bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pretransplant Plasmatic Citrulline and Monocyte-Derived Macrophage Reactivity : Two Useful Surrogate Markers to Predict Acute GvHD
Authors: Hueso Thomas , Magro Leonardo , Jonquel-chevalier Curt Marie , Yakoub-Agha Ibrahim , Seguy David 
Published: October 2019

Abstract
Acute graft versus host disease (aGvHD) represents the first complication after allogeneic stem cell transplantation (allo-HCT). During conditioning regimen (CR) intestinal damage induces microbial translocation, which primes macrophage reactivity and leads to donor-derived T cells alloreactivity. To date, little is reported on intestinal status and macrophage reactivity prior to CR. This study aimed to assess citrulline, an amino acid reflecting intestinal damage, and circulating monocytes-derived macrophage reactivity 30 days before CR.
Blood samples from consecutive patients undergoing allo-HCT for hematological malignancies and from 10 healthy donors were analyzed. Citrulline levels were assessed by high performance liquid chromatography associated to spectrometry. Monocytes were isolated and stimulated with pathogen-associated molecular patterns as LPS, PAM, flagelline, MDP, curdlan and PBS as control. In each condition, cytokines (TNF-α, IL-1β, IL-6, IL-10) releasing was evaluated by multiplex fluorescent immunoassay. Citrulline and cytokine levels were analyzed relatively to aGvHD onset within 100 days post-transplant.
Forty-seven patients underwent allo-HCT for different indications, including acute leukemia (n=26) and myelodysplastic syndrome (n=11). Twenty patients developed an aGvHD I-IV. Among the groups, differences were depicted for levels of citrulline, IL-1β, IL-6 and IL-10. Citrulline levels were lower in aGvHD group than in no-aGvHD group (p<0.05). Among all cytokines, only IL-6 and IL-10 were greater in aGvHD group than no-aGvHD group, especially after curdlan stimulation (p<0.05). Citrulline levels below the normal value (<20 μmol/L), IL-6 and IL-10 above thresholds (326 pg/ml and 78pg/ml after curdlan stimulation respectively) were associated with the aGvHD development (log rank test, p<0.05). Any patients with citrulline>20 µmol/L, IL-6<326 pg/ml and IL-10<78 pg/ml (n=10) declared aGvHD within the 100-days post-transplant.
Our results highlight the role of intestinal damage and macrophage reactivity in aGVHD development. Pretransplant plasmatic citrulline level and monocyte-derived-macrophage reactivity are promising accessible surrogate markers suitable for identifying patients likely to develop aGvHD.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
Expression Pattern of CD55 and CD59 on Red Blood Cells in Sickle Cell Disease
Authors: Al Humood Salah A. , Al-Faris Lama A. , Al-Rukhayes Monera 
Published: October 2019

Abstract
Background: Altered expression of glycosylphosphatidylinositol (GPI)-anchored proteins, might result in increased susceptibility of red blood cells (RBCs) to complement-mediated lysis. Limited information is available on the pattern of CD55 and CD59 expression on RBCs of sickle cell disease (SCD) patients.
Methods: Flow cytometric analysis was performed on RBCs from 71 adult SCD patients and 53 healthy controls, using the commercial REDQUANT kit.
Results: CD59 deficiency was significantly higher among SCD patients than among healthy controls. The proportions of CD55-deficient and CD59-deficient RBCs from SCD patients were significantly higher when compared with those from healthy controls (0.17 vs. 0.09 and 2.1 vs. 1.2, respectively). The MFI of CD55 and CD59 expression on RBCs in SCD was significantly reduced when compared to the expression healthy controls (5.2 vs. 6.4 and 19.4 vs 20.3, respectively). The pattern of CD55 and CD59 expression was not correlated with anemia, biomarkers of hemolysis, erythropoietin level or other pro-inflammatory markers.
Conclusions: There is an altered pattern of CD55 and CD59 expression on RBCs of SCD Patients; however, it does not seem to play a causal role in the pathophysiology of anemia, and is unlikely to be influenced by the level of erythropoietin or other inflammatory mediators.

Disclosures
No relevant conflicts of interest to declare.
Arterio-Venous Fistula Is an Applicable Vascular Access for Erythracytapheresis in Patients with Sickle Cell Disease
Authors: Delville Marianne , Manceau Sandra , Ait Abdallah Nassim , Stolba Jan , Awad Sameh , Damy Thibaud , Gellen Barnabas , Sabbah Laurent , Debbache Karima , Audard Vincent , Beaumont Jean-Louis , Arnaud Cecile , Chantalat-Auger Christelle , Driss Françoise , Lefrere François , Cavazzana Marina , Franco Gilbert , Galacteros Frédéric , Ribeil Jean-Antoine , Gellen-Dautremer Justine 
Published: October 2019

Abstract
Introduction Erythracytapheresis (ER) can improve outcome in patients with sickle cell disease (SCD). A good vascular access is required but can be difficult to obtain for sickle cells patients. Arterio-venous fistula (AVF) has been suggested for ER in SCD supported by limited evidence. We report the largest cohort of ER performed with AVF from three French sickle cell disease reference centers.
Methods Data of SCD patients undergoing ER with AVF in the French SCD reference center were retrospectively collected. The inclusion criteria were: SS or Sβ-Thalassemia and AVF surgery for ER. AVF for hemodialysis were excluded. SCD-related complications, transfusion history, details about AVF surgical procedure, echocardiographic data before and after AVF, AVF-related surgical and hemodynamical complications were collected.
Results Twenty-six patients (mean age 20.5 years; median follow-up 68 months [11; 279]) were included. Twenty-three patients (88.5%) required a central vascular access before AVF. Fifteen AVF (58%) were created on the forearm and 11 (42%) on the arm. Nineteen patients (73%) had stenotic, thrombotic or infectious AVF complications. The median AVF lifespan was 34 months [13; 66]. One patient with severe pulmonary hypertension worsened after AVF creation, and died.
Conclusions We report the first series of SCD patients with AVF for ER demonstrating AVF could be considered as a potential vascular access for ER. Patients with increased risk for hemodynamic intolerance of AVF must be carefully identified, in order to consider alternative vascular accesses.

Disclosures
Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding.
Characterization of B- and T-Cell Immune Repertoires By Anchored Multiplex PCR and Next-Generation Sequencing
Authors: Eberlein Jens , Harrison Thomas , McKittrick Ian , Wemmer Megan , Griffin Laura , Culver Brady P. , Johnson Laura , Kudlow Brian A. 
Published: October 2019

Abstract
The adaptive immune system is involved in various disease conditions including cancer, chronic infection, autoimmune disease and transplant rejection. Adaptive immunity is mediated by B and T lymphocytes, which are activated upon antigen binding to antigen receptors expressed on their surface. Therefore, the spectrum of these antigen receptors, or immune repertoire (IR), provides a means to monitor adaptive immune responses to disease, vaccination and therapeutic interventions. Next-generation sequencing (NGS) of antigen receptor genes is a valuable tool in the study of disease states and responses to various interventions. Traditional amplicon-based NGS assays use opposing primers for targeted amplification of rearranged antigen receptor genes. Thus, large primer panels are required to capture the extensive combinatorial diversity exhibited by the IR. Quantification from such assays requires a complex system of synthetic controls to account for differential amplification efficiency across segment combinations.
Here, we describe an Anchored Multiplex PCR (AMP)-based NGS assay to analyze the IR, employing a minimal set of gene-specific primers in conjunction with molecular barcodes (MBCs) to reduce amplification bias. AMP uses MBCs ligated to cDNA ends and gene-specific primers for amplification, enabling immune chain mRNA interrogation from a single side. This eliminates the need for opposing primers that bind within the highly variable V-segment, eliminating clone dropout due to somatic hypermutation. Furthermore, this facilitates CDR3 sequence capture from highly fragmented RNA inputs. We validated the quantitative reproducibility and sensitivity of AMP-based B- and T-cell IR assays using high-quality mRNA isolated from peripheral blood leukocytes and highly fragmented RNA isolated from formalin-fixed paraffin-embedded (FFPE) samples.
Our data showed high reproducibility between replicates and quantitative clone tracking down to 0.01%. Furthermore, our data indicate that clonal diversity in sequencing data is driven by input quantity, total T-cell number, and, to a lesser degree, mRNA quality. Therefore, AMP-based NGS with MBC quantification and error-correction is a powerful method to characterize the immune repertoire. This enables sensitive clone tracking and measurement of lymphocyte diversity from fragmented RNA samples.

Disclosures
Eberlein: ArcherDX, Inc.: Employment. Harrison:ArcherDX, Inc.: Employment. McKittrick:ArcherDX, Inc.: Employment. Wemmer:ArcherDX, Inc.: Employment. Griffin:ArcherDX, Inc.: Employment. Culver:ArcherDX, Inc.: Employment. Johnson:ArcherDX, Inc.: Employment. Kudlow:ArcherDX, Inc.: Employment.
Circulating Histone Is Elevated in Acute Leukemia and Protects Leukemic Cell Death through Enhancement of Leukemic Cell Adhesion to Endothelium
Authors: Kim Hyunkyung 
Published: October 2019

Background: Leukocytes release nuclear contents into extracellular milieu called by extracellular traps (ET) when they are stimulated by reactive oxygen species (ROS). The nuclear contents mainly compose of histone-DNA complex and neutrophil elastase. This study investigated whether the leukemic cells could release ET and the released histone could induce endothelial activation, finally resulting in leukemic progression.
Methods: The circulating ET biomarkers (histone-DNA complex, cell free DNA, neutrophil elastase) were measured in 80 patients with hematologic diseases and 40 healthy controls by ELISA. ET formation and ROS level were investigated during leukemic cell proliferation in vitro. Histone-induced endothelial adhesion molecule expression and survival were measured by flow cytometry.
Results: Acute leukemia showed high ET biomarkers, which correlated with peripheral blast count. Leukemic cells produced high ROS and release extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, promoting leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, activated protein C), 3 neutralizing antibodies against adhesion molecules, and Toll like receptor(TLR)9 antagonist. When the leukemic cell co-cultured with endothelial cells, the adherent leukemic cells showed better survival than the non-adherent, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death.
Conclusion: Our data for the first time supports that extracellular histone could be released from leukemic cells through ROS dependent mechanism. The released histone could promote leukemic cell adhesion through endothelial adhesion molecule induction and finally protect from leukemic cell death.

Disclosures
No relevant conflicts of interest to declare.
A Prospective Study to Evaluate the Efficacy, Safety and Response Duration of Newly Diagnosed Adult Immune Thrombocytopenia Patients Treated with Low Dose Rituximab and High Dose of Dexamethasone
Authors: Dolai Tuphan Kanti , Mukherjee Soumya , Mandal Prakas Kumar , De Rajib , Chakrabarti Prantar 
Published: October 2019

Abstract
Introduction
Primary immune thrombocytopenia (ITP) is an acquired autoimmune disorder that involves antibody and cell mediated destruction of platelets as well as suppression of their production. Corticosteroids are initial standard therapy in adults. Initially Rituximab (dose375mg/m2) was approved as one of the important second line therapy in chronic ITP. In the present prospective study, we will evaluate the efficacy, safety and response duration of low dose Rituximab (100mg) and high dose Dexamethasone as a front line therapy in new onset adult ITP cases.
Methods
Inj. Rituximab at a fixed dose of 100mg intravenous infusion for four doses (day 1,8,15,22) and Dexamethasone tablet 40 mg/daily PO for four days in a fifteen days' interval (day 1-4 and day 15-18). Response rate evaluated according to published guidelines.
Results
Total no. of cases was 15. Male 5, female 10. Age range 18 to 62 years with median age of 36 years. Course completed in 15 patients. Median follow up of the patients was 6 months. Complete response (CR) was achieved after 1st dose of rituximab in 3 cases, 2 after 2nd dose and 4 after 4th dose of Rituximab. Partial response (PR) was achieved in 2 cases after completion of therapy and no response seen in 4 cases. Seven cases (46.66%) achieving CR maintaining sustained response after 6 months of completion of therapy. Two cases who achieved CR initially reverted to PR after 6 months with dropping of platelet count but without any evidence of bleeding symptoms. Minor side effects like GI intolerance, nausea, vomiting occurred in 3 patients, High blood sugar level during treatment seen in 2 patients and severe adverse effects like pneumonitis and intestinal obstruction seen in 2 patients who needed hospitalization. We did not observe any infusion related reaction.
Conclusions
CR and PR rate was 60% (9/15) and 14%(2/15) respectively. Six months after completion of therapy 77.8% (7/9) showed sustained response after achieving CR. Low dose Rituximab and high dose Dexamethasone is a useful treatment option in adult acute ITP patients.

Disclosures
No relevant conflicts of interest to declare.
Aptamer Mediated Inhibition of Protein S
Authors: Plautz William E. , Layzer Juliana , Sullenger Bruce A. , Majumder Rinku 
Published: October 2019

Abstract
Protein S (PS) is an anticoagulant that acts as a direct allosteric inhibitor of the activated form of factor IX (FIXa); FIXa is a coagulant, in both the presence and absence of its cofactor, factor VIIIa. Inhibition of FIXa by PS results in a retarded rate of activation of factor X; FX plays an important role as the initial protease common to the two pathways of blood coagulation. Factor IX (FIX) deficiencies present in the form of varying severities of hemophilia B; additionally, elevated levels of PS result in lengthened clotting times.
We intend to increase the efficacy and functional lifetime of natural and infused FIXa in hemophilia B patients by creating a high affinity aptamer that selectively inhibits PS in its interaction with FIXa. Although inhibitory aptamers that target pro-coagulant proteins have been produced in a facile manner, the hemostasis field has yet to direct its sights towards a therapeutically viable aptamer that inhibits a naturally occurring anticoagulant.
Through the use of previously selected, and experimentally successful, aptamer pools targeting either Gla-domain or plasma proteomes, we were able to use SELEX to select novel PS ligands. After two rounds of aptamer selection, using the focused pools as an initial state, we have found by nitrocellulose binding assays a strong enrichment from the focused Gla domain proteome, whereas the plasma proteome showed a weaker response.
Following development of an inhibitory PS aptamer, we will perform fluorescence binding and substrate cleavage studies to obtain kinetic constants, and ex vivo studies, including aPTT and thrombin generation assays, to demonstrate aptamer efficacy in a physiological system. Ultimately, we plan to test the aptamer in vivo, to demonstrate its use as a possible adjunct therapy to current treatments of hemophilia B.

Disclosures
No relevant conflicts of interest to declare.
Tracing the Formation of Haematopoietic Stem Cells in Mouse Embryos By Single-Cell Functional and RNA-Seq Analyses
Authors: Tang Fuchou 
Published: October 2019

Abstract
Haematopoietic stem cells (HSCs) are derived early from embryonic precursor cells, such as haemogenic endothelial cells and pre-HSCs. However, the identity of precursor cells remains elusive due to their rareness, transience, and inability to be isolated efficiently. Here we employed potent surface markers to capture the nascent pre-HSCs at 30% purity, as rigorously validated by single-cell-initiated serial transplantation assay. Then we applied single-cell RNA-Seq technique to analyse five populations closely related to HSC formation: endothelial cells, CD45- and CD45+ pre-HSCs in E11 aorta-gonad-mesonephros (AGM) region, and mature HSCs in E12 and E14 foetal liver. In comparison, the pre-HSCs showed unique features in transcriptional machinery, arterial signature, apoptosis, metabolism state, signalling pathway, transcription factor network, and lncRNA expression pattern. Among signalling pathways enriched in pre-HSCs, the mTOR activation was uncovered indispensable for the emergence of HSCs but not haematopoietic progenitors from endothelial cells in vivo. Transcriptome data-based functional analysis revealed de novo the remarkable heterogeneity in cell cycle status of pre-HSCs, with considerable proportion being actively proliferative. By comparing with proximal populations without HSC potential, the core molecular signature of pre-HSCs was identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating the step-wise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical application.

Disclosures
No relevant conflicts of interest to declare.
Influence of Biological Character of Acute Lymphoblastic Leukemia Cell Linet after Overexpression of C/EBPa and PU.1
Authors: Xu Linwei , Hao Xiaoxiao , Wei Yongqiang , Wei Xiaolei , Wei Qi , Feng Ru 
Published: October 2019

Abstract
Objective: To observe the influence of biological character of acute lymphoblastic leukemia cell line Jurkat in vitro and in vivo after overexpression of C/EBPa and PU.1.
Methods: To establish transducing plasmids, using 293T cells to package lentiviral vector and to transfect Jurkat cell line. Flow cytometry is used to analyze the expression of membrane cluster differentiation antigen, apoptosis rate, proliferation and cell cycle of cells. Cell morphology and Q-PCR was used to analyze the changes of cell morphology and molecule gene. Six to eight weeks old immunodeficiency (NSI) mice were transplanted by tail vein injected with 2x106 cells after transduction. To observe the biological behavior and survival time, using Flow cytometric, cell morphology, pathological HE staining, immunohistochemistry to analyze the invasion of leukemia cells.
Results: The expression of CD3 (mark antigen of lymphocyte) fall as low as 20% after overexpression of C/EBPa and PU.1, CD11b (mark antigen of myeloid cell) increase as high as 30%, which has significant difference with Jurkat-gfp group. The apoptosis rate has significantly increase, proliferation rate has significantly decrease after overexpression of C/EBPa, which blocked in S period. Adding myeloid cytokine (GM-CSF, M-CSF, IL-3, FLT-3L,100ng/mL) on the supernatant of transduction cells, Jurkat cells can be reprogrammed to attached cells from suspension after overexpression of C/EBPa and PU.1 in the 5th day. Attached cells can chemotaxis and consuming yeast. Cell multicore and small amount of purple particles in cytoplasm has observed in Jurkat after overexpression of C/EBPa and PU.1, high expression of myeloid gene (CD14, CD64) and low expression of lymphocyte gene (BCL-11B, GATA-3) has observed in cells after transduction. All the mice have leukemia between 1 to 2 months after injection with transfected cells. The survival time is longest in overexpression of C/EBPa group, which shows significant difference (P<0.001). Cells of peripheral blood, liver, spleen and bone marrow can partly transduce to myeloid cells after overexpression of C/EBPa and PU.1, additionally, the expression of CD11b is observed in liver and spleen cells by immunohistochemistry.
Conclusion: Jurkat cells can be partly reprogrammed to monocyte- macrophages, while with the help of myeloid cytokines, it can be reprogrammed to macrophages with chemotactic and phagocytic function. Jurkat cells can be partly reprogrammed to monocyte- macrophages in vivo and prolong the survival time after overexpression of C/EBPa and PU.1.

Disclosures
No relevant conflicts of interest to declare.
Excelent Option Therapy of BONE Marrow Failure in Fanconi Anemia Patients Withouth Full Match Donnor
Authors: Ribeiro Lisandro L , Nichele Samantha , Bitencourt marco Antonio , Petterle Ricardo , Loth Gisele , Pilonetto Daniela , Bonfim Carmem 
Published: October 2019

Abstract
The main cause of morbidity and mortality of FA pts is bone marrow failure (BMF), which usually arises in the first decade of life and progresses to transfusion dependence and severe neutropenia. Androgen treatment has been recommended for FA pts with BMF for whom there is no acceptable hematopoietic stem cell transplant donor. Oxymetholone and Danazol are frequently used in these pts. We retrospectively analyzed data on 67 FA pts who received oxymetholone or danazol for the treatment of their BMF. The starting dose was approximately 1mg/kg for oxy and 2-4mg/kg for danazol. The hematological parameters at the initiation of treatment were hemoglobin (Hb) < 8 g/dL and/or thrombocytes < 30.000/μl. Patients were diagnosed between 01.2005 and 01.2016. The median age was 10.5 ys (2.9 - 40ys). Gender: 39M/27F. The median duration of treatment was 18m (3m - 95m). Fifty-three patients (79%) showed hematological response and became transfusion independence at a median of 3 months after beginning oxymetholone (2-9m) and 5 months after danazol (4-7m). Two adult pts treated with danazol achieved total hematological response with 2.5mg/kg. Seven pts are stable after tapering and stopping androgen with a median follow up of 4 ys (6m-8.5ys). Fourteen pts did not respond to treatment (21%). Eleven pts received an HSCT and seven are alive and well. Three pts were not transplanted and two are alive but transfusion dependent and one pt died from CNS bleeding. All patients developed variable degree of virilization but it was more evident with oxymetholone therapy. Older age at starting therapy was related to less virilization. Conclusion: This study shows the largest number of FA pts treated with androgen up till now. Androgen is an effective and well-tolerated treatment option for FA pts who develop BMF with 79% of them showing transfusion free after 3-5 months. This response may give us time to search for better donors.

Disclosures
No relevant conflicts of interest to declare.