bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional Regulation of Oncogenic MEF2C In T-Cell Acute Lymphoblastic Leukemia (T-ALL).
Authors: Nagel Stefan , Venturini Letizia , Meyer Corinna , Kaufmann Maren , Scherr Michaela , Drexler Hans G. , MacLeod Roderick AF 
Published: October 2019

Abstract
Abstract 3636
Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS-box family which is physiologically expressed in hematopoietic stem cells and during development of B-cells. Recently, we identified ectopic expression of MEF2C in T-cell acute lymphoblastic leukemia (T-ALL) cell lines activated either via chromosomally mediated ectopic expression of homeodomain protein NKX2-5 or via deletion of non-coding exon and promoter regions at 5q14, suggesting loss of negative regulatory elements. Our aim was to identify additional transcriptional regulators of MEF2C in T-ALL. Therefore, we analyzed the sequence of the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix (bHLH) proteins, STAT5 and HOXA9/HOXA10. Overexpression studies demonstrated MEF2C activation by GFI1B (strong), LYL1 and TAL1 leukemic bHLH proteins (weak), and inhibition by STAT5 (strong) and HOXA9/HOXA10 (weak). Chromatin-Immuno-Precipitation analysis demonstrated direct binding of GFI1B, LYL1 and STAT5 but not of HOXA10 to the MEF2C 5′-region in T-ALL cells. However, HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of MEF2C regulation. Chromosomal deletion of the 5′-MEF2C STAT5 binding site in LOUCY cells by del(5)(q14), reduced expression levels of STAT5 protein in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5-signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that ectopic expression of MEF2C in T-ALL cells is mainly regulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory STAT5-signaling.

Disclosures:
No relevant conflicts of interest to declare.
Survey of Current Prophylaxis Practices and Bleeding Characteristics of Children with Severe Hemophilia A In U.S. Hemophilia Treatment Centers.
Authors: Ragni Margaret V. , Fogarty Patrick F. , Josephson Neil C. , Neff Anne T. , Raffini Leslie , Kessler Craig M. 
Published: October 2019

Abstract
Abstract 3653

Introduction:
Every other day (qod) factor VIII prophylaxis prevents joint bleeds in children with severe hemophilia A. Although three times weekly or qod prophylaxis is recommended by the National Hemophilia Foundation (NHF), how widely these practices have been adopted is not known.

Aim:
We sought to define current prophylaxis practices at U.S. Hemophilic Treatment Centers (HTCs).

Method:
An email survey was distributed to U.S. HTCs, utilizing web-based membership rosters of the Centers for Disease Control (CDC) and the Hemophilia Thrombosis Research Society (HTRS).

Results:
Of 62 HTCs responding, prophylaxis is initiated on a three times weekly schedule in 29 (46.8%), twice weekly in 13 HTCs (21.0%), and once weekly in 20 HTCs (32.2%). Central venous catheters are used to infuse factor prophylactically at 55 HTCs (88.7%), including in 100% of children initiating prophylaxis at 19 HTCs (30.6%) and in 50% of those at 41 HTCs (66.1%), but avoided altogether at seven HTCs (11.3%). Prophylaxis is initiated after one or more bleeds in 56 HTCs (90.3%), but after the first bleed in only 28 HTCs (25.2%). Among 226 newborns with severe hemophilia A in 62 HTCs, 1.82 births/HTC/year, the median age at first bleed, excluding circumcision, is 7 months. Of the 113 (53.5%) who underwent circumcision, 62 (54.9%), bled.

Conclusion:
Despite a recommended standard of three times weekly prophylaxis, over half of surveyed HTCs do not follow these guidelines, and nearly one third begin prophylaxis on a once weekly schedule to delay or avoid the need for central venous access.

Disclosures:
Kessler: Grifols S.A.: Research Funding.
A Case of FVII Inhibitor.
Authors: Levitt Michael Joshua , Topilow Arthur A. , Lerner William , Mencel Peter , Henningson Carl , Greenberg David Benjamin , Philipp Claire S. 
Published: October 2019

Abstract
Abstract 3655
Acquired inhibitors of coagulation are bleeding disorders that require prompt recognition, diagnosis, and management. Antibodies against factor VII are extremely rare with only a few cases reported in the literature. We present a case of a patient with an acquired Factor VII inhibitor. This is an 87 year old female with a past medical history of breast cancer, hypertension, and hyperlipidemia who presented to the emergency room with right flank pain and hematuria. A CT scan of the abdomen and pelvis showed bilateral hydronephrosis with no evidence of nephrolithiasis. The patient denied hematemesis or hematochezia but was noted to have hemoccult positive stools. The patient denied any anticoagulant use. Admission laboratories revealed a coagulopathy with a normal partial thromboplastin time (PTT) of 28 seconds and prolonged prothrombin time (PT) of greater than 50 seconds and INR of 19.59. The patient received vitamin K without improvement in coagulation parameters. A mixing study revealed a markedly prolonged PT that did not correct with 1:1 (18.9 seconds) and 4:1 (27.3 seconds) mix normal plasma. Factor assays shows an abnormal Factor VII level of less than 1%, and normal Factor II 121%, Factor V 140%, Factor VIII 201%, Factor IX 99%, and Factor XI 126% levels. A FVII inhibitor was 1.66 Bethesda units per milliliter. Immunosuppressive treatment was initiated with prednisone 1 milligram per kilogram daily and cyclophosphamide 200 milligrams daily. Factor VII levels normalized without evidence of inhibitor. The patient clinically improved and immunosuppressive medications were gradually tapered off. This case emphasizes the importance of prompt recognition in a patient with a rare acquired inhibitor of coagulation. Treatment with immunosuppressive therapy consisting of corticosteroids and cyclophosphamide resulted in normalization of factor VII levels and resolution of bleeding symptoms and should be considered as first-line management for such patients.

Disclosures:
Philipp: Baxter: Research Funding; Wyeth: Research Funding; Octapharma: Research Funding.
Allogeneic Platelet MHC Class I Antigens Prevent CD61 Specific Cytotoxic T Cell (CTL)-Mediated Immune Thrombocytopenia (ITP).
Authors: Guo Li , Aslam Rukhsana , Speck Edwin R , Ni Heyu , Hou Ming , Freedman John J. , Semple John W. 
Published: October 2019

Abstract
Abstract 3686
In the International Consensus Report (Rodeghiero et al. Blood 2009;113:2386-2393), platelet transfusions are only recommended in patients with auto-immune thrombocytopenia (ITP) as an adjunctive therapy for life-threatening bleeding. However, the clinical evidence of post-transfusion increments in ITP suggests that in some cases of ITP, platelet transfusions may be a useful prophylactic therapy. To understand the importance of platelet transfusion in this setting, we utilized our murine model of ITP that demonstrates both antibody- and CTL-mediated thrombocytopenia. BALB/c CD61 knockout (KO) mice were immunized by transfusions of three different platelet populations that were either single antigen positive (e.g. CD61+ or MHC class I+) or double antigen positive (CD61+/MHC class I+). Splenocytes from the immune KO mice were either not depleted (ND) or depleted of CD19+ B cells and then transferred into SCID mice and the development of thrombocytopenia and bleeding diathesis was monitored. When ND splenocytes from MHC class I+ immune KO mice were transferred, no thrombocytopenia or bleeding was observed. In contrast, transfer of ND splenocytes from CD61+ immune mice induced a significant thrombocytopenia with a bleeding mortality of 40% within 3 weeks post-transfer; depletion of B cells was used to analyze CD8+ T cell- mediated thrombocytopenia and these depleted splenocytes also induced thrombocytopenia and bleeding. In addition, compared with healthy mice, the bone marrow of the transferred mice showed elevated numbers of megakaryocytes with some of them appearing to be apoptotic. On the other hand, when splenocytes from the double immunized (CD61+/MHC class I+) mice were transferred, the CD8+ T cell- mediated thrombocytopenia and bleeding mortality was not observed. In addition to preventing the thrombocytopenia and bleeding, histological analysis of the bone marrow showed normal numbers of megakaryocytes. These results suggest that both antibody- and CD8+ T cell-mediated immune thrombocytopenia exist and that when anti-CD61 specific CD8+ T cells interacted with allogeneic MHC antigens on platelets they were prevented from inducing CD8+ T cell-mediated thrombocytopenia. Thus, allogeneic MHC platelet transfusions may be responsible for inhibiting CD8+ T cell-mediated thrombocytopenia and may be a therapeutic option for those patients who suffer from cell-mediated ITP.

Disclosures:
No relevant conflicts of interest to declare.
Study of Polymorphisms in DNMT3B promoter and IL-1 Ra in Childhood Idiopathic Thrombocytopenic Purpura.
Authors: Stiakaki Eftichia , Lourou Marilena , Pesmatzoglou Margarita , Vasjiourakis Vassilis , Dimitriou Helen , Kalmanti Maria 
Published: October 2019

Abstract
Abstract 3705
Idiopathic thrombocytopenic purpura (ITP) or primary immune thrombocytopenia is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease both environmental and genetic factors including polymorphisms of TNF-a, IL-10 and IL-4 genes have been suggested to be involved. Aim: To investigate the association of the single-nucleotide polymorphism (SNP) in DNA methyltransferase 3B (DNMT3B C46359T) gene promoter and the polymorphism of IL-1 receptor antagonist (IL-1 Ra) intron-2 with the risk of immune thrombocytopenic purpura in childhood. Methods-patients: The genomic DNA of bone marrow mononuclear cells (MNC) of 20 children with idiopathic thrombocytopenic purpura, and peripheral blood leykocytes of 20 healthy children and 40 healthy adult volunteers (blood donors), was isolated and the region of interest was amplified by PCR. Furthermore for the DNMT3B polymorphism following digestion with Avr II the products were analyzed on agarous gel. Results: The frequencies of DΝMT3B C/C, C/T, T/T genotypes and C and T alleles in children with ITP were 50%, 35%, 15%, 67.5% και 32.5%, respectively. No significant differences were found in genotype and allele distribution between children with ITP and healthy children or adults (p=0.778 and p=1, respectively). However, there was significant difference in genotypic distribution of IL-1 Ra among children with ITP and control groups: children (p=0.001) and adults (p=0.005) as well as in allelic frequencies of IL-1 Ra between the same groups (p=0.000 and p=0.005 respectively). More specifically, children with Ι/Ι genotype or I allele seem to have decreased risk of development of ITP (OR: 0.07 [0.01–0.4]) compared both to their healthy counterparts and to healthy adults (OR: 0.17 [0.05–0.54]). Moreover the presence of the I/II genotype seems to increase 8.56 times the relative risk for disease development (OR: 8.56 [2.36–30]) while the presence of the II allele 3.8 times (OR: 3.8 [1.45–9.9]). Conclusion: The SNP (C46359T) in the promoter of DNMT3B does not seem to correlate with the diagnosis of ITP whilst the polymorphism of IL-1 receptor antagonist (IL-1 Ra) intron-2 seems to have an association with the development of the disease. This finding has to be further investigated in order its role in the pathogenesis and course of immune thrombocytopenic purpura to be confirmed.

Disclosures:
No relevant conflicts of interest to declare.
Clinical Scale Enrichment and Expansion of Highly Pure CD25+Foxp3+ Regulatory T Cells.
Authors: Conrads Carina , Schmitz Jürgen , Assenmacher Mario , Niemand Claudia , Scheffold Alexander 
Published: October 2019

Abstract
Abstract 3718
CD25+Foxp3+ regulatory T cell (Treg) bear great potential to prevent or treat a variety of immune mediated diseases, including autoimmunity, organ rejection or GvHD. Currently Treg for clinical application can be separated by magnetic cell separation via the CliniMACS® Plus Instrument using CD25 enrichment plus/minus prior depletion of CD8 or CD19 positive cells. With this technology Treg can be enriched to a mean purity of about 50% and first clinical trials for prevention of GvHD show no adverse effects at all. Despite these promising results, concerns have been raised whether in the setting of organ transplantation or autoimmunity higher Treg purities and/or the in vitro expansion of Treg without loss of Foxp3+ expression are required.
Therefore, we have optimized the parameters for CD25 enrichment via CliniMACS to achieve higher purity of the isolated Treg. The purity of Treg could be increased by about 20–30% resulting in an average purity of 70–80% of Foxp3+ Treg. We have also developed a protocol for the in vitro expansion of CliniMACS isolated Treg using CD3/CD28 coated MACSiBead™Particles. In the presence of Rapamycin CliniMACS isolated Treg could be expanded about 20 fold with a single round of stimulation. Importantly Foxp3+ expression was not affected by the expansion but remained constant at about 70–80%. Similarly the expression of effector cytokines by expanded Treg was greatly suppressed by Rapamycin.
These data show that Treg for clinical application can efficiently be isolated with high purity via CliniMACS and subsequently be expanded in vitro without loss of Foxp3 expression.

Disclosures:
Conrads: Miltenyi Biotec: Employment. Schmitz:Miltenyi Biotec: Employment. Assenmacher:m: Employment. Niemand:Miltenyi Biotec: Employment. Scheffold:Miltenyi Biotec: Employment.
VE Cadherin Positive Endothelial Cells Regulate Hematopoietic Reconstitution In Vivo.
Authors: Russell J. Lauren , Doan Phuong , Himburg Heather A , Meadows Sarah K. , Daher Pamela , Helms Katherine , Quarmyne Mamle , Chao Nelson J. , Kirsch David , Chute John P. 
Published: October 2019

Abstract
Abstract 3734
We have recently demonstrated that targeted deletion of the pro-apoptotic genes, Bak and Bax, in Tie2+ bone marrow endothelial cells (BM ECs) causes the protection of the BM sinusoidal vasculature, and the BM hematopoietic stem cell (BM HSC) pool following high dose total body irradiation (TBI). Since Tie2 is expressed on BM ECs and a small subset of quiescent BM HSCs, we developed a complementary model utilizing VE-cadherin Cre mice to more specifically confirm the function of BM ECs in regulating hematopoietic reconstitution in vivo. VE-cadherinCre;Bak−/−;BaxFl/− mice, which bear deletion of Bak and Bax in VE-cadherin+ ECs, and VE-cadherinCre;Bak−/−;BaxFl/+ control mice were generated and we compared the hematopoietic responses of these animals to high dose TBI. At steady state, these mice showed no difference in total BM cells, bone marrow ckit+sca+lineage- (KSL) progenitor cells, BM colony forming cells (CFCs), or BM colony forming unit spleen day 12 (CFU-S12) counts. At 2 hours after exposure to 500cGy TBI, the mice also did not demonstrate any significant differences in total BM cells, BM KSL cells, CFCs, or CFU-S12. However, 7 days after exposure to 500cGy TBI, VE-cadherin;Bak−/−;BaxFl/− mice displayed a 2-fold increase in total BM cells (p=0.006), a 3-fold increase in BM CFCs (p=0.0009), and 4-fold increase in BM CFU-S12 (p=0.079) compared to VE-cadherinCre;Bak−/−;BaxFl/+ control mice. Microscopic examination confirmed severe hypocellularity and corruption of the BM sinusoidal vasculature at day +7 post-TBI in VE-cadherinCre;Bak−/−;BaxFl/+ mice, whereas VE-cadherinCre;Bak−/−;BaxFl/− mice displayed nearly normal cellularity and preserved BM sinusoidal vessels. Taken together, these data demonstrate that VE-cadherin+ BM ECs regulate hematopoietic reconstitution in vivo and that targeted therapies aimed at augmentation of BM EC function can accelerate hematopoietic regeneration in vivo. Currently, we are comparing the cytokine production and gene expression profiles of VE-cadherinCre;Bak−/−;BaxFl/− BM ECs versus VE-cadherinCre;Bak−/−;BaxFl/+ BM ECs to identify candidate BM EC-derived proteins which are responsible for the protection of the BM stem/progenitor cell pool from radiation injury. VE-cadherin+ BM ECs represent an attractive mechanistic target for the identification of signaling pathways that regulate hematopoietic regeneration following injury.

Disclosures:
Chao: Genzyme: Research Funding.
Characterisation of B Cell Receptor-Induced NF-KB Activation In Chronic Lymphocytic Leukaemia Cells
Authors: Talab Fatima , Thompson Victoria , Allen John C , Lin Ke , Slupsky Joseph R 
Published: October 2019

Abstract
Abstract 375
B cell receptor (BCR) signaling promotes survival of the malignant clone in chronic lymphocytic leukaemia (CLL) through its ability to stimulate NFkB pathway signaling. In lymphoid cells, antigen receptor stimulation of this pathway is achieved by engaging the Carma-1 – Bcl10 – MALT1 (CBM) complex for eventual activation of I-kB kinases (IKKs). In B cells, protein kinase C beta (PKCbeta) is an important mediator of CBM complex activation. However, in CLL cells we found that PKCs do not appear to have a role in BCR-mediated NFkB pathway signaling, despite high expression levels of PKCbeta, because the presence of specific inhibitors of this kinase (LY379196 and bisindolylmaleimide-I) has no effect on the induction of IKK phosphorylation during BCR crosslinking. Examination of CBM complex expression suggests an explanation for this phenomenon; the expression levels of Carma-1 and MALT-1 are largely similar in CLL and normal B cells, but the expression of Bcl10 is much reduced in CLL cells. These findings, taken together with the established role of Bcl10 in the pathway of BCR-induced NFkB activation, suggest that CLL cells may employ a different mechanism to activate this pathway during BCR stimulation. Tyrosine kinases are known to play a role in BCR-induced IKK activation in CLL cells because compounds like dasatinib and PP2 inhibit NFkB pathway activation by BCR. One possible tyrosine kinase is c-Abl because we have shown this protein to be overexpressed in CLL cells, where it plays a role in activation of the NFkB pathway. To investigate the role of c-Abl in BCR-induced IKK activation, we used the inhibitor imatinib and found that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells. However, imatinib can also inhibit Lck, a T cell-specific src-family tyrosine kinase that is expressed by CLL cells. To differentiate between Lck- and c-Abl-mediated BCR signals we used the specific inhibitor 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin-7-yl-cyclopentane (Lck-i). We found that the presence of this compound in CLL cell cultures undergoing BCR stimulation almost completely inhibited the induction of IKK activation. Investigation of Lck-i specificity revealed this compound did not inhibit either c-Abl or Lyn at the concentration used to inhibit Lck in CLL cell cultures. Further investigation of the effects of Lck-i showed that this compound was also effective in inhibiting BCR-induced activation of the Akt and ERK signaling pathways. Taken together, these data suggest a major role for Lck in BCR-mediated signaling in CLL cells, and question the existing paradigm on the importance of Lyn.

Disclosures:
No relevant conflicts of interest to declare.
Recombinant Erythroid Kruppel-Like Factor Fused to GATA1 up-Regulates δ-Globin Expression In Erythroid Cells.
Authors: Zhu Jianqiongz , Chin Kyung , Aerbajinai Wulin , Trainor Cecelia D , Perter Gao , Rodgers Griffin P. 
Published: October 2019

Abstract
Abstract 3752
The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2, α2δ2) and fetal hemoglobin (HbF, a2γ2) both inhibit the polymerization of hemoglobin S that results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA, α2β2) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to upregulate δ-globin to increase HbA2 expression, we created a series of EKLF-GATAl fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATAl and then tested their effects on hemoglobin expression. EKLF-GATAl fusion proteins activated δ-, γ-, and β-globin promoters in K562 cells, and significantly upregulated δ- and γ-globin RNA transcripts and proteins expression in K562 and CD34+ cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter, and may represent a potentially new genetic therapeutic approach to β-hemoglobinopathies.

Disclosures:
No relevant conflicts of interest to declare.
CD19 Targeted Cord Blood Derived T Cells for Cancer Immunotherapy.
Authors: Pegram Hollie J. , LaRussa Vincent , Brentjens Renier 
Published: October 2019

Abstract
Abstract 3767
Transplantation of unrelated umbilical cord blood (CB) derived stem cells is often used to treat adult patients with B-cell acute lymphoblastic leukemia (B-ALL). However, many patients relapse and overall prognosis is poor. We hypothesize that additional therapy involving adoptive transfer of CB derived T cells modified to express a CD19-specific chimeric antigen receptor (CAR) could improve patient outcome following allogeneic CB transplant. To this end, we have previously demonstrated that human T cells which express the anti-CD19 19–28ζ CAR, containing the signaling domains of the co-stimulatory CD28 receptor and CD3ζ chain, effectively eradicate CD19+ tumors both in vitro as well as in vivo in SCID-Beige mice. Herein, we demonstrate the ability to effectively isolate and expand T cells from CB samples using magnetic beads coated with agonistic CD3 and CD28 antibodies (Invitrogen) and comparing subsequent T cell expansions in in vitro cultures with varying additions of exogenous stimulatory cytokines, including IL-2, a combination of IL-2 and IL-7, IL-12 or IL-15. We demonstrate that in vitro culture in the context of exogenous IL-12 (10 ng/ml) resulted in optimal expansion of CB T cells (150-fold). In addition, expansion of T cells in the context of exogenous IL-12 resulted in a favorable phenotype for adoptive cell transfer, with T cells expressing high levels of Granzyme B and Perforin while retaining a “memory” phenotype as assessed by persistent expression of CD62L. This combination of cytotoxic and memory phenotype is optimal for adoptive cell therapy. T cells expanded in this manner were subsequently efficiently retrovirally transduced to express the 19–28ζ CAR. The resulting CD19-specific CB derived T cells were able to specifically lyse CD19+ tumor targets as assessed by standard 51Cr release cytotoxicity assays. We are currently investigating the in vivo anti-tumor function of these modified cells in our previously established preclinical SCID-Beige mouse tumor model and ultimately plan to conduct a Phase 1 clinical trial with these modified T cells in patients with B-ALL undergoing CB transplant.

Disclosures:
No relevant conflicts of interest to declare.