bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of Platelet PLC-β2 Expression by NF-κB: Studies in Human Platelet PLC-β2 Deficiency.
Authors: Mao Guangfen , Kunapuli Satya P. , Rao A. Koneti 
Published: April 2020

Abstract
We have previously described a patient with platelet phospholipase C (PLC)-β2 deficiency characterized by impaired platelet responses to activation with multiple G-protein coupled receptor agonists. The PLC-β2 coding sequence was normal and platelet PLC-β2 mRNA levels were decreased in the patient (Blood, 2002, 99:905). Very little is currently known regarding the transcriptional regulation of PLC-β2. PCR-amplification of patient leukocyte DNA and sequencing of the PLC-β2 5′-upstream region revealed a heterozygous 13-bp deletion (−1645 to −1633 bp from ATG) that encompasses a consensus binding site (GGGAATTCCC) for nuclear factor-κB, NF-κB. This deletion was present in the propositus and her affected son, but not in control subjects. PCR amplification of region −1791 to −1606 bp of genomic DNA revealed one band in 5 control subjects (size ~186 bp) on agarose gel electrophoresis but 2 bands in the patient and her son, consistent with a heterozygous defect. Luciferase reporter gene studies were performed in human erythroleukemia (HEL) cells treated with phorbol myristate acetate (PMA, 30 nM) to induce megakaryocytic transformation. Genomic fragment (−1648/−23 nt) of PLC-β2 5′-upstream sequence and its truncated form without the 13 nt region (−1633/−23 nt) were inserted upstream of luciferase gene in a promoterless expression vector PGL3-basic (Promega) and transiently transfected into HEL cells. Truncation of the wild-type −1648/−23 fragment at 1631 bp resulted in a consistent decrease in promoter activity by ~ 25% (6 experiments, p&lt;0.05). Protein binding assay (EMSA) was performed using PMA-treated HEL cell nuclear extracts and oligonucleotide probes (−1652/−1628 bp) with wild-type and mutated NF-κB consensus sites. Specific protein binding to the wild-type oligonucleotide was abolished when the NF-κB consensus sequence was deleted or mutated. Protein binding to wild-type probe was not competed by the unlabeled mutant oligonucleotide lacking NF-κB consensus sequence. In supershift assay, antibody targeted against the p65 subunit of NF-κB abolished protein binding, indicating a role for NF-κB. In summary, our studies demonstrate in the 5′-upstream region of PLC-β2 gene of the patient a 13-bp deletion that has a consensus site for NF-κB. Luciferase gene promoter assays demonstrate loss of activity when the 13-bp site is truncated. These studies provide evidence that impaired regulation of PLC-β2 gene by NF-κB may be the basis for the PLC-β2 deficiency in our patient. They show for the first time that PLC-β2, the most abundant β-PLC in platelets, is regulated by NF-κB. These findings are highly relevant because of the important role of PLC-β2 in platelet function, and of NF-κB in megakaryocytic differentiation and atherosclerosis.
Pharmacogenetics of Oral Anticoagulation - VKORC1-Haplotypes Determine the Inter-Individual and Inter-Ethnical Variability.
Authors: Geisen Christof , Watzka Matthias , Sittinger Katja , Luxembourg Beate , Steffens Michael , Müller Clemens R. , Wienker Thomas F. , Lindhoff-Last Edelgard , Seifried Erhard , Oldenburg Johannes 
Published: April 2020

Abstract
Purpuse: Coumarins are known for their high inter-individual and inter-ethnical variability of the dose-response association (i.e. significant higher maintenance dose in Africans and lower dose in Chinese). Recently, common SNPs of the VKORC1 gene (encoding the molecular target of coumarins, vitamin K epoxide reductase) were shown to be associated with lower warfarin dose requirement (c.-1639G&gt;A [rs17878363], c.173+1000C&gt;T [rs9934438]). In order to clarify whether these SNPs are part of more extended haplotypes, we established a comprehensive haplotype map of the entire VKORC1 gene locus. The association of VKORC1-haplotypes with coumarin dose requirement was analysed in different ethnical populations and patient cohorts.
Methods: In 200 German blood donors the VKORC1 gene locus was analysed by direct sequencing. VKORC1 genotype data in 7 additional populations from Africa, Asia and Europe were retrieved from internet databases. Haplotype frequencies were inferred using EHplus and FAMHAP. Unselected patients receiving phenprocoumon (n=61) and patient cohorts of European origin with either increased coumarin sensitivity (n=14, weekly phenprocoumon dose &lt;6mg) or partial coumarin resistance (n=36, weekly phenprocoumon dose &gt;42 mg or warfarin dose&gt;70mg) were typed for VKORC1 haplotypes.
Results: In 200 controls we identified 28 SNPs within the VKORC1 gene. 6 SNPs formed 3 main haplotypes covering more than 99% of the genetic variability of VKORC1 (VKORC1*2: 42%, VKORC1*3: 38%, and VKORC1*4: 20%). SNPs associated with low warfarin dose (rs17878363, rs9934438) were in complete linkage disequilibrium with the VKORC1*2 haplotype. Haplotype frequency in Africans and Chinese differed significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, Africans 14%). In 61 unselected patients receiving phenprocoumon c.-1639AA genotype patients (homozygous VKORC1*2) required less phenprocoumon (1.40 mg/d) than AG (2.12 mg/d) and GG patients (3.02 mg/d).13 of 14 patients (93%) with increased coumarin sensitivity but none of the patients with partial coumarin resistance were c.-1639AA. Vice versa, the c.-1639G allele (present in the non VKORC1*2 haplotypes) was found homozygous in 31 patients (86%) with partial coumarin resistance but in none of the patients with increased coumarin sensitivity.
Conclusions: Three main haplotypes represent more than 99% of VKORC1’s genetic variability in Europeans. VKORC1 haplotypes are strongly associated with the inter-individual and inter-ethnical variability of oral anticoagulation. In future studies, VKORC1-haplotype testing may provide a clinically relevant predictor of coumarin dosing and bleeding risk in oral anticoagulation.
Dasatinib (SPRYCEL®) 140 mg Once Daily (QD) vs 70 mg Twice Daily (BID) in Patients (pts) with Advanced Phase Chonic Myeloid Leukemia (ABP-CML) or Ph(+) ALL Who Are Resistant or Intolerant to Imatinib (im): Results of the CA180-035 Study.
Authors: Kantarjian Hagop , Ottmann O. , Pasquini R. , Goh Y.T. , Kim D.W. , VanTornout J. , Damokosh A. , Shah N. 
Published: April 2020

Abstract
Dasatinib (SPRYCEL®, formerly BMS-354825), an oral multi-targeted kinase inhibitor of BCR-ABL and SRC kinases, has been shown to be safe and effective at 70 mg BID in pts with CML and Ph(+) ALL who are resistant or intolerant to im. Based on a previous phase I study, in which QD dosing was as effective as BID dosing, a dose-optimization study was performed. CA180035 is a randomized, global multicenter, open-label trial of dasatinib 140 mg QD versus 70 mg BID in pts with ABP-CML (accelerated or blastic phase) or Ph(+)ALL that were resistant to or intolerant of im. Patients were stratified by phase of disease (accelerated, myeloid blast, or lymphoid blast/Ph(+)ALL) and by prior im (resistant or intolerant). The primary objective of the study was to compare the major hematologic response (MaHR) rate between the 2 regimens. Dose escalation to 180 mg QD or 90 mg BID was allowed for inadequate response, and dose reduction to 100 or 80 mg QD or 50 or 40 mg BID for drug toxicity. Evaluations consisted of weekly blood counts for the first 12 wks, and bone marrow cytogenetics every 3 months (mo) in patients with complete hematologic response (CHR) and BCR-ABL mutation analysis at baseline and at end of study. From June 2005 through March 2006, 612 pts were randomized: 56% of the pts were male with median (med) age of 55 years and med time from CML diagnosis of 58 mo. All pts received prior im: 42% had &gt;600 mg/day, and 37% were treated &gt;3 years. Other prior therapy included interferon in 42% of pts, chemotherapy in 57% of pts and stem cell transplant in 14% of pts. With a minimum follow-up of 4 mo, 42% of pts have been discontinued, mostly due to disease progression. The MaHR rate was 35% including 21% CHR and the major cytogenetic response (MCyR) rate was 33%, including 23% complete cytogenetic response (CCyR). The most common non-hematologic drug-related toxicities included diarrhea (24%, gr 3–4: 3%), headache (17%, gr 3–4: 1%), nausea (17%, gr 3–4: 2%), pleural effusion (15%, gr 3–4: 4%), and fatigue (12%, gr 3–4: 3%). Hematologic toxicity included neutropenia gr 3 and gr 4 in 22% and 40% of pts, respectively, and thrombocytopenia gr 3 and gr 4 in 16% and 50% of pts, respectively. Dose reductions were required in 23% of pts and dose interruptions were required in 43% of pts, both due mostly to non-hematologic toxicity. This is the first direct comparison of the 70 mg BID vs 140 mg QD dasatinib schedules in pts with ABP-CML or Ph(+)ALL. Updated efficacy and safety data, including response by baseline mutation analysis, on all pts with a minimum of 6-months follow-up will be presented at the meeting.
A Randomized Trial of Rabbit Anti-Thymocyte Globulin, Given on Day+7 after Alternative Donor Transplants.
Authors: Bacigalupo Andrea , Lamparelli Teresa , Ciceri Fabio , Peccatori Fedro , Locasciulli Anna , Majolino Ignazio , Di Bartolomeo Paolo , Milone Giuseppe , Mazza Patrizio , Lacopino Pasquale , Sacchi Nicoletta , van Lint Maria Teresa 
Published: April 2020

Abstract
Background. We have shown that the risk of transplant mortality (TRM) can be predicted by using laboratory values, on day+7 after an allogeneic hemopoietic stem cell transplant (HSCT) (Sormani et al, Bone Marrow Transpl.2003). TRM in patients with low, intermediate and high risk on day+7, is 15%, 40% and 69%: increased mortality is mainly, but not exclusively, due to increased acute graft versus host disease (GvHD). In a pilot study we have shown that a small dose of rabbit anti-thymocyte globulin (rATG) (Thymoglobulin, Genzyme), was feasible and reduced GvHD and TRM.
Aim of the study. We have therefore opened a randomized trial testing this hypothesis in patient undergoing an unrelated donor HSCT, with rATG (7.5 mg/kg) in the conditioning regimen.
Patients. 170 were eligible: all patients received rATG 3.75 mg/kg x2 pre-transplant, and on day+7 they were scored and randomized to receive an additional dose of rATG (1,25 mg/kg day +7 and day+9) (n=84) or to receive no further treatment (n=86) (control arm). The two groups were balanced for age, disease phase and day+7 score.
Results. The predictive value of day+7 score on TRM was confirmed in the control arm (18% vs 42% for intermediate and high risk patients, p=0.03), whereas in the day+7 ATG arm, there was no difference (29% vs 29%, p=1.0). TRM was overall reduced from 35% in the control arm to 29% in the ATG day+7 arm (p=0.37) : the difference was more pronounced in patients with early disease and high risk on day 7 (35% vs 15% p=0.08). Acute GvHD grade III–IV was reduced overall from 16% to 6% (p=0.04), and chronic GvHD was reduced 32% to 14% (p=0.02). In multivariate COX analysis TRM and overall survival was predicted by risk score day7 and disease phase.
Conclusions. We confirm that patients with different risk of TRM can be identified on day+7 after HSCT. Administration of rATG on day+7 can influence the risk of TRM, by reducing acute and chronic GvHD. Additional intensified supportive care (including anti-infectious therapy) may further reduce TRM in patients with a high risk score on day+7.
Von Willebrand Factor Protects Factor VIII from Endocytosis by Human Monocytes−Derived Dendritic Cells and Subsequent Presentation to Immune Effectors.
Authors: Repesse Yohann , Dasgupta Suryasarathi , Navarette Ana Maria , Delignat Sandrine , Wootla Bharath , Borel-Derlon Annie , Kaveri Srinivas V. , Lacroix-Desmazes Sebastien 
Published: April 2020

Abstract
The role of Von Willebrand Factor (VWF) as a chaperone molecule for procoagulant factor VIII (FVIII) has been extensively documented. Under physiological conditions, VWF binds to FVIII after its release in the circulation. VWF protects FVIII from proteolysis by lipid−bound proteases, stabilizes FVIII and further regulates its elimination by lipoprotein receptors. We investigated whether VWF might modulate FVIII endocytosis by professionnal antigen presenting cells (APCs). Immature dendritic cells (DCs), utilised as model of APCs, were generated from circulating monocytes of healthy blood donors and incubated with FITC−labeled FVIII (FVIII−FITC). T cell assays were performed by co−culturing the human FVIII−specific CD4+ T cell clone, D9E9, with immature DCs from MHC−matched donors in presence of unconjugated FVIII and increasing concentrations of VWF (1:1 to 1:130). Preincubation of FVIII−FITC with VWF at 1 to 130−fold molar excess resulted in a dose−dependent inhibition of FVIII endocytosis (12 ± 2% to 94 ± 18%, respectively). Human serum albumin used in equivalent molar ratios did not prevent FVIII internalization. In contrast, the protective effect of VWF on FVIII uptake was significantly restored by co−incubation of FVIII and VWF in the presence of F(ab’)2 fragments of BO2C11, an anti−FVIII IgG, and Ac418, an anti−VWF IgG, that both disrupt the interaction between FVIII and VWF. In addition, blocking DC entry of FVIII by VWF resulted in a dose−dependent reduction of the activation of D9E9 (up to 75%). Interestingly, D9E9 activation by DCs loaded with a synthetic FVIII−derived peptide, I2144–T2161, was not altered in the presence of equivalent concentrations of VWF, indicating that VWF does not have a direct inhibitory effect on T cell activation. The administration of exogenous FVIII to patients with hemophilia A, results in up to 30% of the cases, in the developement of FVIII−specific antibodies that inhibit FVIII procoagulant activity. In vivo experimental evidences and clinical observations have documented that the presence of VWF in FVIII therapeutic concentrates may be associated with a lower incidence of FVIII inhibitors. Here, we demonstrate that VWF may reduce the immunogenicity of FVIII by preventing its internalization by professionnal APCs, and reducing the subsequent presentation of FVIII−derived peptides to T lymphocytes.
Purification of an MLL Partner Associated Complex (MPAC) Suggests a Common Role for MLL Fusion Partners in Transcriptional Elongation.
Authors: Slany Robert K. , Mueller Dorothee , Bach Christian , Zeisig Deniz , Garcia-Cuellar Maria-Paz , Monroe Sara , Sreekumar Arun , Zhao Ron , Nesvizhskii Alexey , Chinnaiyan Arul , Hess Jay L. 
Published: April 2020

Abstract
Chromosomal translocations at 11q23 are characteristic for a subset of particularly aggressive acute lymphoid and myeloid leukemias. This event fuses the mixed lineage leukemia (MLL) gene to a puzzling variety of seemingly unrelated fusion partners to create novel transcription factors with potent oncogenic activity. Despite the molecular heterogeneity, MLL−associated leukemias show similar clinical presentations and an almost identical gene expression pattern regardless of the translocation partner. We and others previously identified interactions between individual MLL translocation partners suggesting that these might share a common mechanism for transformation. To characterize these interactions in more detail we generated a HEK293−derived producer line expressing a FLAG−tagged derivative of the frequent fusion partner ENL. Double affinity purification of nuclear extracts on FLAG and ENL antibody resins reproducibly yielded a macromolecular complex of 10 proteins we term MPAC, for MLL Partner Associated Complex, which in addition to ENL includes the MLL fusion partners AF4, AF9 and AF5q31, and two components involved in transcriptional elongation: pTEFb and DOT1L. The pTEFb dimer consists of cyclin T1/2 and CDK9 and is responsible for serine−2 phosphorylation of the C−terminal repeat domain of elongating RNA Polymerase II. DOT1L is the only known histone methyltransferase that catalyzes dimethylation of H3K79, a modification introduced during elongation of actively transcribed transcription units. Surprisingly, PC3 and RING1b, members of the polycomb repressive complex I were also identified in the MPAC complex. Further examination demonstrated a direct interaction of ENL with DOT1L through C−terminal interaction domains by yeast 2−hybrid, pulldown and colocalization experiments. Knock−down of ENL diminished global H3K79 dimethylation and also impaired various promoters in transient reporter assays suggesting the MPAC complex is involved in transcriptional regulation mediated by DOT1L, even in the absence of MLL fusion proteins. In summary, our results indicate that the most common MLL fusion partners are associated in an hitherto undescribed complex involved in transcriptional elongation.
Regulation of the PU.1 Gene by Sense and Functional Antisense RNAs Generated through the Same Chromatin Architecture.
Authors: Ebralidze Alex , Zhang Pu , Rosenbauer Frank , Huang Gang , Steidl Ulrich , Dayaram Tajhal , Guibal Florence , Klupp Johanna , Neel Dana , Bonifer Constanze , Tenen Daniel G. 
Published: April 2020

Abstract
The transcription factor PU.1 is an important regulator of hematopoiesis and correct expression levels in specific lineages are critical for normal hematopoietic development. Specifically, PU.1 is maintained or upregulated in specific lineages, and failure to downregulate PU.1 in other lineages can lead to a block in development of that lineage and/or leukemia. In vivo expression of PU.1 is dependent on an upstream regulatory element called the URE. Disruption of the URE leads to downregulation of PU.1 and development of leukemia and lymphoma, but the other distal elements regulating PU.1 have not been defined. Here we show that other phylogenetically conserved elements participate in the initiation of antisense transcription, and that these antisense RNAs function as important modulators of proper dosages of PU.1. Specifically, antisense transcripts originate from specific conserved sites in introns 1 and 3, and that the intron 3 site contains binding sites for transcription factors such as AML1 and Ets factors. The conserved intron 3 element also possesses anti-sense promoter activity. These antisense transcripts are present at about 15% of PU.1 sense transcripts in PU.1 expressing cells. They negatively regulate PU.1 sense RNA, as introduction of siRNA molecules which specifically target antisense transcripts lead to 3–5 fold increases in PU.1 sense RNA and protein. Both sense and antisense PU.1 gene RNAs are dependent on the URE and are transcribed from the same chromatin architecture, in which the conserved elements, including URE and sense and antisense promoters are located in the same nuclear fraction and can be shown to exist in the same nucleoprotein complex by chromosome conformation capture (3C). We are currently testing the mechanisms involved in the formation of such complexes, and specifically whether the complexes are mediated by binding of AML1 to the URE. Since we do not observe significant differences in antisense transcript levels between PU.1 high-expressing and PU.1 low-expressing cells, we hypothesize that the function of these antisense transcripts is to modulate rather than absolutely control PU.1 levels: in PU.1 high-expressing cells, such as myeloid cells, the antisense transcripts trim PU.1 levels to prevent overexpression, while in cell types in which PU.1 is not expressed, such as T cells, the antisense transcripts prevent any expression of PU.1. We propose that such a mechanism will likely be important in fine-tuning the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.
Ferritin Heavy Chain Stimulates HbS-to-HbF Switching in Erythroid Precursor Cells from Sickle Cell Patients.
Authors: Broyles Robert H. , Belegu Visar , Roth Austin C. , Clarkson Emily J. , Williamson Kelly S. , Stewart Charles A. , Pye Quentin N. , Floyd Robert A. , Jani Klodiana , Trudel Marie , Santambrogio Paolo , Levi Sonia , Arosio Paolo , Cain Joan Parkhurst 
Published: April 2020

Abstract
We have found that ferritin heavy chain (FtH), an antioxidant/stress response/iron-storage protein, localizes to the nucleus in K562 cells and represses the human adult beta-globin promoter in transient assays in primate cells (Broyles et al., PNAS98: 9145, 2001). Since other work indicates FtH is also a gene activator of fetal-globin genes, we hypothesize that FtH is a long-sought developmental hemoglobin (Hb) switching factor and that delivery of FtH to human adult erythroid cell precursors will reverse the phenotype to HbF, offering a phenotypic cure for sickle cell disease (SCD). Chromatin immunoprecipitation (ChIP) assays, antisense treatments, and an FtH transgenic mouse have confirmed that FtH is a globin gene regulatory protein in vivo. With erythroid precursor cells from pediatric SCD patients, under an IRB-approved protocol, we have used a two-phase culture system for in vitro maturation of erythroid cells in the presence of FtH, delivered to the cells as pure protein, as an expression plasmid, or as a priority inducer compound that activates the endogenous FtH gene. HPLC with a PolyCAT A column was used to separate and quantify human Hbs. With each mode of delivery, FtH stimulated a complete switch from HbS to HbF. This result was repeatable in multiple experiments using erythroid precursor cells from three different SCD donors. Fluorescently-labeled recombinant human FtH protein was taken into red cell precursors in culture, suggesting that the purified protein can be directly delivered without gene therapy. This method of producing a phenotypic cure in SCD patients should be easy and inexpensive to deliver in vivo.