bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinical & Molecular Analysis of Patients with Type 2 (Qualitative) Hereditary Antithrombin (AT) Deficiency
Authors: Patnaik Mrinal M. , Guenther Jennifer , Pruthi Rajiv , Heit John 
Published: October 2019

Abstract
Abstract 4201

Background:
Hereditary AT deficiency is classified as type 1 (quantitative) or type 2 (qualitative). Type 2 deficiency can be further subdivided into type 2a (reactive center loop [RCL]), type 2b (heparin binding domain), and type 2c (pleiotropic) based on functional and molecular AT analysis. Patients with heterozygous type 2b AT deficiency are thought to be at a lower risk for venous thromboembolism (VTE).

Objectives:
1) To estimate the frequency of type 2 hereditary AT deficiency. 2) To utilize molecular analysis to accurately sub type patients with type 2 defects. 3) To correlate thrombotic and obstetric complications with AT deficiency sub types.

Methods:
Apparently unrelated Mayo Clinic AT-deficient patients (n=20) were categorized as type 1 or 2 based on plasma AT activity and antigen, or by molecular analysis for previously reported type 2a-c mutations. Demographic and clinical characteristics were abstracted from patient medical records. The SERPINC1 putative promoter region, all exons (n=7) and splice junctions, and the 3′UTR were PCR amplified from leukocyte genomic DNA, and sequenced with nested forward and reverse primers. For patients without identifiable mutations, multiplex ligand-dependent probe amplification (MLPA) was performed.

Results:
Out of 20 probands tested 7 (35%) had type 2 AT deficiency. The mean patient age at diagnosis was 36 years (range 18–65) and 5 (69%) were women. There were 2 patients with type 2a, 4 with type 2b (including 1 with a homozygous defect), and 1 with type 2c AT deficiency. The mean plasma AT (range) activity/antigen for these patients were 50% (37-67%)/93% (78-103%) [AT activity & antigen normal range=80-130%]. 6 patients had a previously described mutation, whereas 1 had a novel mutation [S380R] affecting the RCL (Table1). All patients with type 2a and type 2c AT deficiency had unprovoked VTE occurring at a young age. All 3 patients with heterozygous type 2b AT deficiency had no VTE or obstetric complications. One patient with a homozygous type 2b defect (AT Vienna) presented with an unprovoked DVT at age 15. One family with AT Toyama (type 2b AT deficiency) had 5 asymptomatic adult family members with the mutation.

Background:
Molecular testing is important for an accurate subtyping of patients with type 2 AT deficiency. Type 2 defects have a diverse clinical spectrum. Patients with heterozygous type 2b AT deficiency have a low rate of VTE and obstetric complications. Homozygous type 2b AT deficiency can be compatible with adult life.

Disclosures:
No relevant conflicts of interest to declare.
Patients with Cancer Who Develop a First Venous Thromboembolic Event After Surgery Are at High Risk of Venous Thromboembolism Recurrence During the Anticoagulation Period
Authors: Louzada Martha L , Lazo-Langner Alejandro , Carrier Marc , Dao Vi , Zhang Jerry , Rodger Marc A. , Kovacs Michael J. , Wells Philip 
Published: October 2019

Abstract
Abstract 4202

Background:
It is unknown whether patients with cancer who develop VTE after a surgical procedure have the same risk of recurrent VTE as clinical patients cancer-associated thrombosis. VTE recurrence risk in non-cancer patients with VTE after surgery is approximately 1% in the 3 months following completion of anticoagulation. It is unknown whether surgical patients with cancer follow the low risk of recurrence as other provoked VTEs or whether they have the high recurrence risk typical of cancer patients.

Methods:
We performed a post-hoc analysis of a single centre retrospective cohort study conducted at the Thrombosis Unit of the Ottawa Hospital. The charts of patients with cancer and VTE followed from 2002 to 2004 and from 2007 to 2008 were reviewed. We sought to compare the risk of recurrent VTE between patients with cancer who developed a first VTE after major surgery with all other patients with cancer-associated thrombosis. We included patients > or = 18 years of age with active malignancy and objectively diagnosed index VTE [pulmonary embolism (PE), proximal deep venous thrombosis (DVT) of the legs or arms, PE + DVT; unusual site thrombosis]. After the first VTE, all patients received a minimum of 6 months of anticoagulation. In the surgery group, index VTE was considered associated with the intervention if it occurred within the first 3 months after the procedure.

Results:
543 patients were included. 121 patients had VTE after surgery and 17 (13.1%) developed a recurrence during therapeutic anticoagulation. Of 422 clinical patients, 61 (14.7%) had a recurrent VTE (Table). The relative risk of recurrent VTE comparing patients who had and who did not have surgery was non-significant (RR= 0.97 (95%CI: 0.587 – 1.574; p= 1.000) suggesting that patients with cancer who undergo surgery have similar risk of developing a recurrent VTE during anticoagulation as patients with cancer-associated VTE who do not undergo surgery. VTE recurrence occurred predominantly within the first 6 months of anticoagulation [Surgery: 9 of 17 patients (52.9 %); no surgery: 45 of 61 (73.7%) patients (p=0.1377)] (Figure). There was no significant difference in VTE recurrence risk according to anticoagulant strategy, tumor site, histology, TNM stage, age or gender between surgery and no surgery groups.

Conclusion:
Patients with cancer who develop VTE after surgery have similar risk of developing a recurrent VTE during the anticoagulation period as clinical patients with cancer-associated VTE.

Disclosures:
Rodger: Pfizer: Research Funding; Leo Pharma: Research Funding; Sanofi Aventis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Canadian Institutes of Health Research: Research Funding; Heart and Stroke Foundation: Research Funding.
Treatment with the Small Molecule Genistein Increases Hepcidin Expression In Zebrafish Embryos and Human Hepatocytes
Authors: Gibert Yann , Zhen Aileen W. , Nguyen Nancy H. , Fraenkel Paula G. 
Published: October 2019

Abstract
Abstract 4245
Hepcidin, a transcriptionally regulated peptide hormone, produced by hepatocytes in response to iron overload, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes, because its levels are inappropriately low in these patients. Among patients with hereditary hemochromatosis, premenopausal women exhibit less severe iron overload than their male counterparts. While this has been attributed to menstrual blood loss, we hypothesized that estrogens may play a role in regulating Hepcidin expression. We tested estradiol and the phytoestrogens, genistein, apigenin, daizdein, and genistin, for their effects on hepcidin expression in zebrafish embryos. In addition to its estrogenic effects, genistein is known to inhibit multiple protein tyrosine kinases. We found that genistein treatment from 28–52 hours post-fertilization in zebrafish embryos enhanced hepcidin transcript levels relative to liver size, as assessed by whole mount in situ hybridization, while estradiol and the other phytoestrogens did not produce a similar effect. To evaluate whether these effects were conserved in human hepatocytes, we treated HepG2 cells with genistein for 24 hours. We found that genistein treatment was associated with a significant increase in Hepcidin transcript levels, which was abrogated by co-treatment with the bone morphogenic protein (BMP) signaling antagonist dorsomorphin, but not by co-treatment with the estrogen receptor antagonist ICI 182,780. In conclusion, genistein promotes Hepcidin expression in hepatocytes in a BMP-dependent, but estrogen receptor independent manner. Future experiments will address the signaling pathway by which genistein exerts its effect on Hepcidin expression.

Disclosures:
No relevant conflicts of interest to declare.
Nitrogen Monoxide Inhibits Heme Synthesis In Reticulocytes
Authors: Mikhael Marc , Apte Sameer , Soe-Lin Shan , Ponka Prem 
Published: October 2019

Abstract
Abstract 4252
Anemia of chronic disease (ACD) is a condition that often manifests in patients with chronic immune activation due to chronic infections, autoimmune disorders, cancer and other diseases. The pathogenesis of ACD is complex and involves inefficient erythropoietin production, immune-mediated inhibition of erythropoiesis, and retention of iron in hemoglobin-processing macrophages. During their development, erythroid cells are closely associated with macrophages. In inflammatory conditions, activated macrophages generate large quantities of the gaseous molecule, nitric oxide (NO), which has numerous effects on iron metabolism. In this study, we explored the possibility that NO affects iron metabolism in erythroid cells. We treated reticulocytes with the NO donors, sodium nitroprusside (SNP) and S-Nitroso-N-acetyl-D,L-penicillamine (SNAP). We show that NO inhibits 59Fe incorporation from 59Fe-transferrin into reticulocytes and their heme. Significantly, 5-aminolevulinic acid (ALA, the product of ALA synthase, which catalyzes the first step of heme synthesis) reversed the SNP-mediated decrease in 59Fe incorporation into heme but not the cellular 59Fe uptake. In addition, SNP treatment led to an increase in eIF2α phosphorylation (which is known to occur in heme-deficient cells) and decreased globin translation. Importantly, the addition of ALA to SNP-treated reticulocytes prevented the effect of SNP on eIF2α phosphorylation and reversed globin synthesis inhibition. This indicates that in SNP treated reticulocytes, the phosphorylation of eIF2α and inhibition of globin synthesis occur indirectly, via NO's effect on erythroid-specific ALA synthase (ALA-S2). These results led us to conclude that NO has two distinct effects on reticulocytes, namely: a decrease in ALA-S2 activity and a decrease in transferrin-mediated iron uptake. The profound impact of NO on heme synthesis, iron uptake and globin translation in reticulocytes raises the possibility that NO production by macrophages could also contribute to ACD.

Disclosures:
No relevant conflicts of interest to declare.
Molecular and Cellular Specialization of Iron Regulation In Human Erythroid Progenitor Cells Prior to Hemoglobin Production
Authors: Noh Seung-Jae , Byrnes Colleen , Lee Y. Terry , Miller Jeffery L. 
Published: October 2019

Abstract
Abstract 4255
Based upon the importance of iron for erythropoiesis, it was hypothesized that specific molecular and cellular mechanisms may be manifested during early erythropoiesis in preparation for heme and hemoglobin production. Here iron regulation was studied in immature erythroid progenitor cells prior to the increased production of heme or hemoglobin. CD34+ cells were cultured in SCF, IL-3 and FLT-3 ligand for 7 days, and the erythroid progenitor cells were identified and sorted from three donors according to the differential expression of CD36, CD41, and CD71. Among the sorted populations, the CD71(+)CD36(+)CD41(-)GPA(-) cells grew almost exclusively (>95%) into erythroid colonies in methylcellulose culture. Cytospin examination of those erythroid progenitor cells demonstrated large blasts with deep blue cytoplasm and blebbed membranes consistent with pre-proerythroblasts. Furthermore, heme accumulation and hemoglobin were absent. Western analyses revealed that ferritin, transferrin receptor 1 (TfR1), and transferrin receptor 2 (TfR2) expression were regulated by dosed titrations (0, 10, 20, 40, 60, and 100 %) of iron-saturated transferrin in the culture medium. The intracellular iron-storage protein, ferritin, showed increased expression with higher holo-transferrin saturation. Opposite patterns of iron-dependent regulation for the transferrin receptors was identified. TfR1 protein decreased and TfR2 increased with increased transferrin saturation. While TfR1 was detected at high levels on the plasma membranes, immunofluorescence studies showed TfR2 co-localization with lysosomes (correlation with LAMP1, a lysosomal marker, R = 0.53 ± 0.11, n=22). Decreased holo-transferrin in the culture medium resulted in no significant reduction in the proliferation of these cells until hemoglobin production was induced by erythropoietin. Confocal microscopy demonstrated a distinct peri-centrosomal lysosomal compartment surrounded by mitochondria. Coincidentally, upon sulfide-silver staining, it was observed that iron accumulated within the lysosomal compartment in a transferrin-dependent fashion. No similar iron-containing lysosomal-mitochondrial structure was identified in non-erythroid cells in the same cultures. These results suggest that functional, lineage-specific differentiation occurs at the progenitor stage of hematopoiesis, and that iron import and regulation is highly developed during erythropoiesis prior to hemoglobin production.

Disclosures:
No relevant conflicts of interest to declare.
Adoptive Immunotherapy with Haploidentical Kir Ligand-Mismatched Natural Killer Cells In Elderly High Risk Acute Myeloid Leukemia Patients: Biological and Clinical Results of A Pilot Study
Authors: Curti Antonio , Ruggeri Loredana , D'Addio Alessandra , Bontadini Andrea , Giudice Valeria , Dan Elisa , Motta Maria Rosa , Rizzi Simonetta , Trabanelli Sara , Urbani Elena , Martinelli Giovanni , Paolini Stefania , Fruet Fiorenza , Isidori Alessandro , Casadei Beatrice , Bandini Giuseppe , Baccarani Michele , Velardi Andrea , Lemoli Roberto M. 
Published: October 2019

Abstract
Abstract 4287

Purpose:
To evaluate safety, feasibility and anti-leukemia potential of haploidentical KIR-L mismatched natural killer (NK) cell infusion in elderly high risk acute myeloid leukemia (AML) patients.

Patients and Methods:
Thirteen patients (5 active disease, 2 molecular relapse and 6 complete remissions) with median age 62 years (range 53–73) received NK cell infusion after immunosuppressive chemotherapy (fludarabine/cyclophosphamide), followed by interleukin-2. Highly purified CD56+CD3- NK cells from haploidentical KIR-L mismatched donors were used. The median number of infused NK cells was 2.74 × 106/Kg. T cells were less than 105/Kg. NK cell chimerism, phenotyping, and functional assays were performed.

Results:
No significant toxicity, including graft versus host disease, related to NK cell infusion was observed. Among patients with active disease, 1/5 obtained transient complete remission (CR), whereas 4/5 patients had no clinical benefit. Both patients in molecular relapse obtained CR, which lasted 9 and 4 months. Three/6 patients in morphologic CR are disease-free after 34, 32 and 18 months. Donor NK cells were demonstrated in the peripheral blood (PB) of all evaluable patients with a peak at day 10 after infusion and, in some cases, also in the bone marrow (BM). NK alloreactivity was demonstrated in vivo by the detection of donor-derived postinfusion NK clones capable of killing recipient targets.

Conclusion:
Infusion of purified CD56+CD3- NK cells is feasible and safe in elderly high risk AML patients. Adoptively transferred NK cells, which can be detected in PB and BM after infusion, are alloreactive against recipient cells and may induce an anti-leukemic activity.

Disclosures:
No relevant conflicts of interest to declare.
Decreased Expression of KLF2 and KLF4 Induced by Antiphospholipid Antibodies Promotes NF-Kb-Mediated Endothelial Cell Activation and Is Modulated by CBP/p300
Authors: Allen Kristi L , Jain Mukesh K , McCrae Keith R 
Published: October 2019

Abstract
Abstract 4315
Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells.

Disclosures:
No relevant conflicts of interest to declare.
Atypical T Cell Prolymphocytic Leukemia – One Case Report
Authors: He Xuefeng , Depei Wu , Ma Xiao , Zhou Yanan , Fu Chengcheng , Li Weiyang 
Published: October 2019

Abstract
Abstract 4332
Here we reported one case of atypical T cell prolymphocytic leukemia. The 38-year female was admitted to our hospital for fatigue and night sweat. She complained of fatigue and night sweat for one month and accompanied by gum bleeding. Physical examination showed anemia and massive spleen extending to umbilical level. Blood routine test indicated pancytopenia (detailed info: Wbc 0.66×109/L,Hb 82g/L,Plt 60×109/L)and elevated proportion of lymphocytes. Blood biochemistry and routine serum tumor marker were normal. Mutation of JAK2V617F was negative. Marrow film showed many atypical mature lymphocytes which expressed mature T cell markers. Marrow biopsy showed a SLL-like scene and accompanied by local fibrosis, immunochemical stains strong CD3, CD45-R0, CD43 and weak CD5, MPO. Repeated TCR β rearrangement indicated positive. PET/CT scan could not find specific elevated SUV lesions. This patient may be diagnosed as atypical T cell prolymphocytic leukemia despite her lymphocytopenia and unpalpable peripheral lymph nodes. She received one cycle of CVP (cyclophosphamide, Vincristine and prednisone) and another cycle of CHOP(cyclophosphamide, Adriamycin, Vincristine and prednisone). Her blood cell count partly recovered and she was preparing for following high dose chemotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Triptolide Exerts a Cytotoxic Effect on Human Acute Myeloid Leukemia Cell Lines
Authors: McCormack Jennifer M.H. , Feldberg Tanya , Lamba Jatinder 
Published: October 2019

Abstract
Abstract 4352

Background:
Triptolide, a diterpenoid derived from the Chinese herb Tripterygium wilfordii Hook f., has shown anti-tumor effects in various leukemia and solid tumor cell lines in vitro. Previous studies have shown that triptolide synergizes with various chemotherapeutic agents in both leukemia and solid tumor cell lines. Triptolide has been shown to synergize with cytarabine in the THP-1 acute myeloid leukemia (AML) cell line (Pigneux et al 2008). This is of particular interest because cytarabine forms the backbone of AML therapy in both children and adults, and cytarabine resistance poses a significant challenge in the setting of refractory or relapsed AML. Therefore, an agent that works synergistically with cytarabine is of particular interest as a potential new agent for the treatment of relapsed or refractory AML. The aim of this study was to assess the cytotoxicity of triptolide, both as a single agent and in combination with cytarabine, in human AML cell lines representing low-, intermediate-, and high-relapse risk groups.

Methods:
The following cell lines were selected: Kasumi-1 (low risk, t(8;21)), HL-60 and THP-1 (intermediate risk), and MV4-11 (high risk, FLT3 ITD). Kasumi-1 and HL-60 cells were cultured in RPMI 1640 media with 20% fetal bovine serum (FBS). MV4-11 cells were cultured in IMDM media with 10% FBS and THP-1 cells were cultured in RPMI with 10% FBS. We performed the following drug treatments: triptolide alone; cytarabine alone; co-treatment with triptolide and cytarabine. Drug-induced cytotoxicity after 48 hours of treatment was assessed using the MTT assay. The combination index (CI) was determined using Calcusyn software (Biosoft, Cambridge, UK).

Results:
Single-agent triptolide induced cell death in all human AML cell lines tested, including cytarabine-resistant Kasumi-1 cells, at low nanomolar concentrations (average Ic50 ranging from 6–15 nM). The combination of triptolide and cytarabine acted synergistically to induce cell death in THP-1 cells as has been previously reported. Interestingly, the combination of triptolide and cytarabine acted in an antagonistic fashion in all other cell lines tested, except for at low drug concentrations in MV4-11 cells.

Conclusion:
Single-agent triptolide exerts a cytotoxic effect on human AML cell lines, including those that are relatively resistant to cytarbine. The effect of the combination of triptolide and cytarabine was cell-line dependent. The results of this study warrant further investigation on the potential role of triptolide as an anti-leukemic agent in combination with cytarabine.

Disclosures:
No relevant conflicts of interest to declare.