bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transgenic Expression of Bach1 Transcription Factor Results in Down-Regulation of the p45 Target Genes in Megakaryocytic Lineage Cells.
Authors: Toki Tsutomu , Katsuoka Fumiki , Kanezaki Rika , Watanabe Seiji , Kamio Takuya , Tandai Satoru , Kurotaki Hidekachi , Yagihashi Soroku , Sun Jiying , Komatsu Norio , Igarashi Kazuhiko , Yamamoto Masayuki , Ito Etsuro 
Published: October 2019

Abstract
Both p45 and BACH transcription factors can form dimers with one of the small Maf proteins, and these heterodimers bind to Maf recognition elements (MARE). MARE is known to act as a critical cis-regulatory element of erythroid and megakaryocytic genes. While detailed analyses of p45 -null mutant mice and small maf compound mutant mice revealed that these factors are both critical for platelet production, the functional contributions of BACH1 and the relationship or redundancy between BACH1 and p45 in megakaryocytes remain to be clarified. To address these issues, we generated transgenic lines of mice bearing human BACH1 cDNA under the control of the GATA-1 locus hematopoietic regulatory domain. The transgenic mouse lines showed significant thrombocytopenia associated with impaired maturation of the megakaryocytes, and they developed myelofibrosis. The megakaryocytes overexpressing the BACH1 transgene exhibited reduced proplatelet formation. Since the phenotype of the BACH1 transgenic mice resembled that of the p45 -deficient mice, we examined the expression of the p45 NF-E2 target genes in the primary megakaryocytes from fetal liver cells of the BACH1 transgenic mice. RT-PCR analyses showed that expression of the hematopoietic-specific ß1-tubulin, thromboxane synthase ( TXAS), and of the 3ß-hydroxy-steroid dehydrogenase genes was significantly downregulated in the megakaryocytes from BACH1 transgenic mice. The TXAS gene is a well-known MARE-dependent gene containing functional MAREs in its promoter and in the second intron. To ask whether BACH1 actually binds to MARE in the megakaryocytic genes, we then performed chromatin immunoprecipitation (ChIP) analysis with a BACH1-specific antibody. A ChIP assay with a human megakaryocytic cell line, UT-7/TPO, demonstrated that BACH1 bound to the promoter and enhancers region in vivo. As expected, co-transfection with BACH1 or Bach1-MafK fusion protein (B1K) expression plasmids repressed the reporter gene activity driven by the TXAS promoter. These findings thus provide evidence that BACH1 acts as a transcriptional repressor in the regulation of MARE-dependent genes in megakaryocytes.
Inflammatory Disease and Abortive Platelet Shedding Caused by a Mutation in a Pivotal Regulator of Actin Dynamics in the redears Mouse.
Authors: Justice Monica J. , Kile Ben T. , Woodward Lanette S. 
Published: October 2019

Abstract
Mouse mutagenesis using forward genetics is valuable as a gene function discovery tool. We are looking for blood defects in a large ENU mutagenesis screen, and have isolated many new mouse mutants that reveal new mechanisms in hematopoiesis. One mutant mouse strain, called redears, is an intriguing model of inflammatory disease and thrombocytopenia. Animals homozygous for the redears (rd) mutation develop spontaneous inflammatory lesions of the ears and tail characterized by neutrophil infiltration and peripheral neutrophila. Unexpectedly, blood platelet numbers are dramatically reduced in rd/rd animals. A thorough analysis of platelet biogenesis shows that the platelet precursor cell, the megakaryocyte, undergoes abnormal maturation, which results in gross morphological abnormalities, increased ploidy and abortive platelet shedding. Here we report a mutation in a novel gene related to the yeast actin-interacting protein Aip1 in rd/rd mice. In yeast, Aip1 interacts with, and increases the activity of cofilin, a key regulator of actin depolymerization. Our data confirm that actin dynamics are dysregulated in rd/rd megakaryocytes and neutrophils. The massive cytoplasmic reorganization that is required for megakaryocyte maturation and platelet shedding has long been assumed to depend on the actin cytoskeleton. Intriguingly, recent studies suggest the process is caspase-dependent, and represents a form of ‘para-apoptosis’. With this in mind, we found that chemotaxis and apoptosis are perturbed in rd/rd neutrophils, suggesting that neutrophils are playing a key role in driving the inflammation. Disrupted actin depolymerization would provide an explanation for chemotactic deficiencies. Further, recent evidence implicating cofilin and other actin regulators in the initiation of apoptosis would suggest that this novel protein may play an essential role in neutrophil cell death. Thus, the redears mouse not only provides the first in vivo demonstration of the critical role of the actin cytoskeleton in megakaryocyte development and platelet production, but also represents a unique reagent to examine the relationship between actin dynamics, cellular maturation, inflammation and apoptosis.
Our ongoing mutagenesis efforts continue to reveal new developmental mechanisms. New mutants, genetic tools, and resources can be found at www.mouse-genome.bcm.tmc.edu
Persistence of GATA1+ Erythroid Progenitors In Vivo in Absence of Zebrafish SCL.
Authors: Juarez Marianne A. , Su Fengyun , Kiel Mark J. , Lyons Susan E. 
Published: October 2019

Abstract
The stem cell leukemia (SCL) gene encodes a bHLH transcription factor essential to the primitive and definitive hematopoietic systems in vertebrates. In the zebrafish, studies of hematopoiesis have shown significant functional conservation with mammals. In order to assess the functions of scl in zebrafish primitive hematopoiesis, zebrafish embryos were analyzed after ablation of Scl protein by morpholino-modified antisense RNA injection. Embryos injected with morpholinos targeting the scl translation initiation site or two independent splice donor sites lacked circulating blood and exhibited cardiac edema by 28 hours post fertilization. RNA in situ hybridization analysis with a panel of hematopoietic-specific genes showed that myeloid lineage development in the anterior lateral mesoderm was absent upstream of draculin and pu.1 expression. In contrast, erythroid development in the posterior lateral mesoderm occurred with little or no functional Scl, as shown by expression of draculin, pu.1, and gata1 in this PLM region. Primitive erythroid cells failed to mature, as evidenced by the absence of circulating cells after 24 hpf. Only upon titration of scl morpholinos to near toxic levels was gata1 reduced or absent in embryos at 21 somites. These findings show that scl plays a vital role in development of all hematopoietic lineages in zebrafish. However, these data reveal differential effects of reduction or loss of Scl on hematopoietic development in the anterior lateral mesoderm versus the posterior lateral mesoderm. The results reveal that reduction or loss of Scl affects erythroid development in the posterior lateral mesoderm or ICM at a progenitor stage of erythroid development, after initiation of gata1 and embryonic globin expression. These data suggest that early erythroid development in the zebrafish requires little or no Scl, while maturation of erythroid progenitors cannot occur in the absence of Scl. Myeloid development in the anterior lateral mesoderm is dependent upon Scl. This embryonic analysis in the zebrafish is the first to demonstrate a transient, in vivo population of erythroid precursors in embryos with severely reduced or absent expression of Scl.
Core Epo Receptor Signaling Circuits in Adult Bone Marrow-Derived Primary Pro-Erythroblasts.
Authors: Wojchowski Don M. , Menon Madhu , Karur Vinit , Vincent Bethany 
Published: October 2019

Abstract
Knock-in experiments have revealed that Epo receptor-dependent steady-state erythropoiesis is supported by a Jak2-coupled cytoplasmic Epo receptor (EpoR) box-1 domain. However, signals that are transduced by this cytoplasmic phosphotyrosine-null EpoR-HM allele (and by a related EpoR-H allele with a restored PY343-STAT5 binding site) are not well understood. To address this basic question, conditions have been optimized for the serum-free expansion of adult bone marrow- derived primary erythroid progenitor cells, and core signaling circuits for EpoR-HM, EpoR-H and wt-EpoR receptor forms have been defined. As an important baseline consideration, each receptor form proved to activate Jak2 at an essentially equivalent time-course and intensity (but EpoR-HM did not detectably activate Stat5, or appreciably stimulate CIS transcription). In addition, EpoR-HM failed to stimulate AKT, and this outcome discounts possible EpoR-HM plus Jak2 coupling to P13K (eg. via IRS-1/2 and/or GAB-1/2). AKT was stimulated at moderate, yet sustained levels via EpoR-H (and a PY343/Stat5 axis). EpoR-HM, however, did couple to p60Src and ERK1, 2. While p60-Src stimulation was somewhat diminished, ERK1/ 2 activation via EpoR-HM was sharp and sustained (as compared directly to EpoR-H and the wt-EpoR). EpoR coupling to apoptotic factors also was assessed. In EpoR-HM erythroblasts, Bcl-xl expression was clearly (and reproducibly) deficient, but occurred at wild-type levels in Stat5-coupled EpoR-H erythroblasts. Bax expression was less affected, but was reproducibly increased 1.5 to 2-fold in both EpoR-HM and EpoR-H erythroblasts. Finally, expression of an under-studied pro-apoptotic DAPK-1 orthologue, DAPK2 (death associated protein kinase-2) was discovered to be markedly elevated in EpoR-HM erythroblasts, but to be repressed to normal levels in EpoR-H cells. With regards to bioactivity, EpoR-HM pro-erythroblasts faltered in their ex vivo development specifically at a transition from a Ter119negCD71low to Ter119posCD71high stage, and were visibly deficient in their levels of hemoglobinization. Overall, these first-time detailed studies of Epo signaling in primary adult pro-erythroblasts from minimal EpoR allele knock-in mice reveal: 1/ that EpoR plus Jak2 dependent erythropoiesis may depend primarily upon enhanced ERK1/2 stimulation (and not upon collateral Stat5 or AKT circuits); 2/ that EpoR-HM retains coupling to p60-Src; 3/ that an EpoR-H/PY343/Stat5 axis (but not an EpoR/Jak2 only axis) can link to AKT; 4/ that EpoR/PY343/Stat5 signals regulate not only Bcl-xl, but also pro-apoptotic DAPK2 expression at a key stage of Epo-dependent pro-erythroblast development; and 5) that EpoR-PY coupled signals contribute in several important ways to red cell development.
Elevated Estimated Pulmonary Arterial Pressures in Patients with Chuvash Polycythemia.
Authors: Gordeuk Victor R. , Sergueeva Adelina I. , Miasnikova Galina Y. , Polyakova Lydia A. , Okhotin Daniel J. , Castro Oswaldo L. , Prchal Josef T. 
Published: October 2019

Abstract
Chuvash polycythemia is characterized by a homozygous 598C>T mutation in VHL and up regulation of HIF-1α during normoxia. Disorders of chronic hypoxia may be complicated by the development of pulmonary hypertension. Because of the up regulation of the hypoxic response in Chuvash polycythemia, we postulated that there may be a tendency to increased pulmonary artery pressures in this condition as well. To test this hypothesis, we analyzed results for Doppler echocardiography in 15 patients with Chuvash polycythemia and 15 Chuvash individuals without polycythemia. The tricuspid regurgitation velocity (TRV) allows estimation of pulmonary artery systolic pressure. A TRV of 2.5 m/sec or higher corresponds to a pulmonary artery systolic pressure of at least 35 mm Hg (normal up to 32 mm Hg), while a TRV of 3.0 m/sec or higher to a pressure of at least 46 mm Hg. The results are summarized in the Table.
Pulmonary artery pressures as estimated by tricuspid regurgitation velocity (TRV) in Chuvash subjects with and without polycythemia
Chuvash polycythemia (n = 15) Controls (n = 15) P Age in years; mean (SD) 35 (17) 35 (17) 1.0 Female sex in no. (%) 8 (53%) 8 (53%) 1.0 Hemoglobin in g/dL; mean (SD) 16.7 (2.3) 13.3 (1.2) <0.001 TRV in m/sec; mean (SD) 2.2 (0.6) 1.4 (0.6) 0.001 TRV > 2.4 m/sec in no. (%) 4 (27%) 0 (0%) 0.1
Most of the patients with Chuvash polycythemia were receiving phlebotomy therapy and therefore many had hemoglobin concentrations in the upper normal range. Four of the patients with Chuvash polycythemia and none of the others had TRV ≥ 2.5 m/sec (range of 2.5 to 3.0), and mean TRVs were significantly higher in the patients with Chuvash polycythemia. Interestingly, the mean ± SD TRV in these 15 patients with Chuvash polycythemia was identical to the mean ± SD TRV that was recently reported in 195 American patients with sickle cell disease (Gladwin et al, NEJM2004;350:886), another hematological condition with a tendency to pulmonary hypertension. While the pulmonary arterial pressures detected so far in Chuvash polycythemia patients are lower than those in patients with primary pulmonary hypertension, our results suggest that pulmonary hypertension may be an unrecognized complication of Chuvash polycythemia.
Serum Level of Interleukin-10 Correlates Well with the Activity of Chronic Graft-Versus-Host Disease and the Responsiveness to Corticosteroid Treatment.
Authors: Yeh Su-Peng , Chiu Chang-Fang , Liao Yu-Min , Lo Wen-Jyi , Liaw Yu-Ching , Lin Chiao-Lin , Chang Jan-Gowth , Hsueh Chung-Tsen 
Published: October 2019

Abstract
Current evidence suggest the interaction between subset of T cells, the T help1 (Th1) and T help2 (Th2), plays an important role in the pathogenesis of GVHD. Cytokines, either produced by Th1/Th2 or other cells, are important regulators in the whole process and will define the polarization of Th1 or Th2 from naïve T cell. In this study, we used ELISA (R&D, Minneapolis, MN, US) to monitor the serum level of Interleukin-4 (IL-4), IL-10, IL-12, and interferon-γ (INF-γ) in patients receiving allogeneic transplant weekly from day 0 to at least day 200. From Jan. 2003, consecutive 20 patients were enrolled. The GVHD prophylaxis consisted of Cyclosporine-A and short course Methotrexate. Of the 20 patients, 2 had grade 3/4 acute GVHD (aGVHD) and 7 had extensive chronic GVHD (cGVHD). The serum levels of IL-4 and IL-12 were below the detectable level (0.13pg/ml and 0.5pg/ml respectively) in most occasions, even during the period of GVHD. Nevertheless, the serum level of IL-10 correlated well with the activity of cGVHD and we used data gathered from 5 patients to demonstrate this good correlation in figure 1 and 2. Patient 1 (open square, figure 1) had no GVHD and the IL-10 levels were below 5pg/ml during the whole period of follow-up. Patient 2 (solid triangle, figure 1) and 3 (open triangle, figure 2) had de novo cGVHD. The IL-10 level increased gradually when cGVHD developed, however, both clinical manifestations of cGVHD and serum IL-10 level decreased rapidly after the administration of Prednisolone (black bar on figure 1 and 2, with the thickness indicating the relative dosage of Prednisolone). Patient 4 (open circle, figure 2) had grade 2 aGVHD that resolved quickly after Prednisolone treatment. However, IL-10 level increased gradually after discontinuing Prednisolone and cGVHD developed subsequently. After adding Prednisolone again, cGVHD improved and IL-10 decreased to undetectable level rapidly. Patient 5 (solid square, figure 2) had steroid-refractory cGVHD. The IL-10 level kept above 5pg/ml for more than 1 month despite Prednisolone 1mg/kg/day giving at the same time and the patient eventually needed further salvage treatment (thalidomide). The serum INF-γ were undetectable in most occasions. It became detectable during period of cGVHD in patients 3, 4 and 5 but not patient 2. In conclusion, post-transplant serum IL-10 level correlates well with the clinical activity of cGVHD as well as the responsiveness to corticosteroid treatment. This novel finding will allow the functional evaluation of individuals’ immune system after allogeneic transplant and should give insight for the adjustment of immunosuppression necessary for cGVHD control.
Figure Figure Figure Figure
Treatment with Hydroxyurea in Sickle Cell/ ß Thalassemia: A Long-Term Experience.
Authors: Maggio Aurelio , Rigano Paolo , Renda Disma 
Published: October 2019

Abstract
Sickle cell/ß-Thalassemia is a common disease in areas where ß-Thal and ßS genes are endemic, like in Sicily. In the current study we evaluated clinical and hematological data of Sicilian patients with Sickle cell/ß-Thalassemia treated with Hydroxyurea (HU). The endpoint of the study was to evaluate the efficacy of HU in terms of reduction of sickle cell crises after 2 years of treatment in comparison with the 2 years before. Moreover, we evaluated the outcome after long-term treatment. Fortytwo patients (18 males, mean age 36, range 18–53) were treated with HU (mean dosage 15 mg/kg, range 10–30) for an average 6.6 years follow-up (range 3–9 years). Twentytwo were ß0/ßS and 20 ß+/ßS genotype. All had 3 or more crises in the year before starting HU. We observed a significant reduction in sickle cell crises (7.8 ± 6.9 crises per year versus 0.9 ± 1.8 per year, P < 0.0001), hospitalizations (2.5 ± 2.9 per year versus 0.3 ± 1.5, P < 0.0001), and days in hospital (22.4 ± 21.9 per year versus 1.2 ± 2.3, P < 0.0001). Altogether, there was a 86% reduction in vasoocclusive events in comparison with the 2 years before (P < 0.001). Moreover, there was a significant increase of MCV (71.4 versus 97.5fl, P < 0.0001), HbF (7.5 versus 25.2 %, P < 0.0001), and decrease of WBC (11.4 versus 9.2 109/L P < 0.01) and reticulocytes (14.1 versus 10.2%, P< 0.01). Finally, redution of hyperdense cells and increase of erithropoyetin were seen. After a mean follow-up of 6.6 years, 39 patients are alive. Three died (2 end stage HCV related liver disesases, 1 bleeding after ERCP). Nine of the 40 alive patients developed complications: 1 acute chest syndrome, 2 strokes, 2 myocardial infarctions, 4 bone necrosis. Brain MRI of 15 patients after and during a mean of 6.9 years of HU treatment showed 2 new onset strokes, 1 of which in a patient with a previous stroke. Moreover, 4 patients developed new onset asymptomatic ischemic brain lesions. In every case there had been a significant reduction of sickle cell crises. There were two cases of cancer, occurring in two patients who were brother and sister: lung cancer in the former, a proeviously heavy smoker, breast cancer in the latter. No further serious adverse events were seen. Five patients with iron overload were treated with Deferiprone: no drug interaction with HU was noticed. Our study confirms that HU is effective in reducing clinical relevant crises of patients with Sickle cell/ß-Thalassemia. However, our preliminary data suggest that chronic organ damages are not prevented by HU. Safety has to be assessed by more prolonged studies.
Upregulation of Endogenous Erythropoietin (EPO) in Healthy Subjects by Inhibition of Hypoxia Inducible Factor (HIF) Prolyl Hydroxylase.
Authors: Urquilla P. , Fong A. , Oksanen S. , Leigh S. , Turtle E. , Flippin L. , Brenner M. , Muthukrishnan E. , Fourney P. , Lin A. , Yeowell D. , Molineaux C. 
Published: October 2019

Abstract
High-altitude hypoxia stimulates erythropoiesis in anemic hemodialysis patients. Similarly, intermittent exposure to hypobaric hypoxia elevates EPO and stimulates erythropoiesis in normal subjects. HIF is a transcription factor that mediates the body’s response to hypoxia. The stability and activity of HIF are regulated by HIF Prolyl Hydroxylase (HIF-PH). Inhibition of HIF-PH results in the accumulation of HIF leading to the upregulation of erythropoietic genes. A FibroGen HIF-PH inhibitor, FG-2216, is an orally active small molecule with appropriate pharmacokinetic and pharmacodynamic activity for testing in humans. In animal studies, inhibitors of HIF-PH induce erythropoietic changes qualitatively similar to the effects of intermittent exposure to high altitude. Phase 1 studies were initiated in healthy male subjects to determine the safety, tolerability, pharmacokinetics, and biologic activity of FG-2216 dosed orally (0.3 to 20 mg/kg). Serum levels of FG-2216 increased in a dose-dependent fashion; its elimination was characterized by a half-life ~ of 14 hr. Dose-dependent elevation in serum EPO levels was observed with a minimum effective dose of 6 mg/kg. FG-2216 was subsequently administered using a schedule of 2 or 3 times a week for three weeks at doses of 10 and 20 mg/kg or placebo. Increased EPO was observed after each dose of FG-2216. There was no decrement in EPO response to subsequent doses, indicating a resetting of the EPO response during the dosing interval. An increase in reticulocytes and soluble transferrin receptor was accompanied by a modest increase in hematocrit and hemoglobin above normal values at baseline. A total of 54 subjects received FG-2216, which was well tolerated. There were no serious adverse events or dose-limiting toxicities. Adverse events possibly related to FG-2216 were mild and subsided with continued administration. The data provide first proof of concept for upregulation of endogenous EPO and erythropoietic responses in humans by a specific HIF-PH inhibitor.
Early In Vivo Depletion of Suicide Gene-Modified T-Lymphocytes Transplanted in Conjunction with CD34+ Enriched Blood Stem Cells May Be Associated with an Increased Risk of Secondary Graft Failure.
Authors: Fehse Boris , Ayuk Francis A. , Kröger Nicolaus , Fang Lubin , Kühlcke Klaus , Heinzelmann Marion , Zabelina Tatjana , Fauser Axel A. , Zander Axel R. 
Published: October 2019

Abstract
Introduction of a “suicide gene” into donor T-lymphocytes is a promising strategy to prevent severe graft-versus-host disease (GvHD) in the setting of allogeneic blood stem cell transplantation (B-SCT). We initiated a clinical phase I/II-study combining CD34-enriched peripheral B-SCT and Herpes simplex virus thymidine kinase- (HSV-tk) transduced donor T-cell infusions at days 0 and (optional) 60. Before our study was put on hold (after the leukemia cases in the French X-SCID trial) three patients were treated within the protocol each receiving >4x106/kg highly enriched CD34+ and appr. 5x106/kg suicide gene-modified (SGM) donor T-cells. All patients showed fast donor cell engraftment. Patient 2 (without signs of GvHD) received two SGM T-cell infusions and is well >2.5 years thereafter. Patient 1, showed an early (days 11–13) strong increase of SGM cell-counts peaking at 7.2x107/l, but did not develop GvHD. Unexpectedly, his blood was completely cleared from SGM T-lymphocytes a few days later. This patient received a second dose of SGM T-cells (day 65), which vanished within 2 days strongly indicative of their immune rejection. Indeed, MLR data confirmed anti-SGM reactivity of this patient’s PBL possibly related to an HSV reactivation during transplantation. Patient 3 showed a similar early sharp rise in SGM T-cells (days 14–17), in his case associated with acute skin GvHD grade II. Treatment with ganciclovir led to complete resolution of GvHD and rapid disappearance of SGM donor T-cells. Importantly, both patients 1 and 3 developed secondary graft failure at days 156 and 119. In summary, we confirmed previous reports that the use of SGM T-lymphocytes may allow control of acute GVHD. At the same time, we made the worrying observation that early total in vivo depletion of SGM donor T-cells may be associated with an increased risk of transplant rejection. This suggests that minimum numbers of donor CD3+ cells are required post-transplant not only to facilitate engraftment, but also to prevent late rejection.