bluuebunny/crossref_metadata_2025_split huggingface dataset:
CBFA2/RUNX1 Regulates Human Platelet 12-Lipoxygenase: Studies in Runx1 Haplodeficiency.
Authors: Kaur Gurpreet , Jalagadugula Gauthami , Rao A. Koneti 
Published: April 2020

Abstract
Core binding factor 2 (CBFA2), also known as AML1 and RUNX1, is a transcription factor that regulates the expression of genes involved in hematopoiesis, through highly conserved DNA binding region, called RUNT homology domain (RHD). We have previously reported a patient with a mutation (haplodeficiency) in the conserved region of RUNX1/CBFA2 associated with mild thrombocytopenia and impaired platelet function. Expression profiling of patient platelets revealed ∼5 fold decreased mRNA expression of 12-lipoxygenase (12-LO, gene ALOX12) (Sun L. et al. J Thromb Haemost, 5:146–154, 2006). 12-LO catalyzes 12-hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid (AA) upon platelet activation. We have performed studies to determine whether ALOX-12 is regulated by CBFA2. We studied 12-HETE production in patient platelets and the regulation of ALOX-12 by CBFA2 in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA) to induce megakaryocytic (MK) transformation. 12-HETE production was decreased in patient platelets in response to 10 U/ml of thrombin (19.5 ng/10 8 platelets, normal subjects: range 29–306, n=9) and 100 μM of arachidonic acid (5.4, normal subjects: 67– 442, n=10). Three CBFA2 consensus binding sites were identified (−1498/−1493, −708/−70, −526/−521 from ATG) by computer analysis within 2 kb of ALOX-12 5′ upstream region. The binding site at −1489 is a 13nt palindromic sequence with two CBFA2 motifs. The other two CBFA2 binding sites at −708 and −526 bp overlap AP2 binding sites. Luciferase reporter studies in HEL cells using a construct carrying ∼ 1600 bp of 5′ upstream region indicate a greater than 10 fold increase in activity in PMA treated HEL cells relative to untreated cells. Truncation at − 873 bp in PMA-treated HEL cells resulted in a ∼10 fold decrease in activity with only minimal decreases with truncations at −705 or −438 to delete the other CBFA2 binding sites. Mutation of each of the putative CBFA2 binding sites individually resulted in 5–10 fold decrease in activity. Gel shift studies using a 30-mer probe (−1507/−1478) and PMA-treated HEL extracts revealed specific protein binding that was eliminated by CBFA2 antibody and by mutating CBFA2 site from TGGGGT to TGCATT. Specific protein binding was observed with probes containing putative CBFA2 sites at −708 and −526, but it was not altered by the antibody. Chromatin immunoprecipitation (ChIP) analysis using HEL cells demonstrated (PCR amplification) in vivo binding of CBFA2 to ALOX-12 promoter in the region −1507/−1478 but not the other two sites.
Conclusions: CBFA2 haplodeficiency is associated with decreased platelet 12-HETE production and 12-LO activity. ALOX-12 is regulated by CBFA2 in megakaryocytes/platelets. These findings are important because of the role of 12-lipoxygenase in platelet arachidonate metabolism and function.
Prospective Assessment of Pulmonary Hypertension in Children with Chuvash Polycythemia.
Authors: Dham Niti , Sable Craig , Okhotin Daniel , Sergueeva Adelina , Miasnikova Galina , Bushuev Vladimir , Gordeuk Victor R. 
Published: April 2020

Abstract
Background: Chuvash Polycythemia (CP) is an autosomal recessive disorder that is endemic to the Chuvash region of Russia; however, it does occur worldwide. It is characterized by a mutation (598C&gt;T) of the von Hippel Lindau gene, a negative regulator of hypoxia-sensing, and increased levels of hypoxia inducible factor-alpha during normoxia. CP is manifested by vertebral hemangiomas, varicose veins, hypotension, elevated concentrations of products of hypoxia-induced genes (serum vascular endothelial growth factor and endothelin-1), and premature mortality related to cerebral vascular events and peripheral thrombosis. Echocardiography (echo) studies in adults with CP suggest an increased prevalence of pulmonary hypertension (PH), but these findings have not been validated in children with CP.
Methods: This work is supported by an NIH grant evaluating the prevalence, outcomes, pathophysiology, vascular response, and gene polymorphisms of PH in sickle cell disease and CP. We traveled to Cheboksary, Russia to prospectively enroll children with CP and age and gender matched controls to undergo echo for assessment of left ventricular (LV) systolic and diastolic function, LV size and mass, estimated right ventricular (RV) systolic pressure by tricuspid regurgitation velocity (TRV), and the RV to systemic systolic blood pressure (SBP) ratio. Data was compared between CP and control children.
Results: 24 CP and 11 control patients were enrolled in the study. 20 CP and 9 control patients had measurable TRV. 4 of 20 CP and none of the control patients had TRV &gt; 2.5 m/sec (max TRV 2.89 m/sec). There was a trend towards higher TRV and estimated RV pressure in CP patients. Systolic function, LV size, and mass were the same in both groups. Diastolic function was normal in both groups but significantly better in CP patients. SBP was the same in both groups. Systemic mean and diastolic BP, adjusted for age, was significantly lower in CP children.
Conclusions: Preliminary prospective data show a trend towards higher echo derived estimates of RV pressure in children with CP that can not be explained by hypertension or diastolic dysfunction. Rather, congenital upregulation of the hypoxic response may be responsible. Continued patient recruitment and correlation of TRV with other variables will allow for a better understanding of PH in children with CP.
ECHO PARAMETERS CP Controls p TRV (m/sec) 2.24 ± 0.32 2.08 ± 0.14 0.08 RV systolic pressure (mm Hg) 25.4 ± 5.7 22.4 ± 2.4 0.06 RV/systemic pressure ratio 0.26 ± 0.06 0.24 ± 0.03 0.17 LV diastolic dimension z score 0.11 ± 0.91 −0.13 ± 0.79 0.42 LV Mass Index: g/m^2.7 25.8 ± 8.2 24.7 ± 6.5 0.68 Ejection fraction (%) 64.9 ± 3.7 64.7 ± 3.2 0.91 Mitral E/Tissue Doppler E 5.35 ± 0.81 6.48 ± 1.31 0.02
Correction of Anemia with Hematide™, a Synthetic Peptide-Based Erythropoiesis Stimulating Agent (ESA), in Oncology Patients Receiving Chemotherapy.
Authors: Pickering Lisa M. , Cwiertka Karel , Jassem Jacek , Petera Jira , Pettengell Ruth , Ramlau Rodryg , Vorlicek Jiri , Vyzula Rostislav , Duliege Anne-Marie , Lang William , Stead Richard B. , Harper Peter G. 
Published: April 2020

Abstract
Introduction: Hematide™ a novel, synthetic, PEGylated peptidic compound, binds to and activates the erythropoietin receptor. It is in development for treatment of anemia associated with chronic renal failure and cancer.
Objective: To assess the subcutaneous (SC) Hematide™ dose required to increase hemoglobin (Hb) by ≥ 1 g/dL in ≥ 50% of anemic cancer patients (pts) on chemotherapy. Safety, pharmacodynamics and pharmacokinetics were also assessed.
Method: In a phase 2, open label, multi-center, dose finding study, up to four doses of Hematide™ were given SC every 3 wks (Q3W) to 4 cohorts of fifteen pts at 0.05, 0.10, 0.15 or 0.2 mg/kg each. Entry criteria included confirmed solid tumor malignancy or lymphoma, &gt;9 weeks of chemotherapy, baseline (BL) H ≥ 8 and &lt;11 g/dL, and adequate iron, folate and B12 stores.
Results: Sixty patients were enrolled (45% male). Preliminary data show that across cohorts 0.05, 0.10, 0.15 and 0.20 mg/kg, mean (BL) Hb values were 10.1(±1.07), 10.0(±0.89), 9.8(±0.67), 10.1(±0.69); the % of pts completing the study were approximately 79%, 50%, 60%, and 92%. An increase in mean Hb from BL of ≥ 1g/dL in the pharmacodynamic dataset (forty two pts) at Week 7 occurred in approximately 20%, 70%, 55% and 55% of patients. Three pts withdrew due to AEs and six due to SAEs (none attributed to study drug). Three deaths occurred (two disease progression, one renal insufficiency) none attributed to study drug. One SAE, thrombophlebitis, was considered to be possibly/probably related to study drug. Final safety and efficacy data will be provided at the meeting.
Conclusion: In this study, HematideTM dosed SC Q3W resulted in an increase from BL of ≥ 1 g/dL of Hgb in ≥ 50% of patients in the 0.10, 0.15 and 0.20 mg/kg cohorts in the pharmacodynamic analysis. HematideTM appeared to be well tolerated at all doses studied.
NKG2D-Mediated Marrow Injury in Paroxysmal Nocturnal Hemoglobinuria and Its Related Disorders.
Authors: Hanaoka Nobuyoshi , Kawaguchi Tatsuya , Horikawa Kentaro , Nagakura Shoichi , Ishihara Sonoko , Tsuzuki Yasuchika , Yonemura Yuji , Nakakuma Hideki 
Published: April 2020

Abstract
Immune mechanism is considered to exert in the pathogenesis of marrow failure in paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS); however, the molecular events are unknown. We have currently reported the appearance of NKG2D ligands such as cytomegalovirus glycoprotein UL16 binding proteins (ULBPs) and MHC class I-related chains A and B (MICA/B) on granulocytes and CD34+ marrow cells of some patients with PNH and its related diseases (Hanaoka N, et al. Blood. 2006;107:1184–1191). ULBP and MICA/B are stress-inducible membrane proteins that appear in infection and transformation. The ligands share NKG2D receptor on lymphocytes such as NK, CD8+ T, and γδ T-cells and promote activation of the lymphocytes. Cells expressing the ligands are then deadly injured by NKG2D+ lymphocytes (Groh, PNAS 1996; Cosman, Immunity 2001). Indeed, cells expressing NKG2D ligands were killed in vitro by autologous NKG2D+ lymphocytes of our patients (Hanaoka N, et al. Blood. 2005;106:304a; Blood. 2006;108:295a). In further analysis, ligands were detected on granulocytes in 47 (53%) of 88 patients: 11 (58%) of 19 PNH, 28 (60%) of 47 AA, and 8 (36%) of 22 refractory anemia. Ligands were also detected on immature bone marrow cells in all 11 patients (3 PNH, 5 AA, and 3 refractory anemia) who permitted analysis of their marrow cells. In the patients, it is conceivable that blood cells were exposed to a certain stress to induce NKG2D ligands, leading to NKG2D-mediated marrow injury. We also observed a close association of the ligand expression with pancytopenia and favorable response to immunosuppressive therapy by prospective analysis of 5 patients (3 AA-PNH syndrome and 2 AA) for more than one year up to 5 years. Thus, we here propose that NKG2D-mediated immunity, which drives both NK and T-cells, is critically implicated in the pathogenesis of bone marrow failure of PNH and its related disorders.
Outgrowing Endothelial Cells That Are Derived from Human Umbilical Cord Blood Improve Neovascularization in Hind-Limb Ischemia.
Authors: Yoo Eun-Sun , Kwon KiHwan , Ahn Jee-Young , Oh Soo-Ah , Chang Hye-Jung , Choi Moon-Young , Mun Yeung-Chul , Lee Kyoung-Eun , Seong Chu-Myong 
Published: April 2020

Abstract
Backgroud: Human umbilical cord blood (UCB) contains a high number of endothelial progenitor cells (EPCs) and may be useful for the treatment of ischemic disease. Recently, we have isolated EPCs from UCB having different biologic properties for angiogenic capabilities in vitro. In this present study, the aim is to examine the usefulness of OECs in hind-limb ischemia.
Methods: Mononuclear cells from UCB cultured using EGM-2 medium with VEGF, IGF-1 and FGF for 21 days. Early spindle-shaped cells (early OECs), which were grown during the first week of culture and late cobblestone shaped cells (late OECs), which were in peak growth during the third week of culture were found. The hind-limb ischemia was established as follows: Athymic nude mice (BALB/C-nu) 18–22 g in weight were anesthetized with pentobarbital (60 mg/kg) and their left femoral arteries and main extension arteries were operatively resected. To examine the effect of the vasculogenesis of the two types of OECs, the mice were divided into three groups (PBS, early and late OECs). Twenty-four hours after operative excision 5 × 105 OECs in 200 μl and an equal volume of PBS were administered by intramuscular injection into the mice on hind-limb ischemia. To compare the effect of OECs on neovascularization in vivo, the analysis of blood flow of ischemic and healthy hind limbs was performed on days 1 and 21 after surgery using near-infrared (NIR) imaging with incocyanne Green (ICG).
Results: Late OECs expressed a high level of mRNA on endothelial marker genes and formed capillary tubes in Matrigel plates. The early spindle cells excreted more angiogenic cytokines and had more migratory ability. We divided the mice into two groups according to the degree of perfusion; good (22.5–50%/min) and poor (0–22.5%) perfusion. OECs improved the blood flow of the ischemic hind-limb in the ’good’ perfusion group but not in the ’poor’ perfusion group. Early OECs led to a more significant improvement in blood flow than that of the late OECs.
Conclusion: The different types of OECs from UCB have different biologic properties in vitro and different vasculogenic potential in vivo as well. The results might have potential application for the treatment of hind-limb ischemia.
Prostaglandin E2 Stimulates Human Brain Endothelial Cell Migration Via a Signaling Pathway Involving Activation of Rho-Kinase II
Authors: Khan Gausal , Rizzo Maria Theresa 
Published: April 2020

Abstract
Prostaglandin E2 (PGE2) plays a crucial role in angiogenesis as well as in ischemic and inflammatory disorders of the brain associated with breakdown of the blood-brain barrier. However, the effects of PGE2 on brain endothelial cell migration, a key process in the angiogenic response and blood-brain barrier stability, are not well defined. Exposure of human brain endothelial cells (HBECs) to PGE2 elicited a chemotactic response in a time- and dose-dependent manner. The maximum migratory response was detected following 8 hours exposure of HBECs to PGE2 (100 nM). Migration of HBECs in response to PGE2 was accompanied by profound changes in the reorganization of actin filaments. Fluorescence microscopy examination of NBD-phallacidin-labeled endothelial cells showed increased formation of stress fibers, lamellipodia and podosomes after treatment with PGE2 (100 nM) compared to control. Based on these results, we hypothesized that Rho-kinase (ROCK), an enzyme involved in regulation of actin dynamics and cell migration, mediated the effects of PGE2 on HBEC migration. Western blot analyses revealed that ROCK II (type alpha), but not ROCK I (type beta), was expressed in HBECs. To examine ROCK II activation, we performed immunocomplex kinase assays using myosin light chain (MLC) as a substrate. PGE2 (100 nM) induced a 2-fold increase of 32P-incorporation into MLC indicating activation of ROCK II. Pretreatment of HBECs with the selective ROCK inhibitor, Y27632 (150 nM), blunted HBEC migration in response to PGE2 but had no effect on migration induced by fetal bovine serum (10%). Knockdown of ROCK II by siRNA also abrogated the migratory response of HBECs to PGE2. In contrast, similar treatment had no effect of HBEC migration stimulated by hepatocyte growth factor. Taken together, these results are consistent with the hypothesis that stimulation of HBECs with PGE2 leads to activation of ROCK II, reorganization of the actin cytoskeleton and ultimately migration. A better characterization of the molecular events that regulate migration of brain endothelial cells is critical for the development of novel strategies to treat cerebrovascular diseases associated with deregulated angiogenesis.
Genes Involved in Acute Leukemias Are Favored Targets of HIV Vector Integration
Authors: Appelt Jens U. , Giordano Frank A. , Zimmermann Marcel , Weinhard Stephan , Grund Nadja , Hotz-Wagenblatt Agnes , Zeller W. Jens , Allgayer Heike , Fruehauf Stefan , Laufs Stephanie 
Published: April 2020

Abstract
Insertional mutagenesis and development of leukemia following retroviral gene therapy has created intense interest in assessing the safety of viral vectors for further gene therapy trials. Using the gtsg.org database we analyzed more than 14,900 different viral integration sites of ASLV, FIV, FV, HIV, MLV and SIV based vectors in terms of insertions into fragile sites, cancer genes, transcription factor binding sites, CpG islands, and repetitive elements (SINE, LINE, LTR elements). When we compared these data with our newly generated random set, containing 1,000,000 random integrations, we discovered that the gene density on fragile sites strongly correlates to the HIV vector insertion frequency. Furthermore, we report a up to a five fold increased frequency of HIV, MLV and SIV insertions in cancer genes. The majority of cancer genes preferentially hit by HIV viruses were found associated to acute leukemias, while MLV and SIV vector insertion sites are seen more evenly spread over the cancer gene repertoire. When analyzing different cell entities, it turned out that CD34+ hematopoetic stem cells had highest rates of intragenic insertions and hosted significantly more HIV and FV insertions in cancer genes than other cell types, such as HeLa, T cells, 293T cells, macrophages, fibroblasts, or SupT1 cells.
A Prospective Randomised Trial of Intravenous Iron Therapy Versus Oral Iron for Iron Deficiency Anaemia in Pregnant Australian Women
Authors: Khalafallah Alhossain A. , Dennis Amanda , Bates Joan , Bates Gerald , Smith Lauern , Shaw Susie , Seaton David , Berteram John , Robertson Iain , Ball Madeleine , Beamish Michael , Rooney Kim , Brain Terry 
Published: April 2020

Abstract
To date, limited data is available regarding prevalence of iron deficiency during pregnancy in Australia. There is little if any data currently available regarding comparative efficacy of IV iron versus oral iron therapy in pregnant women. In Australia the prevalence of iron deficiency anaemia (IDA) is approximately 5% in the general population with higher rates in pregnant women. At a single site, the Launceston General Hospital (LGH), we prospectively investigated 200 pregnant women between January and July 2007 with FBC and iron studies at the first or second antenatal visit. Among those, 40 women (20%) had iron deficiency anaemia, and were recruited to a prospective randomised trial to determine whether intravenous iron therapy (iron polymaltose) is superior to oral iron (ferrous sulphate) for the management of IDA associated with pregnancy. The patients’ median age was 28 years old (range; 19–40) with a median gestational age at recruitment of 27 weeks (range; 13–29) and a median body weight of 74 kg (range; 47–130). At recruitment median Hb was 106 g/L (range; 90–114, normal range; 120–160 g/L), while median serum ferritin was 11.5 μg/L and mean ferritin was 19 μg/L normal range; 30–460). After four weeks of treatment the Hb level increased by a mean of 6g/L on oral iron and by 10.5 g/L after IV iron. Mean/Median serum ferritin did not increase significantly in women on oral iron, but increased to a median of 96.5 μg/L and a mean of 224 μg/L in those received IV iron. Multi-variate analysis using general linear modelling for continuous variables showed a significant increase in serum ferritin after treatment with IV iron versus oral iron (p=0.017). There was no statistically significant difference in terms of Hb increment, patients’ parity, weight, and date of last pregnancy between groups. Analysis of quality of life questionnaires to assess patients’ well-being, ability to perform activities, and symptoms of anaemia showed improvement in both groups of patients with a trend to greater and faster improvement after IV iron. Both treatments were well tolerated without major side effects. The preliminary data indicate IDA is a common finding during pregnancy in the LGH population, and intravenous iron therapy appears a safe and effective treatment in this cohort of patients. This research received a grant from the Clifford Craig Medical Research Trust, Tasmania, Australia.
HMGB1 Expression in Sickle Cell Disease: A Pro-Inflammatory Cytokine and Potential Therapeutic Target.
Authors: Barginear Myra F. , Chandok Anu G. , Sison Cristina , Yang Lihong , Budman Daniel R. , D’Olimpio James , Tracey Kevin J. , Bradley Thomas 
Published: April 2020

Abstract
Background: HMGB1 is traditionally known as a DNA binding protein involved in gene transcription. When released into the extracellular milieu, HMGB1 activates vascular endothelial cells and macrophages/monocytes to express proinflammatory cytokines, chemokines, and adhesion molecules. Therefore, HMGB1 may be a good marker for disease activity in Sickle Cell Disease (SCD), a chronic inflammatory state.
Methods: After informed consent as per Institutional guidelines, plasma was obtained, frozen, and batch run for HMGB1 levels by ELISA technique in patients with SCD during non-crisis and crisis states. The detection limit of HMGB1 was achieved at concentrations above 1 ng/ml. Each experiment was repeated at least five times to ensure reproducibility.
Results: 12 patients enrolled, 6 patients have gone into crisis. HMGB1 levels were measured on consecutive days for 5 five days during a crisis. We established mean HMGB1 expression levels of 32.83 ± 27.13ng/ml during a non-crisis state and 52.57± 15.22 ng/ml during a crisis, respectively (p&lt; .028). In noninflammatory states, HMGB1 is &lt; 5 ng/ml.
Conclusion: We have demonstrated, for the first time, a persistent expression of HMGB1 in SCD patients. Although recruitment is on going, this data is consistent with pre-clinical data indicating that HMGB1 may be an important diffusible signal of necrosis-mediated inflammation. Additionally, our laboratory has previously demonstrated therapeutic benefit by neutralization of HMGB1 in sepsis. Thus if confirmed in a larger cohort, anti-HMGB1 therapy to reduce the effects of inflammation may be an attractive therapeutic target in sickle cell disease.