bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.
Authors: Zhou Jin , Hou Jinxiao , Li Wen , Zhang Xiaoqian , Fu Yueyue , Li Huibo , Xie Rui , Zhang Zhuo , Gilbert Gary E. , Shi Jialan 
Published: October 2019

Abstract
Abstract 3150
Poster Board III-87

Background
Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs).

Method
PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively.

Results
Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively.

Conclusion
PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time.

Disclosures
No relevant conflicts of interest to declare.
Phage-Displayed Peptide-Q Dot Nanocrystal Combo for High-Sensitivity Bacterial Detection in Plasma.
Authors: Sainath Rao Shilpakala , Ketha Krishna Mohan V. , Atreya Chintamani D 
Published: October 2019

Abstract
Abstract 3153
Poster Board III-90

Introduction
Despite improved phlebotomy practices, refrigeration of red cells, freezing of plasma and improved materials for transfusion product collection and storage, bacterial contamination of transfusion products is still a longstanding problem. Current bacterial detection tests relevant to transfusion medicine, especially for stored platelets have limitations with regard to time, specificity and sensitivity. There is a need for new and improved cost-effective high-affinity detection probes to fill this gap. In this study, using plasma spiked with Bacillus cereus 4342 and B. anthracis-Sterne as an experimental system, we identified peptides from a bacteriophage-displayed random peptide library that selectively bind and detect the Bacillus strains. By labeling the peptides with Q dot-liquid nanocrystals, the detection sensitivity of the peptides was further enhanced.

Methods
A commercially available bacteriophage-displayed random peptide library was screened using B. cereus 4342 as bait, using appropriate controls under stringent conditions. The screening and subsequent sequencing of the phage DNA identified two phages each containing a coding sequence for 12-amino acid peptide that are selectively capable of binding to the Bacillus. Based on the nucleic acid sequence, the two synthetic peptides with biotin tag were prepared for detection assays and to enhance the detection sensitivity further, the peptides were labeled with streptavidin-conjugated fluorescent quantum-dots (Q dots). Fluorescence was measured either by a plate reader or using a fluorescence microscope.

Results
The two synthetic peptides selectively bound to the Bacillus strains in both dot blot and ELISA assays. The membrane-based dot blot assay demonstrated an assay sensitivity of 103 colony forming units/ml (CFU/ml), whereas ELISA demonstrated a sensitivity of 102 CFU/ml detection limit. Fluorometry analysis of spiked plasma samples revealed that the two peptides were able to bind to B. cereus 4342 and B. anthracis Sterne and detect these bacteria in plasma at 102 CFU/ml concentrations. The peptide-Qdot combo even detected a single bacterium in fluorescence microscopy.

Conclusion
Overall, the results reported here validate the usefulness of affinity-selected recombinant filamentous phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity detection probes for bacteria in various platforms and settings relevant to the blood safety and transfusion medicine.
The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

Disclosures
No relevant conflicts of interest to declare.
Screening of Telomere Length in Peripheral Blood Lymphocytes From Patients with Aplastic Anemia by Flow-Fluorescence in Situ Hybridization.
Authors: Nishio Nobuhiro , Takahashi Yoshiyuki , Muramatsu Hideki , Hama Asahito , Nagata Toshihito , Kojima Seiji 
Published: October 2019

Abstract
Abstract 3205
Poster Board III-142
Telomeres in peripheral blood cells from patients with aplastic anemia (AA) are shorter than those from normal individuals. One possible explanation is that telomere shortening reflects the extent and duration of damage to hematopoietic stem cells and the number of compensatory stem cell divisions. Patients with such stressed hematopoiesis probably do not respond to immunosuppressive therapy (IST). Cryptic forms of dyskeratosis congenita (DC) have been recognized in patients with AA who do not have physical abnormalities, but who harbor the gene mutation associated with telomere maintenance. To identify such patients is important because those who have these mutations might respond poorly to IST. The present study evaluates the usefulness of screening telomere length in peripheral blood lymphocytes from patients at the time of diagnosis with AA by flow-fluorescence in situ hybridization (flow-FISH) using Telomere PNA kits (Dako Cytomation, Glostrup, Denmark). After gating diploid cells based on propidium iodine staining, lymphocytes were isolated according to size and granularity. Relative telomere length (RTL) was calculated as the ratio between the telomere signal of each sample and the telomere signal of the control cell line. Because telomeres shorten with age, we obtained age-adjusted measurements for comparison. We used flow-FISH to measure the length of telomeres in peripheral blood lymphocytes from five and two patients with classical and cryptic DC who had the TERT mutation, respectively, to determine whether these two forms of DC can be screened. Significant telomere shortening was detected in all seven patients compared with age-matched normal individuals. To determine the association between telomere length and response to IST, we compared telomere lengths in peripheral blood lymphocytes from 61 normal controls and 27 patients with AA (very severe, severe and non-severe, n = 9 each) at diagnosis who received IST with antithymocyte globulin (ATG) and cyclosporine. The median age of the patients was 9.0 years (range, 1.5 to 14.8). The causes of AA were unknown etiology in 25 patients and hepatitis in 2. In total, 17 of the 27 patients (63%) responded to IST at 6 months after administration of ATG. Age-adjusted delta RTLs did not significantly differ between normal controls and responders (p = 0.181), but those in non-responders were significantly lower than those in normal individuals (p = 0.000). Our findings suggest that screening telomere length in patients with AA at diagnosis is useful for detecting cryptic DC and for predicting responses to IST.

Disclosures
No relevant conflicts of interest to declare.
The Addition of Plerixafor Allows Adequate PBSC Collection in Multiple Myeloma and Lymphoma Patients Poor –mobilizers After Chemotherapy and G-CSF.
Authors: D'Addio Alessandra , Curti Antonio , Giudice Valeria , Motta Maria Rosa , Rizzi Simona , Dan Elisa , Taioli Simona , Baccarani Michele , Lemoli Roberto M 
Published: October 2019

Abstract
Abstract 3233
Poster Board III-170
Peripheral blood stem cell (PBSC) mobilization and subsequent autologous stem cell transplantation (ASCT) is the most effective strategy in many patients with Multiple Myeloma (MM), Hodgkin's Disease (HD) or non-Hodgkin's Lymphoma (NHL). However, a significant proportion of patients are not able to mobilize an adequate number of cells after conventional stem cell mobilization strategies [i.e. chemotherapy and granulocyte colony-stimulating factor (G-CSF) or G-CSF alone], and can not proceed to ASCT.
In the present work, we report, for the first time, of five heavily pre-treated or treatment refractory MM or lymphoma patients (3 males and 2 females, median age 51 years) classified as “poor-mobilizers”, or “no mobilizers” at all, after chemotherapy (DHAP, =2; Cyclophosphamide, =3) and G-CSF mobilization strategy, who received Plerixafor to successfully collect PBSCs. Poor mobilization was defined when the concentration of PB CD34+ cells was consistently lower than 10 cells/μL during the recovery phase after chemotherapy and/or the collection of PBSCs was predicted to be inadequate to perform single or double ASCT. The timing for Plerixafor administration was not planned in advance. The rapid biological activity of Plerixafor (i.e. peak time of CD34+ cell mobilization occurring 10-12 hours after injection) allowed its administration “on demand”, that is when PBSC mobilization was felt to be inadequate, considering WBCs and CD34+ cell kinetics. Plerixafor, at 0.24 mg/Kg, was administered subcutaneously for a maximum of 3 consecutive days, while continuing G-CSF, the evening before the planned leukapheresis.
Overall, the drug proven to be safe and no adverse events were recorded. In all patients we observed a significant fold-increase (mean value= 7.96) in the number of circulating CD34+ cells following Plerixafor administration as compared to CD34+ cell concentration evaluated daily after chemotherapy and G-CSF. All patients were able to collect the minimum required dose for ASCT (2×106 CD34+ cells/Kg) and 4/5 patients collected the target cell dose (4×106 CD34+ cells/Kg for single ASCT in NHL and 6×106 CD34+ cells/Kg for tandem ASCT in MM). Three of 5 patients have already undergone ASCT with Plerixafor-mobilized PBSCs showing a rapid and durable hematological recovery.
Our results demonstrate that the addition of Plerixafor to G-CSF after chemotherapy is safe and allows the rescue of heavily pre-treated or poor-prognosis NHL and MM patients, who need to perform ASCT as consolidation therapy, but are failing PBSC mobilization.

Disclosures
No relevant conflicts of interest to declare.
Deletions of the Normal ABL1 at the Non-Translocated Chromosome 9 in CML Associated with TKI Resistance.
Authors: Virgili Anna , Brazma Diana , Grace Colin , Nacheva Elisabeth P 
Published: October 2019

Abstract
Abstract 3254
Poster Board III-1
Chronic myeloid leukaemia (CML) is characterized by the expression of the BCR/ABL1 fusion gene, a constitutively activated tyrosine kinase that commonly results from the formation of the Philadelphia chromosome (Ph) after a t(9;22)(q34;q11) or variant rearrangement. Some 10% to 15% of CML patients present with cryptic deletions adjacent to the translocation breakpoint junction on the derivative chromosome 9. These deletions are believed to occur during the formation of the Ph translocation and are associated with a worse prognosis for CML patients treated with hydroxyurea or interferon. However there is still conflicting data regarding the significance of these deletions in patients receiving imatinib therapy, which appears to overcome the poor prognosis associated with the genome loss. Here we present data of three BCR/ABL1 positive CML patients on tyrosine kinase inhibitors (TKI) who developed therapy resistance. In all patients loss of the ABL1 sequences was identified. Surprisingly, these deletions affected not the ABL1 sequences of the ABL1/BCR fusion on the der(9) chromosome but the normal ABL1 allele on the non- translocated homologue of chromosome 9. This loss was not present at the time of diagnosis but was a new event developed during disease progression and found along with other additional chromosome rearrangements in the BCR/ABL1 positive cells. Bone marrow chromosome suspensions were studied by array comparative genomic hybridization (aCGH) analysis and/or fluorescent in situ hybridization (FISH) using and a range of commercial and bacterial artificial chromosomes (BAC) probes. While one patient carried a large deletion del(9)(q31q34) that spanned several megabases and was detectable by G-banding analysis, the other two patients had small cryptic losses (from 1.8 Mbp to 2.6 Mbp). The loss involved 3' ABL1 sequences in one patient whereas it affected the whole gene in the other two cases. Since the negative control exercised by the normal ABL1 allele over the BCR/ABL1 expression has been documented, the sensitivity to the TKI might be affected in the BCR/ABL1 positive cell clone bearing the genome loss. In summary, we report a new type of ABL1 loss in Ph positive cells - deletions at the 9q34 associated with TKI resistance in CML patients.

Disclosures:
No relevant conflicts of interest to declare.
Treatment with mTOR Inhibitor, Everolimus (RAD001) Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent or T315I-Mutated Cells.
Authors: Minami Yosuke , Minami Miho , Kuwatsuka Yachiyo , Tanizaki Ryohei , Nomura Yuka , Abe Akihiro , Kiyoi Hitoshi , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 3277
Poster Board III-1
Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells.

Disclosures:
Kiyoi: Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.
Ubiquitin-Mediated Binding and Stabilization of β-Catenin by p210 BCR/ABL.
Authors: Chen Ru , Mahon Gwen , Whitehead Ian P 
Published: October 2019

Abstract
Abstract 3278
Poster Board III-1
Chronic myelogenous leukemia (CML) is a malignant hematopoietic stem cell disorder that is invariably associated with a balanced reciprocal translocation between chromosomes 22 and 9. The major product of the rearrangement is a 210 kD in-frame fusion protein (p210 BCR/ABL) that contains amino-terminal sequences from BCR, and carboxy-terminal sequences from the non-receptor tyrosine kinase ABL. This fusion event leads to the constitutive activation of the ABL-encoded kinase activity, which is the principal driving force behind CML. Thus, inhibitors that target the kinase activity produce durable clinical remissions. Although the ABL-encoded kinase activity is essential for disease progression, several studies have shown that BCR encoded sequences are also necessary for BCR/ABL-mediated leukemogenesis. In this current study we have identified ubiquitin as a binding partner for BCR, and demonstrated that binding is retained in p210 BCR/ABL. We have localized the docking site for ubiquitin to residues 180-191 which is present in all fusion variants of BCR/ABL (p190, p210 and p230). This putative ubiquitin binding domain (UBD) does not conform to any known consensus sequence, and is unique to BCR. Deletion of the UBD does not impair the auto-or trans-kinase activity of p210 BCR/ABL, nor does it impair the interaction between p210 BCR/ABL and GRB2, or the ability of p210 BCR/ABL to activate ERK1/2. A mutation at residue tyr-177 of p210 BCR/ABL also does not impair the interaction with ubiquitin suggesting that the GRB2 and ubiquitin binding sites are adjacent, but separable. β-catenin has been identified as a binding partner for BCR and BCR/ABL, and the docking site has been mapped to a region of BCR/ABL that contains the UBD. Over-expression of p210 BCR/ABL, but not the ubiquitin binding mutant, leads to an accumulation of β-catenin that is phosphorylated on the serine residues that normally trigger ubiquitin-mediated turnover. This difference cannot be attributed to a difference in the activation status of GSK-3β or Pin-1. These results suggest that either β-catenin and ubiquitin share a docking site on p210 BCR/ABL, or that the interaction between β-catenin and p210 BCR/ABL is ubiquitination dependent. The latter possibility is consistent with a previous study which showed that glycine residue in β-catenin is required for Bcr interaction. We propose a model in which p210 BCR/ABL may influence the Wnt signaling pathway by binding ubiquitinated β-catenin, and stabilizing it against degradation.

Disclosures:
No relevant conflicts of interest to declare.
WP1130 Inhibits Signaling through BCR-ABL Ubiquitination and Cytoplasmic to Aggresome Trafficking to Induce Apoptosis of CML Cells.
Authors: Donato Nicholas J , Sun Hanshi , Kapuria Vaibhav , Peterson Luke F , Bartholomeusz Geoffrey , Bornmann William G. , Talpaz Moshe 
Published: October 2019

Abstract
Abstract 3303
Poster Board III-191
BCR-ABL remains the primary target for chronic myelogenous leukemia (CML) therapy but longstanding control and complete suppression of the disease may require additional strategies beyond kinase inhibition. We previously described a small molecule, WP1130, that mediated down-regulation of BCR-ABL, induced apoptosis of CML cells with limited toxicity against normal myeloid progenitors and decreased CML tumor growth in mice (Blood. 2007;109(8):3470-8). In this report we further investigated the BCR-ABL targeting activity and mechanism of action of WP1130 in CML cells. WP1130 rapidly (within 30 to 60 min) reduced cytoplasmic levels of BCR-ABL protein and suppressed Stat5 tyrosine phosphorylation without affecting Jak2, c-Abl, BCR, Erk, Akt, Lyn, Hck or gp130. Down-regulation of BCR-ABL by WP1130 was not affected by point mutations that alter imatinib or dasatinib binding or BCR-ABL kinase inhibitory activity. Examination of the cellular distribution of BCR-ABL in treated cells revealed that BCR-ABL was sequestered into compact, cytoskeletal-enriched aggresomal structures that were recovered in the detergent-insoluble fraction of the cell. WP1130-mediated translocation of BCR-ABL was associated with increased cellular poly-ubiquitination without 20S proteosome inhibition, disruption of Hsp90/BCR-ABL complexes or increased reactive oxygen species. WP1130 induced BCR-ABL trafficking into aggresomes correlated with its poly-ubiquitination (primarily K63) and lysates from WP1130 treated cells demonstrated reduced capacity to hydrolyze ubiquitinated substrate. Together, these results suggest that WP1130 induces poly-ubiquitination and trafficking of BCR-ABL into signaling incompetent aggresomes in CML cells through a unique mechanism that may involve inhibition of deubiquitinase activity and define a unique pathway to suppress BCR-ABL signaling and induce CML cell destruction.

Disclosures
No relevant conflicts of interest to declare.
Allogeneic Stem Cell Transplantation (allo-SCT) for Secondary Acute Myeloid Leukemia (AML) Following Breast Carcinoma.
Authors: Ayari Sameh , Mohty Mohamad , Bilger Karin , Guillerm Gaelle , Guyotat Denis , Michallet Mauricette , Ifrah Norbert , Chevallier Patrice 
Published: October 2019

Abstract
Abstract 3305
Poster Board III-193
Patients with breast carcinoma who received Radio and/or chemotherapy, have an increased risk for developing therapy-related myelodysplastic syndromes/acute myeloid leukemia (1-5%). Such secondary AMLs have often poor prognosis when treated with conventional chemotherapy.
This retrospective series assessed the outcome of 29 female patients who were reported to the Societe Francaise de Greffe de Moelle et de Therapie Cellulaire Registry, and who were treated with allo-SCT for secondary MDS/AML developing after initial therapy for breast cancer. The median age of patients at time of breast cancer treatment was 51 (range, 31-62) years. The median age at time of allo-SCT was 53 (range 31-63) years. Diagnosis included 21 AML and 8 MDS. Cytogenetics: four patients had CBF abnormalities, 10 had 11q23 abnormalities, 5 had an intermediate caryotype and 3 had unfavourable one. At time of allo-SCT, 21 patients were in complete remission (CR), while 8 had a refractory/relapsed disease. PBSCs were used as stem cell source in 18 patients, while 9 patients received classical bone marrow and one patient received an unrelated umbilical cord blood. Patients received a median of 5.2 ×10e6/Kg CD34+ cells. A matched related donor was used in 23 cases (82%) and an unrelated donor in 5 cases (18%). Conditioning regimen was myeloablative (Cy-TBI or Bu-Cy) in 7 cases (24%) and reduced-intensity in 22 cases (76%). Twenty four patients engrafted with a median time of 18 (range, 9-32) days for ANC>500/μL. Seven (24%) patients experienced grade 2-4 acute GVHD. Also, 7 patients (24%) experienced chronic GVHD (5 extensive and 2 limited).
With a median follow-up of 24 (range, 3-129) months, 16 patients (55%) were still alive. Disease progression accounted for 6 deaths while transplant related causes (infection n=4, GVHD n=1, MOF n=1, cardiac failure n=1) occurred in 7 cases. The Kaplan-Meier estimates of overall and disease-free survival at 2 years were at 54 and 38% respectively.
These results highlight the poor outcome of secondary leukemia occurring after therapy for breast cancer, even with the use of allo-SCT. Innovative maintenance approaches such as early use of hypomethylating or immunomodulatory agents after allo-SCT aiming to decrease the relapse are warranted.

Disclosures
No relevant conflicts of interest to declare.