bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increased Capacity Of Monocytes From Patients With Multiple Myeloma Treated With Thalidomide Or Lenalidomide To Generate Tissue Factor
Authors: Kornberg Abraham , Izak Marina , Cohen Yossi 
Published: October 2019

Abstract
Treatment of multiple myeloma (MM) with thalidomide or lenalidomide is associated with increased incidence of thrombosis in contrast to treatment with bortezomib. The mechanism of thrombosis is unknown. Monocytes generate potent tissue factor (TF), the main activator of coagulation, in response to various stimuli (endotoxin, cytokins and others) and in diseases with increased incidence of thrombosis. We investigated the capacity of monocytes from patients with MM to generate TF in relation to different modalities of treatments and activation of coagulation. Peripheral blood mononuclear cells (PBMC) were isolated on Ficoll-Hypaque centrifugation and monocytes by adhesion. TF activity was assayed by modified PT using the cells as source of TF and TF antigen by ELISA, in endotoxin stimulated (10 ug/ml) and unstimulated PBMC ( all patients) and purified monocytes (50% of patients), before and after 3-4 courses of treatment. Monocytes were identified by anti CD14 and Plasma Fragment1.2 was assayed by ELISA.
No TF activity@ (X10-5 U/monocyte) TF antigen@ (X10-5 pg/monocyte) Plasma fragment 1.2@ (nM) Endotoxin - + - + Bortezomib based treat* 12  before 2.2 4.8 22 64 0.28  after 1.8 4.2 18 57 0.36 Thalidomide based treat* 4  before 2.4 5.5 26 60 0.24  after 1.8 34.8# 20 440# 2.48# Lenalidomide based treat^ 6  before 2.1 6.2 28 62 0.30  after 4.1 58.6# 16 312# 3.42# @Mean *First line treatment ^Second line treatment # P<0.05
One patient on thalidomide and 2 on lenalidomide developed thrombosis. The results show that TF was low in unstimulated monocytes from all patients before treatment. It was increased mildely in stimulated monocytes from all patients before treatment and also after treatment with bortezomib (X 2-3). TF in stimulated monocytes and also plasma fragment1.2 were increased significantly after treatment with thalidomide and lenalidomide (x 14-22 and X 10). The phenomenon of enhanced capacity of monocytes to generate potent TF after treatment with thalidomide and lenalidomide but not bortezomib, and the correlation with activation of coagulation suggest a role of monocyte TF in thrombus formation by these drugs. The mechanism of the enhanced capacity is unknown and may be attributed to the immunomodulatory effect of the drugs.

Disclosures:
No relevant conflicts of interest to declare.
Tanshinone IIA has anti-platelet effect and induces apoptosis in megakaryocytes via TNFR and Caspases
Authors: Yang Mo , Wu Fu qun , Zhou Min , Ye Jie yu , Li Xiao jing , Chong Beng , Liu Chang 
Published: October 2019

Abstract
Tanshinone IIA (TIIA) isolated from Danshen (Radix Salviae Miltiorrhiza Bge) is a derivative of phenanthrenequinone, which has been used for centuries to treat hypercoagulation related diseases. Our previous study showed that TIIA has neural protective effect (Xia et al, Pediatric Research, 2005). In this study, we investigated the effect of TIIA on platelets and megakaryocytes in a rabbit model, and its molecular mechanisms. Rabbit immune vasculitis model was established by intravenous injection of bovine serum albumin with TIIA treatment (5 mg/kg/day, iv). The platelet count, platelet aggregation, and inflammatory cytokine IL-1β level were significantly increased in vasculitis model compared with normal group at day 7. There were positive correlations between platelet count and IL-1β level. The number of megakaryocytes and CFU-MK formation in bone marrow were significantly increased in this model. Treatment with TIIA significantly decreased all these parameters, and also significantly reduced the injury vessel in immune vasculitis. Furthermore, the effect of IIA on in-vitro megakaryopoiesis was investigated. TIIA at 10-30 ug/ml significantly inhibited CFU-MK formation (p<0.05, n=6). TIIA also induced the apoptosis of megakaryocytic cell line CHRF. The cell viability was reduced to 81% after 72hrs of TIIA treatment. Using Annexin V/PI staining, TIIA induced apoptosis on CHRF cells in a dose dependent manner. This was verified by Caspase 3 assay which showed the activation of Caspase 3 increased from 5% to 16% after TIIA treatment. Using JC-1 assay, we found that the depolarized cells increased from 9% to 17% after TIIA treatment suggesting the involvement of intrinsic apoptotic pathway. To further determine the molecular mechanisms involved in the pro-apoptosis effect, Microarray studies using Affymetrix 133 plus genechips were conducted to identify the genes that were differentially expressed after TIIA treatment. Several groups of genes involved in apoptosis, calcium regulation and cell cycle checkpoints were found to be differentially expressed after the treatment. The differential expressions of these genes were validated using quantitative PCR. The most significantly upregulated gene (6.0±0.333 folds) was TNF Receptor Super-Family 9 (TNFRSF9). In addition, Receptor Interacting Protein Kinase (RIPK), a protein that likely interacts with TNFRSF9, was up-regulated to 2.0±0.167 folds. These results suggest that TIIA has an anti-platelet effect in this model. Its mechanism may be related to its inhibiting megakaryopoiesis and inducing apoptosis of megakaryocytes via TNFR and caspases.

Disclosures:
No relevant conflicts of interest to declare.
Development of HemSol-P(TM), a Novel Platelet Storage Solution Permitting Storage at 4o C
Authors: Ho David H. , Stefanson Steingrimur , Ershler William B 
Published: October 2019

Abstract
The current method of storing platelets as platelet rich plasma (PRP) limits storage life to five days. As many as 10% of platelet units are discarded at a cost of approximately $500 million dollars annually in the U.S. alone. Platelets stored at 22°C remain metabolically active and thus require nutrients supplied in PRP. These nutrition rich platelet preparations promote bacterial growth, demonstrable in approximately one of 3000 platelet units and one of six contaminated units result in clinical sepsis. Also of critical importance, the transfusion of plasma has been associated with transfusion-related acute lung injury (TRALI), which is now the leading cause of transfusion related deaths.
We have developed an innovative preservation technology to address these issues. This process involves mixing cells with a non-toxic proprietary mixture of simple and complex carbohydrates that protect cells from oxidative damage, reduce metabolic activity and allow extended storage. Preliminary studies demonstrate that platelets preserved in this solution (HemSol-P) for up to 14 days at 22°C respond normally to platelet agonist in aggregation assays and had low active GPIIb-IIIa expression and demonstrated low Annexin V binding.
Furthermore, platelets stored in HemSol-P can be maintained at 4oC for at least 15 days and remain in circulation in a mouse transfusion model. Mouse platelets were isolated from 0.1 ml of blood collected by retro-orbital puncture. Washed platelets were biotinylated and then resuspended in either plasma or HemSol-P to a final concentration of 2x109 platelets/mL and stored at 4°C for up to 15 days. Our results show that platelet survival in the circulation 24 hours after transfusion held steady at 65%, even after 15 days of cold storage in HemSol-P.
HemSol-P has excellent potential for clinical application in transfusion medicine. The potential for cold storage extends shelf life of donated platelets beyond five days. The absence of a plasma component in HemSol-P will likely reduce the incidence of contamination/infection and TRALI, and also reduce overall transfusion costs by increasing the platelet storage time and simplifying procurement and preparation logistics.

Disclosures:
Ho: HeMemics Biotechnologies Inc: Employment, Equity Ownership, Patents & Royalties. Stefanson:HeMemics Biotechnologies Inc: Employment, Equity Ownership, Patents & Royalties. Ershler:HeMemics Biotechnologies Inc: Consultancy, Equity Ownership.
Glucocorticoids Reduce Expression Of p53 Target Genes and Reverse Anemia In a Mouse Model Of Diamond-Blackfan Anemia
Authors: Sjögren Sara , Jaako Pekka , Karlsson Stefan , Flygare Johan 
Published: October 2019

Abstract
Diamond-Blackfan anemia (DBA) is a rare congenital disease of macrocytic anemia, increased cancer predisposition and progressive bone marrow failure. Mutations in ribosomal protein genes are responsible for most cases and the most commonly mutated gene, RPS19 is affected in around 25% of DBA patients. Although glucocorticoid (GC) administration has been used to stimulate erythropoiesis in DBA patients for decades, the therapeutic mechanism of GC treatment is poorly understood. Since it has been shown that bone marrow failure in DBA is caused by p53 activation we hypothesized the therapeutic mechanism of GC involves reduced p53 activity. To assess possible connections between p53 activity and GC administration in DBA we used a doxycycline-inducible mouse model for RPS19-deficient DBA (Jaako et al, Blood 2011). In these mice, doxycycline administration leads to reduced Rps19 levels, defective ribosomal biogenesis, proliferative arrest, increased apoptosis of erythroid progenitors and p53-dependent bone marrow failure.
In this study we show that Rps19-deficient mice respond to treatment with the GC prednisolone, with a significant increase in red blood cell count as well as in hemoglobin concentration and hematocrit compared to Rps19-deficient controls (p-values<0.05). Also, prednisolone treated Rps19-deficient mice show a significant increase in the Ter119+ erythroid cell fraction in the bone marrow compared to Rps19-deficient controls. Gene expression analysis of c-Kit+ fetal liver cells from E14.5-15.5 of Rps19-deficient embryos cultured in vitro in the presence of doxycycline for 3 days, showed an increase in p53 responsive gene expression of multiple p53 targets such as p21, Bax, Ccng1, Phlda3 and Prl-3. However, upon administration of the synthetic GC dexamethasone all these genes significantly failed to up-regulate, even in the presence of doxycycline (p-values< 0.02). Also, dexamethasone treated RPS19-deficient c-Kit+ fetal liver cells show an increase in proliferation compared to RPS19-deficient controls (p-value< 0.05), that starts already after 2 days of in vitro culture, being more pronounced over time. These results indicate that administration of GC dampens p53 responsive gene expression of genes known to promote cell cycle arrest as well as apoptosis. Although GCs are known to regulate many functions within an organism, our results indicate that the therapeutic effect of GC in DBA patients at least in part is explained by inhibition of p53 responsive gene activation, leading to increased proliferation and survival of erythroid progenitors.

Disclosures:
No relevant conflicts of interest to declare.
FANCD2 and RAD51 Regulate PCNA Monoubiquitination and Translesion Synthesis
Authors: Kupfer Gary M. , Chen Xiaoyong 
Published: October 2019

Abstract
FANCD2 is a key player in Fanconi anemia (FA) pathway. It has been shown that PCNA and Rad18 can interact with FANCD2 and regulate FANCD2 monoubiquitination. Rad18 is the E3 ubiquitin ligase responsible for PCNA monoubiquitination, which is critical for PCNA function in translesion synthesis (TLS). Previous work by us and others has shown that the FA pathway interacts with PCNA and RAD18. Even though FA cells demonstrate unequivocal sensitivity to crosslinkers such as mitomycin C (MMC), we find surprisingly that they are largely resistant to hydroxyurea (HU), except for cells containing no expression of FANCD2. In order to understand the mechanism for this finding, we explored the interaction of FANCD2, Rad51, Rad18, and PCNA, which form a complex and is enhanced upon HU exposure. While FANCD2 is required, monoubiquitination of FANCD2 is not. In addition, PCNA monoubiqutination follows exactly the same pattern. The monoubiquitination of PCNA in response to HU in FANCA deficient mutant confirms that non-ubiquitinated FANCD2 can still support PCNA monoubiquitination. Rad51, but not BRCA1 is also required in regulating PCNA monoubiquitination in response to HU, suggesting that this function is independent of homologous recombination (HR). We further show that translesion polymerase Pol H chromatin localization is decreased in FANCD2 deficient cells, FANCD2 siRNA knock down cells and Rad51 siRNA knock down cells. Our data suggest that FANCD2 and Rad51 play an important role in PCNA monoubiquitination and TLS in a FANCD2 monoubiquitination and HR-independent manner in response to HU. This effect is not observed with mitomycin C (MMC) treatment, indicating that DNA repair systems differentially respond to different types of DNA damage.

Disclosures:
No relevant conflicts of interest to declare.
NSGS Mice Develop a Progressive, Myeloid Cell Dependent Aplastic Anemia and Bone Marrow Failure Upon Engraftment With Human Umbilical Cord Blood CD34+ Cells
Authors: Wunderlich Mark , Sexton Christina , Mizukawa Benjamin , Mulloy James C. 
Published: October 2019

Abstract
One key limitation of hematopoietic xenograft mouse models is the relative lack of effective myelopoiesis compared with human hematopoiesis. NSGS mice expressing human myelo-supportive cytokines allow for improved myeloid cell output upon human hematopoietic cell transfer. Surprisingly, we found that NSGS mice developed a severe, eventually fatal anemia upon humanization with umbilical cord blood (UCB) CD34+ cells. Anemia was characterized by a consistent, progressive drop in red blood cell numbers and hemoglobin that was correlated with higher MCV and an increased reticulocyte production at early time points. Eventually total white blood cell and platelet counts were also affected and reticulocyte numbers declined to or below normal. These effects were only apparent in mice with human grafts, and were also not observed in NSG mice with human grafts. Bone marrow cellularity was 40% of control humanized NSG mice and spleens were significantly larger, implying bone marrow dysfunction and extra medullary hematopoiesis. Bone marrow sections from anemic mice clearly showed a general hypocellularity with abundant fatty tissue filling the majority of the intramedullary space. We were able to recover red blood cell counts and hemoglobin to some degree upon transfusion of non-humanized mouse blood, however, the effect lasted only 7 days. CFSE labeling of anemic blood allowed us to detect a decreased half-life of red blood cells of 20 days compared to 30 days for humanized NSG mice. Treatment of mice with Campath, a CD52 antibody, completely reversed the phenotype at the expense of the entire human graft, demonstrating clearly that this was a graft-mediated process. However, specific ablation of B and T cells with rituximab and OKT3 did not alter the phenotype, even when administered soon after CD34+ cell transplant to suppress development of lymphocytes. Additionally, dexamethasone and IV/IG were ineffective in reversing the disease. These data, coupled with negative findings from tests to detect mouse reactive human antibodies, point towards myeloid cell involvement. Indeed, further experiments revealed that specific ablation of human myeloid cells effectively reversed the anemia. In summary, these data highlight a potential pitfall for effective myelopoiesis in the xenograft setting. More broadly, these findings demonstrate the possibility for myeloid driven bone marrow dysfunction and may warrant investigation of myeloid cells in cases of idiopathic aplastic anemia that fail to respond to therapies directed at autoimmune lymphocyte activity.

Disclosures:
No relevant conflicts of interest to declare.
PU.1 and Spi-B Oppose Transformation Of Pre-B Cells Through Activation Of Key Genes Involved In B Cell Receptor Signalling
Authors: Christie Darah A. , Turkistany Shereen A. , Xu Li S. , Li Stephen K. H. , Welch Ian , Broughton Heather C. , Bell Gillian I. , Hess David A. , DeKoter Rodney P. 
Published: October 2019

Abstract
B cell development is controlled by stage-specific expression of transcription factors. Aberrant expression of such factors can lead to B cell acute lymphoblastic leukemia (B-ALL). Deletion of genes encoding the E26 transformation-specific (ETS) transcription factors, PU.1 and Spi-B, in B cells (CD19+/CreSfpi1lox/loxSpib-/- mice, abbreviated to CD19-CreΔPB) leads to B-ALL at 100% incidence and with a median survival of 21 weeks. However, little is known about the target genes of PU.1 and Spi-B that explain leukemic transformation in these mice. In the current study, we investigated the developmental origins and mechanisms of leukemogenesis in CD19-CreΔPB mice. We found that B-ALL cells in CD19-CreΔPB mice had frequently rearranged both their heavy and light chain genes, but retained cell surface expression of interleukin-7 receptor (IL-7R), suggesting aberrant pre-B cell differentiation. Preleukemic CD19-CreΔPB mice had increased frequencies of pre-B cells compared to wild type mice. Pre-B cells, but not mature B cells, purified from the bone marrow of preleukemic CD19-CreΔPB mice could rapidly transfer disease to transplanted recipient mice. B-ALL cells from established tumors had uniform expression of markers indicating a pre-B cell phenotype and contained a high-frequency of leukemia-initiating cells as measured by transplantation assays. Genome-wide analysis of gene expression showed that B cell receptor signalling was the top impaired pathway in B-ALL cells from CD19-CreΔPB mice. Bone marrow cells from CD19-CreΔPB mice had increased responsiveness to IL-7R signalling and could be cultured as IL-7-dependent cell lines. Preleukemic or leukemic cells from CD19-CreΔPB mice expressed reduced levels of the gene encoding Bruton’s tyrosine kinase (Btk), which we show is a target gene of PU.1 and/or Spi-B that in combination with reduced BLNK is sufficient to explain increased IL-7R responsiveness. We conclude that mutation of PU.1 and Spi-B predispose developing B cells to leukemogenesis by impairing expression of key genes, such as Btk, that are required for BCR signalling and are involved in attenuation of IL-7 receptor signaling.

Disclosures:
No relevant conflicts of interest to declare.
NRF2 Signaling Promotes Survival and Drug Resistance Of Acute Myeloid Leukaemia Through Induction Of Mir-125b-1
Authors: Shah Niraj M , Rushworth Stuart A , Zaitseva Lyubov , Bowles Kristian M , Macewan David J 
Published: October 2019

Abstract
We have previously shown that Nrf2, an important regulator of several biologic processes involved in the progression of cancer, has abnormal constitutive expression in AML. As post-transcriptional regulation by micro RNAs (miRNAs) affects the malignant phenotype of a number of diseases, in this study, we aimed to identify and characterise NRF2 regulated miRNAs in AML.
Initially a qRT-PCR miRNA array was utilised to investigate miRNA expression in response to NRF2 lentiviral mediated knockdown in the AML cell line THP-1. To verify these results analysis of miRNA identified in the array were performed in response to transfection of siRNA against the NRF2 inhibitors Keap1 and Bach1. Subsequently we performed bioinformatic analysis to identify putative NRF2 binding sites upstream of the miRNA coding regions. From this we identified miR-125b as a putative NRF2 regulatory miRNA.
There are two miR125b paralogs coding for the same mature sequence in humans, miR-125b-1 on chromosome 11 in a cluster containing let-7a-2 and miR-100 and miR-125b-2 on chromosome 21 in a cluster with miR-99a and let-7c. To identify which paralog is upregulated by NRF2 qRT-PCR of the miRNA in each cluster was performed and referenced to the immature miR125b sequences which do differ between each paralog. Results show that NRF2 regulates miR-125b-1, but does not regulate miR-125b-2 or the other miRNAs within the miR125b-1 or the miR125b-2 clusters, implying that mir-125b-1 contains an alternative promoter which is regulated by NRF2.
To locate the NRF2 promoter site in miR125b-1 electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChiP) were carried out which confirmed NRF2 binding to the putative ARE site 946bp upstream of the 5’ of the miR-125b-1. To confirm NRF2 capacity to transcriptionally regulate miR-125b-1, we cloned the miR-125b-1 promoter into the PGL4 luciferase vector (PGL4/p125b) and found that NRF2 knockdown significantly inhibited PGL4/p125b luciferase activity compared to control knockdown.
Finally to identify the functional relevance of miR-125b-1 in a broad range of AMLs, miR-125b-1 mimic and antagomiRs were transfected into 3 AML cell lines and 11 primary AML samples. Results show a 10-25% increase in annexin V staining in the miR-125b-1 antagomiR experiments compared with the mimic or controls. Moreover miR-125b antagomiR increased the cytotoxicity of conventional AML therapy (daunorubicin and cytosine arabinoside). These results confirm the cytoprotective role of miR-125b-1 in AML.
In summary we find that NRF2 regulation of miR-125b-1 acts as a novel survival pathway in human AML.

Disclosures:
No relevant conflicts of interest to declare.