bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Effective Program to Resolve Ambiguous Results from State Newborn Hemoglobinopathy Screening.
Authors: Hoppe Carolyn , Azimi Mahin , Aslanian Sharon , Lubin Bertram , Vichinsky Elliott , Therrell Brad 
Published: October 2019

Abstract
Newborn screening (NBS) for hemoglobinopathies is an important mechanism for identification of affected individuals so that prophylactic treatment and comprehensive care are delivered before life-threatening complications occur. Whilst more common Hb variants, such as Hb S and Hb C, are readily identified, many children with other potentially significant Hb variants never have a conclusive diagnosis made by state newborn screening programs. In conjunction with the National Newborn Screening and Genetics Resource Center (NNSGRC), we investigated whether unconfirmed or ambiguous samples could be rapidly diagnosed and reported, using only a portion of the original dried blood spot (DBS) specimen.
State NBS programs were approached about sending DBS samples from newborns with ambiguous Hb results for further testing. Using a single 6 mm hole punch from the original DBS, the primary screening results were first confirmed with HPLC, IEF and citrate agar electrophoresis. With the same sample, genotyping for Hb S, C, E, Knossos, D-LA, and O-Arab, and > 95% of the most common β-thalassemia mutations, was performed using a novel linear array platform. Samples revealing Bart’s Hb were tested for common deletional and non-deletional α-thalassemia mutations using gap-PCR or DNA sequencing. The test results, along with a clinical interpretation, were reported back to the NBS programs within 2 weeks.
During a one-year period (08/03–08/04), a total of 309 newborn samples from 30 participating states were received for definitive diagnostic testing. Of these, 115 (37%) revealed a clinically significant genotype. Specifically, 102 newborns had sickle cell disease (55 HbSS, 27 HbSC, 16 HbS/β-thal, 3 HbSE, 1 HbS/O-Arab); 6 newborns had β-thalassemia (2 β0-thal, 4 E/β-thal); and 7 had α-thalassemia (5 HbH, 1 HbH-CS, 2 α-triplication). Compound heterozygosity for 2 Hb variants was found in 15 cases and interpreted as clinically insignificant. Another 14 samples revealed a clinically benign HbEE genotype. A Hb variant trait was identified in 124 samples and 41 samples had a normal Hb genotype.
This NNSGRC-sponsored pilot study demonstrates that a centralized referral laboratory providing definitive diagnostic testing for hemoglobinopathies is not only feasible, but a much needed resource for many state newborn screening programs. Using rapid and efficient molecular methods, our laboratory identified a clinically significant sickling of thalassemic disorder in 37% of previously unconfirmed newborn samples. Based on these results, the implementation of a referral laboratory to provide prompt definitive diagnosis of clinically relevant hemoglobin variants would be an invaluable extension of existing newborn screening programs.
Increased GM-CSF Levels in Sickle Cell Disease Are Associated with Increased Leukocyte Counts and Are Reversed by Hydroxyurea.
Authors: Conran Nicola , Ikuta Tohru , Saad Sara T.O. , Costa Fernando F. 
Published: October 2019

Abstract
The hematopoietic cytokine, GM-CSF, stimulates the proliferation and differentiation of progenitor cells to macrophages, eosinophils and megakaryocytes. Increased levels of GM-CSF have previously been reported in sickle cell disease (SCD) patients with low fetal hemoglobin (HbF) levels (Croizat, Br J Haematol. 87:592; 1994); however, a clearcut association between GM-CSF and HbF levels in SCD has yet to be established, since there is some evidence that levels may depend on other factors (Haider et al., Acta Haematol. 102:140; 1999). To further investigate the possible role of GM-CSF in SCD, plasma samples were collected from normal individuals (AA), steady-state sickle cell anemia patients (SS) and sickle cell patients on hydroxyurea therapy (SSHU; 20-30 mg/kg/day). Plasma GM-CSF levels were measured using a specific immunoenzymatic assay. HbF levels were quantified by ion-exchange HPLC and hematological measurements obtained using an Advia Hematology System. Plasma GM-CSF levels seem to be higher in SS patients than in normal controls (0.828 ± 0.215 pg/ml, n=39; 0.230 ± 0.081 pg/ml, n=9; respectively). In contrast, GM-CSF was significantly lower in SSHU patients than in SS without HU (0.288 ± 0.104 pg/ml; n=14; P = 0.045 compared to SS). A negative correlation between plasma GM-CSF and HbF levels was seen in patients (r = −0.332; P = 0.026; n=45; SS and SSHU groups) and, notably, a positive correlation between GM-CSF and white blood cell counts was observed (r = 0.302; P = 0.037; n= 48; SS and SSHU groups). Furthermore, GM-CSF (2.5 ng/ml) abolished the production of γ globin during erythropoietin-stimulated differentiation of TF-1 hematopoietic cells. These results provide further support to the hypothesis that plasma GM-CSF correlates negatively with HbF in sickle cell disease. Interestingly, in vitro data suggest that increased GM-CSF may, in fact, diminish HbF levels rather than reflect increased haematopoietic stress as a consequence of low HbF. Importantly, GM-CSF appears to correlate with leukocyte numbers in SCD, and levels were significantly decreased in patients taking HU, indicating that this cytokine may contribute to the leukocytosis seen in some SCD patients and may play a role in leukocyte count reduction by HU.
Generation of Transgenic Mice Expressing Human Hemoglobin E.
Authors: Chen Qiuying , Bouhassira Eric E. , Besse Arnaud , Suzuka Sandra M. , Fabry Mary E. , Nagel Ronald L. , Elison-Hirsch Rhoda 
Published: October 2019

Abstract
HbE (β26 Glu → Lys) is the most common pathogenic Hb variant in the world and is found with the greatest frequency in Southeast Asia. The HbE mutation at codon 26 of the βE-globin leads to an alternative splicing site, making this globin both a structural as well as a thalassemic mutation; furthermore, βE globin chains are unstable. HbE trait is asymptomatic and the HbEE genotype has only mild clinical manifestations. On the other hand, HbE/thalassemia presents a panoply of phenotypes from very severe to a mild beta/+ thalassemia. To date, there is no animal model of HbE disease. An animal model of this disorder could lead to better understanding of the effects of modifier genes and some of the pathogenic features of this disease. In addition, an animal model can facilitate the development of gene therapy. We report here the creation of four lines of transgenic mice that are PCR positive for HbE: two with founders that express high levels of HbE (32% human α, 26% human βE), one with low expression of HbE (9% human α, 8% βE), and one without detectable expression, but PCR positive. Knockouts (KOs) for mouse α and β globins have been bred into the founders. To date, the most severe mouse generated has a full KO of mouse α and a partial KO of mouse β. RBCs from all of the partial KOs generated to date have normal MCHC and minimally elevated reticulocyte counts (less than 1% greater than the C57Bl background). We previously demonstrated that HbEE RBCs are microcytic, but have normal MCHC. We found that HbE mice with full α-KO and partial β-KO, are microcytic (MCH 10.9 vs 14.2 for C57Bl and MCV 35.4 vs 47.5 for C57Bl), but have normal MCHC (MCHC 30.0 vs 30.5 for C57Bl). Smears from these mice exhibit numerous target cells that are not necessarily associated with low MCHC. As reported for human red cells containing HbE, elevated levels of hemoglobin oxidation products were detected in founder mice expressing HbE, but not in negative litter mates, and were found, at an even higher level, in partial KO mice expressing HbE. The next step will be to generate a mouse similar to the HbE/thalassemia phenotype by generating mice expressing exclusively HbE from either the lines expressing high levels of HbE or the line expressing low levels of HbE. These models will help elucidate several of the challenging features of the phenotypes described in humans and allow for the development of the potential cure or amelioration of HbE/thalassemia.
Histone Acetyltransferase Activity of p300 Is Required for Transcriptional Repression by the Promyelocytic Leukemia Zinc Finger Protein.
Authors: Guidez Fabien , Howell Louise , Isalan Mark , Cebrat Marek , Alani Rhoda M. , Ivins Sarah , Pierce Sarah , Cole Philip A. , Licht Jonathan D. , Zelent Arthur 
Published: October 2019

Abstract
The Promyelocytic Leukemia Zinc Finger (PLZF) gene was identified in a rare case of acute promyelocytic leukemia (APL) with translocation t(11;17)(q23;q21) and resistance to therapy with all-trans-retinoic acid. Recent studies have indicated a critical role of PLZF in maintenance of spermatogonial stem cells. Prominent expression of PLZF in hematopoietic stem cells, suggest that it may also play a similar role in this compartment. The wild type PLZF protein is a DNA sequence-specific transcription repressor containing nine Krüppel-like C2-H2 zinc fingers and an N-terminal BTB/POZ repression domain. Transcriptional repression by PLZF is mediated through recruitment of the nuclear receptor co-repressor (N-CoR/SMRT)/histone deacetylase (HDAC) complexes to its target genes, such as c-MYC and HOX genes. We now show that transcriptional repression by PLZF is surprisingly also dependent on the histone acetyl transferase (HAT) activity of the p300 protein. PLZF associates with p300 in vivo and its ability to repress transcription is specifically dependent on acetylation of PLZF on lysines in its C-terminal C2-H2 zinc-finger motifs. Acetylation of PLZF enhances its ability to bind its cognate DNA binding site in vitro as determined by EMSA and in vivo as measured by chromatin immunoprecipitation. An acetylation site mutant of PLZF fails to repress transcription despite retaining its abilities to interact with co-repressor/HDAC complexes, due to inefficient DNA binding. Inhibitors of p300 abolish transcriptional repression by PLZF and mutants of PLZF that mimic acetylation were insensitive to these inhibitory effects. Acetylation of PLZF by p300 was specific since over-expression of another HAT, p/CAF or the selective inhibition of p/CAF had no effect on PLZF activity despite the ability of the proteins to associate with each other. Taken together, our results indicate that a histone deacetylase dependent transcriptional repressor can be positively regulated through acetylation and point to an unexpected role of a co-activator protein in transcriptional repression.
Generation of Robust Numbers of Functional Human Hepatocytes by Human Hematopoietic Stem Cells in the Non-Injury Fetal Sheep Model.
Authors: Torabi Alireza , Almeida-Porada Graca , Zanjani Esmail D. 
Published: October 2019

Abstract
We have previously shown that human hematopoietic stem cell preparations (HSCs) are able to engraft and transdifferentiate into human hepatocyte in the human/sheep xenograft model (Blood. 2004 Jul 1 Epub). In the present study 1x106 human CD34+ cells were transduced with GFP expressing RD114 pseudotyped spleen fociforming retroviral vector in vitro and injected into pre-immune fetal sheep. Two months post-transplantation, immunohistochemistry confirmed production of human hepatocytes that were double positive for GFP and Hepar1. These data indicate that transduced human CD34+ cells are able to transdifferentiate into human hepatocyte and still be able to express GFP. In order to retrieve human hepatocytes from the liver of chimeric sheep, we used c-met antibody to sort out human hepatocytes. Flowcytometric analysis demonstrated 8% c-met+ cells in the liver of chimeric sheep. Preliminary data using PCR analysis from c-met+ cells for human beta-2 microglobulin demonstrated presence of human cells in c-met+ population. Livers from chimeric sheep were treated with collagenase to obtain single cell suspension and 3x105 cells/ml were placed in T75 flask covered with 0.2% gelatin and cultured in optimized media designed to support the proliferation and maintenance of human and sheep hepatocytes. In this system, using a weekly passage protocol, we were able to maintain/expand human (and sheep) hepatocytes for up to 3 months. The cells are able to produce human albumin in culture and are CK19 positive, a-fetoprotein negative, and cytochrome P450 negative. These results demonstrate that in this non-injury large animal model human HSCs generate robust numbers of functional human hepatocytes apparently via a “transdifferentiation” mechanism.
Immune-Mediated Hepatitis Drives Bone marrow to Hepatocyte Plasticity.
Authors: Dahlke Marc H. , Popp Felix C. , Piso Pompiliu , Schlitt Hans J. , Bertolino Patrick 
Published: October 2019

Abstract
Liver dysfunction is a major health burden world-wide. Future cell-based therapies for liver regeneration may benefit from the fact that bone marrow cells can fuse with or transdifferentiate into hepatocytes. All models demonstrating bone marrow to hepatocyte plasticity presented so far, however, have used highly artifical conditions of liver regeneration - applying toxins, genetic pressure models or liver resection. We have set up a model of transgenic T cell induced bystander hepatitis in bone marrow chimeras to assess the effect of hepatitis, a common liver pathology in humans, as an enhancer of bone marrow to hepatocyte plasticity events.
MHC haplotype (Kb) transgenic bone marrow from 178.3 mice or control bone marrow from B10.BR (Kk) mice was transplanted into sublethally irradiated B10.BR (Kk) mice. Hepatitis was induced by repeated injections of Des Kk T cell receptor transgenic T cells against the Kb antigen. In additonal groups Retrorsine was used as an agent inhibiting endogenous hepatocyte proliferation and GCSF for mobilisation of bone marrow stem cells.
Repeated injections of transgenic T cells induced subsequent waves of hepatitis in recipients of MHC haplotype transgenic bone marrow but not in control animals confirmed by serum ALT levels. Hepatocyte single cell suspensions from animals suffering from hepatitis revealed an increased expression of donor bone marrow derived antigen. This could be further enhanced by either increasing the number of circulating stem cells or by inhibiting the endogenous response of resident hepatocytes. FISH analysis showed fusion nuclei on a single cellular level.
T cell receptor transgenic T cells induce bystander hepatitis in an antigen specific manner. This inflammatory response drives the plasticity of bone marrow cells to hepatocytes and their potential contribution to liver regeneration. Fusion between donor cells and resident hepatocytes is the underlying mechanism of liver regeneration in this model mimicking a common liver pathology.
Low Bone Mass in Thalassemia: The Thalassemia Clinical Research Network (TCRN) Experience.
Authors: Vogiatzi Maria G. , Lane Joseph , Fleisher Martin , Macklin Eric A. , Fung Ellen B. , Kirby Melanie , Kwiatkowski Janet , Holm Ingrid , Olivieri Nancy , Vichinsky Elliott , Giardina Patricia J. 
Published: October 2019

Abstract
Low bone mass is emerging as a frequent and debilitating morbidity in Thalassemia (thal). The TCRN conducted a cross-sectional observational study to determine the prevalence and factors contributing to bone disease among North American thal patients (pts). Spinal (L1-L4) Bone Mineral Density (BMD) Z- and T-score measurements by DXA (Hologic 4500 and Delphi models) were read centrally. Each subject’s weight, height, hematologic, endocrine and genetic parameters, iron chelation and transfusion regimens, dietary calcium intake, history of fractures and bone pain, self-reported physical activity and bone turnover markers were assessed. BMD was measured in 302 pts: 207 Thal Major (TM), 37 Thal Intermedia (TI), 35 Beta E, 7 Hemoglobin H disease (HbH), 2 homozygous alpha (α) and 14 HbH/Constant Spring (HbH/CS). Among all diagnostic groups, the prevalence of low bone mass (LBM; Z/T <-2), reduced bone mass (RBM; Z/T -2 to -1) and normal bone mass (NBM; Z/T >-1) was 52%, 27% and 21%, respectively. LBM prevalence was 55% in TM, 53% in TI, 51% in Beta E, 0% in HbH, 50% in α and 43% in HbH/CS. RBM prevalence was 26% in TM, 22% in TI, 31% in Beta E, 71% in HbH, 50% in α and 29% in HbH/CS. Pt groups aged: 6–11 yrs, 11–20 yrs, 20+ yrs had Z/T-scores mean±SD[n] were: −1.32±0.82[51], −1.73±1.08[77] and −2.43±1.14[174], respectively. Z/T-scores were significantly lower among older pts (p<.001) and significantly higher among heavier pts after controlling for Tanner stage. Mean age-adjusted Z/T-scores of thal diagnostic groups and their slopes vs. age did not differ significantly although the samples of some groups were small. Among TM and TI pts, those with genotype β°/β° tended to have lower age- and weight-adjusted Z/T-scores (mean [95% CI]: −2.25 [−2.65 to −1.86], n=39). Less than 1% had hypoparathyroidism, 4% vit D deficiency, 8.5% diabetes mellitus, 8.5% hypothyroidism and 11.5% growth hormone deficiency (GHD). Only GHD was significantly correlated with decreased Z/T-scores after controlling for age and diagnosis. Urinary N-telopeptide (NTx) is elevated across all three age groups (median[IQR] mM BCE/mM creatinine: 664[456–930], 302[90–624], 69[34–124]), respectively. Preliminary analysis of bone turnover markers in a subset of subjects (n=114) suggests that NTx, urinary or serum was a significant independent predictor of spine Z/T-scores controlled for age and age-adjusted weight. There was no relationship between Z/T-score and serum osteocalcin. This large and comprehensive study of thal bone disease has demonstrated that decreased bone mass occurs with high frequency, worsens with age, is affected by weight and GHD and is associated with elevated NTx, i.e. increased bone resorption. Future studies are needed to identify efficacious long-term therapies to improve thal bone disease.
Stabilization of Human Erythrocytes for Lyophilization: Recovery of Biochemical Activities and Membrane Filterability.
Authors: Xu Shang-Zhi , Bode Arthur P. 
Published: October 2019

Abstract
It has much more advantages to preserve human red blood cells (RBC) in freezing and drying form than conventional blood storage, even though it is still under developing. In the present study, we successfully used 0.045% glutaraldehyde(GA) to fix RBCs under appropriate conditions prior to lyophilization and regained the bulk of RBCs membrane filterability. The phlebotomized whole blood was anti-coagulated with CPDA-1 and the plasma was removed by centrifugation. White blood cells were removed using Leucocytes filter (Pall Corporation). 1 x 10 9 cells/ml were separately incubated with 0.03%, 0.045% and 0.05% GA at 37 °C for 10 min; RBCs at concentrations 0.5 x 10 9, 1 x 10 9 and 2 x 10 9 cell/ml were incubated with 0.045% GA at 37 °C for 10 min; Bovine serum albumin (BSA), trehalose, hydroxyethyl starch and dextran were used as protective additive in freezing and drying RBCs, respectively; ethanolamine, glycine, lysine, BSA, glutamic acid and homocysteic acid were used to recover membrane deformability due to GA fixation. Recovery RBCs yield after reconstituion was determined by a cell counter. Fee hemoglobin , glutathione (GSH), 2,3-diphosphoglycerate (2,3-DPG), ATP, and Glucose-6-phosphate dehydrogenase (G-6-PDH) in RBCs were determined before and after lyophilization. Membrane flexibility was assayed by osmotic fragility test. RBCs filterability was determined using positive filter apparatus through 5 micrometer pore size nitrocellulose membrane. Our results suggested that RBCs at 1 x 10 9 cells/ml fixed by 0.045% GA for10 min at pH 7.8 were enough to resist freezing and drying damages and the bulk of RBCs’s membrane filterability were remained. The effective fixation of GA is dependent on GA’s concentration, RBCs concentration, incubation period and pH etc. BSA is the most potential additive in preserving RBCs. More interestingly, the following reagents can recover the reduced RBCs membrane filterability by GA fixation. The potency of recovery is in the order of : 10 mM ethanolamine > 5 mM lysine > 0.5%BSA > 10 mM glutamatic acid > 5 mM homcysteic acid > 2% glycine. After reconstitution, 85 ± 2.3% RBCs yield is achieved, 71 ± 4.1% of these RBCs can freely pass through 5 micrometer pore size filter membrane. Biochemical function indexes as GSH, 2,3-DPG, ATP, and G-6-PDH are saved by 20 ± 1.2%.
Lyophilized human red blood cells yields from reconstitution by different treatment.
1x 109 cells/ml fixed at 0.03% glutaraldehyde 44.7 ± 4.1 0.045%glutaraldehyde 84.7 ± 5.8 0.05%glutaraldehyde 88.7 ± 7.6 0.045% glutaraldehyde fix: 0.5x 109 cells/ml 91 ± 4.3 1x 109 cells/ml 84.7 ± 5.8 2 x 109 cells/ml 35.2 ± 2.3
Effects of various treatment on lyophilized RBCs after reconstitution.
Group Filterability(%) 1x9 cells/ml fixed by: N = 5. 0.03%glutaraldehyde 88.3 ± 1.2 0.045%glutaraldehyde 73.3 ± 1.8 0.05%glutaraldehyde 44.7 ± 2.8 0.045%glutaraldehyde fixation at: 0.5x109 cells/ml 0 1x109 cells/ml 73.3 ± 1.8 2x109 cells/ml 83.9 ± 4.6
The High Frequency of HLA-A*0206 Allele in Japanese Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and Its Significance.
Authors: Ikeda Kazuhiko , Shichishima Tsutomu , Akutsu Kazuko , Maruyama Yukio 
Published: October 2019

Abstract
PNH is an acquired hematologic disorder which is characterized by complement-mediated hemolysis, thrombosis, and bone marrow failure. Also, PNH is one disorder of bone marrow failure syndromes, including aplastic anemia (AA) and myelodysplastic syndrome (MDS). It is well known that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) contribute to pathophysiology of these disorders. In fact, some reports (Maciejewski et al, Blood, 2001; Shichishima et al, Blood, 2002) showed that HLA-DR*1501 is related with clinical pathophysiology of PNH. In this study, to clarify significance of CD8+ CTLs in pathophysiology of PNH, we investigated HLA class I (A and B) alleles in Japanese patients with PNH (female: male=7:17), AA (female: male=14:15), and MDS (female: male=6:16) by high-resolution method using polymerase-chain reaction after obtaining informed consent and approval from the institutional Human Research Committee. Mean age ± standard deviation of PNH, AA, and MDS patients was 52 ± 16, 54 ± 20, and 59 ± 18, respectively. HLA genotyping showed that the frequency of HLA-A*0206 allele in PNH patients (22.9%) was significantly different from those in 309 unrelated Japanese individuals (Saito et al, Tissue Antigens, 2000) (7.7%; p<0.02) and AA patients (5.2%; p<0.01). In contrast, we found no significant differences in the frequencies of the other alleles between PNH, AA, or MDS patients and the controls or between these disorders, except for high frequency of HLA-B40 alleles in AA patients (Nakamura et al, Blood, 2003). Then, various clinical parameters, including peripheral blood, bone marrow blood, and laboratory findings, proportions of glycosylphosphatidylinositol protein-negative population in erythrocytes, granulocytes, and monocytes, findings of chromosomal analyses and HLA-DR alleles, transfusion requirements, past history of AA, and history of thrombosis, were statistically compared between HLA-A*0206-positive group (n=10) and -negative group (n=14) in PNH patients. Statistical analyses showed that the reticulocyte counts , the values of lactate dehydrogenase, and the frequency of PNH patients with over 30% of CD59− erythrocytes in HLA-A*0206-positive group were significantly higher than in HLA-A*0206-negative group (121 ± 49 x 109/L vs 76.9 ± 43.9 x 109/L, p<0.03; 2866 ± 2606 IU/L vs 938 ± 775, p<0.02; and 80.0 % vs 28.6 %, p<0.02, respectively). Moreover, we found no AA (n=3) and MDS (n=5) patients with both the HLA-A*0206 allele and more than 1% of CD59− granulocytes. In conclusion, our findings suggest that the HLA-A*0206 allele in PNH may be correlated with the grade of the disease by complement-mediated hemolysis during negative selection of PNH clones, probably due to immunologic mechanisms by CD8+ CTLs.