bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Robust Ex Vivo culture System for Production of Mature Human Reticulocytes From a Single Donor
Authors: Cogan Nicola , Kupzig Sabine , Parsons Steve F , Anstee David J 
Published: October 2019

Abstract
Abstract 2096
We have formulated a robust culture system simulating human erythropoiesis, producing mature reticulocytes from adult peripheral blood CD34+ progenitor cells in 20 days. Expansion of cell numbers over the course of the culture exceeded 104-fold and enucleation efficiency was up to 90% without the use of feeder layers. Mature reticulocytes and erythrocytes were isolated from culture by passage through a standard leucocyte filter. Scanning and transmission electron microscopy of the reticulocytes revealed morphological features indistinguishable from those of mature reticulocytes isolated from the peripheral blood of normal donors. Post-filtration cells expressed adult haemoglobin and had a capacity to bind and release oxygen comparable to that of adult peripheral blood erythrocytes. Examination of blood group active- and other cell surface antigens revealed a normal glycosylation profile and normal cell surface protein expression. Most notably, the cells did not express cryptantigens T and Tn. LORCA analysis indicated that cultured cells had slightly reduced membrane stability and deformability compared to peripheral blood erythrocytes. Haematology parameters demonstrated a typical mean cell volume of 131fL and mean cell haemoglobin of 37.7pg. This, scalable approach has allowed us to culture cells from a single apheresis cone to produce 1010 reticulocytes, which represents a packed cell volume of 1ml. These results clearly demonstrate the feasibility of producing cultured red cell products suitable for transfusion therapy ex vivo from adult peripheral blood stem cells.

Disclosures:
No relevant conflicts of interest to declare.
Functional Evaluation of HbF-Associated Region of BCL11A Locus
Authors: Bauer Daniel E. , Xu Jian , Fujiwara Yuko , Stamatoyannopoulos John A. , Orkin Stuart H. 
Published: October 2019

Abstract
Abstract 2148
The severity of the major β-globin disorders, sickle cell disease and β-thalassemia, is modified by the residual level of fetal hemoglobin (HbF, α2γ2). Reactivation of HbF by derepression of γ-globin gene expression constitutes a major therapeutic goal, though an incomplete understanding of globin gene regulation has limited such efforts. The postnatal level of HbF is a highly heritable quantitative trait. Genome-wide association (GWA) studies have identified several HbF-associated loci, including the BCL11A gene. BCL11A has been demonstrated to be a bona fide repressor of γ-globin expression in mouse and man. However, the mechanisms by which common genetic variation modulate BCL11A expression remain poorly understood. Numerous GWA studies, conducted in both healthy individuals and those with hemoglobinopathies, and including populations of European, Asian, and African ancestries, have consistently found that HbF-associated variants cluster within a 14-kb region of the second intron of BCL11A. At least twenty HbF-associated SNPs have been identified within this region, with various of these genetic markers considered as “tag SNPs” most strongly associated with HbF levels depending on the particular GWA study. Based on patterns of linkage disequilibrium among SNPs, several common haplotypes have been identified. The tag SNP-dense intronic region, more than 55 kb downstream of the BCL11A transcription start site, demonstrates evolutionary sequence conservation. Here we show that this region of BCL11A is decorated by epigenetic features in human erythroid precursors indicative of distal regulatory potential. Specifically, this intronic region is marked by the presence of the histone modifications H3K4me1 and H3K27ac and the absence of H3K4me3, indicating an enhancer signature. The erythroid transcription factors GATA1 and SCL/TAL1 co-occupy discrete sequences within this region, with the binding peaks of these master erythroid regulators overlapping those of the histone marks. Furthermore, this tag SNP-dense intronic region of BCL11A displays CpG hypomethylation and exhibits DNase I hypersensitive sites restricted to the erythroid lineage but not other lineages that express BCL11A. Moreover, in in vivo transient transgenic assays, this region displays erythroid-restricted enhancer activity. A minimal 1.8-kb sequence possessing erythroid enhancer function has been mapped. This region includes GATA1 and SCL/TAL1 binding sites. We hypothesize that common polymorphisms are in linkage disequilibrium with functional variants that modulate erythroid enhancer activity. These studies further delineate pathways critical for hemoglobin switching and serve as an example of how common genetic variation may influence traits by affecting non-coding regulatory elements.

Disclosures:
No relevant conflicts of interest to declare.
Relationship Between Pulmonary Artery Pressure and Spleen Volume in Non-Splenectomized Hemoglobin E/β-Thalassemia Patients
Authors: Atichartakarn Vichai , Chuncharunee Suporn , Yamwong Sukit , Yingchoncharoen Teerapat , Jongjirasiri Sutipong , Archararit Napaporn , Udomsubpayakul Umaporn , Aryurachai Katcharin 
Published: October 2019

Abstract
Abstract 2156

Introduction:
Clinically significant pulmonary arterial hypertension (PAH) has been reported in splenectomized hemoglobin E/β-Thalassemia patients. Like primary PAH, the underlying basis is hypercoagulability related thrombotic pulmonary arteriopathy, which results in an increased pulmonary vascular resistance. In the non-splenectomized counterparts with no clear evidence of hypercoagulability only mild PAH is found. To evaluate role of splenomegaly and the accompanying increased blood volume in the pathogenesis of PAH in this group of patients, we studied the relationship between spleen volume and PA pressure.

Patients and Methods:
Nineteen clinically stable hemoglobin E/β-Thalassemia outpatients, on no medication aside from folic acid and who received no blood transfusion in the preceding 4 weeks were first analyzed in this ongoing study. All gave written informed consent, and study protocol was approved by the institution ethics committee on studies in humans (ID 07–53-01). PA pressure (PAP) was estimated by a single echocardiologist (T.Y.) using Philips iE33 echocardiographic system with a S5 probe (Philips Medical Systems, Andover, MA). Spleen volume was determined by CT scan with non-contrast, 320-slice, 3 mm axial cut (Aquilion one, Toshiba, Japan). Statistical analysis of clinical and laboratory data was done by SPSS version 18 (SPSS Inc, Chicago, Ill), considering a P value <0.05 as statistically significant. Results: Patients' characteristics are shown. Results are expressed as mean ± SD or median (range) where appropriate. All had normal left ventricular systolic ejection fraction and no valvular heart disease. There was good correlation (r=0.609, P=0.006) between spleen volume and mean PAP.

Conclusions:
Non-splenectomized patients with hemoglobin E/β-Thalassemia have elevated PAP in proportion to spleen size, likely secondary to elevated blood volume. This situation is distinct from the pathologic PAH that results from increased pulmonary vascular resistance in splenectomized patients.

Disclosures:
No relevant conflicts of interest to declare.
Onset of Cardiac Iron Loading in a Large and Homogeneous Cohort of Thalassemia Major Paediatric Patients
Authors: Pepe Alessia , Meloni Antonella , Favilli Brunella , Capra Marcello , D'Ascola Domenico Giuseppe , Palazzi Giovanni , Quarta Antonella , Casini Tommaso , Putti Maria Caterina , Cianciulli Paolo , Franceschi Lucia De , Positano Vincenzo , De Marchi Daniele , Lombardi Massimo , Filosa Aldo 
Published: October 2019

Abstract
Abstract 2157

Introduction:
Magnetic Resonance Imaging (MRI) by the T2* technique allows highly reproducible and non invasive quantifications of myocardial iron burden and it is the gold standard for quantifying biventricular function parameters. It is important to determine the appropriate age to start MRI screening, because its high cost. Few data are available in the literature and they are contrasting. So the aim of this study was to address this issue in our paediatric patients with thalassemia major (TM).

Methods:
We studied retrospectively 72 patients (47 males, 4.2–17.9 years old, mean age 13.03 ± 3.70 years), enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network. Myocardial iron overload was measured by T2* multislice multiecho technique. Biventricular function parameters were quantitatively evaluated by cine images.

Results:
The global heart T2* value was 29.7 ± 11.2 ms (range 6.2 – 48.0 ms). No significant correlation was found between global heart T2* value and age (see figure). The global heart T2* value did not show significant differences according to the sex (male 30.2 ± 11.0 ms versus female 28.7 ± 11.8 ms, P=0.568). Sixteen patients (22%) showed an abnormal global heart T2* value (<20 ms) and none of them was under 8 years of age. Global heart T2* value was negatively correlated with mean serum ferritin levels. Odds Ratio for high serum ferritin levels (≥ 1500 ng/ml) was 8.4 (1.01–69.37, OR 95%CI) for abnormal global heart T2* values (< 20 ms). The global heart T2* value did not show a significant difference with respect to the chelation therapy (P=0.322). No significant correlations were found between the global heart T2* values and the bi-atrial areas or the LV and RV morphological and functional parameters. Eight patients showed a left ventricular (LV) ejection fraction (EF) < 57% and none of them was under 7 years of age. Two patients showed a right ventricular (RV) EF < 52% and none of them was under 14 years of age.

Conclusion:
The MRI screening for both cardiac iron overload and function assessment can be started for TM patients at the age of 7 years. At this age not sedation is generally needed. If the availability of cardiac MRI is low, the serum ferritin levels could be used as a discriminating factor.

Disclosures:
No relevant conflicts of interest to declare.
Leukocyte Integrin Activation and Alterations in Parathyroid Hormone Levels Following G-CSF Administration
Authors: Donahue Robert E. , Tuschong Laura , Bauer Thomas R. , Hickstein Dennis D. , Yau Yu Ying , Leitman Susan F. 
Published: October 2019

Abstract
Abstract 2164
Following administration of granulocyte colony-stimulating factor (G-CSF) there is a marked but transient drop in circulating neutrophils. To determine the contribution of integrin activation to these events, a monoclonal antibody (mAb 24) that recognizes the activated α-subunit cation binding domain of the integrin family of receptors was used to evaluate human leukocytes following G-CSF administration. A single 480 mcg dose of human recombinant G-CSF (Neupogen, Amgen) was administered in the upper arm of six healthy human subjects (3 male, 3 female). EDTA and heparinized blood samples were collected prior to and at 15, 30, 60, 90, 120 and 240 minutes after injection. Mean absolute neutrophil count decreased from 2,557 ± 654/μl at baseline to 227 ± 193 (p<0.001) at 30 and 45 minutes. Flow cytometric analysis revealed a dramatic increase in binding of mAb24, which recognizes the occupied divalent cation binding site of the activated integrin α subunit, to neutrophils but not other circulating nucleated cells within 15 to 30 minutes after G-CSF administration, concurrent with a dramatic decrease in circulating neutrophil numbers. This activation, like the drop in neutrophil count, was transient, with a return to baseline status by 60 minutes. Intact parathyroid hormone (iPTH) levels were also evaluated. Within 30–90 minutes following G-CSF injection there was a significant drop in iPTH from a baseline of 52.5 ± 31.1 to a nadir value of 30.6 ± 20.9 pg/ml (p=0.01). A rebound in iPTH levels to 63.4 ± 27.0 pg/ml (p<0.001) occurred at 90 to 240 minutes. There were no significant changes in ionized calcium or magnesium levels. These results demonstrate that the integrin on circulating neutrophils is transiently activated following G-CSF administration and that this activation may mediate the transient neutropenia seen in this setting. In addition, alterations in PTH levels occur following G-CSF administration. As it is known that iPTH influences the hematopoietic stem cell (HSC) niche, fluctuations in iPTH levels following G-CSF administration may play a role in HSC expansion.

Disclosures:
No relevant conflicts of interest to declare.
Non-AIDS-Defining Hematologic Malignancies in HIV-Infected Patients: A Nationwide Epidemiologic Study in Japan
Authors: Hagiwara Shotaro , Yotsumoto Mihoko , Okada Seiji 
Published: October 2019

Abstract
Abstract 2183

Background.
The prevalence of HIV infection is estimated to be less than 0.1% in Japan; however, the number of newly reported HIV-infected persons has gradually increased, and the estimated number of HIV-infected patients now ranges from 6300 to 10000. Since the introduction of highly active antiretroviral therapy (HAART), the morbidity and incidence of AIDS-defining cancers has decreased. However, the incidence of non-AIDS-defining cancers has increased in Japan. To elucidate the incidence and clinical outcome of non-AIDS-defining hematologic malignancies, excluding non-Hodgkin's lymphomas, in HIV-infected patients, we conducted a nationwide epidemiologic study.

Methods.
We surveyed 429 regional AIDS centers and 497 educational hospitals certified by the Japanese Society of Hematology. Epidemiologic data for HIV was sourced from the Joint United Nations program on HIV/AIDS.

Results.
We obtained data from 511 institutes (55.4%). From 1991 to 2010, 47 cases of non-AIDS-defining hematologic malignancies were found—19 cases of Hodgkin's lymphoma (HD), 13 of AML, 7 of ALL, 4 of CML, 2 of multiple myeloma, 1 of CLL, and 1 of MDS-RAEB1. The mean age was 49.3 years (21–70 years), and 93.6% patients were male. The mean CD4-positive cell count was 303.7/μL (1–1371/μL). The mean duration from the diagnosis of HIV infection to hematologic malignancy was 45.0 months (0–204 months). Before diagnosis, 68.1% patients had been treated with HAART and 51.1% patients had AIDS. Sixteen of 19 HD patients were treated with ABVD or radiation therapy. Of these, 80% patients achieved complete remission, with a 2-year overall survival of 62.3%. Twelve of AML and 6 of ALL patients were treated with standard induction chemotherapy. Eight of twelve patients (66.7%) with AML and five of six patients (83.3%) achieved complete remission. The median survival for both AML and ALL patients was 16 months, with 42.8% ALL and 30.8% AML patients alive after 4 years. Comparison of the last 2 decades (1991–2000 and 2001–2010) showed a 4.7-fold increase in the incidence of hematologic malignancies (11.5 vs. 54.6/100000 person-years).

Conclusion.
A nationwide epidemiologic study revealed that HIV-infected patients are at high risk of hematologic malignancies, and that the incidence of hematologic malignancies has increased in the past decade. The prognosis was equivalent to that of HIV-negative patients, and standard chemotherapy may be a feasible treatment option for HIV-infected patients with hematologic malignancies.

Disclosures:
No relevant conflicts of interest to declare.
In Vitro Generation of “Long-Lived” Human Plasma Cells
Authors: Stephenson Sophie , Cocco Mario , deTute Ruth , Rawstron Andy , Doody Gina , Tooze Reuben 
Published: October 2019

Abstract
Abstract 2187
Plasma cells, the terminal effectors of the B-cell lineage include both short- and long-lived cells. The latter persist for extended periods in the absence of cell division, supported in niche environments. No model system has successfully recapitulated the function of the niche to allow the in vitro generation of long-lived plasma cells. This limits investigation into the factors controlling and targeting plasma cell populations. Here we describe the generation of mature human plasma cells with extended lifespan from peripheral blood B-cells. Cell division accompanies phenotypic maturation between plasmablasts and plasma cells. These cells then persist in the absence of cell division, remaining functional and viable in extended culture, currently limited solely by elective termination. Extended survival is accompanied by maturation to a phenotype consistent with human bone marrow plasma cells. By establishing a set of conditions sufficient to allow the development and persistence of mature human plasma cells in vitro, we recapitulate the essential function of the plasma cell niche. We definitively link phenotypic maturation to lifespan and provide the first platform with which to explore and manipulate the full trajectory of human plasma cell differentiation.

Disclosures:
No relevant conflicts of interest to declare.
Pathological Shear Regulates ADAM10 Activity on Circulating Platelets
Authors: Gardiner Elizabeth E , Qiao Jianlin , Tan Cheewee , Arthur Jane Frances , Al-Tamimi Mohammad , Ludwig Andreas , Ward Christopher M , Andrews Robert K 
Published: October 2019

Abstract
Abstract 2194
Metalloproteinase-mediated ectodomain shedding of platelet-specific receptors for collagen (GPVI) and von Willebrand factor (GPIbα of the GPIb-IX-V complex) is triggered by either ligand-induced platelet activation-dependent pathways, or by activation-independent pathways mediated by Factor Xa or induced by the thiol-modifying agent, N-ethylmaleimide. We recently reported that shed soluble GPVI (sGPVI) was elevated in plasma of 159 ischaemic stroke patients compared with 159 community-based controls (P=0.0168), and in 29 patients with disseminated intravascular coagulation compared with healthy donors (n=25, P=0.002), consistent with a pathophysiological role for GPVI shedding from human platelets. Our new studies now show that transient exposure of human platelets to arterial or pathological shear rates of 3000–10,000 s−1 for 1–5 min ex vivo in a cone-plate viscometer, in the absence of GPVI ligand or platelet activation, activated sheddases producing a 2- to 3-fold increase in plasma sGPVI and a corresponding loss of surface GPVI from sheared platelets. Shear-induced GPVI shedding was blocked by GM6001 or GI254023, a selective inhibitor of ADAM10. In contrast to shear-induced platelet aggregation, shedding was unaffected by inhibitors of aggregation (VWF-blocking anti-GPIbα mAb, AK2, or the αIIbβ3 antagonist, RGD peptide) or by the absence of VWF in a patient with von Willebrand's disease Type III (VWF antigen levels <1%). Further, shedding of GPVI increased for up to 10 min after cessation of a short exposure of platelets to shear even when signalling, secretion and aggregation was blocked by inhibiting intracellular kinases (PP2, piceatannol), thromboxane generation (aspirin), ADP (apyrase) and calcium flux (BAPTA). Together, the combined results provide the first evidence that receptor sheddase activity can be regulated by hydrodynamic shear stress independent of cellular activation. This may represent a novel protective mechanism for down-regulating platelet reactivity as a response to pathological shear.

Disclosures:
No relevant conflicts of interest to declare.
Trypsin Causes Platelet Activation Independently of Known Protease-Activated Receptors
Authors: Mao Yingying , Kunapuli Satya P. 
Published: October 2019

Abstract
Abstract 2197
In platelets, Thrombin is the major agonist for PAR1 and PAR4, although PARs can be activated by other proteases. The physiological agonist of PAR3 has not been identified to date. To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, Gq inhibitor YM-254890 or calcium chelator BAPTA-AM had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was completely abolished by ROCK inhibitor Y27632 or H1152, suggesting that trypsin-induced PAR4 null murine platelet shape change is mediated by G12/13, but not Gq pathway. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. To investigate whether Trypsin activated PAR3, we desensitized PAR4 in both WT and PAR3 null murine platelets. Surprisingly, trypsin caused similar shape change in PAR4-desensitized PAR3 null murine platelets as in PAR4 null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, a Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signaling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signaling pathway in platelets that is independent of PARs. This protease-induced novel signaling pathway regulates platelet shape change through SFKs and p160ROCK.

Disclosures:
No relevant conflicts of interest to declare.
Long-Term Outcomes of Renal Function in Patients with TTP Associated with Severe ADAMTS13 Deficiency
Authors: Reese Jessica A , Deford Cassandra C , Al-Nouri Zayd , Terrell Deirdra , Vesely Sara K. , Strebel Johanna A. Kremer Hovinga , Lammle Bernhard , George James N. 
Published: October 2019

Abstract
Abstract 2215

Introduction:
Among survivors of TTP associated with severe ADAMTS13 deficiency, long-term outcomes other than relapse are not well documented. The recent report of persistent minor neurocognitive abnormalities attributed to subcortical microvascular thrombosis (Transfusion 2009;49:1092) suggested that other organs may also be affected by subclinical abnormalities. Therefore we documented renal function in patients in the Oklahoma TTP-HUS Registry who survived acute episodes of TTP associated with severe ADAMTS13 deficiency (activity <10%).

Methods:
ADAMTS13 activity has been measured in 299 of 321 (93%) patients, 1995–2010. Glomerular filtration rate (GFR) was calculated by the Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equation (Ann Int Med 2009;150:604). Urine protein was measured by dipstick. Patient GFRs were compared to GFRs for the US population using data from the National Health and Nutrition Examination Survey (NHANES), 1998–2006, that had been analyzed in the CKD-EPI study.

Results:
68 of 299 (23%) patients from the Oklahoma TTP-HUS Registry had severe ADAMTS13 deficiency. Fifty-five of the 68 (81%) patients with severe ADAMTS13 deficiency survived their initial episode of TTP. Among the 55 survivors, 48 (87%) have had ≥1 year of follow-up (median, 6.4 years; range, 1.0 – 14.9 years). The 7 patients whose follow-up was less than 1 year included 3 patients whose initial episode was within the past year; 2 with systemic lupus erythematosus who died within one year; 1 who relapsed and died at one year; 1 lost to follow-up. Sixteen (33%) of the 48 patients had 1–4 relapses. None of the 48 patients developed end stage renal disease and none developed severe chronic kidney disease (GFR <30 ml/min/1.73m2). Three of the 48 patients had chronic kidney disease with moderately decreased GFR (GFR 30–59 ml/min/1.73m2); two of these patients had hypertension and one had hypertension and diabetes prior to their initial episodes of TTP. The median GFR was 97 ml/min/1.73m2 (range, 30 – 139 ml/min/1.73m2). The GFR for the ADAMTS13 deficient TTP patients was not different from the GFR of the U.S. population (p=0.289) (Figure 1). Urine protein was measured on 34 patients (median follow-up, 6.7 years; range, 1.2–14.9 years): 32 (94%) were negative; 2 (6%) were trace, equivalent to <30 μg albumin/mg creatinine.

Conclusion:
Renal function of TTP patients with severe ADAMTS13 deficiency who had been followed for a median of 6.4 years following recovery from their initial episode was not different from the US population.

Disclosures:
No relevant conflicts of interest to declare.