bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Polymorphism in the XPNPEP2 Gene Is Associated with the Development of Hypotensive Transfusion Reactions without ACE Inhibitors.
Authors: Hui Yiang , Tormey Christopher , Wu Yanyun 
Published: October 2019

Abstract
Abstract 3140
Poster Board III-77

Introduction
Hypotensive transfusion reactions (HTRs) have been associated with the transfusion of different blood products. They are characterized by the rapid onset and disappearance of hypotension upon starting and finishing the transfusion respectively. The use of ACE inhibitors has been associated with HTRs. However, there are reported HTRs in patients not taking ACE inhibitors. An inability to break down bradykinin (BK) generated during the infusion/transfusion is suggested to be responsible for the hypotensive effect. Angiotensin-converting enzyme (ACE) and aminopeptidase P (APP) are primarily responsible for degradation of BK. We hypothesize that the C-2399A single-nucleotide polymorphism in the membrane-bound APP gene XPNPEP2 (SNP rs3747343) may be linked to HTRs. This polymorphism is present upstream of the coding sequence of the APP gene and has already been associated with severe hypotension in an angioedema patient treated with an ACE inhibitor. This suggests that it may have an important regulatory role.

Patients and Methods
With the approval of our IRB, patients' samples were collected, de-identified, and then used for testing. Specimens from 4 patients who presented with classic HTRs while not taking ACE inhibitors were analyzed through sequencing of DNA acquired from pre-transfusion blood samples. The same primers were used for PCR and sequencing (5'-GAGTATTATGTGGGGACCATCC-3' and 5'-ATGCCTCGCAGAGACAAGAG-3'). Samples were sequenced in both sense and antisense directions.

Results
Among these patients, two (50%) were found to possess the C-2399A mutation. One was found to have changes in both reference and complementary strands while the other had a C/A mutation only in one strand.

Conclusions
These results indicate that the C-2399A polymorphism may be a contributing factor in the development of HTRs and that it may promote HTRs in the absence of ACE inhibitors. The hypotensive effect may be due to a structural or regulatory change caused by C-2399A though it also seems that other factors may be involved since not all patients assayed possessed the mutation. Further screening of additional contributing factors responsible for HTRs is necessary to fully comprehend the pathogenesis of this type of transfusion reaction.
Keywords: XPNPEP2, C-2399A, ACE Inhibitors, Hypotensive Transfusion Reactions, aminopeptidase P.

Disclosures
No relevant conflicts of interest to declare.
Epitope Mapping of FIX Inhibitors Identify Contact Residues in the Protease Domain.
Authors: Hoehl Sebastian , Kreuz Wolfhart , Krause Nicole , Stichel Diana , Martinez-Saguer Inmaculada , Kessel Christoph , Klingebiel Thomas , Oldenburg Johannes , Irving James , Königs Christoph 
Published: October 2019

Abstract
Abstract 3172
Poster Board III-112
Hemophilia B is an X-linked bleeding disorder that requires regular substitution of FIX preparations to restore coagulation. A rare but severe complication is the development of FIX inhibitors affecting about 3% of hemophilia B patients. Possible allergic reactions and challenging but often unsuccessful immune tolerance protocols complicate the treatment of affected patients. In the present study, five samples from patients with inhibitors against FIX (including one acquired inhibitor) were screened against random peptide libraries displayed on phage. A biopanning protocol was established to isolate phagotopes specific for FIX inhibitors and thus small peptides that mimic the epitopes of the inhibitors. Binding was confirmed by ELISA and the peptide sequences were determined by translating the DNA sequence of the phage. In total 65 peptide sequences were determined. For all patients except for the patient suffering from acquired FIX inhibitors consensus motifs were identified. These motifs include the amino acids FTK(T), TSL(T) and LRQ. In order to compare the motifs to the surface of FIX for epitope identification a novel structural model of human FIX was generated. Using this model epitopes were identified on the surface of the protease domain of FIX. The residues comprising the conformational epitopes are found in the regions AA290 - 330 and 380 - 390. Small peptides have been identified that mimic the epitopes for FIX inhibitors in hemophilia B patients. These mimotopes mimic conformational epitopes on the surface of the protease domain on FIX. Identifying inhibitor epitopes and isolating molecules that can possibly interfere with inhibitor binding might help to understand and to develop novel approaches to treat this rare but life-threatening complication.

Disclosures
Königs: Octapharma AG: Research Funding; Baxter AG: Research Funding.
Prelinical Evaluation of Lyn Kinase Inhibition for the Treatment of B Cell Chronic Lymphocytic Leukemia (B-CLL).
Authors: Velcheti Vamsidhar , Li Li , Philips Joseph , Fuloria Jyotsna , Rodwig Francis R , Cole John T. , Samaniego Felipe , Prakash Om 
Published: October 2019

Abstract
Abstract 3447
Poster Board III-335
B-CLL is characterized by the accumulation of mature B lymphocytes incapable of undergoing apoptosis. Although several mechanisms have been implicated in the apoptotic defects in B-CLL cells, the signal transduction pathways underlying these defects remain unresolved.
Lyn kinase is known to be a negative regulator of apoptosis, and is linked to chemo-resistance. Our preliminary study indicated that Lyn activity was 4- to 7-fold higher in primary B cells from six randomly-selected B-CLL patients than in normal B cells from healthy donors. Treatment of the B-CLL cells for 4 h with 10 μM concentration of a Lyn-specific inhibitor peptide targeting a unique interaction site within Lyn (Cancer Res. 2004;64:1058) resulted in 40 to 50% inhibition of Lyn kinase activity compared to negligible inhibition with control peptide. Further, treatment of the B-CLL cells for 16 to 20 h with the Lyn inhibitor peptide at 5 μM and 10 μM concentrations, decreased the cell viability by ∼25% and ∼50%, respectively, compared with no peptide or the control peptide. Fludarabine is one of the key chemotherapeutic agents for B-CLL and is known to induce cell death by apoptosis. The combined treatment with Lyn inhibitor peptide (5 μM) and fludarabine (2 μg/ml), decreased cell viability by ∼50% compared to ∼25% decrease with fludarabine or Lyn inhibitor peptide alone. In addition, the combined treatment showed ∼75% increase in caspase-3/7 activity compared to ∼25% increase with the Lyn inhibitor peptide or fludarabine alone. Because, overexpression of antiapoptotic proteins is correlated with apoptotic resistance of B-CLL cells, we examined the effect of Lyn inhibitor peptide on the changes in the expression of antiapoptotic genes, myeloid cell leukemia-1 (Mcl-1), x-linked inhibitor of apoptosis (XIAP), and B-cell leukemia/lymphoma-2 (Bcl-2). Treatment of B-CLL cells with 10 μM Lyn inhibitor peptide for 16 to 20 h resulted in more than 50% decreases in the expression of all the three antiapoptotic genes. Further, the Lyn inhibitor peptide markedly inhibited NF-κB activation (∼60%) and VEGF production (∼80%), both strongly implicated in the apoptotic resistance of B-CLL cells. Collectively, our results suggest that targeting Lyn kinase pathway with a clinically relevant Lyn kinase inhibitor may have a therapeutic potential for B-CLL.

Disclosures
No relevant conflicts of interest to declare.
Apoptosis Gene Expression Profiling in Chronic Lymphocytic Leukemia Using Multiplex Ligation-Dependent Probe Amplification.
Authors: Norberg Maria , Sevov Marie , Mansouri Mahmoud , Tobin Gerard , Merup Mats , Åleskog Anna , Nilsson Kenneth , Rosenquist Richard 
Published: October 2019

Abstract
Abstract 3457
Poster Board III-345
One of the hallmarks of the clinically heterogeneous disease chronic lymphocytic leukemia (CLL) is defective apoptosis, which is considered to contribute not only to cell accumulation but also to disease progression and resistance to therapy. In an effort to further investigate the ‘apoptotic profile’ in CLL, we have utilized the multiplex ligation-dependent probe amplification (MLPA) method to analyze the expression profile of 35 apoptosis-related genes in 67 CLL cases including 49 peripheral blood, 9 lymph node, 7 bone marrow and 2 spleen samples. CLL cells derived from peripheral blood displayed a different overall expression profile when compared to peripheral blood mononuclear cells (PBMC) from five healthy controls, with several genes including the anti-apoptotic genes BCL2A1 and MCL1 and the pro-apoptotic genes BAX and BMF all being significantly differently expressed (>2 fold difference in median expression). Few differences were detected when comparing peripheral blood CLL samples (25/49 IGHV unmutated/IGHV3-21) to lymph node samples (6/9 unmutated/IGHV3-21) or to bone marrow samples (5/7 unmutated/IGHV3-21), with lower relative expression of BIK and BAX in peripheral blood samples. Furthermore, we correlated the expression profiles of all genes included in the assay with IGHV mutation status, IGHV3-21 gene usage and genomic aberrations, but overall few differences were evident between prognostically different subgroups. An exception to this was the BIK gene which showed significantly higher expression in unmutated/IGHV3-21 compared to mutated CLL. Additionally, MCL1 and BNIP3L displayed higher expression in 17p-deleted cases compared to 13q deletion/no aberration cases. High expression of HRK and BAX indicated a correlation with poor overall survival and shorter time to treatment. However, when performing real-time quantitative PCR on an extended patient cohort (n=112) only prediction of time to treatment could be confirmed for HRK and BAX (p=0.0035 and p=0.024, respectively). Finally, in order to validate our MLPA approach, we compared the gene expression levels of four genes in 19 CLL samples using both MLPA and real-time quantitative PCR, which revealed a significant correlation for all genes tested. In summary, MLPA is a quick and cost-effective method for pathway specific gene expression analysis and the present analysis of 67 CLL cases highlights the rather homogenous apoptosis gene expression profiles among different clinical subsets of the disease.

Disclosures
No relevant conflicts of interest to declare.
Effects of Danggui and Its Component Ferulic Acid On Hematopoiesis and Platelet Production.
Authors: Deng Rui Xia , Ye Jie Yu , Liu Chang Charlie , Chan Godfrey ChiFung , Chen Jian Liang , Shen Jian Gang , Yang Mo 
Published: October 2019

Abstract
Abstract 3509
Poster Board III-446
Chinese herbs have been used for the treatment of myelosuppression and thrombocytopenia for centuries and some herb mixtures have been shown to have favorable effects in cancer patients with myelosuppression. Our recent publication has shown that Danggui Buxue Tong, a traditional Chinese medicine decoction containing Radix Angelicae Sinensis (Danggui) and Radix Astragali (Huangqi), promotes hematopoiesis and thrombopoiesis in mouse model (Yang et al, J Ethnopharm, 2009). However, reports about the pharmacological effects of each herb ingredient have been scanty. The root of Danggui is an important ingredient of many commonly used prescriptions in Chinese herbs for promoting blood production. The chemical constituents and the pharmacological action of Danggui have been investigated using modern scientific methods at the beginning of this century. The objective of this study was to compare the effect of Danggui and TPO on hematopoiesis and platelet production in radiation-induced hematocytopenic mouse model. Danggui extracts (10 mg/kg/day) and TPO (10 ug/kg/day) were given by injection daily for 21 days starting from the day after radiotherapy. Peripheral blood platelet, WBC and RBC from Danggui, TPO and water control groups were analyzed at different time-points. On day 21, the mouse was sacrificed and bone marrow cells were harvested for CFU-MK and CFU-GM assays using the plasma clot and methylcellulose methods. The results showed that both Danggui and TPO significantly enhanced the recovery of platelet and WBC count, as well as CFU-MK and CFU-GM formation (n=6). Morphological examination of bone marrow also showed that Danggui treatment significantly increased the recovery of hematopoietic progenitor cells and megakaryocytic cell series. In addition, Danggui also increased the recovery of the body weight and organ weight (liver and spleen) in mouse model. We further analyzed the in vitro effect of Ferulic acid (major component of Danggui) and Astragaloside IV (major component of Huangqi) on hematopoiesis using CFU assay. Ferulic acid plus Astragaloside IV significantly promoted mouse CFU-GEMM formation (n=4). Ferulic acid together with Astragaloside IV also significantly reduced serum free-induced apoptosis in K562 cells by annexin V assay using flow cytometry (n=3). This anti-apoptotic effect can be mediated by reducing the activation of Caspase-3 pathway. In summary, our studies showed that Danggui extracts as well as TPO enhances hematopoiesis and thrombopoiesis in a radiation-induced hematocytopenic mouse model. Ferulic acid, in combination with Astragaloside IV, also has promoting effect on hematopoiesis and anti-apoptotic effect on hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.
Transcriptional Silencing of the Wnt-Antagonist Dickkopf-1 (DKK1) by Promoter Methylation Unleashes Aberrant Wnt Signaling In Advanced Multiple Myeloma
Authors: Kocemba Kinga A , Groen Richard W , van Andel Harmen , Mahtouk Karene , Kersten Marie Jose , Spaargaren Marcel , Pals Steven T 
Published: October 2019

Abstract
Abstract 1919
Aberrant activation of the Wnt/β-catenin pathway is implicated in driving the formation of various human cancers. Recent studies indicate that the Wnt pathway plays at least two distinct roles in the pathogenesis of multiple myeloma (MM): i) Aberrant, presumably autocrine, activation of canonical Wnt signaling in MM cells promotes tumor proliferation and metastasis; ii) Overexpression of the Wnt inhibitor Dickkopf1 (DKK1), contributes to osteolytic bone disease by inhibiting osteoblast differentiation. Since DKK1 itself is a target of TCF/β-catenin mediated transcription, these findings suggests the presence of a negative feedback loop in MM, in which DKK1 acts as a potential tumor suppressor. In line with this hypothesis, we show here that DKK1 expression is lost in most MM cell lines and in a subset of patients with advanced MM. This loss is correlated with activation of the Wnt pathway, as demonstrated by increased nuclear accumulation of β-catenin. Analysis of the DKK1 promoter revealed CpG island methylation in several MM cell lines as well as in MM cells from patients with advanced MM. Moreover, demethylation of the DKK1 promoter restores DKK1 expression, which results in inhibition of β-catenin/TCF-mediated gene transcription in MM lines. Taken together, our data identify aberrant methylation of the DKK1 promoter as a cause of DKK1 silencing in advanced stage MM, which may play an important role in the progression of MM by unleashing Wnt signaling.

Disclosures:
No relevant conflicts of interest to declare.
Bioluminescent Monitoring of Microenvironmental Effects on Multiple Myeloma Engraftment In a Humanized Mouse Model
Authors: Wu Christina , Leu Heather , Ma Wenxue , Shih Alice , Carson Dennis , Kulidjian Ana , Jamieson Catriona 
Published: October 2019

Abstract
Abstract 1927
Multiple myeloma (MM) is characterized by clonal proliferation of CD138+ plasma cells in the bone marrow (BM) and remains an incurable disease. Recent identification of a rare population of MM cancer stem cells (MM CSC) is phenotypically similar to memory B cells (CD138- CD34- CD19+) but differs in that they have the self-renewal capacity within the BM and may be responsible for drug resistance. Preclinical testing of novel therapeutic strategies that target MM CSC requires animal models that closely resemble human disease and allow quantitative evaluation of the applied therapy. We have previously reported results on establishing a MM animal model by transplanting MM CSC from autologous mobilized peripheral blood of primary MM patients, transduced with lentiviral luciferase GFP (GLF) and transplanted intrahepatically (IH) into neonatal RAG2/gc double knock-out (RG-KO). Here we evaluate engraftment efficiency in consideration of BM microenvironment by comparing CD45+ human cell engraftment in mice transplanted either IH to neonates or intrafemorally (IF) to gamma-irradiated young adult mice. MM CSC were selected from isolated PBMC after Ficoll gradient centrifugation of fresh BM biopsy from two primary MM patients or from a human MM cell line, H929, followed by immunomagnetic bead depletion of CD34+ and CD138+ cells. The cells were transplanted into RG-KO mice ranging from 53,000 to 10⋀6 cells per mouse either IH or IF. Mice transplanted with GLF-transduced MM CSC were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that bioluminescence signal levels were detected in mice transplanted IF with 53,000 MM CSC per mouse even before 3 weeks by ventral view and as early as 5 weeks by lateral view. To date, tumor growth was only discovered in mice transplanted IH with 2 × 10⋀6 unselected MM PBMC from a fresh BM biopsy as early as 10 week post-transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+, CD19+ and CD138+ population in tumor, bone marrow, spleen and liver. In addition, expression of clonal light chain restriction in myeloma cells confirmed myeloma engraftment. Future studies will focus on expression of genes involved in sonic hedgehog pathway as analyzed by PCR to confirm the self-renewal capacity. Moreover, investigations on the effect of B cell-activating factor (BAFF) in BM microenvironment by transplanting MM CSC into BAFFxRG-KO mice are in progress.

Disclosures:
Jamieson: Bristol-Meyers Squibb: Research Funding.
High Mitochondrial Membrane Potential Identifies Patients with Myeloproliferative Neoplasms with a More Aggressive Natural History
Authors: Gardner Jeffrey R , Abdel-Wahab Omar , Frattini Mark , Jurcic Joseph G , Knapp Kristina , Rosenblat Todd L. , Soff Gerald A. , Levine Ross , Heaney Mark 
Published: October 2019

Abstract
Abstract 1992
The myeloproliferative neoplasms (MPN) can have a variable natural history. Polycythemia vera and essential thrombocythemia, in particular, are conditions that can extend over decades, but some patients have clinical progression to myelofibrosis or acute myeloid leukemia. As first articulated by Warburg, cancers are metabolically distinguished from normal tissues by the use of glycolysis under aerobic conditions. To metabolically characterize the blood cells of patients with myeloproliferative neoplasms, we measured the mitochondrial membrane potential using the cyanine dye, JC-1. In examining cells derived from the blood and/or marrow of 159 patients with primary myelofibrosis, polycythemia vera and essential thrombocythemia, we found that the mitochondrial membrane potential (FL2/FL1=electrochemical potential/mitochondrial mass) was elevated compared to the blood cells of normal individuals. Thirty five percent of patients with polycythemia vera and essential thrombocythemia had normal MMP. In contrast, 97% of patients with primary myelofibrosis, post-polycythemia myelofibrosis, post-essential thrombocythemia myelofibrosis and acute myeloid leukemia following an MPN had evidence of cell populations with higher mitochondrial membrane potential. Cells with distinctly higher mitochondrial membrane potential could be indentified in platelets and polymorphonuclear leukocytes; however the MMP of lymphocytes was normal, indicating that the alteration in metabolic state likely occurred in a multipotential myeloid stem cell. Cell populations were confirmed by co-staining with anti-CD19, -CD45, -GlycophorinA and -β3-integrin antibodies. Sequential analysis of patient samples found that the acquisition of higher mitochondrial membrane potential was stable and persistent over 2 years or more of follow up and that elevated membrane potential predisposed patients to disease progression. The balance of patients (65%) with ET had evidence of increased MMP suggesting the possibility of disease in an early state of evolution to a more aggressive condition. The increased MMP did not correlate with the presence of mutation in JAK2. These results indicate that clinically advanced MPN can be characterized by changes in mitochondrial physiology that might be identified non-invasively by flow cytometric staining with JC-1. In addition, the early nature of these changes may help to identify therapeutic targets.

Disclosures:
No relevant conflicts of interest to declare.
Thrombocytopenia Induced-Tumor Vascular Leakiness Increases Drug Efficacy
Authors: Demers Mélanie , Ho-Tin-Noé Benoit , Schatzberg Daphne , Yang Janie J , Wagner Denisa D. 
Published: October 2019

Abstract
Abstract 2105
Important research goals in cancer therapy include delivering high doses of drug to tumor sites for maximum treatment efficacy while minimizing side effects to healthy organs. We have recently demonstrated that platelets contribute to tumor vascular homeostasis through prevention of severe tumor hemorrhage. In order to define a new way of increasing drug delivery to the tumor site, we hypothesized that the selective induction of tumor vascular leakiness by thrombocytopenia may facilitate the delivery of chemotherapeutic agents to tumors. Using a mammary carcinoma murine model, we showed that platelet depletion induced hemorrhage specifically at the tumor site and favored the accumulation of fluorescently-labeled microspheres in the tumor. Moreover, the induction of thrombocytopenia in tumor-bearing mice before injection of the chemotherapeutic drug paclitaxel significantly reduced tumor growth compared to mice with normal platelet count that were treated with paclitaxel. Analysis of the tumors ex vivo revealed an increase in tumor apoptosis and a reduction in tumor cell proliferation in thrombocytopenic mice that received therapy. No increased toxicity was seen on other organs and blood cells. Taken together, our results indicate that low platelet count selectively induces leakiness of tumor vessels and allows better access of a chemotherapeutic agent to the tumor, enhancing its tumoricidal effect.

Disclosures:
No relevant conflicts of interest to declare.
Heparin Prevents Histone-Induced Thrombocytopenia and Mortality
Authors: Fuchs Tobias A. , Wagner Denisa D. 
Published: October 2019

Abstract
Abstract 2106
Histones and DNA are released from activated neutrophils to form extracellular traps (NETs) which bind and kill bacteria. Histones contribute to this antimicrobial activity, but they are a double-edged sword because they are a major mediator of death in sepsis. Histones are cytotoxic and intravenous infusion of histones into mice can lead to death. Recently we have shown that NETs provide a scaffold supporting platelet adhesion, activation and thrombus formation. NETs and histones were found in deep vein thrombi. Here we show that platelets in blood are preferentially targeted by histones. Histone binding to platelets induces calcium influx, recruits fibrinogen and causes platelet aggregation which depends on the interaction of histones, fibrinogen and αIIβ3-integrins. Mice challenged intravenously with histones at sublethal concentrations developed a rapid and profound thrombocytopenia reflecting platelet aggregation in vivo. Heparin administration abolished histone interaction with platelets and prevented thrombocytopenia. Heparin also prevented histone induced cytotoxicity towards endothelial cells and rescued mice after a challenge with a lethal dose of histones. The biological function of heparin stored in granules of mast cells is not known. We propose that it may be to counteract histone-mediated thrombosis and injury during innate immune response.

Disclosures:
No relevant conflicts of interest to declare.