bluuebunny/crossref_metadata_2025_split huggingface dataset:
The C-Terminal Region of the G-CSF Receptor Is Required for Induction of Neutrophil Elastase Expression in Myeloid Cells: Implication for Understanding CSF3R Mutations in Acute Myeloid Leukemia Evolving from Severe Congenital Neutropenia.
Authors: Dong Fan , Qiu Yaling , Friedman Alan D. , Liu Qingquan 
Published: April 2020

Abstract
Severe congenital neutropenia (SCN) is characterized by early onset of bacterial infections and maturation arrest of myeloid cells at early stages of differentiation in the bone marrow. Point mutations in ELA2 encoding neutrophil elastase (NE) have been identified in 60% to 80% of patients with SCN. SCN patients are predisposed to acute myeloid leukemia (AML), which occurs in approximately 15 % of cases. With rare exceptions, leukemic cells from these patients carry mutations in CSF3R encoding the G-CSF receptor, leading to C-terminal truncation of the receptor. Notably, the nonsense mutations in CSF3R are present only in SCN/AML patients, particularly those with ELA2 mutations, but not in other types of neutropenias and de novo AML. The mechanism for the exclusive presence of the nonsense CSF3R mutations in SCN/AML is unknown. In myeloid 32D cells transfected with the wild type (WT) G-CSF receptor (32D/WT), G-CSF treatment induced the expression of NE. However, NE expression was not upregulated by G-CSF in 32D cells expressing the truncated G-CSF receptor d715, derived from an SCN patient. It has been shown that myeloid cells from patients with SCN/AML express both the wild type and the truncated G-CSF receptors. Indeed, the d715 mutant acted in a dominant negative manner to suppress NE upregulation by the WT G-CSF receptor. In luciferase reporter assays, the WT G-CSF receptor, but not the d715 mutant, activated a 1.8-kb fragment of the mouse Ela2 promoter. Significantly, forced expression of an SCN-associated NE mutant G185R caused premature apoptosis of differentiating 32D/WT cells in response to G-CSF with no significant effect on IL-3-stimulated survival. To address whether the d715 mutant may abolish the proapoptotic effect of the G185R mutant via suppressing its expression, we transfected 32D/WT and 32D/d715 cells with an expression construct in which the expression of the G185R mutant was driven by the 1.8-kb fragment of the Ela2 promoter. G-CSF treatment induced the expression of the G185R mutant and subsequent apoptosis in 32D/WT cells. In 32D/d715 cells, however, the expression of the G185R mutant was not induced by G-CSF and accordingly its proapoptotic activity was not evident. We propose that acquisition of the nonsense mutations in CSF3R may represent a mechanism utilized by the myeloid cells harboring the ELA2 mutations to evade the proapoptotic effect of the NE mutants. However, expression of the truncated G-CSF receptors has other biological consequences: they transduce strong proliferative signals but are defective in inducing granulocytic differentiation, which may initiate the leukemogenic process.
Erythropoietin Fusion Proteins Comprised of Identical Head-to-Tail Repeats with Enhanced Biological Activity.
Authors: Jeong Jee-Yeong , Chen Changmin , Davis Kerry L. , Breidbach Andreas , Catlin Don H. , Sytkowski Arthur J. 
Published: April 2020

Abstract
Recombinant human erythropoietin (EPO, epoetin) is used widely for treatment of chronic anemia due to renal failure, cancer, and other causes. However, considerably high and frequent doses of EPO are required to maintain therapeutic effectiveness, since it has a relatively short in vivo half-life. Thus, alternatives with higher efficacy and/or longer half-life are being developed. We have shown previously that EPO-dimers, either produced by chemical cross-linking of monomeric EPO or expressed as a recombinant fusion protein from COS cells, exhibit enhanced biological properties in vitro and in vivo (Sytkowski, et.al. Proc. Natl. Acad. Sci. USA 95, 1184; Sytkowski, et.al. J. Biol. Chem. 274, 24773). We now report increased activities of EPO-dimer fusion protein and EPO-trimer fusion protein comprised of identical head-to-tail repeats and a 15 or 20-amino acid linker (for dimer), or 17-amino acid linkers (for trimer) produced from stably transfected CHO cells. EPO-fusion proteins were expressed under a CMV promoter with a signal peptide present on the first monomer coding sequence. The EPO-dimer fusion protein was connected with either three or four repeats of Gly-Gly-Gly-Gly-Ser as a 15 or 20-amino acid linker sequence, respectively. The expression levels of EPO-dimer fusion protein from cloned CHO cells to supernatant of protein-free medium ranged from 4 to 40 mg/L determined by EPO-ELISA, and from 2.0×105 to 4.5×106 IU/L determined by in vitro bioassay. We selected clones producing EPO-dimer fusion protein with the greatest extent of glycosylation, as indicated by SDS-PAGE and isoelectric focusing. Subcutaneous injection of mice with three doses of EPO-dimer fusion protein resulted in percent increases in mean hematocrit of 32.6% (300 IU/kg) or 18.2% (100 IU/kg), while equivalent unit doses of EPO-monomer increased mean hematocrit by 12.5% (300 IU/kg) or 6.4% (100 IU/kg). Moreover, a single dose of EPO-dimer fusion protein (100 IU/kg) increased their mean hematocrit by 4.3% within 7 days, while an equivalent unit dose of EPO-monomer had no effect. Importantly, three doses of EPO-trimer fusion protein increased their mean hematocrit by 8.83% per IU injected, which was much greater than that observed with EPO-monomer (0.69%) or EPO-dimer fusion protein (1.81%). The results show that EPO-fusion proteins exhibit biological activities superior to those of EPO-monomer, suggesting important therapeutic advantages.
Transgene Insertion into the Proximity of the c-myb Gene Disrupts Erythroid-Megakaryocytic Lineage Bifurcation.
Authors: Mukai Harumi Yamamoto , Motohashi Hozumi , Nagasawa Toshiro , Yamamoto Masayuki 
Published: April 2020

Abstract
The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We established three lines of erythropoietin receptor transgenic mice and found that one of them exhibited anemia, thrombocythemia and splenomegaly. These abnormalities were independent of the function of the transgenic erythropoietin receptor, and were observed exclusively in mice harboring the transgene homozygously, suggesting transgenic disruption of a certain gene. The transgene was inserted 77-kb upstream of the c-myb gene and c-Myb expression was markedly decreased in megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs) of the homozygous mutant mice. In the bone marrows and spleens of the mutant mice, megakaryocytes were increased, but erythroid progenitors were decreased. These abnormalities were reproducible in vitro in a co-culture assay of MEPs with OP9 cells, but eliminated by the retroviral expression of c-Myb in MEPs. The erythroid-megakaryocytic abnormalities were reconstituted in mice in vivo through the transplantation of mutant mouse bone marrow cells. To better understand the transcriptional program that accompanies the decline of c-myb gene expression, we performed DNA microarray analysis of MEPs. We identified 74 genes that are upregulated and 36 genes are downregulated in the homozygous mutant mice. Of these genes, expression levels 10 genes are actually changed in bone marrow cells of the homozygous mutant mice, and harbor c-Myb recognition elements in the upstream region. These results thus demonstrate that the transgene insertion into the c-myb gene far upstream regulatory region affects the gene expression at the stage of MEPs, leading to an imbalance between erythroid and megakaryocytic cells, and suggest that c-Myb is an essential regulator of the erythroid-megakaryocytic lineage bifurcation. Elucidation of transcription network based on the c-myb gene in erythroid and megakaryocytic lineages will take a new turn of lineage specific regulation.
Figure Figure
Gfi1-N382S Mutants from Human Severe Congenital Neutropenia Patients Function through a Transcriptional Dominant Negative Mechanism.
Authors: Zarebski Adrian P. , Baktula Avinash M. , Basu Sudeep , Trent John O. , Grimes H. Leighton 
Published: April 2020

Abstract
The Growth factor independence 1 (Gfi1) zinc finger transcriptional repressor is a T cell leukemia oncoprotein that also plays a crucial role in granulopoiesis in both mice and humans. A single point mutation in the amino terminal SNAG repressor domain (P2A) is able to ablate both Gfi1 transcriptional repression activity and linked oncogenic activity in T lymphoctyes. Mice deleted for Gfi1 are lymphopenic, but also lack mature neutrophils. Gfi1−/− mice display a profound block to myeloid differentiation and abnormal promyelocytes accumulate in the blood. Humans with Severe Congenital Neutropenia (SCN) with heterozygous mutations in Gfi1 have similar abnormal promyelocytes. We introduced the SCN patient Gfi1N382S DNA-binding-deficient mutation into murine Gfi1 and overexpressed it in primary murine bone marrow cells. While expression of the wild type Gfi1 resulted almost exclusively in mature granulocyte differentiation, forced expression of the N382S mutant resulted almost exclusively in monocytic differentiation. Flow cytometric analysis revealed a population of N382S-expressing cells with markers of both monocytes and neutrophils resembling the atypical Gfi1−/− promyelocytes. To determine if mutation of the N382 residue is uniquely able to block Gfi1 function, we constructed a virtual model of Gfi1 zinc fingers 3, 4 and 5 interacting with DNA. The model revealed several possible protein-DNA interactions. In order to validate the model we mutated those residues to alanine and performed EMSA with in vitro transcribed/translated proteins. The same alanine substitution mutants were expressed in primary murine bone marrow and tested for their ability to control myelopoiesis. Lack of DNA binding in EMSA tightly correlated with impaired granulopoesis in our in vitro model, suggesting the necessity of intact DNA binding for proper Gfi1 function. These data suggested that the non-DNA binding mutants were able to inhibit repression by wild type endogenous Gfi1, perhaps through the sequestration of limiting corepressor proteins. The Gfi1P2A mutant is unable to repress transcription. We therefore tested the effect of Gfi1P2A expression on myelopoiesis and found that it blocked granulopoiesis equivalently to Gfi1N382S. To rigorously determine whether the titration of limiting corepressors was the cause of N382S neutropenia, we constructed a compound mutant containing both N382S and P2A and expressed it in primary murine bone marrow cells. Expression of Gfi1P2A-N382S had little effect on myelopoiesis. We conclude that SCN patients with heterozygous Gfi1 mutations have blocked granulopoiesis because the non-DNA binding mutant protein competes with the wild type allele for titratable associated cofactors.
Identification of Oxidative Post-Translational Modifications on Plasma Albumin in Patients with Pulmonary Hypertension of Sickle Cell Anemia.
Authors: Klings Elizabeth S. , Odhiambo Adam , Perlman David , Huang Hua , Costello Catherine , Farber Harrison W. , McComb Mark , Steinberg Martin H. 
Published: April 2020

Abstract
Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in patients with SCA and PH, oxidative post-translational modifications occur on plasma proteins and are important in disease pathogenesis. To examine this hypothesis, we chose to determine if oxidative post-translational modifications occur in a large-abundance protein, albumin, as a reflection of the presence of more widespread protein oxidative damage. Plasma was obtained from subjects with:SCA and PH (n=5);SCA steady-state without PH (n=4);Pulmonary Arterial Hypertension (PAH) (n=4);no evidence of cardiopulmonary disease (n=4).
Platelet-poor plasma was separated into albumin-enriched and albumin-depleted fractions. The albumin-enriched fraction was subjected to proteolytic digestion by trypsin and studied by matrix assisted laser desorption ionization (MALDI) mass spectroscopy (MS) and liquid chromatography (LC)-MS/MS tandem mass spectrometry. Proteomics analyses were performed on all samples and post-translational modifications characterized by MS/MS. Results were confirmed by Western and dot-blot analysis using commercially available antibodies. In the albumin fraction, we identified peaks of differential post-translational modification on albumin peptide 146–159 only in PAH and SCA and PH samples; this PTM was identified to be a malondialdehyde adduct. The presence of a malondialdehyde adduct on albumin was confirmed by LC-MS/MS and Western analysis. We have identified a malondialdehyde adduct in plasma albumin that appears to be a common link between PH related to SCA and PAH. This finding supports the notion that oxidative stress modulates the pathogenesis of PH of SCA and suggests that this and other post-translational modifications may be important biomarkers of disease.
New Approach for Finding Novel Antisickling Agents at the NIH NHLBI Sickle Cell Disease Reference Laboratory.
Authors: Asakura Toshio , Yang Jisheng , Chen Qiukan , Evans Greg , Abdulmalik Osheiza 
Published: April 2020

Abstract
Although numerous antisickling agents that chemically bind with sickle hemoglobin (Hb S) and inhibit sickling of sickle erythrocytes (SS cells) in vitro were reported and some of them were brought to clinical trials, none were satisfactory for clinical use. The major problem with this type of drugs is the difficulty of delivering a sufficient amount of antisickling drug to intracellular Hb S because:chemicals that bind with the side chains of hemoglobin also bind with the side chains of other proteins in the gastrointestinal tract, blood and red blood cells (RBCs) prior to binding with intracellular Hb S; andthe concentration of Hb S in SS cells is very high (5 mM of tetrameric Hb S, 20 mM of Hb subunits, or a total amount of approximately 500 grams of Hb S in an adult patient), and the amount of chemical that reaches SS cells upon oral administration cannot modify a significant portion of Hb S to inhibit sickling of SS cells.
For these reasons, drugs that chemically bind with Hb S including those that form Schiff base adducts with Hb S seem to have no clinical value. We found that certain aromatic aldehydes that bind with Hb S allosterically do not have these problems. Since these compounds combine with Hb S physically like a key-and-lock relationship, a low concentration (such as less than 2 mM) of such compound in the blood can bind with a large portion of intracellular Hb S, shift the oxygen equilibrium curve toward the left and inhibit sickling of SS cells (Brit. J. Hematol.128:552–561, 2005). This type of allosteric binding between aromatic aldehydes and Hb is analogous to the binding between 2,3-diphosphoglycerate and Hb. Quite interestingly, our investigations indicate that NIPRISAN (NICOSAN), an herbal medicine that shows a strong antisickling effect both in vitro and in vivo (in transgenic sickle mice) (Brit. J. Haematol.118:337–343, 2002; Brit. J. Haematol.122:1001–1008 2003), contains various aromatic aldehydes (unpublished data). Allosteric drugs designed to interact with specific proteins in the body may be useful as a new type of therapeutic modality for the treatment of sickle cell disease and other diseases. [Researchers with candidate drugs that may have beneficial effects for SCD are welcome to contact the SCD Reference Laboratory for free in vitro evaluation of their agents (for more information, please see our website at www.tatcom.com/sickle-cell).
Placental Growth Factor Prevents Erythroid Suppression by Inflammatory Mediators.
Authors: Means Robert T. , Dallalio Gail , Law Erin R. 
Published: April 2020

Abstract
Although sickle cell anemia is the result of an abnormality in the beta globin chain of hemoglobin, its clinical manifestations cannot be explained solely on that basis. Inflammatory mechanisms contribute to the clinical syndromes associated with sickle cell disease. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family and is associated with inflammation and with pathologic angiogenesis. Perelman and colleagues have reported that PlGF is released from marrow erythroid cells (Blood2003;102:1506–13). These and other investigators have reported that serum PlGF concentration distinguishes patients with sickle cell disease from healthy controls, although results vary depending upon disease activity at the time of study (Blood2003;1514–24 and Brit J Haematol2006;134:116–9). In studies we reported previously, we measured PlGF and other cytokines in the marrow of stable homozygous sickle cell (SS) patients and controls. Plasma PlGF did not differ between homozygous sickle cell SS patients and controls, but bone marrow PlGF concentrations were significantly higher in SS patients. This was not a phenomenon general to cytokines, and did not reflect increased erythroid activity but rather represented an increase per erythropoietic unit (J Invest Med2005;53:S305–6). In this report, we address the consequences of exposure to PlGF for erythropoiesis in the context of inflammation. We observed that CFU–E from SS patients were less sensitive to inhibition by recombinant human gamma interferon (rhgIFN) than those from healthy controls, with this effect being most pronounced at lower gIFN concentrations. The potential contribution of PlGF to this process was evaluated. At PlGF concentrations 10 – 1000 pg/mL, no inhibition or enhancement of CFU-E colony formation was observed, and there were no differences between the responses of progenitors from SS patients or from normal volunteers to PlGF. However, PlGF 100 pg/mL reversed the inhibitory effects of rhgIFN on CFU-E colony formation (p = 0.04). This effect was most apparent at lower rhgIFN concentrations. Using the HCD57 erythroleukemia cell line as a model, it was found that PlGF produced significant attenuation of Fas ligand induction by rhgIFN. Both HCD57 and CD36+ human marrow mononuclear cells were shown to express Flt-1, a receptor for PlGF. Neutralizing antibody against Flt-1 attenuated the gIFN-protective effect of rhPlGF on CFU-E colony formation. In conclusion, increased PlGF concentrations in the marrow of stable SS patients may contribute to protecting erythroid progenitors from cytokine-induced apoptosis, and may be a mechanism by which erythropoiesis in sickle cell disease is preserved despite concurrent inflammation.
Platelets from Patients with Sickle Cell Disease and Pulmonary Arterial Hypertension Are Hypersensitive to Agonist-Induced Activation.
Authors: Villagra Jose D. , Nichols James T. , Gladwin Mark T. , Kato Gregory J. 
Published: April 2020

Abstract
Pulmonary arterial hypertension (PAH) in sickle cell disease (SCD) is associated with chronic hemolysis and nitric oxide (NO) consumption and characterized by abnormal vascular tone, vascular proliferation and thrombosis. In previous studies, we demonstrated that increased platelet activation is linked to the severity of PAH in SCD. In addition, we reported that patients with SCD and secondary PAH have decreased platelet activation when given sildenafil, a finding that highlights the role of nitric oxide inhibition by hemolysis in this pathological mechanism. Since the vascular homeostasis is altered by the release of hemoglobin in plasma, we hypothesized that platelets of patients with PAH may show an enhanced degree of reactivity to platelet agonists released during hemolysis.
Whole-blood samples were used to determine the in vivo baseline and stimulated platelet activation from SCD patients with PAH, without PAH during steady state (not in pain crisis) and healthy controls. PAH was defined by a tricuspid regurgitant jet velocity ≥ 2.5 m/sec by Doppler echocardiography. Upon withdrawal, citrated venous whole blood was mixed gently with either phosphate-buffered saline (PBS) or escalating doses (1 – 10 μM final concentration) of adenosine diphosphate (ADP), a platelet agonist present in red blood cells. Platelet activation was measured by the flow cytometry detection of activated fibrinogen receptors (activated GPIIb/IIIa) and surface expression of P-selectin. Patients with SCD and PAH reached high degrees of activation with significantly lower concentrations of ADP (EC50 0.88 μM, 95% confidence interval 0.64 to 1.23, n=5) compared to controls (1.3 μM, 1.00 to 1.64, p &lt; 0.01, n=8), and particularly to patients without PAH (1.7μM, 1.31 to 2.27, n=5)(Figure below, left panel). Similar results were observed in parallel experiments with thrombin-receptor activating peptide, another strong platelet agonist.
In separate experiments on platelets from healthy subjects, the addition of cell-free plasma hemoglobin induced platelet activation in dose-dependent fashion (p&lt;0.05), supporting intravascular hemolysis as a mechanism of platelet activation in SCD (Figure below, right panel). Furthermore, cell-free hemoglobin blocked the ex vivo platelet inhibitory effects of an exogenous NO donor, MAHMANONOate (p&lt;0.05), consistent with NO scavenging by cell-free hemoglobin.
These findings suggest that platelets from patients with SCD and PAH are unusually sensitive to activation. Our data also suggests that most of the variability in platelet hyperreactivity observed by other groups in SCD is primarily associated with PAH. Our data support a role for intravascular hemolysis and NO scavenging by cell-free hemoglobin in platelet hyperreactivity. Additional research is needed to define this mechanistic pathway in more detail.
Figure Figure
CCR5 and RANTES/CCL5 Gene Polymorphisms Affect the Risk of EBV Reactivations after Allogeneic HSCT.
Authors: Bogunia-Kubik Katarzyna , Kuzyk Anna , Jaskula Emilia , Lange Andrzej 
Published: April 2020

Abstract
Epstein-Barr virus (EBV) reactivation is a serious complication affecting the recipients of allogeneic hematopoietic stem cell transplants (HSCT). Chemokines and their receptors play a major role in the inflammatory and immune responses that mediate allograft outcome. CC-chemokine receptor-5 (CCR5) is known to be involved in perpetuation of HIV infection (as a co-receptor for HIV entry) and the clinical course of this infection is favored by the presence of the 32-nucleotide deletion within the CCR5 gene (CCR5Δ32 polymorphism). The CCR5 deletion mutation (CCR5Δ32) results in a non-functional chemokine receptor. In the present study the CCR5Δ32 polymorphism and two single nucleotide substitutions (−28 C/G; −403 G/A) within, one of the CCR5 ligands, the CCL5/RANTES gene were analyzed in 75 HSCT recipients, 75 donors and related with EBV load. The control group constituted 99 healthy individuals. DNA was extracted from peripheral blood taken into EDTA using silica membranes. Viral load were assessed 2-3 moths after transplantation in blood cells employing a real-time PCR technique. The detection threshold for viral reactivation equaled 10 EBV-DNA copies/105 peripheral blood cells. EBV reactivation was detected in 26 patients. The CCR5Δ32 and RANTES (−28 C/G; −403 G/A) polymorphisms were analyzed by PCR and PCR-RFLP technique, respectively. Distributions of CCR5 and RANTES −28 genotypes were similar in patients, donors and healthy individuals. RANTES −403 AA homozygosity was more frequent in donors than controls (0.61 vs 0.47, p=0.036). The higher number of EBV copies was detected in patients lacking CCR5Δ32 deletion (0.92 vs 0.71, p=0.031). No significant correlation between EBV reactivation and RANTES −28 C/G polymorphism. However, patients transplanted with donors homozygous for RANTES −403 AA (genotype associated with an increased expression of the RANTES gene) more frequently presented with EBV reactivation (0.85 vs 0.49, p=0.002). In conclusion, the presence of the CCR5 deletion-mutation in the recipient and RANTES-403 G in the donor of HSC appeared to lower the susceptibility for EBV reactivation.