bluuebunny/crossref_metadata_2025_split huggingface dataset:
Bleeding Events and Factor IX Levels in Female Carriers from a Large Kindred of Hemophilia B.
Authors: Salter John T. , Hardesty Brandon , Heiman Meadow , Sulkin Matthew , Shapiro Amy D. , Greist Anne 
Published: October 2019

Abstract
Introduction: Factor IX (FIX) deficiency (FIXdef) is an X-linked disorder where affected males manifest various hemorrhagic tendencies based on the severity of deficiency. Women who carry the causal mutation exhibit a range of FIX activity levels (FIXact) and may experience bleeding when the FIXact is below the minimum hemostatic level. There is a paucity of reported data regarding the range of FIXact and bleeding events in FIX carriers. We report the FIXact and associated bleeding symptoms in the female carriers of a large kindred of moderate FIXdef caused by a common point mutation (31008 C>T [thr296met]).
Methods: Since 2000, our facility has compiled a database containing information on over 1300 patients with bleeding disorders, including 402 males with FIXdef representing 98 families, 469 confirmed FIX carriers, and 406 at-risk females not yet tested for carrier status. Overall there are 121 FIX carriers with FIX act; this kindred represents 108 documented carriers of which 83 have had FIXact. In this kindred, data on FIXact and bleeding symptoms were collected on carriers. A bleeding symptom questionnaire was developed specific to this population and administered to the target population; at this time questionnaires on 42 of the known carriers is available. Responses on the questionnaire were compared to the FIXact to determine if a correlation existed between reported symptoms/events to FIXact.
Data: For 83 female carriers included in this kindred’s analysis, the mean FIXact was 52% (range 14–114%, median 46%). Of the 42 women completing the bleeding questionnaire, the mean FIXact was 50.2% (range 21–114% median 41%). Typical bleeding events reported included epistaxis, postpartum bleeding, menorrhagia, and easy bruising. By comparing reported bleeding events to FIXact we found that women with FIXact <40% reported increased rate of easy bruising/hematoma formation (9.8% vs. 2.4%) and postoperative bleeding (14.6% vs. 2.4%) compared to women with FIXact >40%. While other bleeding events demonstrated no direct correlation with FIXact, our limited sample available at this time likely impacted this association.
Conclusions: This effort represents the largest report of data from a single kindred of FIX deficiency to date. Female carriers in this kindred have an identical mutation causing moderate FIXdef, yet exhibit a wide range of FIXact; the etiology for this variability is not known and requires further investigation. Ongoing data collection in our FIX carrier population will provide additional information to determine an association between the range of FIXact and bleeding symptoms. FIX carriers require documentation of levels (regardless of the level of deficiency for which the specific mutation codes) and monitoring of symptoms, particularly related to childbirth or surgery, as treatment with exogenous FIX may be required for prevention of hemorrhage and optimal care.
Gene Dosage of the Cell Cycle Regulatory Phosphatases Cdc25C and PP2A Determines Sensitivity to Lenalidomide in del(5q) MDS.
Authors: Wei Sheng , Rocha Kathy , Williams Ann , Chen Xianhong , Burnette P.K. , Djeu Julie Y. , Liu Qing , Byrd John , Sokol Lubomir , Lawrence Nick , Pireddu Roberta , Dewald Gordon , Maciejewski Jaroslaw , List Alan 
Published: October 2019

Abstract
MDS with chromosome 5q deletion [del(5q)] is uniquely sensitive to clonal suppression by lenalidomide (LEN), yielding high rates of cytogenetic and erythroid response. Although haploinsufficiency for a candidate tumor suppressor gene within the common deleted segment (CDS) has been implicated in the pathogenesis of del(5q) MDS, no specific gene products account for the karyotype-dependent LEN sensitivity. Using primary AML cells, we showed that LEN is selectively cytotoxic to del(5q) cells, inducing cell cycle arrest at the G2 > G1 junctures and apoptosis in a concentration-dependent fashion. Now we show that the CDS-encoded Cdc25C and PP2A phosphatases (PPTs) serve as molecular targets for LEN. Cdc25C and PP2A are dual specificity PPTs that regulate cell cycle. The genes encoding the catalytic subunit alpha (PP2ACa), the dominant catalytic isoform, and the regulatory subunit B (beta isoform PR 52) of the PP2A holoenzyme reside within the del(5q) CDS. Real-time RT-PCR showed that Cdc25C and PP2ACa gene expression is significantly reduced in primary del(5q) AML and MDS BM-MNC compared to non-del5q and normal controls. A Cdc25C FISH probe confirmed presence of only a single allele in del(5q) specimens [n=5] vs. normal karyotype [n=5] with no overlap with the EGR1 locus. We found that LEN inhibited rHu-Cdc25C activity with IC90 inhibitory concentrations ≤10nM. Similarly, PP2A PPT activity was inhibited by LEN in enzyme immunoprecipitates from Ramos cells at sub-micromolar concentrations. In U937 cells, we confirmed intracellular Cdc25C and PP2A inhibition, showing concentration-dependent retention of the inhibitory p-Tyr15 -Cdc2 substrates for Cdc25C with corresponding phosphorylation of the PP2A substrate Ser216 Cdc25C, findings reproduced by treatment with the PP2A selective antagonist, fostreicin. Cdc25C immunoprecipitation showed that phosphorylation of Ser216 Cdc25C increased binding to the 14-3-3 protein with consequent cytoplasmic sequestration confirmed by fluorescence microscopy. Phosphorylation of histone H2A.X Ser139 was unchanged after LEN treatment, indicating that phosphorylation of the inhibitory Cdc25C serine does not arise from upstream DNA damage activated kinases. Importantly, siRNA suppression of Cdc25C gene expression in U937 cells promoted LEN-specific apoptosis in 45% of cells, which increased to 68% after fostreicin inhibition of PP2A. These data indicate that haplodeficiency for the Cdc25C and PP2ACa genes promotes selective sensitivity of del(5q) cells to LEN-induced apoptosis. Haplodeficiency of critical drug-able targets may represent a novel strategy for tumor selective therapy.
Erythropoietin (Epo) Secreting Mesenchymal Stromal Cells and Autocrine Signalling Via the Epo Receptor. A Novel, Cell-Based Paracrine Epo Delivery System for Cardiovascular Therapy.
Authors: Copland Ian B. , Jolicoeur Marc E. , Gillis Marc-Antoine , Cuerquis Jessica , Eliopoulos Nicoletta , Annabi Borhane , Calderone Angelo , Tanguay Jean-Francois , Ducharme Anique , Galipeau Jacques 
Published: October 2019

Abstract
Separately, the roles of MSCs and erythropoietin (Epo) in cardiovascular regenerative therapy have expanded rapidly. However, poor in vivo survival/engraftment of MSCs and Epo’s short half-life and bioavailability, limit their individual effectiveness. We propose that a combinatorial approach would be synergisitic. We therefore determined whether administration of Epo-secreting Mesenchymal stromal cells (MSCs) could represent a better therapeutic platform in cardiovascular medicine than MSCs in their primeval form. MSCs from C57Bl/6 mice were retrovirally transduced to express murine Epo (Epo+MSCs) and compared to wild type (WT) MSCs in relation to their abilities to promote positive heart tissue remodelling following myocardial infarction (MI). Relative to WT-MSCs, we found that via an Epo/EpoR autocrine loop, Epo+MSCs have an enhanced proliferative capacity and are more resilient to apoptotic stimuli. In vivo, Epo+MSCs also have enhanced survival and can initiate a more robust host-derived angiogenic response. Injection of WT-MSCs or Epo+MSCs into the borderzone of the MI following ligation of the left coronary artery demonstrated engraftment within the borderzone seven days post-implantation, while sequential (7 and 14 day post-MI) echocardiograms and invasive hemodynamic measurements (14 days post-MI) showed that the Epo+MSC group exhibited significantly improved LV systolic and diastolic function compared to WT-MSCs injected animals and PBS controls (Fractional shortening 29.7% ± 8.5 versus 19.9% ± 6.6 and 19.1% ± 7.4 respectively, p=0.02). Myocardial capillary density was significantly higher in the Epo+MSC group and was coupled to reduced neutrophilic infiltration by direct actions of Epo on endothelial and neutrophil chemotaxis. In conclusion, we show that Epo overexpression enhances the cellular regenerative properties of MSCs by both autocrine and paracrine pathways.
Novel Direct Evidence That Adult Bone Marrow-Derived Very Small Embryonic Like (VSEL) Stem Cells Are Mobilized into Peripheral Blood - Leukopheresis as a Potential Tool To Isolate Pluripotent Stem Cells for Therapeutic Pruposes.
Authors: Kucia Magda , Wysoczynski Marcin , Wan Wu , Ratajczak Mariusz Z. 
Published: October 2019

Abstract
Recently, we identified in adult bone marrow (BM) a population of very small embryonic like (VSEL) stem cells (Leukemia2006:20;857) that are
very small in size (∼3.5 μm), CXCR4+Oct-4+ SSEA-1+Sca-1+CD45−lin−, respond to SDF-1 gradient and contain large nuclei with open type of euchromatin.
We asked if VSEL are mobilized into peripheral blood (PB) similarly as BM-derived hematopoietic stem cells. To address this question, we evaluated VSEL level in PB after
pharmacological (G-CSF) and stress/organ damage-induced mobilization (toxic liver and skeletal muscle damage after exposure to CCl4 and cardiotoxin respectively).
The number of circulating VSEL in PB was measured by
FACS, direct histochemical staining of PB-cells for SSEA-1 and Oct-4 expression, and RQ-PCR analysis of pluripotent genes (Oct-4, Nanog).
Finally, VSEL sorted from the PB were tested for their pluripotency in our established assay based on formation of embryoid-like spheres by VSEL in co-cultures with C2C12 cells. We report that in steady state conditions, VSEL circulate at very low levels in PB (∼100–200 cells/ml) and that the number of these cells/ml of PB is five times higher in two month old mice as compared to one year old animals. Furthermore, we found that VSEL are mobilized into PB both by G-CSF- and tissue injury and the number of circulating VSEL increases 5–6 and 2–3 times after G-CSF and after liver or skeletal muscle injury, respectively. Furthermore, the mobilization of VSEL could be additionally enhanced by blocking CXCR4 receptor by T140 antagonist. Based on this, we provide the evidence that VSEL
is a mobile pool of primitive CXCR4+SSEA-1+Oct-4+ stem cells, is released into circulation during tissue/organ injury and their trafficking is regulated by SDF-1-CXCR4 axis.
Further studies will asses if the level of mobilized VSEL
correlates with overall regenerative potential of an organism and could become a helpful prognostic indicator for the clinical outcome from organ damage/injury (e.g., stroke, heart infarct).
Finally, mobilized VSEL could also be employed in regenerative medicine and we are testing this possibility in appropriate animal models.
Differential Transactivation Activities of Sumoylated CCAAT Enhancer Binding Protein alpha (C/EBPα) p42 Versus p30 May Contribute in Part, to Aberrant C/EBPα Activity in Acute Leukemias.
Authors: Khanna-Gupta Arati , Silver Matthew , Hankey William , Sun Hong , Berliner Nancy 
Published: October 2019

Abstract
CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific genes at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for C/EBPα, including a selective early block in the differentiation of granulocytes. Recently mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting normal karyotypes. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. Since the molecular basis for this observation remains unknown, a complete understanding of the regulation of this key transcription factor during myelopoiesis is critical. C/EBPα was recently shown to be post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) which lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. Sumoylation at K159 in the C/EBPα protein is thought to prevent association of the SWI/SNF chromatin remodeling complex with C/EBPα, thereby hampering transactivation. In order to demonstrate the role of sumoylation in mediating C/EBPα activity, we examined the effect of both the full length (p42) and the dominant negative (p30) isoforms of the C/EBPα protein on myeloid gene expression. We demonstrate that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation, and that transactivation of the myeloid-specific lactoferrin promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells while loss of sumoylation correlates with loss of p42C/EBPα binding and LF expression in more mature cells. Based on these observations we conclude that sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. We are now in the process of confirming this finding in leukemic cell lines. This observation leads us to hypothesize that the differential transactivation potential of sumoylated p30 versus the p42 C/EBPα proteins may contribute, in part, to the aberrant activity of C/EBPα in acute leukemias.
The First Zinc Finger Domain of EVI1 Enhances Self-Renewal of Hematopoietic Progenitors.
Authors: Laricchia-Robbio Leopoldo , Nucifora Giuseppina 
Published: October 2019

Abstract
EVI1 was first identified as a preferential integration site of ecotropic retroviruses in the MDS1/EVI1 genomic locus leading to myeloid tumors in susceptible mice. Later studies showed that retroviral integration in the MDS1/EVI1 locus results in the emergence of a non-malignant dominant hematopoietic stem cell clone in non-susceptible mice strains, in non-human primates, and in patients, suggesting that a gene encoded by the locus could be deregulated by the retrovirus and affect the self-renewal potential of the cell. The locus encodes two genes. One of them, EVI1, has long been associated with myeloid leukemia and myelodysplastic syndrome. To understand whether EVI1 has a role in self-renewal control, we forcibly expressed EVI1 in the bone marrow progenitors of two mouse strains that differ in their proliferation and self-renewal potential. By comparing the response of the hematopoietic cells to EVI1, we show that EVI1 has a role in prolonging the self-renewal potential of the cells and that this ability of EVI1 is however limited and modulated by inherent strain-specific characteristics. To identify the region of EVI1 mediating this effect, we infected the bone marrow progenitors of the two murine strains with EVI1 wild type or with specific alternative EVI1 point mutants and compared the self-renewal potential of the cells. This approach allowed us to show that the first zinc finger domain of EVI1 is required to enhance the self-renewal of the hematopoietic progenitors. This function is mediated by two specific zinc finger motifs and their disruption by point mutations abolished this effect. These results suggest that the motifs interact with factors that regulate self-renewal. The cooperation between EVI1 and these unknown proteins in deregulation of self-renewal occurs inappropriately when EVI1 is deregulated, but based on EVI1 gene knock out studies it is likely that this function of EVI1 is required during embryonic development.
Mesodermal Patterning Activity of the Transcription Factor SCL.
Authors: Ismailoglu Ismail , Yeamans Gabriel , Daley George Q. , Perlingeiro Rita , Kyba Michael 
Published: October 2019

Abstract
The transcription factor SCL is critically required for the establishment of the hematopoietic lineage as well as for proper endothelial development, however it is not required for maintenance of HSCs or endothelial cells in the adult. Conflicting data exists regarding the developmental function of SCL, namely whether it acts as a master regulator, actively patterning mesoderm toward hematopoietic/endothelial development at the expense of other lineages, or is merely necessary to maintain the earliest committed hematopoietic and endothelial precursors. To answer this question in a mammalian model, we have engineered murine ES cells with a conditional doxycycline-inducible SCL transgene, and evaluated the effects of pulses of SCL expression at defined time points during in vitro development. During embryoid body differentiation, a pulse of SCL expression during mesodermal patterning results in enhanced hematopoiesis at later time points, as assayed by CFC activity and FACS analysis, as well significantly increasing the frequency of bipotent hematopoietic/endothelial hemangioblast CFCs. Concomitantly, SCL represses cardiac development, assayed by beating cardiomyocyte frequency, and molecular markers of the cardiac lineage. SCL also represses paraxial mesodermal developmental potential, severely reducing both the earliest PDGFαR+/Flk-1neg paraxial progenitors, as well as repressing levels of later markers including Pax3. Through chimeric embryoid bodies in which SCL cells are mixed with GFP-labelled ES cells, we show formally that this effect is cell-autonomous. Finally, we tested whether expression of SCL would enable hematopoiesis during ES cell monolayer differentiation. Hematopoiesis requires inter-germ layer inductive signaling which occurs in 3 dimensions but not in monolayer culture, thus ES cells do not spontaneously differentiate into blood in this 2 dimensional culture. We show that when mesoderm is produced during monolayer culture of wild-type ES cells, or control cells not expressing SCL, it is biased towards paraxial or cardiac differentiation. However when given a pulse of SCL expression, these pathways are repressed, PDGFαRneg/Flk-1+ presumptive lateral plate progenitors are produced, and abundant hematopoietic differentiation occurs. This data in the mammalian system thus strongly supports the former hypothesis, of an active role for SCL in mesodermal patterning.
Loss of Ku70 Results in a HSC Repopulating Defect.
Authors: Qing Yulan , Gerson Stanton L. 
Published: October 2019

Abstract
Genomic integrity is essential for organism development and longevity and in large part is mediated by DNA repair proficiency. Non-homologous end-joining (NHEJ) is a specific pathway essential for DNA double-strand break repair in all cells and VDJ processing in B and T cells; and it is crucial for maintenance of mammalian genome. We hypothesize that NHEJ is also critical for hematopoietic stem cells (HSC) maintenance and function. Ku70 is a key component of the NHEJ pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. Ku70-deficient mice were used to assess the importance of NHEJ pathway in HSC function. Ku70-deficient mice have decreased bone marrow cells (BMC), and significant reductions in the frequency of colony-forming units in culture (CFU-C) in the presence of cytokines including IL-3, IL-6, SCF and Epo. SKL (Sca1+, c-Kit+, Lin−) cells from Ku70-deficeint mice showed a similar proliferation rate compared with SKL cells from WT mice when cultured in vitro in the presence of cytokines; single cell culture experiment showed that SKL cells from Ku70-deficient mice and WT mice have similar clonogenic ability. Thus, Ku70-deficient SKL cells retain proliferation capacity although there is a deficiency in overall hematopoietic homeostasis. To address Ku70-deficient HSC function in vivo, Ku70-deficient BMCs were serially transplanted into lethally irradiated congenic wild-type mice. These Ku70-deficient marrow cells were able to reconstitute the myeloid lineages in both primary and secondary recipients, while over 95% of the T cells in the recipients were derived from the host. However, the tertiary whole marrow recipients died within 2 weeks of transplantation, while the WT BMCs recipients survived tertiary transplantation. Competitive repopulation assays were also performed between WT and Ku70-deficient BMCs. The results showed that when Ku70-deficient BMCs were mixed with WT competitor BMCs at a 1:1 ratio, Ku70-deficient BMCs were completely outcompeted by the WT BMCs. These data demonstrate that Ku70, a key component of NHEJ, is required for HSC function. Ku70 deficiency in the hematopoietic compartment results in a long-term repopulating defect and loss of competitive repopulation ability.
The Expression of Polycomb Group Genes Products, Bmi1 and Mel-18, Regulate the Function of Murine Primitive Hematopoietic Cells.
Authors: Kajiume Teruyuki , Sato Takashi , Kobayashi Masao 
Published: October 2019

Abstract
The Polycomb group (PcG) genes (bmi1 and mel-18) known as negative control factors of the Hox gene is thought to regulate the differentiation and self-renewal of hematopoietic stem cells (HSCs). The loss of mel-18 results in the promotion of HSC self-renewal, and the increase of mel-18 expression inversely leads to the differentiation of HSCs. On the other hand, the loss of bmi1 does not lead to self-renewal activity of HSCs. In this study we examined the effect of expression of bmi1 and mel-18 on the role of function in murine HSCs. Lineage-negative, Sca1-positive, and cKit-positive primitive hematopoietic cells were purified and the expression of PcG protein was evaluated from the intra-nuclear distribution of PcG proteins. The Bmi1-positive hematopoietic cells barely contained Mel-18, and the Mel-18-positive cells barely contained Bmi1. the frequency of positive cells for both Bmi1 and Mel-18 was less than 0.5% of purified primitive hematopoietic cells. The expression levels of the PcG genes, bmi1 and mel-18, in HSCs were knocked down by siRNA and then gene expression was assessed by quantitative real-time PCR. The introduction of siRNA against bmi1 or mell-18 resulted in approximate 50 to 60% decrease of each gene expression without affecting another gene expression. Primary colony-forming activity of knocked down cells in response to stem cell factor, thrombopoietin and the ligand for flt3 was not affected by the induction of siRNA. However, secondary colony-forming activity from primary colony-forming cells in bmi1-knockdown cells was significantly decreased when compared with that of control cells. Conversely, the mel-18-knockdown cells significantly increased, suggesting that mel-18-knockdown cells are capable of proliferating activity. Finally, bone marrow reconstitutive activity was examined by using Ly5.1 and Ly5.2 system. While the bmi1-knockdown marrow cells decreased the reconstitutive activity, the mel-18-knockdown marrow cells showed the increase of engraftment activity after 6 months of transplantation. From these results, we consider that mel-18 and bmi1 have reciprocal functions in HSCs. Mammalian PcG protein complexes can be classified into two distinct types, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). The Mel-18 protein is a constituent of mammalian PRC1 together with M33, Bmi1 or rae28, and Scmh1. The Mel-18 protein is composed of 342 amino acids and the N-terminal region of the 102 amino acid, which includes the RING finger motif, shares 93% homology with Bmi1 protein. In addition, its secondary structure shows high homology with the Mel-18 and Bmi1 proteins. We hypothesized that the opposite function is expressed in HSCs because Mel-18 and Bmi1 share the same structure and compete when in the complex form. These results suggest that mel-18 and bmi1 have inverse function in HSCs and that the balance of Bmi1 and Mel-18 may regulate the fate of self-renewal and differentiation in HSCs.