bluuebunny/crossref_metadata_2025_split huggingface dataset:
AML FAB Subtypes Shows Different Proportion of Leukemia Initiating Cell Which Has Lower Single Cell Dividing Property.
Authors: Kim Hye Ran , Shin Myung-Geun , Kim Yester , Kim Hyeoung Joon , Cho Duck , Kee Seung-Jung , Kim Soo-Hyun , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook 
Published: October 2019

Abstract
Abstract 4568

Background
Acute myeloid leukemias (AML) are maintained by a subpopulation of leukemia initiating cell that can regenerate themselves as well as give rise to more differentiated and less proliferative cells that constitute the bulk of the disease. Recent studies discovered both the nature of leukemia stem cells and their origin. Immunophenotype of AML stem cell (ASC) is known as CD34+CD38– leukemic cells. Yet, despite their critical importance, much remains to be learned about the frequency, proportion and dividing activity of ASC in a single cell levels. To address these issues, the current study investigated the frequency, proportion according to AML FAB subtypes and single cell plating efficiency of ASC from bone marrow (BM) at the initial diagnosis.

Materials and methods
The proportion and frequency of ASC were examined on BM samples of twenty AML patients using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). Growth and proliferation capacities of single normal hematopoietic stem cells and LSC were determined using plating efficiency (number of the wells in which more than two cells grew / total number of cells in 96-well plate culture x 100). Individual single cells were cultured in 96-well plates with each well containing 100 uL of serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF for five days.

Results
The frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of remaining other subtypes was 13.6±16.2%. CD34 percentage in BM at the initial diagnosis closely correlated with the proportion of ASC. ASC proportion was statistically significant different between patient's group with more than 20% of CD34 and those with less than 20% (P=0.003). Single cell plating efficiency of ASC was markedly reduced compared with non-ASC (CD34+CD38+ leukemic cell population) (7.2 ± 4.7% in ASC vs. 12.6 ± 7.6% in non-ASC).

Conclusion
ASC proportion and frequency in BM samples were very diverse according to FAB subtypes. This study directly proved lower dividing activity of single ASC, and also implicated strategies for single ASC cloning to develop new therapeutics targeting ASC.

Disclosures:
No relevant conflicts of interest to declare.
Mesenchymal Stem Cell Grafting to Treat Cutaneous Radiation Syndrome: Developement of a New Minipig Model.
Authors: Agay Diane , Forcheron Fabien , Scherthan Harry , Grenier Nancy , Meineke Viktor , Herodin Francis , Drouet Michel 
Published: October 2019

Abstract
Abstract 4574
Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiations. Recent grafting of 3 burned patients has suggested the benefit of local injection of bone marrow multipotent mesenchymal stem cells to favour wound healing. However, further studies are required to standardize recommendations. The aim of this study was to set up a pig model of severe CRS in order to clarify some of these points. Göttingen minipigs were locally irradiated (20 cm x 3 cm area at the back) using a 60Co gamma source. This model corresponds to a 50-60 Gy entrance and a 15-25 Gy exit dose delivery. Skin damage was associated to deeper lesions especially muscular. Experimental groups were as followed: ungrafted 50 Gy (n=2) and 60 Gy (n=1); grafted 50 Gy (minipig GM5) and 60 Gy (minipig GM4). Humeral bone marrow collected before irradiation was put into culture in minimal essential medium with 10% Fetal Calf Serum and Fibroblast Growth Factor (2ng/ml) at 37° in air with 5% CO2. Pig GM5 was intradermally injected at days 48, 69 and 96 for a total amount of 128.5 ×106 MSC, pig GM4 at days 48 and 69 for a total amount of 99 ×106 MSC (range 2 to 3×106 MSC per point). All animals exhibited an early erythema on day 1 and a hair loss marked within 28 days. Minipig GM4, grafted after the onset of macroscopic damage (day 40), exhibited a similar evolution to controls. In minipig GM5, local inflammation and macroscopic damages were delayed in entrance area when compared with ungrafted 50 Gy animals (thermal camera signal at day 96 versus day 53; no macroscopic damage in entrance area until day 222 euthanasia versus day 85).
Immunolabellings showed severe late skin damages in all animals (reduced keratinisation, deep ingrowth of epidermis, small cavities and apoptotic cells). Finally this study establishes a new minipig model close to human for the improvement of cell therapy strategies to treat high local irradiations. Work is going on to document the homing of injected cells (eGFP labelled MSC) and the molecular response of grafted tissues in order to optimize graft efficacy.

Disclosures:
No relevant conflicts of interest to declare.
Human Mesenchymal Stem Cells Elicit Complement Activation in Human Blood.
Authors: Le Blanc Katarina , Moll Guido , Rasmusson Ida , Ekdahl Kristina Nilsson , Elgue Graciela , Sundberg Berit , Nilsson Bo 
Published: October 2019

Abstract
Abstract 4580
Infusion of third-party mesenchymal stem cells (MSCs) appears to be a promising therapy for steroid-refractory acute graft-versus-host disease (GvHD). Little is known about how MSCs interact with the innate immune system after clinical infusion. In this study, we show that exposure of MSCs to ABO-compatible human blood activates the complement system, which triggers complement-mediated effector cell functions, and correlates with the immunosuppressive properties of MSCs. We found deposition of the complement component 3 (C3) derived opsonins iC3b and C3dg on MSCs, and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. These events triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation; but could be prevented by culturing MSCs in human ABserum or by blocking complement function. Our study demonstrates the important role of the complement system as a possible mediator of immune modulation in clinical applications using MSCs, and implies that complement activation may substantially affect the treatment efficiency.

Disclosures:
No relevant conflicts of interest to declare.
The Critical Function of c-Myb and CD9 On Megakaryopoiesis.
Authors: Mukai Harumi Yamamoto , Kouno Tomoko , Motohashi Hozumi , Kozuma Yukinori , Kojima Hiroshi , Ninomiya Haruhiko , Yamamoto Masayuki 
Published: October 2019

Abstract
Abstract 4587
The nuclear proto-oncogene c-myb plays crucial roles in the growth, survival and differentiation of hematopoietic cells. We previously reported that the c-Myb knock down (KD) mice exhibited anemia and thrombocytosis. To better understand the transcriptional regulatory program that accompanies the decrease of c-Myb, we performed a transcriptome analysis of megakaryocyte-erythrocyte lineage-restricted progenitors (MEPs). We found that CD9 expression was dramatically increased in the MEPs from c-Myb KD mice. CD9 belongs to a tetraspanin family and regulates cell motility and adhesion. Several reports described that CD9 is involved in platelet function by forming a complex with integrin family members including platelet fibrinogen receptor integrin aIIb-b III. To elucidate the functional contribution of CD9 to the thrombogenesis within the regulatory hierarchy mediated by c-Myb, we first examine the relation between c-Myb and CD9. We found the recruitment of c-Myb to the promoter region of CD9 by chromatin immunoprecipitation assay. A reporter assay showed that c-Myb represses CD9 promoter activity. These results indicate that CD9 is directly repressed by c-Myb. Since an agonistic antibody against CD9 stimulated megakaryocytic colony formation, we investigated the role of CD9 during megakaryopoiesis in vivo by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Consistent with this result, the recovery phase of platelet counts were delayed in the CD9-null mice after the bone marrow suppression with 5-fluorouracil, indicating the impaired platelet production. In CD9-null mice, the proliferation of megakaryocytes was promoted and circulating megakaryocytes in the peripheral blood were increased, which might compensate the PPF impairment of CD9-null megakaryocytes. Our study suggests that c-Myb suppresses CD9 expression under the steady state, while, in the stress megakaryopoiesis, CD9 is derepressed and acts to enhance platelet production. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.

Disclosures:
Ninomiya: Alexion: Research Funding.
Filamin A Controls Embryonic Stem Cell Differentiation.
Authors: Kanaji Taisuke , Okamura Takashi , Newman Peter J. 
Published: October 2019

Abstract
Abstract 4599
Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells.

Disclosures:
Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.
Hedgehog Signaling and Hematopoiesis Regulation: Preliminary Study in Myelosuppressed Adult Non Human Primate model.
Authors: Drouet Michel , Garrigou Philippe , Grenier Nancy , Delaunay Christophe , Mayol Jean-François , Herodin Francis 
Published: October 2019

Abstract
Abstract 4603
The precise role of hedgehog signaling (Shh) in adult hematopoiesis is still debated. One one hand its implication in differentiation and proliferation of hematopoietic stem cells has been reported by different teams. Thus Bhatia et al showed using an immunocompromized mouse model that Shh allows in vitro amplification of CD34+/38- pool. Shh may be especially invoved in megakaryocytopoiesis. In addition transgenic mice (ptc1/-) exhibiting an increased Shh activity has been shown to recover more rapidly than controls following myelosuppression and prolonged Shh stimulation appeared to deplete the stem cell pool due to cell cycle acceleration. On the other hand, recent studies also suggest that hedgehog signaling could be dispensable for adult hematopoieisis (Hoffmann-Zhang, Blood 2008 abstr 1391. The goal of this study was to set up an adult non human primate model to clarify some of these points. We chose a gene therapy strategy based on short term expression of Shh in bone marrow of highly irradiated monkeys (efficient stimulation and reduction of side effects). We first demonstrated the feasibility of transducing multipotent fibroblastic stem cells as vector cells using a piRES-2-Shh-eGFP plasmid and Nucleofector technology (Amaxa). As bone marrow nonhuman primate mesenchymal stem cells are known to be frequently contaminated by retroviruses, we used adipocyte derived stem cells as vector cells. In this model, Shh protein was detected during 5 days in the transduced cells and was secreted during 3 days in accordance with our short term secretion model. Then Shh-ASC were injected to rhesus monkeys the day after a global and frontal irradiation at the dose of 8Gy 60Co gamma. At this dose, monkeys exhibited a severe and prolonged neutropenia and thrombocytopenia allowing us to evaluate the therapeutic benefit of the grafts. In a first grafted monkey (9kg), 2×106 Shh-ASC (about 43% GFP positive cells) were injected in the femur. Blood cell counts did not significantly differ from irradiated control animals (n=4). Further experiments will contribute to determine whether Hedgehog signaling is involved in adult primate recovery following myelosuppression.

Disclosures:
No relevant conflicts of interest to declare.
Activity of CNTO 530 in a Murine Model of Sickle Cell Anemia.
Authors: Makropoulos Dorie , Achuthanandam Ram , Rafferty Patricia , Chroscinski Denise , Brosnan Kerry , Ogunkua Lawrence , Miller Andrew , Avery Justin , Walker Mindi , Bugelski Peter 
Published: October 2019

Abstract
Abstract 4606
CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia) and in a model of beta-thalassemia. Here we report on the activity of CNTO 530 in murine globin KO-human HbS transgenic (sickle cell) mice. Berkeley mice, as originally described by Pászty et al (Pászty et al. Science 1997), are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Thus, these mice express exclusively human hemoglobin A (HbAsickle) (or sickle hemoglobin, HbS) and can also express human fetal hemoglobin (HbF). To test the effectiveness of CNTO 530 in this model, mice received a single subcutaneous (s.c.) dose of CNTO 530 (0.3 mg/kg), epoetin-alpha (0.1 mg/kg) or darbepoetin-alpha (0.03 mg/kg) (as comparators) at 10,000 U/kg, a dose previously shown to increase hemoglobin in normal mice. Hematology, histology of the spleen, F-cells and expression of HbF were then evaluated over time. We show that these mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. In addition, splenomegaly and extramedullary erythropoiesis were observed. Ten days after dosing, CNTO 530 but not epoetin-alpha or darbepoetin-alpha caused an increase in reticulocytes, red blood cells (RBC), F-cells, total hemoglobin (Hgb) and HbF. In conclusion, these results demonstrate that a single dose of CNTO 530 can increase expression of HbF in sickle cell mice and suggest that human treatment with CNTO 530 may have beneficial effects in sickle cell anemia patients.

Disclosures:
Makropoulos: Centocor, R&D, Inc: Employment, Equity Ownership. Achuthanandam:Centocor, R&D, Inc: Employment, Equity Ownership. Rafferty:Centocor, R&D, Inc: Employment, Equity Ownership. Chroscinski:ACE Animals, Inc: CRO. Brosnan:Centocor, R&D, Inc: Employment, Equity Ownership. Ogunkua:Chroscinski: Employment, Equity Ownership. Miller:Centocor, R&D, Inc: Employment, Equity Ownership. Avery:Centocor, R&D, Inc: Employment, Equity Ownership. Walker:Centocor, R&D, Inc: Employment, Equity Ownership. Bugelski:Centocor, R&D, Inc: Employment, Equity Ownership.
Asthma IN SICKLE CELL DISEASE as A RISK FACTOR for Acute Chest Syndrome IN Pediatric PATIENTS.
Authors: Borns Andrea P , Marquez Kristy , Amendah Djesika , Whiteman Alan , Jordan Lanetta B , Grant Althea 
Published: October 2019

Abstract
Abstract 4610

OBJECTIVES
Both asthma and sickle cell disease are major Public Health concerns. Previous studies have demonstrated that asthma among children with sickle cell disease (SCD) may increase the risk of developing acute chest syndrome (ACS), which can be life threatening. These respiratory complications may increase emergency department (ED) utilization, increase health care costs, and reduce life span among persons with SCD. The purpose of this study is to determine whether children with SCD and asthma have significantly more ED visits and if they are at higher risk of developing ACS compared to children with SCD who do not have asthma.

METHODS
We used MarketScan® Multi-State Medicaid Databases from Thompson Reuters (Ann Arbor, Michigan) for the years 2001 to 2005. These are proprietary datasets covering eight unidentified states. We used International Classification of Disease, 9th Division, Clinical Modifications (ICD-9-CM) codes to identify SCD, asthma and ACS.

RESULTS
In 2005, there were 2428 children with SCD continuously enrolled in Medicaid. Among those, 369 (15.2%) patients were identified as having asthma. The mean number of emergency department visits was significantly higher among children with both SCD and asthma compared to children with SCD without asthma (p<0.05). In addition, children with SCD and asthma were more likely to have at least one episode of ACS than children with SCD without asthma (28.2% vs. 7.8%, respectively).

CONCLUSIONS
Among children with SCD who are enrolled in Medicaid, asthma is a common comorbidity, which is associated with high incidence of ACS. Therefore asthma should be aggressively managed among SCD patients. Proper management of asthma may result not only in reduced pulmonary complications, but also reduced costs related to emergency department utilization and progression of lung disease into adulthood.

Disclosures:
No relevant conflicts of interest to declare.
Adding Hydroxyurea to Chronic Transfusion Therapy for Stroke in Sickle Cell Disease.
Authors: Claster Susan , Quirolo Keith C. 
Published: October 2019

Abstract
Abstract 4620

Introduction
Transfusions have clearly been shown to prevent stroke in patients with Sickle Cell Disease(SCD). The usual goal of transfusion therapy is to keep the percent sickle Hgb (%S) less than 30. %S greater than 30 has been associated with stroke reoccurance. Inability to transfuse adequately may result from poor vascular access or high hemolytic rate. Hydroxyurea, a drug which requires active erythropoeisis, is usually not given to patients who are chronically transfused. However, the presence of an elevated reticulocyte count in a transfused patient indicates ongoing sickle hematopoiesis. We added Hydroxyurea therapy to two patients who were unable to achieve %S levels less than 30 and who had elevated reticulocyte counts to assess whether this drug could improve their hematological parameters.

Results
We treated two post stroke patients, one receiving pheresis therapy, and the other on straight transfusion therapy who were unable to achieve 30% Hgb S despite regular transfusions. Patient 1, a 33 y/o female with Hgb SS had been on transfusions since childhood for multiple CVA's and resulting moya-moya. Her last stroke had occurred while she was being chronically transfused and had a % S of 40. Due to venous access issues the patient was receiving straight transfusion only and was unable to adequately suppress her marrow. In 8/07 her %S was greater than 40 and her reticulocyte count was 11.3 %. Hydroxyurea was started at 1500 mg daily(22 mg/kg). Over the next 24 months her Bilirubin dropped from 8.7mg/dl to 1.9 mg/dl, her reticulocyte count dropped to 4%(figure below) and her MCV increased to 104.6 from 90. Her Hgb F rose to 30%.Her %S has not changed. She has remained clinically stable with no new neurological findings. The second patient, a 17 y/o male had been on chronic transfusions since childhood for a stroke which resulted in a dense left hemiparesis. He has been on a pheresis program for 13 years. Despite this his % S remained greater than 40% and had been as high as 60% prepheresis. Hydroxyurea was added in 5/08 at a dose of 25mg/kg. Within 6 months his pretransfusion %S had dropped to 22-27%. No increase in fetal Hgb was detected, possibly due to the exchange transfusion process. His bilirubin, which had been running between 12-16 mg/dl dropped to 7.6-8.9 mg/dl and his reticulocyte count went from an average of 19.6% to 11.2% in the same time frame. He has also remained stable.

Discussion
These two patients demonstrate that the addition of Hydroxyurea to patients on chronic transfusion can be useful in decreasing hemolytic rate and improving the efficacy of transfusions by decreasing sickle erythropoiesis and increasing fetal hemoglobin synthesis. Further studies of the combination of these two modalities may be warranted in those patients who cannot achieve adequate suppression of sickle erythropoeisis.

Disclosures:
No relevant conflicts of interest to declare.