bluuebunny/crossref_metadata_2025_split huggingface dataset:
HbF-Associated Genetic Variation Marks an Erythroid Regulatory Element Essential for BCL11A Transcription and Subsequent Stage-Specific Globin Expression
Authors: Bauer Daniel E. , Kamran Sophia C , Xu Jian , Lin Carrie , Shao Zhen , Fujiwara Yuko , Voit Richard A. , Porteus Matthew H. , Stamatoyannopoulos John A. , Orkin Stuart H. 
Published: October 2019

Abstract
Abstract 828
Genome-wide association studies (GWAS) have identified common genetic variation within a 14-kb segment of intron-2 of the BCL11A gene that is associated with the level of fetal hemoglobin (HbF) and the clinical severity of the β-globin disorders sickle cell disease and β-thalassemia. Functional studies have validated BCL11A as a major repressor of HbF. Here, we explore the consequences of genetic variation on BCL11A expression. The HbF-associated variants overlap BCL11A sequences that possess an evolutionarily conserved enhancer chromatin signature, including the presence of H3K4me1 and H3K27ac and the absence of H3K4me3 histone modifications. Moreover, these sequences show a unique pattern of DNase I hypersensitivity in erythroid precursors compared to other BCL11A-expressing lineages including fetal brain and B-lymphocytes. Occupancy of the major erythroid transcription factors GATA1 and TAL1 is observed at these DNase I hypersensitive sites (DHS). Chromosome conformation capture studies demonstrate that these DHSs form a long-range interaction with the BCL11A promoter.
To address the function of the putative regulatory element, we generated transgenic mice with the 12-kb intronic region attached to a minimal promoter and reporter gene. The element enhanced reporter gene expression in an erythroid-lineage restricted, definitive erythroid stage-specific manner. The minimal enhancer activity was mapped to the +58-kb DHS. Similar enhancer activity was observed in primary human erythroid precursors. To address the possible requirement of this element for BCL11A expression, we deleted the corresponding region in adult-stage murine erythroleukemia cells with TALEN-mediated gene targeting. Expression of BCL11A was dramatically reduced and embryonic globins Hbby and Hbb-bh1 were markedly derepressed with concomitant reduction of adult globin Hbb-b1. Analysis of heterozygous primary human erythroid precursors revealed that low-HbF haplotype alleles are preferentially occupied by GATA1 and TAL1 at sequences surrounding rs1427407. A composite half-E-box–GATA motif is disrupted at this SNP in the high-HbF allele. Furthermore, the low-HbF haplotype allele of BCL11A is expressed at a 1.7-fold higher level relative to the high-HbF allele. These data demonstrate that HbF-associated genetic variation in BCL11A modulates the function of a critical erythroid enhancer of BCL11A, and consequently expression of BCL11A itself. The quantitative level of BCL11A in turn regulates the relative expression of the stage-specific globin genes. The essential erythroid enhancer of the BCL11A gene might itself serve as a target for genetic manipulation as a novel approach to the human β-globin disorders.

Disclosures:
Porteus: Alex's Lemonade Stand Foundation: Research Funding.
Activation of A1 Domain Adhesiveness in von Willebrand Factor by Elongational Force
Authors: Springer Timothy A. , Kim Jongseong 
Published: October 2019

Abstract
Abstract SCI-16
Multiple mechanisms may contribute to activation of von Willebrand factor (vWF) adhesiveness by elongational flow at sites of hemostasis, including enhancement of A1 domain exposure within vWF concatamers and conformational change within the A1 domain or its complex with GPIbα. A receptor and ligand in a single molecule (ReaLiSM) containing the A1 domain, a flexible linker, and GPIbα fused in a single polypeptide and suspended between beads using DNA handles was interrogated with a laser trap. Two pathways for unbinding representing flexed and extended states were previously reported, with the flexed, more stable state predominantly at forces above 10 pN. vWD type 2B mutations in the A1 domain selectively stabilize the extended, high affinity state, whereas platelet-type vWD mutations in GPIbα stabilize both states. With ReaLiSM, we can also measure the kinetics and force-dependence of receptor-ligand binding. Remarkably, we also see two on-rates for receptor-ligand binding, with the faster on-rate predominating above 10 pN and the slower on-rate predominating below 10 pN. vWD type 2B mutations in the A1 domain selectively increased the fast on-rate, whereas platelet-type mutations in GPIbα increased both on-rates. Our results support force-dependent conformational change as one of the mechanisms that activates vWF in hemostasis.

Disclosures:
No relevant conflicts of interest to declare.
Biology of Myelodysplasia Clonality: Clinical Implications of Whole-Genome Sequencing
Authors: Walter Matthew J. 
Published: October 2019

Abstract
Abstract SCI-32
The discovery of acquired genetic mutations associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has rapidly expanded with the use of next-generation sequencing technologies. The distinction between MDS and de novo or secondary AML (arising after MDS) and therapeutic recommendations are largely based on the presence of morphologic dysplasia and the percentage of bone marrow myeloblasts. Using whole genome sequencing to define the allele burden of hundreds of mutations, our group has shown that nearly all the bone marrow cells in seven patients with MDS and secondary AML were clonally derived (∼85%), regardless of the myeloblast count. We now have experimental evidence suggesting that the vast majority of mutations in these samples are randomly acquired during aging (passenger mutations) and carried forward in a cell when it is transformed. By using all the validated mutations in a sample (passengers and drivers) we found that five of the seven MDS samples harbored two or more malignant clones and that all seven contained a founding clone that gave rise to subsequent daughter clones. Thus, the MDS bone marrow samples are a mosaic of several tumors with unique sets of mutations each shaped by the acquisition of a series of mutations and clonal diversification. Each clone contained at least one coding gene mutation, and we identified 11 recurrently mutated genes in the seven samples, including recently described mutations in U2AF1, a spliceosome gene that is typically mutated in the MDS founding clone. In addition, the MDS founding clone always gave rise to the dominant secondary AML clone, suggesting that therapies targeting mutations in the founding clone may have benefit in treating both MDS and secondary AML. It is possible that therapeutic response and disease progression is driven not only by the presence of recurrent mutations, which have prognostic value, but also by the clone (i.e., founding and daughter) in which they arise. We are currently testing this hypothesis by monitoring the dynamic change in clone size, including driver mutation allele burdens, during treatment of patients with AML and MDS.

Disclosures:
No relevant conflicts of interest to declare.
Microbial Impact on Hematologic Homeostasis
Authors: Manz Markus G. 
Published: October 2019

Abstract
Abstract SCI-40
During systemic infection and inflammation, immune effector cells are in high demand and are rapidly consumed at sites of need. While adaptive immune cells have high proliferative potential, innate mature immune cells are mostly postmitotic and need to be replenished from bone marrow hematopoietic stem and progenitor cells. Indeed, severe clinical infection, particularly infections challenging the innate immune response, lead to an increase in hematopoietic differentiation and throughput in bone marrow, involving subsequent differentiation stages from hematopoietic stem cells, multipotent progenitors, as well as early-lineage and late-lineage restricted hematopoietic progenitors. A fundamental question is how the increased need is sensed and translated in enhanced production and how adequate levels of response are guided. Recent research has shed light on conserved intracellular and extracellular pathogen recognition receptors, such as Toll-like receptors, that are expressed on nonhematopoietic and hematopoietic effector cells and cause activation upon ligation. This activation results in production of hematopoietic growth, survival, activation, and migration factors operating at site on effector cells, but also at remote primary hematopoietic sites to increase production upon need. Recent research by several groups, including ours, surprisingly revealed that conserved pattern-recognition receptors are also expressed on hematopoietic stem and progenitor cells in bone marrow, implying a direct effect of systemically available ligands on these cellular populations. Indeed, it has been demonstrated that, for example, ligation of Toll-like receptor 4 by its cognate agonist lipopolysaccharide can lead to divisional activation, proliferation, lineage-directed differentiation, and migration of hematopoietic stem and lineage-restricted progenitor cells, all aimed at efficient contribution to immune responses and rapid reestablishment of hematopoietic homeostasis. The relative contribution of pathogen sensing by hematopoietic and diverse nonhematopoietic cells to appropriate hematopoietic responses, as well as the subcellular translation of the signals, is the focus of ongoing research. Also to be discussed will be how chronic infectious and inflammatory processes, which are frequently associated with aging, might impinge on hematopoiesis, potentially fostering hematopoietic stem cell diseases as exhaustion or transformation.

Disclosures:
No relevant conflicts of interest to declare.
Cyclophosphamide Related Cardiac Complication in Hematopoietic Transplantation: Analysis of 1091 Cases in Fukuoka Blood and Marrow Transplantation Group
Authors: Kohno Kentaro , Takase Ken , Kadowaki Masanori , Yamasaki Satoshi , Kamimura Tomohiko , Ohno Yuju , Eto Tetsuya , Miyamoto Toshihiro , Okamura Seiichi , Akashi Koichi 
Published: October 2019

Abstract

<Background and Method> Cyclophosphamide (CPM) is one of key drugs for conditioning therapy in hematopoietic transplantation in terms of anti-tumor effect as well as immune-suppressant activity. On the other hand, high dose CPM administration is related to rare but life-threatening cardiac complications, of which incidence and pathogenesis are not fully understood. We retrospectively reviewed the hematopoietic transplantation cases in Fukuoka Blood and Marrow Transplantation Group (FBMTG) during 2000/1/1 to 2013/7/31 and analyzed the cases with the cardiac complications.
<Results> There were 1091 cases, which contained CPM administration in their conditioning regimens. Of which, 23 cases (2.1%) were considered as cardiac complications related to CPM. The mean age at transplantation was 51 (20-74). All of the cases underwent cardiac function surveillance with ultrasound echocardiogram and were administrated more than 45mg/kg/day of CPM. The clinical cardiac manifestation occurred one to seven (mean 3) days after the first administration of CPM. The electrocardiogram showed the statistically significant diminishment of SV1 + RV5 and increase of heart rate. Lowered ejection fraction was observed in ultrasound echocardiogram. Myocardial enzymes such as CPK, Troponins and H-FABP were not very reliable because some cases were negative especially in early phase. Fourteen cases (1.3%) developed acute circulatory failure soon after the first presentation of clinical manifestation and all of them end up with poor prognosis. On the other hand, of the 9 cases (0.8%) who survived first 20 days, 6 of them were alive at 100 days after the transplantation. The incidence of cardiac complication tends to be high in elderly patients especially when they were over 70.
<Conclusion> Cardiac complication related to CPM is rare but it remains to be fatal in present. The development of supportive care has provided higher limit of age that undergo hematopoietic transplantation. However, the administration of high dose CPM in conditioning regimen should be aware especially in elderly recipients.

Disclosures
No relevant conflicts of interest to declare.
Ineffective Erythropoiesis Is the Major Cause of Microcytic Anemia in the TSAP6/Steap3 Null Mouse Model
Authors: Blanc Lionel , Papoin Julien , Vidal Michel , Amson Robert , Telerman Adam , An Xiuli , Mohandas Narla 
Published: October 2019

Abstract
STEAP3 (Six-Transmembrane Epithelial Antigen of Prostate 3) is the major ferrireductase in the erythroblast. Also named TSAP6 (Tumor Supressor Activated Pathway 6) after it had been found to play a role in cancer, its total ablation in the mouse leads to severe microcytic and hypochromic red cells with moderate anemia. The protein function appears conserved among mammals, as patients carrying a nonsense mutation in the TSAP6/STEAP3 gene have been reported with hypochromic anemia. Here, we investigated the mechanism leading to the anemia. In the present study, using the TSAP6/Steap3 knockout mice, we undertook a comprehensive hematologic characterization of the red cell compartment. Red cell indices derived using ADVIA 120 blood counter confirmed the hypochromic microcytic anemia phenotype with a marked reduction in the mean corpuscular volume (MCV; 21.5fL ± 1.3fL in knockout vs 45.2fL ± 1.5fL in wild-type X ± SD, p<0.001) and mean cell hemoglobin content (MCH; 5.8pg ± 0.1pg vs 14.5pg ± 0.1pg, p<0.001). The reticulocyte count was marginally elevated (5.7% ± 1.2% vs 2.6% ± 0.4%, p<0.05) indicating that the anemia is proliferative. By phase contrast microscopy and transmission electron microscopy, we observed marked anisocytosis as well as the presence of fragmenting erythrocytes. Consistent with this observation, we found by ektacytometry decreased membrane mechanical stability of knockout red cells. Interestingly, we were unable to document significant changes in the expression levels of the major skeletal and transmembrane proteins to account for this decrease in the membrane stability.
As defects in either the production or destruction of red cells can lead to anemia, we measured red cell survival and erythropoiesis in these mice. No differences in red cell survival could be documented using biotin labeled red cells implying that decreased survival cannot account for the anemia. However, when we monitored erythropoiesis using Ter119, CD44 and Forward Scatter (FSC) as markers of terminal differentiation, we found a decreased number of proerythroblasts in the bone marrow of TSAP6/Steap3-/- animals (2.20% ± 0.49% vs 4.07% ± 0.77%, p<0.01). In addition, progression from the proerythroblastic to the orthochromatic stage was also affected, with accumulation of cells at the polychromatic stage. These findings imply that ineffective erythropoiesis is the dominant cause of anemia in these mice and that TSAP6/Steap3 may play a role during erythropoiesis beyond its role as ferrireductase. It is interesting to note that while the phenotype of microcytic red cells with decreased hemoglobin content in the TSAP6/Steap3 null mouse is similar to red cells in thalassemic and porphyria mice, there are distinct differences in the etiology of the hematologic phenotype.

Disclosures
No relevant conflicts of interest to declare.
Age As an Independent Clinical Predictor for Vitamin D Deficiency in Sickle Cell Disease
Authors: Han Jin , Saraf Santosh L. , Abbasi Taimur , Zhang Xu , Molokie Robert E. , Hassan Johara , Gowhari Michel , Alhandalous Chaher , Machado Roberto F. , Gordeuk Victor R. 
Published: October 2019

Abstract
Background: Vitamin D deficiency (VDD) is highly prevalent among patients with sickle cell disease (SCD). Although little is known about the risk factors for VDD in SCD, it has been shown that VDD is associated with chronic pain, bone fragility, and pulmonary function in SCD. In this study we investigated the potential clinical predictors for VDD in patients with SCD.
Method: In a retrospective, cross-sectional analysis, a total of 167 adults with SCD treated at the University of Illinois Medical Center with a baseline 25-hydroxy vitamin D (25-OHD) measurement were screened. Clinical variables were recorded from a clinic visit at least four weeks from a vaso-occlusive pain episode or red blood cell transfusion. Statistical association between 25-OHD and other clinical parameters were investigated.
Results: After stratifying the patients based on 25-OHD levels, we observed the median age was significantly younger in patients with lower 25-OHD levels (Table 1). When analyzing different age groups by Kruskal Wallis analysis, 25-OHD levels were significantly elevated in patients ≥ 40 years old (Figure 1). When using Spearman correlation analysis, the 25-OHD levels as a continuous variable positively correlated with increasing age (p<0.001); they also showed a significant negative relationships with creatinine clearance, total bilirubin, platelet count, and white blood cell count (Table 2). Using ordinal logistic regression, age was an independent predictor of 25-OHD levels, as a three-categorical variable, in SCD (OR 0.55, 95% CI: 0.38 – 0.81; p =0.002) after adjusting for gender, creatinine clearance, and vitamin D supplementation (Table 3), which means that younger patients has higher chance of VDD. In the patients with VDD (25-OHD <20 ng/mL), weekly supplementation with oral ergocalciferol (50,000 units for twelve weeks) substantially improved 25-OHD levels (9.9 vs 23.7 ng/mL, p<0.0001, N=24). During a median of 40-month follow-up (range 0 to 96 months), thirteen patients died, but the log rank test or multivariate Cox regression analysis failed to show statistical significance between 25-OHD levels and mortality after adjusting ESRD and baseline vitamin D supplementation, likely due to a short follow-up period and a small sample size.
Summary: Lower 25-OHD levels were associated with younger age in patients with SCD, especially patients younger than 40 years old. One possible explanation is that lower 25-OHD levels may be linked to higher mortality in SCD, but future research is needed to clarify the association between VDD and mortality in SCD.

Disclosures
No relevant conflicts of interest to declare.
Posttranslational Modification in Megakaryocytes Regulates β1 Integrin Expression, Migration and Platelet Production in Vivo
Authors: Giannini Silvia , Adelman Max , Begonja Antonija Jurak , Hoffmeister Karin M 
Published: October 2019

Abstract
Platelet recovery following bone marrow transplant and radiochemotherapy is crucial to prevent bleeding complications. Defects in glycosylation have been associated with decrease in blood platelet counts. However, the role of posttranslational modification in platelet production remains elusive. We here investigated the role of β1,4 galactosyltransferase 1 (β4GalT1), the key glycosyltransferase regulating lactosaminoglycan (LacNAc or βGal1,4 GlcNAc) expression by adding galactose (Gal) to terminal N-acetylglucosamine (GlcNAc), in platelet production. Stromal cell-derived factor 1 (SDF-1), but not thrombopoietin promotes megakaryocyte (MK) migration towards the bone marrow sinusoids, thereby increasing platelet production. SDF-1 (CXCL12) upregulated LacNAc expression in fetal liver wild type (WT), but not β4galt1-null megakaryocytes (MKs) lacking β4GalT1, suggesting that SDF-1 promotes LacNAc expression in vivo to regulate MK migration and platelet production. In support of this hypothesis, β4galt1-null mice had severe macrothrombocytopenia with increased bone marrow MK numbers, but normal platelet clearance. Ploidy, expression of the major glycoproteins (GPIbα/V/IX and αIIbβ3) and the surface expression of the SDF-1 receptor CXCR4 were normal in β4galt1-null bone marrow MKs. However, β4galt1-null bone marrow MKs had increased surface and total β1 integrin expression, as determined by flow cytometry, immunoblot and immunofluorescence. Mature CD42b/CD41 positive β4galt1-null MKs co-localized poorly with endoglin positive bone marrow sinusoids (49.9 ± 2.1 %), compared to WT MKs (72.4 ± 0.6 %). Expression of laminin, the major β1 integrin ligand, was upregulated in β4galt1-null MKs, suggesting that loss of LacNAc expression on β1 integrin increased its function. To exclude an extrinsic contribution to the failure of β4galt1-null MKs to migrate, β4galt1-null fetal liver (E14.5) hematopoietic cells (FLHCs) were transplanted in lethally irradiated WT mice. Transplanted β4galt1-null FLHCs restored bone marrow MKs, but failed to migrate to sinusoids and produce circulating platelets. In marked contrast, production of β4galt1-null white blood cells was normal. Together, our results suggest that SDF-1 upregulates β4GalT1-dependent LacNAc expression to promote MK migration and interaction with BM sinusoids, likely regulating MK β1 integrin expression and interaction with components of the extracellular matrix, specifically laminin. Understanding of the mechanisms regulating posttranslational modifications induced by various hematopoietic stimulating chemokines and cytokines will contribute to better platelet recovery following bone marrow transplant and radiochemotherapy.

Disclosures
No relevant conflicts of interest to declare.
Neutrophil Mobilization into Peritoneal Fluid
Authors: FRUHMAN GEORGE J. 
Published: October 2019

Abstract
Rats were injected intraperitoneally with various solutions, and the local cellular responses were studied using quantitative methods. Contrary to reports in the literature, pure saline did not evoke a local neutrophilia after it was injected intraperitoneally. Under similar conditions, bacterial lipopolysaccharides proved to be highly effective. Trace doses were most effective; larger doses caused a marked diminution of the response. Furthermore, a variety of nonbacterial substances were impotent when tested under comparable conditions.
THE TREATMENT OF LYMPHOBLASTIC LEUKEMIA WITH CRUDE MYELOKENTRIC ACID
Authors: MILLER F. R. , HERBUT P. A. , JONES H. W. 
Published: October 2019

Abstract
Eight cases of blastic lymphoid leukemia have been treated with myelokentric acid in crude form, because hypothetically in blastic lymphoid leukemia there is a deficiency of this material. The crude myelokentric acid was used because it was more easily obtained than partially purified material. Purification of biologically active materials by methods of extraction and precipitation necessarily results in a considerable loss of material. Thirteen partial remissions occurred following the administration of crude myelokentric acid. Seven of the 8 patients have died, and 5 necropsies were performed.
The necropsy material adds further weight to the belief that the remissions were induced by the myelokentric acid in that in all 5 necropsies there was a definite alteration in the histologic morphology as contrasted with the findings in the necropsies of the controls.
It seems inadvisable, however, to treat a large number of patients with this material because it is crude, it is relatively unavailable, and no standard dose has yet been devised.