bluuebunny/crossref_metadata_2025_split huggingface dataset:
Zoledronic Acid Induces the Proliferation of Human Cord Blood Gamma Delta T Cells Ex Vivo
Authors: Tomchuck Suzanne L , He Jin , Perko Ross W. , Evans Scarlett , McKenna Amy , Dash Pradyot , Thomas Paul , Dallas Mari H 
Published: October 2019

Abstract
Cord blood (CB) T cells are known to be naïve cells, but can be activated to respond similar to adult peripheral blood (PB) T cells. Reports indicate that culture with aminobisphosphonate (NBP) stimulates CB gamma delta T cell proliferation ex vivo, specifically the TCRγ9δ2 subset, which has been extensively studied in PB gamma delta T cells. As CB gamma delta T cells are not well described, we compared CB gamma delta T cell proliferation, phenotype and genotype to PB gamma delta T cells when culturing cells with the NBP, Zometa (zoledronic acid), and IL-2. Fourteen days in culture resulted in significant fold increase in the proliferation of gamma delta T cells and in the percent of lymphocytes in both sample types. PB gamma delta T cells proliferated more robustly than CB with a 288.60 versus 21.32 fold increase, respectively. Additionally, in freshly isolated samples, CB gamma delta T cells comprised an average of 1.404% of the lymphocyte population, which was similar to PB gamma delta T cells, with an average of 2.319%. However, by day 14, PB gamma delta T cells increased to 70.15% of lymphocytes whereas CB gamma delta T cells increased to 12.49%. Phenotypically, both CB and PB had similar percent of CD45RA+ and CD45RO+ gamma delta T cell memory subsets in freshly isolated samples. Following culture, PB gamma delta T cells were mostly CD45RO+ memory cells, with significantly fewer CD45RA+ naïve cells, whereas more CB gamma delta T cells were of the intermediate CD45RA+CD45RO+ subset. Further phenotypic analysis of the memory subsets indicated that cultured PB gamma delta T cells were either effector memory cells (CD27-CD45RA-) or central memory cells (CD27+CD45RA-), while CB gamma delta T cells were mostly naïve (CD27+CD45RA+). The cytokines secreted by these cells were also assessed and the culture of PB and CB gamma delta T cells resulted in differing cytokine secretion profiles. After 14 days of culture, PB gamma delta T cells secreted more IFNγ and TNFα, while CB gamma delta T cells secreted more IL-10 and RANTES. We also examined TCRγ9 and TCRδ2 phenotypic expression and found that the TCRγ9δ2 was a common clone in freshly isolated PB gamma delta T cells, which predominated after 14 days in culture. However, while the TCRγ9δ2 variant was expressed in CB gamma delta T cells, it was low before and after culture, suggesting that Zometa may not stimulate gamma delta T cells in CB the same as PB. As limited TCRγδ phenotypic reagents are available, we developed a single cell PCR assay for genotypic analysis of the TCRγδ repertoire. PCR analysis suggests that the TCRγδ repertoire is diverse in both samples, yet TCRγ9δ2 is most prevalent. Further analysis of the variant subsets is warranted and may give insight into how each of these receptor pairings affects function.

Disclosures
No relevant conflicts of interest to declare.
Site-Directed Pegylation of BDD FVIII (BAY 94-9027) Does Not Alter the Ability of the Molecule to Generate Thrombin, Activate Factor X or Become Inactivated By Activated Protein C
Authors: Yegneswaran Subramanian , Jiang Xiaoqiao , Yesantharao Pooja , Kim Ji-Yun , Leong Lilley , Sim Derek S , Patel Chandra , Kauser Katalin Zsofia 
Published: October 2019

Abstract
Introduction: BAY 94-9027 is a rationally designed B Domain Deleted (BDD) FVIII (Mei , B. et al Blood 2010). A single 60 kDa PEG molecule was attached to amino acids 1804 to increase its circulating half-life, reduce the exposure to epitopes reported to cause immunogenicity in the A3 domain (Shima M. Int. J. Haematol. 2006), while preserving full biological function. The PEGylation technology allows for an extended duration of action with the goal of reducing the number of infusions needed while maintaining protection from bleeds. In a recent clinical trial the molecule has shown significant efficacy for prophylactic treatment in hemophilia A patients (NCT01775618). The objective of this study was to further characterize in vitro BAY 94-9027 using Xase kinetics, activated protein C-dependent inactivation of FVIIIa, and thrombin generation assays.
Methods and Results: Biochemical assays to determine apparent binding of the Factor IXa and Factor VIIIa complex and the Factor X activation kinetics revealed identical in vitro properties of BAY 94-9027 to the BDD FVIII comparator, Refacto AFÒ. The molecules also performed similarly in activated protein C (aPC) FVIIIa inactivation studies.
BAY 94-9027 was further characterized by thrombin generation assay (TGA) in human hemophilic plasma. The TGAs were triggered using 1pM TF or 100pM XIa to simulate extrinsic and intrinsic activation. With XIa activation no differences were seen in peak thrombin, ETP or lag time between BDD FVIII and BAY 94-9027. The lag time, peak and ETP values also remained similar using TF activation. The XIa initiated TGA gives a more potent thrombin generation response compared to TF and is more sensitive to FVIII activity as judged by the left shift of the dose response curves. The TGAs were repeated while titrating thrombomodulin (TM) to generate aPC. Both BAY 94-9027 and BDD FVIII showed similar inactivation profile in the TM-TGA.
Conclusion: The data demonstrates that the rationally designed site specific PEGylation of FVIII in BAY 94-9027 does not alter the in vitro coagulation properties, tested by the ability of BAY 94-9027 to generate thrombin, activate FX or inactivation by aPC. BAY 94-9027 represents the next generation of treatment in hemophilia, using PEGylation technology to reduce the number of infusions while maintaining FVIII activity.

Disclosures
Yegneswaran: Bayer Healthcare Pharmaceuticals: Employment. Jiang:Bayer Healthcare Pharmaceuticals: Employment. Yesantharao:Bayer Healthcare Pharmaceuticals: Employment. Kim:Bayer Healthcare Pharmaceuticals: Employment. Leong:Bayer Healthcare: Employment. Sim:Bayer Healthcare: Employment. Patel:Bayer Healthcare Pharmaceuticals: Employment. Kauser:Bayer Healthcare: Employment.
One Year Survey and Implications of an Hemophilic Computerized Alert System in an University Clinic
Authors: Blum Sabine , Guida Linda , Wasserfallen Jean-Blaise , Angelillo-Scherrer Anne 
Published: October 2019

Abstract
Hemophilia patients are confronted to several issues when being treated in urgent and non-urgent situations. Immediate factor substitution can be life or limb preserving. We installed a computerized haemophilia alert system so as to be informed when one of our patients is treated in another department. Patients admitted to the hospital with a bleeding disorder trigger an alert in the hematology department as well as in the department where the patient is actually located.
We defined alerts as being false positive when an alert was triggered by the admission of a patient where interaction with the hematologist wasn’t necessary and as being false negative when a triggered alert for a patient with a bleeding problem or high bleeding risk in need for interaction with the hematologist didn’t reach the hematologist.
We follow 307 patients in our hemophilia consultation. From 01.01.2011 through 31.12.2011, admission of 128 hemophilic patients to our hospital triggered an alert on 232 occasions. Forty-three percent of presenting patients suffered from von Willebrand’s disease type 1, 11.5% from thrombopathies, 9% from severe hemophilia A, 9% from mild hemophilia A, 9% were hemophilia A carrier and 8% had factor XI deficiency.
Out of 232 alerts in 2011, most were identified as false positive. Eighteen alerts were not followed by a contact between the treating physician and the hematology service (8%). Sixteen alerts (7%) were followed by the establishment of a substitution protocol for an intervention that wasn’t planned before, preventing, in consequence, bleeding complications thereafter. Two alerts (1%) were triggered by 2 patients, 1 suffering from von Willebrand’s disease type 1, the other from von Willebrand’s disease type 2B, with massive bleeding, where intervention by the hematologist was potentially life- or limbsaving, even if one of the alerts was only discovered the following morning.
In conclusion, follow up of 232 alerts during 1 year permitted to intervene possibly life- or limbsaving in only 2 cases. In 16 cases a protocol of substitution could be established that wouldn’t have been made without the alert system. All other cases of alerts were followed up without consequences and thus increased work charge unnecessarily. We continue to follow up our patients with the alert system described.

Disclosures
No relevant conflicts of interest to declare.
Gas6 Promotes Pro-Inflammatory (Ly6Chi) Monocyte Recruitment in Venous Thrombosis
Authors: Laurance Sandrine , Bertin Francois-Rene , Ebrahimian Talin , Lehoux Stephanie , Lemarie Catherine A , Blostein Mark D 
Published: October 2019

Abstract
Recent studies have demonstrated that innate immune cells, i.e. neutrophils and monocytes, provide the initiating stimulus for venous thrombus development. Gas6 was found to promote inflammation by inducing interactions between the endothelium and innate immune cells. In addition, we recently showed that Gas6 was involved in venous thrombosis by inducing tissue factor expression in the endothelium. Since Gas6 is expressed in monocytes, we hypothesize that Gas6 may be involved in monocyte recruitment during venous thrombosis. Venous thrombosis was induced in the inferior vena cava of wild type (WT) and Gas6 deficient (-/-) mice using 5% FeCl3. Using ultrasonography, we found that global monocyte depletion by clodronate resulted in the formation of smaller thrombi. Selective depletion of the pro-inflammatory (Ly6Chi) monocyte subset, using an anti-CCR2 antibody, also induced the formation of smaller clots. In addition, MOMA-2 (monocyte-macrophage marker) staining showed a reduced number of monocytes in thrombi from Gas6-/- mice. More importantly, immunofluorescent staining revealed that fewer Ly6Chi monocytes were recruited to the thrombi of Gas6-/- mice compared to WT. However, Ly6Clow (patrolling) monocytes were equivalently recruited between Gas6-/- and WT mice. In vitro, mRNA expression of CCR2 was increased by thrombin in WT but not in Gas6-/- monocytes. The mRNA and protein expression of the CCR2 ligand, CCL2, was also increased by thrombin in WT but not in Gas6-/- endothelial cells. CCL2 secretion, as demonstrate by ELISA, was induced by thrombin treatment in WT but not in Gas6-/- endothelial cells. Conditioned media from WT or Gas6-/- endothelial treated by thrombin was used for monocyte migration experiments. The conditioned media from WT endothelial cells treated with thrombin increased migration of WT monocytes compared to media from untreated or Gas6-/- endothelial cells. Our results demonstrate an important role for Ly6Chi monocytes in thrombus formation and that Gas6 is specifically involved in the recruitment of these monocytes through the expression of CCL2 and CCR2.

Disclosures
No relevant conflicts of interest to declare.
Evaluation of Blood Group Conversion Following ABO-Incompatible Hematopoietic Stem Cell Transplantation (HCT)
Authors: Chan Esther HL , Christofer Thiow , Lim Susan TT , Tan Lip Kun , Poon Michelle 
Published: October 2019

Abstract
Title: Evaluation of blood group conversion following ABO-incompatible Hematopoietic Stem Cell Transplantation (HCT).
Background:
While there have been a number of reports concerning the influence of AB0 incompatibility on hematopoietic transplantation (HCT) outcomes, data evaluating post-HCT blood group conversions following ABO incompatible transplants remains extremely limited.
Methods:
We performed a single centre retrospective analysis of 97 patients undergoing major or minor or bidirectional ABO-mismatched bone marrow (BM), peripheral blood (PB), and cord blood (CB) hematopoietic stem cell transplantation (HCT) between January2005 – April 2014, in whom engraftment had occurred and blood group data was available. The aim of the study was to analyse the red cell ABO phenotype (forward grouping) and levels of anti-A/B antibodies (reverse grouping) in these patients following the ABO-mismatched transplants.
Results:
97 patients transplanted between January 2004 and April 2014 were included in the analysis. There were 40 (41%) matched sibling, 32 (33%) matched unrelated and 25 (26%) CB transplants. Indications for HSCT included both haematological malignancies (n=87) and benign conditions (n=10). Of these donor recipient pairs, there were 44 (45%) minor, 39 (40%) major and 14 (15%) bidirectional ABO mismatches. All patients had achieved full donor type chimerism at the time of analysis.
Analysis of post –HCT blood group conversion was performed in each of the three groups. Amongst the major mismatch patients (n=39), all had conversion of RBC phenotype to donor ABO phenotype and loss of their host derived anti-donor ABO antibodies. Interestingly, in the minor mismatch patients who were tested (n=44), all the patients had conversion of their RBC phenotype, but 43 out of 44 patients failed to produce recipient directed anti-ABO antibodies. Similarly in the bidirectional mismatch group, all 14 patients converted to donor ABO phenotype, but none produced recipient directed anti-ABO antibodies.
Conclusion:
In one of the largest study looking at this issue, our study demonstrated the lack of a development of recipient directed anti A/B antibodies despite complete donor chimerism in the majority (1 out of 58) of patients undergoing minor or bidirectional ABO-mismatched HSCT. We postulate that this phenomenon may be due to the donor B cell tolerance against host ABO antigen due to the presence of host A and B antigens within other body tissues.

Disclosures
No relevant conflicts of interest to declare.
Dissection of BET Protein Function in a Hematopoietic Differentiation Model
Authors: Stonestrom Aaron J , Hsu Sarah C , Jahn Kristen S , Hsuing Chris CS , Huang Peng , Hardison Ross C , Blobel Gerd A. 
Published: October 2019

Abstract
The mammalian Bromodomain and Extra-Terminal motif (BET) proteins associate with acetylated histones and transcription factors including the master erythroid regulator GATA1. Pharmacologic inhibitors that broadly target BET family proteins are being evaluated in clinical trials for hematologic malignancies. Additionally, a rapidly growing number of studies in model organisms and in vitro systems suggests BET modulation is a therapeutic avenue in diverse disorders. However the mechanisms through which BET proteins act as well as the contributions of individual BET family members to biological processes remain mostly uncharacterized.
Previously we showed that BET family members Brd3 and Brd4 can bind to GATA1 in an acetylation-dependent manner and that pharmacologic BET inhibition impairs erythroid maturation (Lamonica et al PNAS 2011). Using a combination of genome-wide occupancy analysis, pharmacologic inhibition, genome editing and knockdown we characterized the mechanism and function of BET proteins in the context of GATA1-driven erythroid differentiation. BET inhibitors prevented GATA1-mediated transcriptional activation, but not repression, genome-wide. Mechanistically, GATA1 required BETs both for initial chromatin association and for transcriptional activation following the establishment of GATA1 occupancy. As BET proteins associate with chromatin during mitosis when transcription is globally disrupted, they have been implicated as mitotic “bookmarks”. BET inhibition specifically during mitosis failed to elicit a measurable impact on post-mitotic gene activation, calling into question a role of BET proteins as mitotic bookmarks in this system.
To determine the BET protein(s) most relevant to GATA1-activated transcription, we genomically disrupted BET family members Brd2, Brd3 and Brd4 using the CRISPR/Cas9 tool. Despite being present at nearly all GATA1-occupied sites genome-wide, Brd3 disruption had little impact on GATA1-dependent transcription. In contrast, both Brd2 and Brd4 were individually required for efficient gene activation by GATA1. A Brd3 requirement was only revealed in the setting of Brd2 deficiency, indicating functional compensation among select BET proteins. These results provide a comprehensive definition of the functions of BET proteins in a model of cellular differentiation. They further suggest that pharmacologic BET inhibition should be viewed in the context of distinct steps in transcriptional activation and overlapping functions among BET family members.

Disclosures
No relevant conflicts of interest to declare.
A Proteomics Screen Identifies MDM2 Binding Partners Specific to Ribosomal Protein Haploinsufficiency
Authors: Raiser David M. , Schenone Monica , Hartman Emily , Carr Steven A. , Ebert Benjamin L. 
Published: October 2019

Abstract
Ribosomopathies such as 5q- syndrome and Diamond-Blackfan anemia are broadly characterized by haploinsufficiency of one or more ribosome-associated proteins leading to an aberrant activation of the p53 pathway. The prevailing hypothesis for the mechanism of p53 stabilization and activation in the context of ribosomal protein haploinsufficiency suggests that increased binding of a subset of free ribosomal proteins (including RPL5, RPL11, RPL23a, and RPS7) to MDM2 abrogates the negative regulatory control of MDM2 on p53. In order to understand more fully how the binding partner profile of MDM2 changes as a consequence of ribosome dysfunction, we performed a SILAC-based IP/MS proteomics screen on MDM2 immunoprecipitates to identify proteins that were differentially bound to MDM2 with and without shRNA-mediated knockdown of RPS14, a commonly haploinsufficient gene in 5q- syndrome. A V5-tagged MDM2 was expressed in A549 cells, and lentivirally-delivered shRNAs were expressed that targeted either luciferase (for control) or RPS14. Immunoprecipitations were performed on whole cell lysates using a V5 antibody and analyzed by mass spectrometry to identify peptides associated with MDM2 in each condition. We found that RPL5, RPL11, and RPL23a were strongly associated with MDM2 in the context of RPS14 knockdown, but were also abundant in MDM2 immunoprecipitates when a control hairpin was used. Another ribosomal protein, RPL38, was also strongly associated with MDM2 in both conditions. In contrast, RPS14 knockdown appeared to abrogate binding of RPS7 to MDM2, which was strongly associated in the control knockdown condition only. These data corroborate previous findings that a subset of 60S subunit ribosomal proteins can physically associate with MDM2, and they further suggest that this binding may commonly occur even in the absence of ribosomal haploinsufficiency. While differential binding of free ribosomal proteins to MDM2 may indeed contribute to p53 pathway activation in ribosomopathies, these data suggest that the difference in binding is subtle and may be a part of a larger set of p53-activating signals. Additional non-ribosomal proteins were identified that were specifically associated with MDM2 in the context of ribosomal protein deficiency, and follow-up experiments are ongoing to interrogate the biological consequences of these context-specific interactions.

Disclosures
No relevant conflicts of interest to declare.
Hepatitis B e Antibody Positive before Rituximab Combination Chemotherapy Is Associated with a Lower Risk of Hepatitis B Virus (HBV) Reactivation in HBV Carriers with Diffuse Large B-Cell Lymphoma
Authors: Cai Qingqing , Chen Kailin , Geng Qirong , Zhong Guangzheng , Li Jianping , Huang Huiqiang , Lin Tongyu , Jiang Wenqi , Li Zhiming 
Published: October 2019

Abstract
Background: With antivirus prophylaxis, reactivation of hepatitis B virus (HBV) also occurred in HBV carriers (hepatitis B surface antigen [HBsAg] positive) undergoing rituximab combination chemotherapy. It was reported that most HBV carriers with improved long-term outcomes were seropositive for hepatitis B e antibody (HBeAb). In this study on HBsAg-positive lymphoma patients with prophylaxis, the objectives were to determine the HBV reactivation rate in patients with HBeAb positive compared with the patients with HBeAb negative.
Methods: We retrospectively analyzed the medical records of 113 HBV carriers with CD20(+) diffuse large B-cell lymphoma (DLBCL) received R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy with antivirus prophylaxis between August 1, 2002 and November 31, 2011 at Sun Yat-sen University Cancer Center, China.
Results: Among 113 HBV carriers with CD20(+) DLBCL, 68(60.2%) were hepatitis B e antibody (HBeAb) positive. There was no significant difference in terms of sex distribution, age, ECOG PS, stage, baseline HBV DNA detectable rate, or ALT, AST, bilirubin, or LDH between the HBeAb positive group and the HBeAb negative group. However, there were a significantly higher proportion of HBeAg seronegativity (94.1%v 24.4%; P< .001), and HBcAb seropositivity (100.0%v 82.2%; P< .001) in the HBeAb positive group.
All the patients received antiviral therapy before chemotherapy. The antiviral treatments were used as follows: lamivudine in 68 (60.2%) patients, entecavir in 18 (15.2%) patients, others in 27 (23.8%) patients. Other antiviral treatments were including adefovir, lamivudine plus entecavir, lamivudine at beginning and then entecavir, and so on. No significant difference was observed in the antiviral drugs between the two groups.
After R-CHOP chemotherapy, HBV reactivation was found in 23.0% of HBV carriers (26/113). And its incidence rate was lower in HBsAg- positive / HBeAb positive patients than in HBsAg-positive/ HBeAb negative patients (16.1% versus 33.3%; P =.034). Multivariate analysis showed that patients positive for HBeAb before chemotherapy was the only significant and independent protective factor associated with hepatitis due to HBV reactivation after chemotherapy.
Chemotherapy was continued when the serum HBV-DNA level reduced or liver function improved. Survival was not affected by the occurrence of HBV reactivation or the status of HBeAb.
Conclusion: HBeAb positive before rituximab combination chemotherapy was associated with a lower risk of HBV reactivation in HBV carriers with CD20(+) DLBCL. A more effective antivirus prophylaxis may be considered in HBV carriers with HBeAb negative in order to reduce HBV reactivation.

Disclosures
No relevant conflicts of interest to declare.