Yasuhiko , Ishiguro Mamiko , Oyake Tatsuo , Sugawara Takeshi , Ishida Yoji 
Published: April 2020

Abstract
TPO is believed to be the physiological humoral hematopoietic factor in megakaryopoiesis and thrombopoiesis. Some investigators reported the inverted relationship between platelet mass and serum TPO levels, resulting in the conclusion that the platelet mass may directly play a role of regulating the circulating TPO levels. In the previous ASH meeting, we reported that serum TPO levels were not elevated in Immune thrombocytopenic purpura (ITP) patients. In this study, we measured platelet count, the serum TPO level, % reticulated platelet (%RP) and megakaryocytes mass in patients with refractory anemia in myelodysplastic syndrome (MDS-RCMD). The mean plasma TPO level in 20 healthy persons was 57.3±15.2 pg/ ml, the percentage of RPs was 1.51±0.71%.
Plasma TPO levels were widely distributed (799.9±883.0 pg/ ml, n=35) in MDS-RCMD, while they were less than 131.0 pg/ml in ITP (48.5±37.0 pg/ ml, n=37). There were no significant relationship between platelet counts and plasma TPO levels in MDS-RCMD or ITP. These results indicated that plasma TPO level was not regulated by the platelet mass at least in thrombocytopenic patient with MDS-RCMD or ITP. We evaluated the thrombopoiesis in thrombocytopenic patients with MDS-RCMD or ITP. %RPs were 1.9±0.2 % in MDS-RCMD (n=34), indicating that increased thrombopoiesis was not observed in MDS-RCMD, in which serum TPO levels were increased. While %RPs were 6.6±4.9 % in ITP (n=45), indicating that thrombopoiesis was observed in ITP, in which serum TPO levels were not increased. These results indicated that thrombopoiesis was not regulated by plasma TPO levels but some thrombopoiesis stimulating factor(s). Therefore, we evaluated the thrombopoiesis stimulating activity in plasma of patients with MDS-RCMD or ITP as the indicator of PPF stimulating activity of murine megakaryocytes. PPF stimulating activity was measured as the ratio of PPF bearing megakaryocytes/ viable megakaryocytes after 48 hrs serum-free culture of murine megakaryocytes.
As the plasma have some inhibitory effects of PPF activiy, the HDL fraction, isolated from plasma, was used as the methods described previously in Ishida Y et al. (Thrombosis and Haemostasis, 2001; 85:349–55).
The higher PPF stimulating activity was observed in patients with ITP (n=15) or MDS-RCMD (n=15) than that with healthy donors (n=10) {healthy donors 26.1±5.4 % versus ITP 38.0± 6.0% (p&lt;0.01), MDS-RCMD 41.9±5.3%(p&lt;0.01)}. There was a significant correlation between the PPF activity and platelet counts (R=0.68; p&lt;0.01) in these thrombopenic patients. These data strongly suggest that thrombopoiesis may not be regulated by TPO but some PPF stimulating factor(s).
Lenalidomide and CC-4047 Inhibit the Proliferation of Namalwa Cancer Cells While Expanding CD34+ Progenitor Cells. New Insights on the Combination Therapy with Certain HDAC Inhibitors for Hematological Cancers.
Authors: Verhelle Dominique , Corral Laura G. , Wong Kevin , Mueller Jessica H. , de Parseval Laure Moutouh , Jensen-Pergakes Kristen , Glezer Emilia , Ferguson Gregory D. , Brady Helen A. 
Published: April 2020

Abstract
Clinical studies involving patients with Myelodysplastic Syndrome and Multiple Myeloma have demonstrated the efficacy of lenalidomide (CC-5013), a drug recently approved by the FDA under the commercial name of Revlimid®, by reducing and often eliminating malignant cells while restoring bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide, and its analog CC-4047, on the proliferation of two different hematopoietic cell models: the Namalwa cancer cell line and CD34+ progenitor cells. We found that both compounds have anti-proliferative effect on Namalwa cells and pro-proliferative effect on CD34+ cells while p21WAF−1 expression was upregulated in both cell models. In Namalwa cells, we determined that p21WAF−1 is responsible for the inhibition of CDK2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest. In contrast, in CD34+ progenitor cells, despite upregulated p21WAF−1 expression, we observed an increase of the cell division rate, leading to the enhancement of CD34+ expansion. Finally, we found that CC-4047 and lenalidomide have synergistic effects with two different HDAC inhibitors (Valproic acid and Trichostatin A) in both increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Taken together, our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus progenitor cells and could explain, at least in part, the reduction of malignant cells and the restoration of the bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, and proposes new potential clinical uses such as bone marrow regeneration. Finally, our vitro experiments showing the efficacy of the combination of CC-4047 and lenalidomide with Valproic acid and Trichostatin A suggest that HDAC inhibitors might be ideal candidates for combination therapy by elevating the therapeutic index, versus monotherapy, to treat hematological malignancies.
Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.
Authors: Wu Tong , Kim Hyeoung Joon , Sellers Stephanie E. , Meade Kristin E. , Agricola Brian A. , Metzger Mark E. , Kato Ikunoshin , Donahue Robert E. , Dunbar Cynthia E. , Tisdale John F. 
Published: April 2020

Abstract
Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.
Structure of Erythropoietin in African Clawed Frogs, Xenopus laevis, and Its Role in the Liver Erythropoiesis.
Authors: Nogawa Nami , Kosaka Nobuyoshi , Aizawa Youichi , Miyazaki Hiroshi , Komatsu Norio , Kato Takashi 
Published: April 2020

Abstract
Erythropoietin (EPO) is a main regulator of erythropoiesis ensuring oxygen supply in mammalian species. However the functions of EPO in nonmammalian vertebrates remain unclear. In this study, EPO was identified in Xenopus laevis (X. laevis), and its contribution to definitive erythropoiesis was studied. The X. laevis EPO (xlEpo) cDNA revealed that the deduced amino acid sequence had only 38% identity to human EPO (hEPO), while all four cysteine residues were conserved. xlEPO mRNA was expressed predominantly in the liver and lung. In order to assess the biological activity, recombinant xlEPO was produced by transfecting COS-1 with CMV promotor-driven vector. A mouse FDC/P2 cells stably expressing xlEPOR cDNA, that is a putative EPO receptor, showed proliferation in response to recombinant xlEPO in a dose dependent manner. This confirmed the ligand-receptor relationship of nonmammalian xlEPO and xlEPOR. To our surprise, xlEPO stimulated proliferation of EPO-dependent human cell line UT-7/EPO as well as murine EPOR expressing FDC/P2 cell lines. The cross-reactivity suggests the tertially structure is conserved through xlEPO to mammalian EPOs. In addition, the amino acid residues that are essential for hEPO binding to hEPOR are highly conserved in xlEPO. Since potent N-glycosylation site is absent in xlEPO, the glycosylation characteristics of recombinant xlEPO was studied by fractionation using wheat germ aggulutinin (WGA) and concanavalin A (ConA) lectin affinity chromatography. XlEPO activity was seen in flow-through fractions indicating the absence of O-glycosylation as well as N-glycosylation in xlEPO molecule. The absence of glycosylation suggests the high affinity of xlEPO to xlEPOR, and the shorter blood half-life. In order to investigate the biological function of xlEPO, in vitro colony forming assay of X. laevis erythroid progenitors was developed. Magnetic cell sorting analysis showed that xlEPOR-positive cells reside in the liver possessing typical erythroblastic morphology with high nucleus-to-cytoplasm ratio containing hemoglobin. The formation of erythroblast colonies from liver cells on addition of recombinant xlEPO was observed. The colonies formed were erythroblast colonies composed of hemoglobin-synthesising erythroblasts, confirming the erythropoietic function of xlEPO in X. laevis erythropoiesis. These results and the detection of xlEPO mRNA in liver hypothesized the paracrine regulation of xlEPO. In the colony assay, erythropoietic activity was observed in the serum of phenylhydrazine (PHZ) induced anemic X. laevis. The highest erythropoietic activity was observed 4 days after PHZ-administration, prior to the peripheral erythrocyte number reaches a nadir at day 8. These results proved that xlEPO is a functional ortholog of mammalian EPO and its role in vertebrate hematopoietic system, providing new insights into the basis of erythropoietic regulations.
NE-180, a Novel glycoPEGylated Erythropoietin Demonstrates Dose-Dependent Activity in a Phase 1 Single Dose, Dose Escalating Study in Normal Human Volunteers (NHV).
Authors: Wallin Bruce A. , Ramjit Denise , Seiberling Michael , Zopf David 
Published: April 2020

Abstract
NE-180 is a glycoPEGylated recombinant human erythropoietin that binds to and activates the erythropoietin (EPO) receptor. It has demonstrated in vitro activities comparable to EPO and an extended serum half-life in animal studies. This may allow less frequent dosing in patients being treated with chronic anemia.
METHODS: A single center, open-label study of NE-180, administered as single escalating doses given by the SC or IV route, was conducted to assess the safety, tolerability, PK and PD. Subjects (male or female NHV) were planned to be assigned to one of 4 dose groups, 10 subjects per dose with 5 SC and 5 IV subjects per group: 0.5, 1.5, 3, or 4.5 mg/kg. Each dose group was planned to be initiated in an ascending, sequential fashion unless or until stopping rules were met.
RESULTS: 25 NHV (16 females) were enrolled in the first two dose cohorts and have completed 56 day follow-up. The 1.5 mg/kg IV cohort met the protocol-specified Hb rate of rise stopping rule (change in Hb greater than 1 g/dL during any 14 day period). Injections were generally well tolerated with no discontinuations for adverse events or serious adverse events. Reticulocyte increases were dose proportional. Average reticulocyte count at baseline was 1.0±0.3%. The maximal increase occurred at day 7. The mean change from baseline for the 0.5 and 1.5 mg/kg SC group was: 0.9±0.4% and 2.2±0.9%, respectively. The mean change from baseline for the 0.5 and 1.5 mg/kg IV group was: 1.7±0.8% and 2.3±0.8%, respectively. PK data will be presented.
CONCLUSIONS: Single doses up to 1.5 mg/kg of NE-180 administered to NHV were generally well tolerated and demonstrated potent dose-dependent erythropoietic activity.
Macrophage-Stimulating Protein Stimulates Erythropoietin Receptor Phosphorylation To Promote Enhanced Erk1/2 and Akt Activation in a Gab2-Dependent Pathway.
Authors: Chohan Manprit , Halupa Adrienne , Barber Dwayne L. 
Published: April 2020

Abstract
Friend-virus mediated erythroleukemia is a well-established model of murine oncogenesis. Several host factors have been shown to be necessary for Friend-mediated infection including a productive interaction between the Erythropoietin Receptor (EPO-R) and the 55 kDa glycoprotein product of the retroviral envelope gene (gp55). The Fv2 locus has been mapped and shown to be a truncated form of the STK tyrosine kinase (Sf-STK). Sf-STK expression and kinase activity is required for Friend virus infection of sensitive mouse strains. While the EPO-R and Sf-STK are required for Friend disease progression, it is unknown whether Sf-STK modulates EPO-R mediated signaling. EPO-R co-immunoprecipitated Sf-STK and STK specifically in COS-7 and Ba/F3 cells expressing both receptors. While the association between EPO-R and STK was shown to be constitutive, intriguingly, we discovered that co-stimulation with the STK ligand, Macrophage-Stimulating Protein (MSP) and EPO resulted in enhanced tyrosine phosphorylation of the EPO-R, compared to stimulation with EPO alone. Interestingly, STK kinase activity was required for EPO-dependent STK tyrosine phosphorylation, but was not required for MSP dependent EPO-R phosphorylation. Analysis of downstream signaling revealed STK and EPO-R co-operated in ligand induced dose-dependent phosphorylation of Erk1/2 and Akt. Interestingly, MSP and EPO also co-operated to stimulate tyrosine phosphorylation of the adaptor protein Gab2, leading to the recruitment of Shp2 and the p85 subunit of Phosphatidylinositol 3 kinase. These data suggest that Gab2 can couple to enhanced phosphorylation of Erk1/2 and Akt. However, no synergy was observed in MSP co-stimulation of JAK2, STAT1, STAT5, Jnk or p38 phosphorylation, demonstrating that STK activates specific EPO-dependent signaling pathways. EPO-dependent regulation of Socs family members was examined in Ba/F3-EPO-R cell lines expressing various STK constructs. The presence of STK induced Cis and Socs3, but not Socs1, transcript levels by 2.0-fold upon EPO stimulation. STK multi-functional docking sites (Y1330/Y1337) were required to observe optimal induction of Socs3. In contrast, Cis induction required STK tyrosine kinase activity. Our data demonstrate that EPO-R and STK interact at a biochemical level leading to augmentation of downstream signaling. These data suggest that Sf-STK contributes to Friend disease through delivery of mitogenic and/or survival signals to target erythroblast progenitor cells.
Recapitulating the Expression Pattern of Jak2 in Transgenic Zebrafish Using Jak2 Promoters and a GFP Reporter Gene.
Authors: Zhang Jing , Lin Hui-Feng , Handin Robert I. 
Published: April 2020

Abstract
The non-receptor tyrosine kinase Jak2 plays an important role in regulating erythro and thrombopoiesis. There has been intense interest in Jak2 since the observation that an activating mutation V617F is present in almost all patients with Polycythemia vera and many patients with Essential Thrombocytosis and Myelofibrosis. The analysis of Jak2 function in vivo has been limited as the murine jak2 knockout is lethal at day 10.5 of embryogenesis. Our laboratory has taken advantage of an ancestral partial duplication of the zebrafish genome, which has yielded two jak2 alleles --- jak2a and jak2b to study jak2 expression and function. Whole mount in situ hybridization studies confirm that the jak2a gene is only expressed in hematopoietic tissues, while jak2b is expressed in the developing lens and nephritic ducts. We have cloned and characterized the full-length jak2a and jak2b cDNAs and characterized the jak2a and 2b genomic loci. The jak2b gene has 24 exons spanning 79kb of genomic DNA. We amplified a 4kb zebrafish genomic fragment upstream of the first exon of the jak2b gene and linked it to the enhanced green fluorescent protein (EGFP) cDNA reporter and then microinjected the construct into single-cell zebrafish embryos. At 24 hours post fertilization (hpf), we observed fluorescence in the lens and nephritic ducts of developing embryos, with some expression in skin, muscle and notochord. The jak2a gene locus is complex as the jak2a gene is linked to a gene of unknown function, STARD4, in a head-to-tail manner with a small intergenic region of 1kb. As observed with jak2b, the first exon contains the jak2b 5′-UTR and the second exon contains the translation initiation site. We cloned a 1.9kb DNA fragment that included exons 1 and 2 the intervening first intron and an additional 800bp upstream of exon 1. This 1.9 kb promoter fragment was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that recapitulated the native expression pattern of jak2a. In injected embryos 24hpf, GFP+ cells were present in the anterior intermediate cell mass (ICM) and the lens. Fluorescent circulating blood cells, largely erythrocytes, were detected in 12 of 124 microinjected embryos 48 hours after fertilization. The jak2-EGFP transgenic zebrafish strains should be useful in the study of normal and pathologic hematopoiesis and in future studies of the pathogenesis of the MPDs.
STAT1 Promotes Megakaryocytic Differentiation in a GATA-1-Independent Manner.
Authors: Huang Zan , Crispino John D. 
Published: April 2020

Abstract
GATA-1 is a critical transcription factor that governs megakaryocyte development and differentiation. GATA-1-deficient megakaryocytes proliferate excessively and fail to undergo terminal differentiation, likely as a consequence of an aberrant gene expression program. In our studies to identify key signal transduction pathways that are dysregulated in the absence of GATA-1, we observed that STAT1, a gene of unknown function in megakaryocytes, was significantly downregulated in GATA-1-deficient megakaryocytes. Surprisingly, ectopic expression of STAT1, or IRF-1, a downstream effector of STAT1 signaling, promoted megakaryocytic differentiation of G1ME cells, a GATA-1-null and TPO-dependent erythromegakaryocytic cell line. Both STAT1- and IRF-1-transduced G1ME cells, but not GFP-expressing control cells, displayed multiple markers of terminal differentiation, including expression of CD42, as well as a significant fraction of polyploid cells. BrdU labeling assays confirmed that these transduced cells continued to effectively synthesize DNA, suggesting that STAT1 signaling switches these cells from mitosis to endomitosis. STAT1 and IRF-1 also modulated TPO signaling evidenced by enhanced STAT3 and STAT5 phosphorylation. Since STAT1 signaling is activated by IFN-g, we further investigated whether IFN-g-treatment also led to polyploidization of G1ME cells. IFN-g effectively promoted polyploidization of both G1ME cells and primary bone marrow-derived CD41+ cells in the absence of TPO. Finally, overexpression of STAT1 in primary bone marrow cells isolated from GATA-1 knockdown mice also promoted polyploidization in the absence of TPO. Taken together, these data reveal a GATA-1-independent mechanism by which STAT1 signaling promotes megakaryocyte differentiation.