bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ex Vivo Prothrombin Complex Concentrates and a Specific Antidote Are Effective In Reversing Dabigatran-Induced Coagulopathy In Pigs
Authors: van Ryn Joanne , Spronk Henri M.H. , Rossaint Rolf , Grottke Oliver 
Published: October 2019

Abstract

Introduction
New oral anticoagulants are effective alternatives to warfarin; however, no specific reversal agents are currently available for life-threatening bleeding or emergency surgery. This study measured the effects of prothrombin complex concentrate (PCC), activated PCC (aPCC) and recombinant factor VIIa (rFVIIa) and compared this to reversal with the specific antibody fragment for dabigatran after high-dose dabigatran etexilate (DE) treatment in a porcine model of trauma, to assess reversal of anticoagulation.

Methods
Studies were performed in 5 male pigs after ethical approval. DE was given orally for 3 days (30 mg/kg bid) and, on the 4th day, dabigatran was infused at 0.77mg/kg/h for 30 min followed by 0.52 mg/kg/min for 60 min. The infusion was administered immediately before trauma (blunt liver insult). Blood samples were taken before dabigatran infusion and 60 min post-injury. Two doses of PCC (30 and 60 IU/kg), aPCC (30 and 60 IU/kg) and rVIIa (90 and 180 μg/kg) and two doses anti-Dabi Fab (30 and 60 mg/kg) were added to blood samples ex vivo. Coagulation was assessed by thromboelastometry (ExTEM assay) and PT and dabigatran levels with diluted TT. Data were analyzed by ANOVA (± SEM).

Results
Oral DE prolonged clot formation (CT: 389 ± 128 sec; CFT: 159 ± 39 sec) and PT (27 ± 9 sec), but coagulopathy was more severe after achieving high dabigatran plasma levels (943 ± 147 ng/ml) and inducing trauma (CT: 2378 ± 480 sec; CFT: 3374 ± 625 sec; PT: 167 ± 40 sec). At this time point, 30 IU/kg PCC and 30 IU/kg aPCC significantly accelerated clot formation (PCC: CT 416 ± 70 sec, CFT 250 ± 91 sec; aPCC: CT 525 ± 126 sec, CFT 309 ± 60 sec; p<0.05 vs. untreated samples). These interventions also shortened PT (PCC: 53 ± 14 sec; aPCC: 66 ± 16 sec; p<0.05 vs. untreated samples). Higher doses of PCC and aPCC had no further effect on hemostatic parameters. rFVIIa had no impact on coagulopathy at any time point. Addition of the specific antidote to dabigatran completely normalized all coagulation parameters to baseline levels.

Conclusion
The ex vivo addition of either PCC or aPCC is effective in reducing coagulopathy in a porcine model of high-dose dabigatran therapy and trauma. However the specific aDabi-Fab is the most promising agent to reverse the anticoagulant effects of dabigatran. The potential for reducing blood loss remains to be investigated.

Disclosures:
van Ryn: Boehringer Ingelheim Pharma GmbH & Co. KG: Employment. Spronk:Boehringer Ingelheim Pharma GmbH & Co. KG: Research Funding. Rossaint:Boehringer Ingelheim: Research Funding. Grottke:Boehringer Ingelheim: Research Funding; CSL Behring: Consultancy.
Chromatin Remodeling Enzyme CHD7 Negatively Regulate Hematopoietic Stem Cell Function
Authors: Hsu Jingmei , Huang Hsuan-Ting , Lee Chung-Tsai , Yu Shuqian , Zon Leonard I. , Speck Nancy A. 
Published: October 2019

Abstract
ATP-dependent chromatin remodeling enzymes alter histone/DNA interactions, and are involved in the regulation of transcription, chromosome segregation, DNA replication and repair. We identified Chromodomain Helicase DNA binding protein 7 (CHD7) as a negative regulator of hematopoietic stem cell function. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Although several cases of T and B cell immunodeficiency in a subset of CHARGE syndrome patients have been reported, no previous role for CHD7 in hematopoiesis had been proposed.
We show that morpholino knockdown of Chd7 in zebrafish embryos results in an increased number of runx1 and c-myb expressing hematopoietic stem and progenitor cells in the aorta/gonad/mesonephros (AGM) region, and this effect is cell autonomous as determined by blastula transplantation. Heterozygous germline Chd7 deletion in mice also results in an increased number of phenotypic hematopoietic stem and progenitor cells (Runx1+c-kit+CD31+) in the AGM region. Downstream lineages such as myeloid and erythroid cells are expanded in zebrafish, and similarly conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre results in myeloid lineage expansion and increased granulocyte/monocyte progenitors. Consistent with these results, microarray analysis of murine CD48- CD150+ Lin- Sca-1+ c-kit+ (SLAM LSK) phenotypic long term repopulating HSCs shows up-regulation of genes in several subclasses of the myeloid lineages. Interestingly, although CHD7 deficient mouse bone marrow had a normal frequency of SLAM LSK cells, it had a two-fold higher frequency of functional LT-HSCs as determined by whole bone marrow, purified LSK and SLAM LSK limiting dilution transplants. ChIP-seq performed in human CD34+ hematopoietic stem and progenitor cells show CHD7 localizes to genes encoding many key hematopoietic transcription factors including MYB and RUNX1. The most abundant transcription factor motif under CHD7 genomic binding sites is a RUNX motif, indicating that CHD7 and Runx1 function together. We show CHD7, Runx1 and c-Myb interact both physically and genetically. CHD7 function in hematopoietic stem cells is dependent on both Runx1 and c-Myb since the increase in hematopoiesis in fish upon morpholino knockdown of Chd7 is abolished when either Runx1 or c-Myb is mutated. In summary, our study identifies CHD7 as a novel evolutionarily conserved negative epigenetic regulator of HSCs and progenitors through its interaction with Runx1 and c-Myb.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Depletion Causes Rapid But Transient Activation Of Mouse Bone Marrow Long-Term Hematopoietic Stem Cells To Participate In Stress Thrombopoiesis
Authors: Shao Lijian , Hou Di , Feng Wei , Chang Jianhui , Ware Jerry , Hauer-Jensen Martin , Cheng Tao , Zhou Daohong 
Published: October 2019

Abstract
Hematopoietic stem cells (HSCs) are responsible for the production of various lineages of blood cells throughout life. To ensure the longevity of HSCs and prevent their premature exhaustion, a small population of HSCs termed long-term HSCs (LT-HSCs) is maintained in quiescence under homeostatic conditions. Whether LT-HSCs can exit quiescence rapidly in response to hematopoietic stress to replenish blood cells is not known, and thus was investigated in the present study in an acute but transient platelet depletion mouse model induced by intravenous injection of anti-GPIbα antibody. The results from our study show that in response to platelet depletion LT-HSCs can exit quiescence promptly and then proliferate rapidly to participate in stress thrombopoiesis probably via an alternative differentiation pathway. The mechanism by which platelet depletion causes LT-HSC activation is not due to a direct effect of the antibody on LT-HSCs or activation of platelets, but is attributed to a transient reduction of thrombopoietin (TPO) resulting from the acute depletion of platelets. However, the activated LT-HSCs return to quiescence when blood platelet counts are almost back to normal levels without compromising their function. These findings suggest that there is a very efficient and sensitive feedback regulatory circuitry between quiescent LT-HSCs and platelets, which allows LT-HSCs to directly and promptly respond to hematopoietic stress resulting from an acute loss of platelets. In this response, TPO may function as a switch that can rapidly turn on LT-HSCs to participate in stress thrombopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Oridonin Upregulates All-Trans Retinoic Acid Receptor Alpha and Induces Differentiation Of NB4 Cells Though NF-Kb Pathway
Authors: Yan Hua , Cao Yang , Wei Wei , Zhang Nan , Wu Ying-Li , Chen Guo-Qiang 
Published: October 2019

Abstract
Nuclear retinoic acid receptors (RARs) function as ligand-dependent transcriptional regulators, often heterodimerized with retinoid X receptors. They play critical roles in a variety of biological processes, including development, differentiation and homeostasis. However, it is still not very clear about the mechanism of RARα catabolism. Almost all cases of acute promyelocytic leukemia (APL) express oncogenic fusion protein involving the RARα due to chromosome translocations. Despite the great success of ATRA therapy, it is still important to search for a variety of promising compounds to enhance the pharmacologic activity of ATRA and alleviate its adverse effects. And some of these compounds may be useful as a probe to investigate the mechanism of RARα catabolism.
Our previous studies showed that oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, could have an ability to accumulate RARα, enhance ATRA-induced differentiation in ATRA-sensitive as well as -resistant APL cells, and induce apoptosis which is initiated by reactive oxygen species (ROS) production. Based on those discoveries, we supposed that inhibition of RARα degradation may be responsible for these serial actions. Herein, we used Ori as a probe to explore how Ori stabilized RARα and its potent mechanisms. Firstly, our results showed that Ori upregulated RARa in a dose- and time- dependent manner in NB4 cells while anti-oxidant agent N-acetyl-L-cysteine (NAC) addition completely blocked this effect, indicating that RARa was stabilized via Ori-induced intracellular ROS formation. Secondly, we found H2O2, which could increase ROS level also, upregulated RARa in a dose- and time- dependent manner. More of interest, we found that both Ori and H2O2 upregulated RARa through NF-kB-signaling, proved by the accumulate evidence that either NF-κB pathway inhibitor or knockdown of P65 or inhibition IkBa phosphorylation could block the RARa stabilization. Whereas, P65 overexpression or knockdown of IkBa or some NF-κB pathway activating agents like tumor necrosis factor alpha (TNFa) and lipopolysaccharide ( LPS) could increase the RARa level.
Collectively, these findings suggested that Ori is an extraordinary tool to enhance the pathophysiological and potential therapeutic roles of RARα expression though NF-kB pathway. Moreover, an approach combining ATRA with Ori warrants further investigation as a therapeutic strategy for APL patients.

Disclosures:
No relevant conflicts of interest to declare.
Histones To The Cytosol: Role Of Nuclear Protein Export In Erythroid Nuclear Condensation
Authors: Figueroa Amalia A , Venkatesan Srividhya , Hattangadi Shilpa M 
Published: October 2019

Abstract
Erythropoietin-refractory anemias and red cell aplasias are rare but devastating disorders in children, with no known treatment other than chronic transfusions and bone marrow transplant, with high morbidity and mortality. New, improved treatments for such anemias are severely limited by incomplete understanding of critical terminal erythroid processes such as enucleation. Our long-term goal is to understand the regulation of erythroid nuclear condensation culminating in enucleation in order to increase our basic knowledge of important cell biology concepts like nuclear export and nuclear condensation during erythropoiesis and ultimately help optimize new treatments for anemia.
Proteomic examination of normal pycnotic nuclei from mouse fetal liver (definitive adult erythropoiesis) revealed a striking decrease in almost all nuclear proteins, suggesting that nuclear protein export occurs prior to extrusion. Expression of the nuclear exportin, Exportin 7 (Xpo7) is highly erythroid-specific—utilizing a tightly-regulated erythroid-specific promoter, induced during erythropoiesis, and abundant in late erythroblasts. Knockdown of Xpo7 in primary mouse fetal liver erythroblasts resulted in severe disruption of chromatin condensation and enucleation but otherwise had little effect on erythroid differentiation, including hemoglobin accumulation (manuscript submitted), suggesting that protein export is crucial to the process of nuclear condensation. Nuclei in Xpo7 knockdown cells were larger and less dense than controls. Strikingly, DNA binding proteins such as histones H2A and H3 accumulated in the cytoplasm of normal late erythroblasts prior to and during enucleation, but not in cells lacking Xpo7.
We have confirmed by chromatin immunoprecipitation and RACE-PCR that there is an erythroid-specific promoter (and start-site) for Xpo7, whose expression is not conserved in lower vertebrates or in primitive mouse erythroblasts. We hypothesize that the erythroid-specific function of Xpo7 therefore may account for the fact that red cells are not enucleated in these other biological systems. We have also performedimmunoprecipitation on the erythroid and non-erythroid forms of Xpo7 and determined several erythroid-specific cargo proteins of Xpo7 that are involved with other types of chromatin condensation (including Rcc1 which is involved in chromosome condensation). We are starting to build a model for how Xpo7 facilitates the removal and replacement of histones in the erythroblast nucleus in order to allow adequate nuclear condensation for extrusion.

Disclosures:
No relevant conflicts of interest to declare.
Direct Cell-To-Cell Interactions Between Osteocytes and Multiple Myeloma (MM) Cells Upregulate Sost and Downregulate OPG Expression In Osteocytes: Evidence For Osteocytic Contributions To MM-Induced Bone Disease
Authors: Delgado Calle Jesus , Bellido Teresita , David Roodman G. David 
Published: October 2019

Abstract
Osteocytes are the most abundant bone cells, comprising more than 95% of the cells in bone. They are embedded into the bone matrix, but extensively communicate among themselves and with cells on the bone surface and the bone marrow through the osteocytic lacunar-canalicular network. Osteocytes secrete sclerostin, the product of the Sost gene, an antagonist of Wnt signaling that potently inhibits bone formation. Osteocytes are also a major source of pro- and anti-osteoclastogenic cytokines that regulate osteoclastogenesis and bone resorption, including RANKL and osteoprotegerin (OPG). Recent evidence suggests that the bone remodeling compartment is disrupted in multiple myeloma (MM) allowing close contact of MM cells with bone cells including osteocytes. However, the consequences of these interactions and the contribution of osteocytes to MM bone disease are unclear. Therefore, we determined if interactions between MM cells and osteocytes regulate osteocytic gene expression. We found that co-culture of murine MLO-A5 osteocytic cells with human JJN3 MM cells up-regulated murine Sost mRNA expression 2-3 fold as early as 4h, which remained elevated up to 24h. Consistent with Sost upregulation induced by MM cells, the expression of OPG, a Wnt target gene, was decreased by 30-50% in MLO-A5 cells, resulting in an increased RANKL/OPG at 4h. Culture of JJN3 cells in the top and MLO-A5 cells in the bottom of Boyden chambers abolished both upregulation of Sost and downregulation of OPG mRNA expression in osteocytic cells, demonstrating the requirement of direct contact between MM cells and osteocytic cells. Human Sost and OPG mRNA transcripts were not detected in any of these experiments, demonstrating lack of contribution of MM JJN3 cells. These findings demonstrate that direct interactions between osteocytes and MM cells upregulate the expression of the bone formation inhibitor Sost in osteocytes, which in turn decreases Wnt signaling, reduces osteocytic OPG expression increasing the RANKL/OPG ratio. We propose that increased Sost/Sclerostin expression contributes to the exacerbated bone resorption and the decreased bone formation that characterizes MM induced bone disease.

Disclosures:
No relevant conflicts of interest to declare.
Effect Of Low Dose Bortezomib On Bone Formation In Smoldering Myeloma Patients
Authors: Zangari Maurizio , Berno Tamara , Salama Mohamed E , Sana Sherjeel , Talamo Giampaolo , Pena Karen , Miller Scott , Zhan Fenghuang 
Published: October 2019

Abstract

Background
Bortezomib has been shown to produce an anabolic bone effect (increase bone ALP and osteocalcin) in relapsed/refractory patients. This study examined the bone anabolic effect in patients with smoldering multiple myeloma (SMM) who, with a historical median age of 67 years, have frequent evidence of osteopenia not associated with lytic bone disease. SMM is usually followed expectantly without therapy. The overall risk of progression to active MM has been estimated up to 20% in the first year from diagnosis (Kyle et al, 2007). The primary aim of this trial is to determine the effect of a course of low-dose Bortezomib on bone remodeling and on disease progression.

Methods
The dose of bortezomib used in this trial of 0.7mg/m² is the lowest dose which has shown efficacy in the 3 largest monotherapy trials with bortezomib. Patients enrolled in this study had serum M protein ≥ 3 g/dL and/or bone marrow plasma cells ≥ 10% with absence of anemia, renal failure, hypercalcemia, and lytic bone lesions. Patients received 9 cycles of bortezomib given on days 1, 8, 15, 22 every 42 days. No bisphosphonates were allowed during the trial, Vitamin D supplements were allowed.

Results
Seventeen patients (9 males) with a median age of 61 years were enrolled in the study. Fourteen had IgG paraprotein and 3 had an IgA. Two participants did not complete the treatment; the first because of development of a skin rash and the second for personal reasons. A total of 11 patients completed the protocol. Four are still receiving treatment. At the time of this analysis with a median follow-up of 20 months, all patients are alive and none has progressed (Figure 1). The mean M component at baseline, end-of-study, and at most recent follow-up are shown in Figure 2. The treatment was well tolerated by the patients. One patient developed reversible grade 3 neuropathy and a drug related rash and only 3 grade 3 hematological adverse events were recorded (15%). Bone densities by DEXA scan were obtained at baseline, end of study, and yearly thereafter. Out of 17 patients, 13 had bone density T-scores before and after treatment. Six patients (46%) showed an improvement in hip T-score (mean T-score improvement 0.41, range 0.1-1.35). In the overall group, mean T scores improved by 38% (range, 3-200%). T score in lumbar spine improved in 3 patients (23%), who had a mean T-score improvement of 0.2 (range, 0.05-0.43).

Conclusions
The use of low dose Velcade in smoldering myeloma patients was well tolerated and produces significant increases in bone density in 46% of participants.

Disclosures:
Zangari: Millenium Pharm.: Research Funding. Off Label Use: Use of Bortezomib in smoldering myeloma. Salama:Eli Lilly and Co: Research Funding.
The Impact Of Thymoglobulin Prior To Pediatric Unrelated Umbilical Cord Blood Transplantation On Immune-Reconstitution and Clinical Outcome
Authors: Lindemans Caroline A , Chiesa Robert , Amrolia Persis J , Rao Kanchan , Nikolajeva Olga , de Wildt Arianne , Gerhardt Corinne E , Gilmour Kimberley , Bierings Marc , Veys Paul , Boelens Jaap Jan 
Published: October 2019

Abstract

Introduction
In vivo T-cell-depletion generated by the use of Thymoglobulin (ATG) within the conditioning regimen might contribute to the delayed immune-reconstitution observed after unrelated umbilical cord blood transplantation (UCBT). We studied the impact of early, late and no ATG on immune reconstitution and clinical outcome.

Methods
127 Children receiving an unrelated UCBT in London or Utrecht between 2006 and 2011 were included and divided in 3 groups: late ATG (day -5 to 0, n=48), early ATG (day -9 to -5, n= 33) and no ATG(n=46). The no ATG group received MMF and CsA as GVHD prophylaxis, while the ATG groups both received CSA and prednison as prohylaxis, Endpoints studied were survival, early and late immune-recovery, infections and GvHD. Subset analysis CD3+, CD4+ and CD4+naïve, B and NK cell numbers were prospectively measured during post-transplant follow-up at 1, 2, 3, 6 and 12 months post-UCBT.

Results
The probability of survival was not significantly different between the groups: 71% +/- 8% (no ATG), 68% +/- 9% (early ATG) and 61% +/- 7% (late ATG). There were no significant differences in engraftment and primary or secondary graft failure. Immune reconstitution as defined by CD3+, CD4+ and CD4+ naïve T-cell counts were significantly higher (p<0.001) in the no ATG group at 1, 2, 3, 6, and 12 months post-UCBT with a log more T cells than in the ATG groups. The median CD3+ T-cell count in the no ATG group at 1, 2, 3, 6 and 12 months post UCBT was 340 x10e6/L, 720 x 10e6/L, 535 x 10e6/L, 940 ± 194 x 10e6/L, and 1860 x 10e6/L respectively. In the no ATG group significantly less viral reactivations (p=0.021) were noted. A higher probability (p<0,001) of severe acute GvHD (31%+/-9%) was found in the no ATG group compared to 18% +/- 9% for the early ATG and 5% +/-4% for the late ATGgroup. This was not associated with a higher probability of chronic GvHD or non-infectious lung injury in the no ATG group.

Conclusion
Cord blood transplantation without Thymoglobulin (ATG), in the context of MMF and CsA post-transplant prophylaxis, is associated with higher GVHD risk but with a surprisingly good immune reconstitution and with an engraftment and survival similar to the groups with ATG. The findings of improved immune-reconstitution, associated with lower viral reactivations, albeit at the cost of increased rates of acute GvHD (but not chronic GvHD), suggest that omitting ATG in cord blood transplantation may be important to prevent viral reactivations, especially in those at high risk for these.

Disclosures:
No relevant conflicts of interest to declare.