bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Role of Recipient Platelets In the Prevention of Antibody-Mediated Transfusion Related Acute Lung Injury (TRALI)
Authors: Chen Yuhan , Kim Michael , Tabuchi Arata , Kuebler Wolfgang M. , Aslam Rukhsana , Freedman John J. , Semple John W. 
Published: October 2019

Abstract
Abstract 3351
Transfusion related acute lung injury (TRALI) is a serious complication of transfusion. The pathogenesis of TRALI is not fully understood but previous findings have suggested that platelet depletion can protect mice in a two-hit model of TRALI (Looney et al J Clin Invest 119:3450, 2009). To further understand the role of platelets in preventing antibody-mediated TRALI, two mouse models of immune thrombocytopenia (ITP) were utilized. In the passive ITP model, SCID mice were injected with a monoclonal anti-platelet antibody (MWReg30) intraperitoneally (ip, 18 h before TRALI induction) or intravenously (iv, 2 h before TRALI induction). In the active ITP model, SCID mice were transferred with splenocytes from anti-CD61 immune GPIIIa-knockout mice and thrombocytopenia occurred within 2 weeks post transfer (Chow et al Blood 115;1247, 2010). TRALI induction was performed by injecting the various thrombocytopenic SCID mice with a murine monoclonal MHC class I antibody (mAb, 34-1 -2s) iv and several parameters were observed for up to 2 h post antibody injection. In control, non-thrombocytopenic SCID mice, 34-1 -2s injection caused severe systemic shock as noted by reduced rectal temperatures which was associated with significant lung damage and mortality (45%) within 1 hour of 34-1 -2s infusion as previously shown (Fung et al. Blood DOI 10.1182/blood-2010-05-284570). In contrast, while SCID mice depleted of platelets by the passive ip route had systemic shock, lung damage and a 60% mortality rate, those mice made thrombocytopenic by the iv route were completely protected from mortality. On the other hand, in the active ITP model, where the induced thrombocytopenia is associated with a proinflammatory anti-platelet immune response, no mortality was observed in those mice made thrombocytopenic by antibody-mediated immune mechanisms whereas 80% of mice rendered thrombocytopenic by CD8+ T cell-mediated immunity were dead within 1 hr post 34-1 -2s infusion. These results suggest that thrombocytopenia in itself does not protect against antibody-mediated TRALI severity but the nature of the thrombocytopenia induction (e.g. acute passive iv infusion or active ITP immune transfer) is important. In fact, depending on the inflammatory milieu associated with the thrombocytopenia, platelets may actually increase the severity of TRALI.

Disclosures:
No relevant conflicts of interest to declare.
α-Enolase From Injured Patients and Stored Packed Red Blood Cells Activates Pulmonary Endothelium and Serves as the First Event In a Two-Event Model of Neutrophil Cytotoxicity
Authors: Tucker Nicole , Dzieciatkowska Monika , Hansen Kirk , Khan Samina , Kelher Marguerite , Banerjee Anirban , Moore Ernest , Silliman Christopher C 
Published: October 2019

Abstract
Abstract 3355
A significant number of injured patients with intermediate injury severity scores (15<ISS<30) develop multiple organ failure (MOF), which clinically begins with acute lung injury (ALI). Transfusion of >6 units of stored PRBCs (≥28 days) is associated with the development of ALI/MOF on day 3 post injury (Am J Surg 178:502-4, 1999). The pro-inflammatory mediators, e.g. cytokines, responsible for MOF/ALI in the injured have remained elusive; therefore, we hypothesize that “moonlighting” proteins, which have defined intracellular function when released in the circulation, activate innate immunity and are etiologic in the development of ALI/MOF post-injury.

Methods:
Proteomics on the field blood (plasma) of 3 patients with blunt trauma who later developed ALI/MOF and the plasma from 3 units of packed red blood cells (PRBCs) on day 1 and day 42 were completed using 2-dimensional gel electrophoresis/mass spectroscopy (MALDI/TOF) with computer analyses of the resultant peptides. The proteins from whole cell lysates from Human pulmonary microvascular endothelial cells (HMVECs) were separted by SDS-PAGE, transferred to nitrocellulse and immunoblotted with antibodies to protease activated receptor-1 (PAR-1) and PAR-2. HMVECs were also incubated for 6 hours and 1) ICAM-1 was measured by flow cytometry, 2) isolated neutrophils (PMNs) were added allowed to settle and in selected wells PMN adherence to these activated HMVECs was measured by myeloperoxidase content in the lysate, or 3) after the PMNs settled, lysophosphatidylcholines (lyso-PCs) [4.5μM], lipids from stored platelets implicated in TRALI, were added and the number of viable HMVECs/mm2 were counted by microscopy.

Results:
HMVECs display immunoreactivity for both PAR-1 and PAR-2. Of the 243 proteins identified in the injured patients and the stored vs. fresh PRBCs, α-enolase increased by 10.8-fold and 4.4-fold respectively (p<.05 & p<.005). Both thrombin and α-enolase induced ICAM-1 expression in HMVECs (Table 1) which was inhibited (60±8%) by pre-treatment with the anti-protease leupeptin. α-Enolase also induced significant PMN adhesion vs. media control: Media: 3.1±1.5; α-enolase (50 μg/ml): 14.4±4.7; LPS: 35.5±0.7*. The α-enolase-activated HMVECs vs. buffer-treated lyso-PCs induced significant PMN-mediated cytotoxicity (Table 2). We conclude that α-enolase from the injured and stored but not fresh PRBCs causes pro-inflammatory activation of HMVECs resulting in PMN adherence and PMN cytotoxicity in a two-event in vitro model through activation of PARs receptors. Moonlighting proteins like the glycolytic lyase α-enolase may have unexpected pro-inflammatory activity, which predispose the injured patient to increased morbidity and mortality.

Disclosures:
No relevant conflicts of interest to declare.
The Spectra of Chromosomal Aberrations In Acute Leukemia and Implications for the Development of Optimal Profiles of Leukemic Fusion Genes In Multiplex RT-PCR System.
Authors: Kim Hye Ran , Choi Hyun-Jung , Baek Hee-Jo , Kim Hyeoung Joon , Kook Hoon , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook , Shin Myung-Geun 
Published: October 2019

Abstract
Abstract 3362

Background:
Cytogenetic aberrations have been identified as important prognostic parameters for acute leukemia, in addition to diagnosis and treatment. The aim of the current study was to investigate the spectrum of chromosomal abnormalities in East Asian (Korean) patients with acute leukemia. Based on the spectrum of genetic aberrations found in our study, we proposed a revised list of chromosomal aberrations for the development of optimal profiles of leukemic fusion genes in multiplex RT-PCR system.

Patients and Method:
We prospectively analyzed blood or bone marrow samples from 348 patients (260 adults, 88 children) with leukemia from 2006 to 2009 at the Chonnam National University Hwasun Hospital (Hwasun, Korea). Simultaneous analyses of conventional cytogenetics, FISH and a commercially available multiplex RT-PCR system (HemaVision) were performed.

Result:
Thirty-six types of chromosomal abnormalities were found in the total patient population. Twenty types of chromosomal abnormalities were detected in 42% of all patients by multiplex RT-PCR system and in 33% of all patients by conventional cytogenetics including FISH. Within the group of 213 AML patients, the common cytogenetic abnormalities were t(15;17)(q21;q22) (n=35), t(8;21)(q22;q22) (n=31), 11q23 abnormalities (n=7), inv(16)(p13;q22) (n=3), t(9;9)(q34:q34) (n=2), and the abnormalities t(9;22)(q34;q11), t(9;12)(q34;p13), t(3;21)(q26;q22), t(11;17)(q23;q21), t(12;22)(p13;q11) and t(16;21)(p11.2;q22.3) were each involved in one case. In the group of 104 ALL patients, the following fusion transcripts were detected: 21 t(9;22)(q34;q11), 15 t(12;21)(p13;q22), nine 11q23 abnormalities, four t(1;19)(q23;p13), with t(9;9)(q34:q34), t(9;12)(q34;p13) and del(1p34) each involved in one case. In addition, cryptic translocations were detected in 9% of patients with normal karyotypes or numerical aberrations by conventional cytogenetics. Among 53 patients showing negative results for multiplex RT-PCR, one child patient with early pre-B-ALL harbored MLL1/AF4 fusion genes and 5 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10).

Conclusion:
The current study demonstrated the spectrum and frequency of chromosomal abnormalities in Korean leukemia patients, which were slightly differed from previous studies including western and some Asian countries. These results may offer important implications in understanding characteristics of molecular pathophysiology, the development of a new RT-PCR system as well as revision of the current commercial available RT-PCR system.

Disclosures:
No relevant conflicts of interest to declare.
Adaptor Protein Lnk Negatively Regulates Bcr-Abl-Induced Cell Proliferation through Inhibition of the Stat5 Signaling Pathway.
Authors: Tabayashi Takayuki , Gery Sigal , Koren-Michowitz Maya , Koeffler H.Phillip 
Published: October 2019

Abstract
Abstract 3406
Adaptor protein Lnk negatively regulates not only several hematopoietic cytokine receptors including MPL, EpoR and c-Kit, but also non-receptor tyrosine kinases such as JAK2 and Src. Our previous studies demonstrated that Lnk, when expressed in hematopoietic cell lines, binds and regulates the mutant proteins, JAK2V617F and MPLW515L. Recent in vivo studies have shown that Lnk has an important role in the development of myeloproliferative neoplasms. These data suggest that Lnk may have the ability to inhibit constitutively activated signaling pathways in hematopoietic malignancies. However, how Lnk can attenuate the activity of Bcr-Abl is unclear.
In the present study, we tested the hypothesis that Lnk might play a role in regulating Bcr-Abl function. In order to assess if Lnk can inhibit the proliferation of Bcr-Abl-positive hematopoietic cells, Bcr-Abl-expressing BaF3 cells were stably transfected with either Lnk (BaF3/Bcr-Abl/Lnk) or vector only (BaF3/ Bcr-Abl). Colony-formation assays revealed that Lnk significantly inhibited the proliferation of Bcr-Abl-expressing BaF3 cells. Similarly, overexpression of Lnk inhibited growth in the human CML cell line, K562. To determine the cause of growth inhibition by Lnk, assays for apoptosis were performed. Annexin V staining demonstrated that Lnk overexpression induced apoptosis in Bcr-Abl-expressing BaF3 cells. Western blotting analysis of protein lysates from BaF3/ Bcr-Abl /Lnk cells and BaF3/ Bcr-Abl cells found that Lnk-mediated growth inhibition was associated with downregulation of the Stat5 signaling pathway, but not associated with MAPK and PI3K signaling pathways. In addition, experiments in 293T cells expressing Bcr-Abl and Stat5 with either wild-type Lnk or SH2 mutant Lnk revealed that wild-type Lnk, but not SH2 mutant Lnk, inhibited phosphorylation of Stat5. Interestingly, Lnk inhibited Bcr-Abl-induced Stat5 phosphorylation in a dose-dependent manner. These data suggest that the SH2 domain of Lnk is essential for Lnk–mediated downregulation of the Stat5 signaling pathway in Bcr-Abl-positive cells.
Taken together, our data suggest that Lnk inhibits Bcr-Abl-induced cell proliferation by attenuating the Stat5 signal transduction and may become a therapeutic target for Bcr-Abl-positive leukemias such as chronic myeloid leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia.

Disclosures:
No relevant conflicts of interest to declare.
Effects of Single-Agent, Low Dose Exogenous Erythropoietin In a Long-Term In Vitro serum-Free 3D Culture of Human Cord Blood Mononuclear Cells for Directed Erythropoiesis
Authors: Blanco Teresa Mortera , Mantalaris Athanasios , Santiapillai Joseph , Bismarck Alexander , Panoskaltsis Nicki 
Published: October 2019

Abstract
Abstract 341
Ex vivo expansion of cord blood mononuclear cells (CBMNCs) could provide a safe, flexible and ample supply of blood components for cellular therapies. Traditionally, hematopoietic cell expansion has been performed in 2D tissue culture flask or well-plate static cultures using abnormally high concentrations of cytokines which is expensive, reduces the self-renewal capacity, and skews normal differentiation. We have previously developed a 3D bone marrow biomimicry through the use of a synthetic scaffold made of polyurethane (PU) coated with collagen type I which could expand CBMNCs in a cytokine-free environment for at least 28 days ex vivo, with or without the addition of serum to the media. We hypothesised that the addition of near physiological concentrations (0.2U/mL and 1.845U/mL) of exogenous erythropoietin (EPO) to these established 3D CBMNC ex vivo cultures at day 14 in a serum-free and cytokine-free environment would be sufficient to enhance erythropoiesis. CBMNCs were separated by Ficoll-Paque density gradient and seeded onto collagen-coated PU 3D scaffolds at a cell density of 2.5×106cells per scaffold (5×5×5mm3). Cultures were established in serum-free conditions and only EPO was added at days 14–28, with full-medium exchange every 2 days. Culture output was evaluated at days 14, 21 and 28 both by physically extracting cells from the scaffolds and by in situ analysis. Over 28 days, most stages of maturation, from erythroid progenitors to enucleated erythrocytes were observed by light microscopy of cytospins and by immunophenotypic analysis of extracted cells (CD45−/CD71+/CD235+), with more maturation occurring by day 28 of culture, after the addition of EPO. Although both concentrations of EPO produced comparable erythroid differentiation of cells, even by CFU assay, the viability (75% vs. 61%, p<0.05) and proliferative capacity at day 28 of culture was enhanced in the higher concentration of EPO compared with that in the lower concentration (p<0.05). In contrast, standard 2D control cultures (without serum or cytokines) collapsed within 5 days. In situ, scanning electron microscopy showed maturation of erythrocytes within central sections of the scaffolds to enucleation by day 28 and multiphoton microscopy confirmed the presence of structures resembling erythroid islands as early as day 14 of culture, prior to the addition of EPO. In conclusion, 3D PU-collagen scaffolds may provide a good model to study erythropoiesis ex vivo, using physiological concentrations of EPO, and has the potential to expand red cells in response to higher levels of exogenous EPO in a culture system that would be suitable for clinical applications.

Disclosures:
No relevant conflicts of interest to declare.
Milestones of Post Transplant Monitoring of JAK2 p.V617F Positive MPN Patients.
Authors: Lange Thoralf , Edelmann Anja , Siebolts Uwe , Nehring Claudia , Jäckel Nadja , Cross Michael , Jacqueline Maier , Niederwieser Dietger , Wickenhauser Claudia 
Published: October 2019

Abstract
Abstract 3462
Detection of JAK2 p.V617F point mutation plays an important role in the initial diagnosis of myeloproliferative neoplasms (MPN) and also in the monitoring of JAK2 p.V617F positive MPN following allogeneic stem cell transplantation (SCT). Therefore it is of great interest to optimize molecular monitoring techniques and to define milestones for clinical decisions after SCT. We analyzed 146 probes of 14 patients with a median age of 61 (range 52–70) and with JAK2 p.V617F positive MPN prior and post SCT after reduced (n=10) or myeloablative conditioning (n=4) using related (n=6) or unrelated (n=8) donors. DNA from 70 peripheral blood (PB) samples and 76 bone marrow biopsies (BMB) were analyzed both by a quantitative allele-specific oligonucleotides (ASO) PCR assay (sensitivity 0.01% JAK2 p.V617F mutated alleles per HCK) and a allele specific quantitative real-time (Q) PCR (sensitivity 1%) followed by a JAK2 p. V617F wild type blocking (WTB) PCR in case of a negative result (sensitivity 0.001%). 144 samples has been analyzed with both assays and showed 100% and 89% concordance in samples containing ≥1% and <1% JAK2 p. V617F, respectively. In order to select the optimal compartment, 26 BMB and simultaneously drawn PB samples showed 77%, 92% and 100% concordance for negative or positive results by ASO, (WTB) Q-PCR or both methods. A total of 146 samples from 14 patients, among them 40 samples prior allo-SCT, were analyzed for molecular follow-up. A cut-off of >1% (ASO) and/ or a JAK2 p.V617F positive result in the QPCR was seen in 7 patients. Six (86%) died due to relapse and disease persistence (n=5) or TRM with significant persistence of JAK2 p.V617F. In contrast, all patients with engraftment post allo-SCT exhibited episodes of molecular negativity if the JAK2 p.V617F allele burden was < 1% one month after SCT. We conclude that (1) adequate monitoring post allo-SCT requires a sensitivity of at least 0.01%, (2) monitoring can be performed on full blood samples as peripheral blood and bone marrow biopsies showed identical results, (3) a cut-off of > 1% JAK2 p.V617F allele burden one month post allo-SCT indicates an adverse course of the disease

Disclosures:
No relevant conflicts of interest to declare.
Allogeneic and Autologous Transplantation In Patients with Aggressive B- and T-Cell Lymphomas
Authors: Schueler Frank , Hoffmann Madelaine , Pfrepper Christian , Daneschnejad Said , Lange Thoralf , Pönisch Wolfram , Al-Ali Haifa K. , Niederwieser Dietger W , Basara Nadezda 
Published: October 2019

Abstract
Abstract 3544
The prognosis of patients with aggressive relapsed or refractory B- or T-cell lymphomas is poor with conventional chemotherapy. Therefore, high-dose chemotherapy followed by autologous or allogeneic transplantation is the treatment of choice in these patients.
We investigated the outcome of patients with aggressive B- or T-cell lymphomas transplanted between 1998 and 2009 in one centre. A total of 104 patients with diffuse large B-cell lymphoma (DLBCL) (n=69) and T-lymphoblastic lymphoma (T-LBL) (n=35) received autologous (auto) (n=66; DLBCL: 68%; T-LBL: 32%) or allogeneic (allo) (n=38; DLBCL: 63%; T-LBL: 37%) HSCT. Patients ineligible for auto transplantation, who did not respond to prior chemotherapy or had bone marrow involvement, were assigned to allo transplantation. Allo HSCT recipients were more likely to have high risk disease (higher disease stage, more prior chemotherapy regimens, resistant disease). Recipients of auto HCT were more likely to have chemosensitive disease at HSCT compared to patients at allo HCT (70% vs. 30%). Median age at transplantation was 52 years for auto and 42 years for allo HSCT. Median follow up was 12.7 (0.2-106) months after auto and 6.4 (0.4-111) months after allo transplantion. All patients with auto HSCT received BEAM and patients with allo HSCT TBI-based preparative regimen (myeloablative: n=23; reduced intensity: n=15).
In the cohort of 104 patients the estimated 5-years overall (OS) and progression free survival (PFS) were 38% and 44%, respectively. There was no difference in OS and PFS between DLBCL and T-LBL (p=0.44). The estimated OS and PFS after allo HCT were significant lower (25% and 37%) than after auto HCT (45% and 47%; p=0.008 and p=0.03) caused by outcome differences in patients with DLBCL (p=0.02). Interestingly, OS and PFS were not different in patients with T-LBL transplanted with auto or allo HSCT (p=0.55 [OS] and p=0.34 [PFS]). The estimated PFS was significantly better in chemosensitive than chemorefractory patients (p=0.0001), however the OS and PFS were similar in chemosensitive group of patients regardless auto or allo HSCT (PFS: 50% vs. 51%; p=0.57; OS: 49% (auto) vs 32% (allo); p=0.19). Treatment related mortality (TRM) at day 100 was 18% after allo HSCT compared to 8% after auto.
In conclusion, allo HSCT in patients with aggressive B- or T-cell lymphoma ineligible for auto transplantation is an attractive treatment option for these patients with high risk of relapse with a considerably low TRM. The response status at the moment of HSCT is the most important parameter affecting either OS or PFS.

Disclosures:
Niederwieser: Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Haploidentical Hematopoietic Stem Cell Transplantation In Hematologic Malignancies with G-CSF Mobilized Bone Marrow Plus Peripheral Blood Stem Cells Grafts without T Cell Depletion: a Single Center Report of 29 Cases
Authors: Wang Hengxiang , Yan Hongmin , Wang Zhidong , Xue Mei , Zhu Ling , Liu Jing , Ding Li 
Published: October 2019

Abstract
Abstract 3552

Introduction:
Haploidentical Hematopoietic stem cell transplantation (Haplo-HSCT) has provided an alternative option since virtually all patients have an immediately available donor. In order to reduce the occurrence of severe acute GVHD, initially we used donor bone marrow with G-CSF mobilization as hematopoietic stem cell source and achieved good effect. However, as this work carried out widely, it is difficult to collect enough bone marrow cells from donor due to the much differences body weight between the recipient and donor, and lead to patients with hematopoietic recovery slowly. From February 2003 we started trying to use bone marrow and peripheral blood stem cell as stem cell source.

Patients and Methods:
Twenty-nine patients with hematologic malignancies were enrolled in this study between February 2003 and August 2007 at the general hospital of air force. Conditioning regimen consisting of high-doses of cytarabine and cyclophosphamide with total body irradiation, while 6 cases were preconditioned with busulfan, cytarabine and cyclophosphamide. aGVHD was prevented by a combination of immunosuppressive drugs including a monoclonal antibody against human CD25 (basiliximab), cyclosporine A (CsA), methotrexate (MTX), mycophenolate mofetil (MMF), and a rabbit anti-thymocyte globulin. Donors were given G-CSF at a dose of 300μg daily for 6 consecutive days prior to marrow harvesting. Peripheral blood stem cell was collected on the 7th day.

Results:
All patients attained successful neutrophil and platelet recovery. The median time to neutrophil engraftment was 17.1days, and that of platelet recovery was 20.9 days. The incidence of grade II-‡W GVHD was 31.03% and grade III-‡W GVHD was 13.79%. The GVHD-related death was 3.45%. The incidence of cGVHD was 48.2%. The incidence of extensive cGVHD was 23.3%. The incidence of diseases relapsed was 13.79%. A median follow-up of 54 months noted that 13 patients died, while 16 survived. The total disease-free survival rate longer than 3 years was 55%.

Conclusion:
G-CSF mobilization bone marrow and peripheral blood stem cell as stem cell source for Haplo-HSCT provided rapid and sustained engraftment without increase of severity GVHD. The rate of disease relapse was reduced. This treatment was particularly suitable for patients with heavier weight.

Disclosures:
No relevant conflicts of interest to declare.