bluuebunny/crossref_metadata_2025_split huggingface dataset:
Searching for Disease Modifiers Genes in Thalassemia.
Authors: Fucharoen Suthat , Sripichai Orapan , Winichagoon Pranee , Abel Kenneth , Braun Andreas 
Published: April 2020

Abstract
β0-thalassemia/HbE disease is one of the most common thalassemias in Southeast Asia. Patients with compound heterozygote for HbE and β0 mutant alleles display remarkable variability in clinical expression, ranging from nearly asymptomatic to severe, transfusion-dependent disease. It is believed that additional genetic factors modifying disease severity may account for this variability. A universal platform technology (MassARRAY) based on mass spectrometry for analyzing nucleic acids at a high level of precision and accuracy has been developed. To identify genetic modifiers influencing severity among 1060 β0-thalassemia/HbE patients, we are utilizing this technology to conduct a genome-wide association study involving approximately 110,000 gene-based single nucleotide polymorphisms (SNPs). This assay panel corresponds to SNPs with a median spacing of 10.4 kilobases, in approximately 99% of all known and predicted human genes. DNAs from approximately 200 regionally matched patients representing the extremes of mild and severe cases were included in each DNA pool. Allele frequencies for all SNPs were estimated in both pools, and those showing suggestive significant differences (p values &lt;0.02) were selected for verification by repeated pooled DNA analysis. Approximately one-fourth of these showed reproducible allelic differences at p&lt;0.05, and more than 600 were selected for genotyping the individual patient DNAs in order to determine precise allele and genotype frequencies. A number of SNPs showed evidence for association with disease severity, including several in reported quantitative trait loci (QTLs) associated with fetal hemoglobin HbF levels. However the most strongly associated SNPs were within a region on chromosome 11 distinct from the beta globin gene cluster, within which most analysis to date has focused.
Oxidative Stress of RBC in a Murine Model of Beta-Thalassemia Can Be Reversed by Treatment with Antioxidants.
Authors: Fibach Eitan , Amer Johnny , Rachmilewitz Eliezer , Guy Ella , Rivella Stefano 
Published: April 2020

Abstract
Oxidative stress is a prominent contributor to the premature destruction of RBC as well as anemia in thalassemia and sickle cell anemia. The oxidative status within RBC is maintained by the balance between oxidative systems, such as Reactive Oxygen Species (ROS), and antioxidative systems, such as reduced glutathione (GSH). Using flow cytometric methods, we previously showed that RBC obtained from patients with thalassemia (Amer et al. Eur J Haematol70:84,2003; Cytometry60:73,2004) or sickle cell anemia (Amer et al. Blood, 104:972a,2004) exhibit oxidative stress. In the present study, we assessed the extent of RBC oxidative stress and the effects of antioxidant administration, using the thalassemic mouse model Th3/+. This model closely mimics the phenotype observed in patients affected by beta-thalassemia intermedia, such as low hemoglobin levels (7 to 9 gr/dL), splenomegaly and iron overload. In addition, the mature RBC have a shorter life-span and are characterized by anisocytosis, poikilocytosis and hypochromatism.
RBC were derived from normal and thalassemic mice before and 4 hours after i.p. injection of the antioxidants N-acetyl cysteine (NAC), vitamin C (Vit. C) or tocotrinol - a mixture of vitamin E derivatives, at a dose of 150 mg/kg. Intracellular ROS was determined in dichlorofluorescin diacetate-stained RBC following stimulation with 2 mM H2O2; GSH content was assessed in RBC stained with mercury orange. Cells were analyzed by flow cytometry: RBC were gated according to size (forward light scatter) and granularity (side light scatter), their fluorescence was measured and the Mean Fluorescence Channel (MFC) was calculated. Fig. 1 shows the average MFC of ROS and GSH of normal and thalassemic mice treated or not treated with anti-oxidants (N=6 in each group).
The results show a significantly higher (2.6-fold) production of ROS and lower (three-fold) levels of GSH in RBC from the thalassemic mice versus those in RBC from normal mice. Administration of antioxidants decreased the ROS of normal and thalassemic RBC by 1.4-fold and 2.6-fold, respectively, whereas GSH levels were significantly increased both in the normal (2.7 fold) and in the thalassemic (9.4-fold) RBC. The results show that the RBC of thalassemic mice are under oxidative stress that could be ameliorated by a short antioxidant treatment. Hence, this mouse model recapitulates the oxidative stress found in thalassemic patients and can serve as a model for studying the effects of antioxidant therapy. The flow cytometry methodology used is helpful in following up the results of the treatment and in evaluating its efficacy in reducing oxidative stress.
Figure Figure
Developmental and Differential Changes of Gene Expression in Erythroid Progenitor Cells.
Authors: Cokic Vladan P. , Bhattacharya Bhaskar , Puri Raj K. , Schechter Alan N. 
Published: April 2020

Abstract
During erythropoiesis and human development different globin genes (α, β, γ, δ and ε) are expressed as a result of globin gene switching. We investigated globin gene expression in comparison to the expression of other genes in erythroid progenitor cells (EPC) during ontogenesis using in-house produced microarrays containing 16,659 oligonucleotides. Human primitive CD34+ cells were isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and mobilized peripheral blood (mPB), and developed into EPC in the presence of erythropoietin and other cytokines. The differentiation to EPC was confirmed by flow cytometry as 100% cells were CD71+. In microarray studies, a total of 2996 genes were highly expressed in FL, 2673 genes in CB, 2580 in mPB, 1465 in PB and 1259 in BM derived EPC. 661 of these genes were common for all type of cells. The high level of expression, beside globin genes, was observed for the following genes: transferrin receptor, proteoglycans, ALAS2, Charcot-Leyden crystal protein, nucleophosmin, eosinophil peroxidase, myeloperoxidase and ribonucleases. Most of the analyzed genes demonstrated down-regulation during ontogenesis (elastase 2, glutathione peroxidase 1, SERPINB1, nudix, mitochondrial proteins, ribosomal proteins, enthoprotin, serine proteinase inhibitor), but some showed up-regulation (hexokinase, superoxide dismutase 2, spectrin). Besides developmental changes of globin gene expression during ontogenesis, we also analyzed changes in their expression during erythropoiesis in these different tissues by quantitative PCR. Beta-globin gene expression reached the maximum levels in cells of adult blood origin: BM (176 fmol/μg) and PB (110 fmol/μg). Gamma-globin gene expression, of FL origin, had steady levels during erythroid differentiation (20 fmol/μg), whereas cord blood derived EPC demonstrated consistent up-regulation (60 fmol/μg) in contrast to cells originated from adult blood (3–15 fmol/μg at day 14th). G protein related genes and histone deacetylases were elevated in CB derived EPC, concomitant with increased gamma-globin gene expression. We also analyzed the gamma-globin induction by hydroxyurea, a well known inducer, and established which G protein-coupled receptors involved pathways are activated in PB derived EPC: dopamine receptors D1, D2 and D5, beta 2 adrenergic receptor, human DP prostanoid receptor and prostaglandin E receptor 1, as well as genes activated by cAMP/PKA, PI-3 kinase, MAP and NO/cGMP pathways. This study establishes concomitant changes in expression of globin genes and other known and/or previously unrecognized genes, which appear to be involved in erythropoiesis.
Human Beta-Globin Expression during Erythroid Cells Development in Transgenic Mice Bearing Human Beta-Globin Gene with Mutated BP1 Binding Site.
Authors: Zoueva Olga P. , Bodine David , Rodgers Griffin P. 
Published: April 2020

Abstract
Binding of beta protein 1 (BP1) to its site on the promoter of adult beta-globin gene has silencing effect on beta-globin transcription in vitro. To better understand the mechanism of the negative regulation of beta-globin expression by BP1 we have developed transgenic mice. Specifically, we introduced a mutated BP1 binding site into the promoter of beta-globin gene sequence of 35 kb cosmid construct. This construct containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into the single cell mouse embryos. To detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we studied the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5. In addition, we analyzed adult erythroid cells. To minimize experimental error, samples from individual animals of three transgenic lines were analyzed independently using real-time PCR assays. Levels of expression of murine alpha-globin mRNA were used as internal controls. The BP1 gene and its mouse analog Dlx4 belongs to the Distal-less family of homeobox genes, which are expressed during early development. We found that the mRNA levels of human beta-globin in transgenic mice containing mutated BP1 binding site were higher at all stages of erythroid cells development as compared with control transgenic mice bearing cosmid construct with wild type sequence of BP1 site. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our data indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice. These results may help develop the novel clinic approaches for the inhibition of the expression of abnormal beta-globin genes, such as sickle (hbs) and hbc.
Allogeneic Myelo-Ablative Stem Cell Transplant (SCT) as Salvage Therapy of Reduced-Intensity Conditioned (RIC) SCT: Toxicity and Outcome.
Authors: Cordonnier Catherine , Hamladji Rose-marie , Gratwohl Alois , Ljungman Per , Martino Rodrigo , Bordigoni Pierre , François Sylvie , Deconninck Eric , Buzyn Agnes , Bay Jacques-Olivier , Fouillard Loic , Kuentz Mathieu 
Published: April 2020

Abstract
RIC regimens are mainly used for patients who cannot benefit from the myelo-ablative (MA) approach, due to age, previous SCT, or comorbidities, based on an expected early toxicity of MA regimen. Some centers may also choose the RIC strategy in young patients because they have no facilities for the management of prolonged neutropenia. The development of RIC has been so encouraging that programs now compare MA and RIC in patients who could tolerate both approaches. However, RIC may fail, and MA SCT be secondary considered.
Objective: The goal of this retrospective survey was to assess the toxicity and outcome of MA after RIC SCT.
Patients: 17 patients (14–63 y; 6 CML, 4 AML, 2 CLL, 2 myeloma, 1 NHL, 1 CMML, 1 myelodysplastic syndrome) received a MA SCT after RIC, for relapse (n=12), graft rejection (n=4), or in a planned program (n=1), from an HLA-id sibling (n=12), a familial mismatched donor (n=1), an unrelated donor (n=3) or a cord blood (n=1). The reason for initial RIC approach was age in 4, a research program in 6, comorbidities in 3, and previous autologous SCT in 4 patients. RIC included Fludarabin (Flu)-Busulfan (BU) in 8, Flu-2Gy irradiation in 4, Cyclophosphamide (CPM)-antithymocyte globulin (ATG) in 1, and various Flu- regimens in 4. One patient had 2 consecutive RIC SCTs before MA SCT. The median delay between RIC and MA SCT was 9 months (range: 2.5–36 months). The same donor was used for the 1st and 2nd SCT in 12/17 cases. The MA transplant was conditioned with BU-CY (±VP16) in 9, Cyclo-TBI in 4, and other regimens in 4 patients. The median follow-up after MA SCT was 9 months
Results: After the MA transplant, 1 patient developed veno-occlusive disease, 2 developed interstitial pneumonia. Five patients died from relapse between 8 and 21 mo after the MA SCT. Seven (41%) died from transplant-related causes at a median of 8 months (GVHD:3; infection:1; multi-organ failure:1; thrombotic microangiopathy:1; EBV proliferative disease:1). 5/17 (29.5%) patients are alive and well 4.5 to 26 months after MA transplant. Three of them have extensive chronic GVHD while they did not develop any GVHD after RIC. The delay between the 2 transplants did not influence the outcome.
Conclusion: This survey shows that MA after RIC SCT is feasible, and considering that most of these patients have been initially eligible for RIC because of their age or comorbidities, MA SCT had an acceptable toxicity. It may be considered for patients who relapsed or rejected after RIC transplant.
Inhibition of Pathological Retinal Neovascularization by α-Defensins.
Authors: Chavakis Triantafyllos , Bdeir Khalil , Cines Douglas B. , Fogt Franz , Bdeir Yasmina , Lubkowski Jacek , Preissner Klaus T. , Hammes Hans-Peter , Economopoulou Matina 
Published: April 2020

Abstract
Proliferative retinopathies, such as those complicating prematurity and diabetes, are major causes of blindness. A prominent feature of these retinopathies is excessive neovascularization, which is orchestrated by the hypoxia-induced vascular endothelial growth factor (VEGF) stimulating endothelial cells, and the integrin-mediated adhesive interactions of endothelial cells with extracellular matrix components like fibronectin (FN). Recently, we demonstrated that α-defensins interfere with α5β1-FN interactions and dependent endothelial cell functions (FASEB J., 2004, 18:1306–8). Here, α-defensins were studied in hypoxia-induced proliferative retinopathy. In vitro, α-defensins specifically inhibited α5β1-integrin dependent migration of bovine retinal endothelial cells (BREC) to FN, attenuated the VEGF-stimulated increase in endothelial permeability, and blocked BREC proliferation and capillary sprout formation in three-dimensional fibrin-matrices. An upregulation of β1-integrin and FN was observed in the retinal vessels in the mouse model of hypoxia-induced retinal angiogenesis. Systemic and ocular administration of α-defensins reduced retinal neovascularization by 45% and 60%, respectively, and this effect was comparable to the inhibitory effect of α5β1-blocking antibody. α-defensins were detected in human diabetic retinas but were absent in retinas of eyes removed because of trauma. Together, these data show that α-defensins inhibit pathological retinal neovascularization in vivo and may provide a clinically efficient strategy against proliferative retinopathies.
Role of Platelets in Homing of Endothelial Progenitor Cells to the Site of Vascular Injury.
Authors: Lev Eli I. , Estrov Zeev , Aboulfatova Khatira , Harris David , Granada Juan F. , Kleiman Neal S. , Dong Jing-fei 
Published: April 2020

Abstract
Endothelial Progenitor Cells (EPCs) are undifferentiated bone marrow cells that are rapidly mobilized upon vascular or tissue injury. The circulating EPCs home to and differentiate at the site of vessel injury to enhance repair of damaged endothelium. Much has been learnt of how EPCs adhere to and transmigrate on endothelial cells during angiogenesis, but little is know about how EPCs home to the site where endothelial cells are denuded and subendothelium is exposed following vascular injury. We hypothesized that platelets mediate EPC homing because they are the first cells that tether and adhere to subendothelium exposed by vascular injury. To test this hypothesis, we examined the platelet-EPC interaction under static and flow conditions. Human EPCs were purified from buffy coats by cell sorting using CD133 and VEGFR-2 as the cell markers. The EPCs were then incubated with washed platelets that were either at resting state or activated by 5 mg/ml of collagen. EPC-platelet interaction was measured in the form of heterotypic aggregation between two types of cells by flow cytometry (dual labeled with FITC-CD42a and PE-CD133). We found the EPCs attached to resting platelets, however, the percentage of platelets binding to EPCs significantly increased when platelets were activated by collagen (1.02% vs. 2.76%, p &lt; 0.05). EPC-platelet aggregation was similarly detected in whole blood and platelet-rich plasma. When perfused over a monolayer of activated platelets under a shear stress of 2.5 dyn/cm2, the CD133 positive EPCs tethered to the platelet monolayer. The tethered EPCs either firmly adhered immediately or rolled a short distance before becoming adherent. This EPC-platelet interaction was calcium-dependent and inhibited by up to 60±11.5% by a polyclonal P-selectin antibody. In comparison, the monoclonal GP Iba antibody AK2, which blocks GP Ib-VWF interaction, had no effect. These results demonstrate for the first time a significant interaction between EPCs and activated platelets both at static and flow conditions. Our findings suggest that platelets may function as carrier cells that direct EPCs to the site of vascular injury. In addition to homing, platelets, which release several growth factors known to affect EPCs, may provide a supporting matrix on which adherent EPCs proliferate and differentiate.
GAS-6 Rescues Endothelial Cells from Apoptosis through FOXO1 Inactivation.
Authors: Ganopolsky Jorge , Varnum Brian , Blostein Mark 
Published: April 2020

Abstract
Growth Arrest Specific gene product 6 (GAS-6), a γ-carboxylated protein expressed in quiescent fibroblasts and endothelial cells, exerts an anti-apoptotic function by binding to the receptor tyrosine kinase Axl. Recently, our laboratory has demonstrated that gas6-Axl interactions activate PI3-kinase with subsequent Akt phosphorylation during gas6-mediated protection from apoptosis. The current study explores further the mechanism by which this survival mechanism is achieved. FOXO1 is a member of the Forkhead family of transcription factors that plays a role in the expression of pro-apoptototic genes. Phosphorylation of FOXO1 at Thr24, Ser256 and Ser319 results in phospho-FOXO1 translocation from the nucleus to the cytoplasm, with consequent suppression of FOXO1 transcriptional activity and inhibition of apoptosis. In the present study we show, for the first time, that the treatment of serum-starved endothelial cells with 100 ng/ml of GAS-6 induces FOXO1 phosphorylation in a time dependent manner. Phosphorylated FOXO1 is translocated from the nucleus to the cytoplasm as evidenced by Western blot analysis of both nuclear and cytoplasmic extracts. Using fluorescence microscopy, FOXO1 is found predominantly in the nucleus during apoptosis induced by serum starvation. Upon gas6 stimulation, phosphorylated FOXO1 is translocated to the cytoplasm (see Figure 1). It is suggested that anti-apoptotic genes are then released from suppression and are thereby able to mediate cell survival. Both FOXO1 phosphorylation and translocation are suppressed by Wortmannin, a PI3-kinase inhibitor demonstrating that FOXO1 phosphorylation is PI3-kinase dependent. These results provide mechanistic insight of how gas6 rescues endothelial cells from serum-starvation-induced apoptosis.
Foxo1 distribution Foxo1 distribution
In Vitro Characterization of Umbilical Cord Blood CD133+ and In Vivo Functionality in Response to Ischemia in a Murine Hind-Limb Injury Model.
Authors: Finney Marcie R. , Greco Nicholas G. , Joseph Matthew E. , Winter Daniel G. , Bhakta Shyam , Fanning Laura R. , Kozik Margaret , Banks Jason , Huang Ying , Pompili Vincent J. , Laughlin Mary J. 
Published: April 2020

Abstract
Previous reports have demonstrated efficacy of cellular therapy in mediating therapeutic angiogenesis in response to ischemia. We sought to determine the potential efficacy of adult umbilical cord blood (UCB) derived selected CD133+ cells in the murine hind limb ischemia model and to characterize these cells by surface phenotype and functionality prior to injection.
Methods and Results: Mononuclear cells (MNC) from UCB were labeled with CD133+ conjugated magnetic beads, followed by automated sorting through magnetic columns (Miltenyi). Routine yield of CD133+ cells was 0.51 ± 0.2% of MNC, with a purity of 78.7 ± 2.4% (n=30). Surface expression in the UCB CD133+ population was 3.59 ± 1.49% KDR(VEGFR2), 8.66 ± 3.79% CXCR4 and 22.74 ± 2.84% CD105 compared to 7.14 ± 2.15% KDR, 28.54 ± 5.81% CXCR4 and 6.74 ± 2.07% CD105 in the UCB MNC population. Transwell plates with 5μm collagen coated filters (Costar) were used to observe chemotactic migration of MNC or CD133+ cells towards SDF-1 (100ng/mL) compared to control wells containing media alone. Following a 3 hour incubation, the cells migrating to the bottom wells were counted by flow cytometry with TruCOUNT™ tubes (BD Biosciences). MNC and CD133+ cells migration to SDF showed a 4.9 ± 2.9 and 1.8 ± 0.7 fold increase over the negative control respectively. To test vasculogenic functionality of these selected cell populations, NOD/SCID mice underwent ligation of the right femoral artery and were randomized into 3 study groups: control (endothelial media with cytokines), non-selected MNC (1 x 106 cells/mouse) or CD133+ (0.5 x 106 cells/mouse) given via intracardiac injection immediately after injury. Doppler flow measurements were taken on both limbs each week for 4 weeks and the ratio of perfusion in the ischemic/healthy limb was calculated. At 28 days, perfusion ratios were statistically higher in study groups receiving CD133+ cells from UCB, 0.55 ± 0.07 (n=8) compared to cytokine controls 0.39 ± 0.02 (n=10, p=0.019). Mice receiving MNC did not show statistically significant improvement over control animals 0.42 ± 0.06 (n=7, p=0.27).
Conclusion: Surface phenotyping was notable for increased expression of the receptor for SDF-1, CXCR4 on MNC when compared to CD133+ cells. In vitro functional assays showed that CD133+ and MNC exhibited increased chemotactic migration to SDF-1. In vivo studies showed that injection of UCB CD133+ cells improved blood flow compared with cytokines alone in the murine hind limb injury model, highlighting the vasculogenic potential of CD133+ cells from UCB.