bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extensively Self-Renewing Erythroid Precursors Emerge from the Embryo but Not from the Adult
Authors: England Samantha , McGrath Kathleen E. , Palis James 
Published: April 2020

Abstract
The only cell in the hematopoietic hierarchy thought to be capable of long-term selfrenewal is the stem cell. An erythroid progenitor derived from mammalian hematopoietic tissue, fetal or adult, is capable of limited proliferation (103–106 fold expansion; Bauer, 1999; Panzenböck, 1998; von Lindern, 1999). Here we report that an erythroid precursor derived from the mouse embryo is capable not only of limited, but also of extensive proliferation (~1030 fold expansion). These cells resemble proerythroblasts and basophilic erythroblasts based on their morphology, their globin gene expression profile, and their immunophenotype. While aneuploidy is not necessary for extensive proliferation, it sporadically begins to accumulate after prolonged culture. These cells are capable of massive (&gt;100 days) daily proliferation in vitro in the presence of Epo, SCF, IGF-1, and dexamethasone. Examination of cultures lacking each of these factors support that glucocorticoids play an important role in this expansion by uncoupling erythroid precursor proliferation from maturation. Despite prolonged in vitro culture, these cells preserve their potential to fully differentiate into enucleated red blood cells with the removal of dexamethasone. Differentiation occurs over 2–3 days and is characterized by the accumulation of adult (α, β1, and β2), but not embryonic (ζ, εy, and βH1), globins. The retention of full differentiation potential despite &gt;1030 fold expansion indicates that this proliferation represents self-renewal. To determine the developmental origin of these extensively self-renewing erythroblasts (ESREs), we initiated in vitro cultures from staged mouse embryos as well as adult tissues. E7.5 embryos, that contain primitive but not definitive erythroid progenitors, failed to generate ESREs. In contrast, ESREs can be derived from E8.5–E10.5 yolk sac and E11.5–E14.5 fetal liver. These findings along with the globin expression pattern indicate that erythroblast self-renewal is associated with definitive, but not primitive, erythropoiesis. Surprisingly, marrow from adult steady-state hematopoiesis failed to yield ESREs. Furthermore, despite the characteristics shared by stress erythropoiesis (adult spleen) and fetal erythropoiesis (liver), stress erythropoiesis only yielded erythroblasts with limited, and not extensive, self-renewal capacity. This result suggests that extensive self-renewal potential is linked either to the transient yolk sac-derived definitive erythroid lineage or to the fetal hematopoietic microenvironment. We are currently investigating the mechanisms responsible for the extensive self-renewal capacity of such lineage-restricted and mature hematopoietic precursors. Our findings raise the possibility that the expansive cellular output of the erythron within the midgestation mammalian embryo may be regulated, in part, at the level of late stage erythroid precursors.
Notch1 Expression Is Regulated at Post-Transcriptional Level by 3′UTR Region in Hematopoietic Stem Cell Development
Authors: Mizuno Shin-ichi , Ozawa Hidetoshi , Iino Tadafumi , Arinobu Yojiro , Yong Chong , Shigematsu Hirokazu , Akashi Koichi 
Published: April 2020

Abstract
In hematopoietic stem cell development, the expression of critical genes is precisely regulated in a stage specific manner, which supports normal hematopoietic development through adequately regulating timing of cell division, self-renewal, and lineage commitment. Regulation of gene expression is known to take place at least at transcriptional level. In addition to the transcriptional regulation, there are growing evidences that post-transcriptional control of critical genes may play an important role, suggesting an interesting possibility that post-transcriptional control may also play a role in hematopoiesis. Here, we provide the evidence that the expression of Notch1, a key factor in lymphoid development, is controlled at post-transcriptional level in hematopoietic stem cell (HSC). By quantitative PCR, Notch1 mRNA is substantially expressed at HSCs as well as common lymphoid progenitors (CLPs) or double negative (DN) thymocytes. However, Notch1 protein is detected at very low level in HSCs compared to CLPs or DN thymocytes, suggesting that Notch1 expression is regulated at post-transcriptional level in HSC. To investigate the effect of 3′UTR (untranslated region) on post-transcriptional regulation, we prepared a retrovirus sensor vector, in which 3′UTR of target gene is placed between the GFP coding region and the retrovirus 3′LTR, and found that induction of the sensor vector with the 3′UTR sequence of Notch1 showed marked suppression of the GFP intensity at the HSC stage. This effect was not observed when we introduced the vector into DN thymocytes. Suppression of Notch1 by its 3′UTR was further confirmed by using a retrovirus vector which has two distinct markers of YFP and GFP-3′UTR fusion genes under bi-directional EF1 promoter. Deletion mutant analysis showed that the responsible region required for this post-transcriptional suppression is confined to 120-bp sequence within Notch1 3′UTR so far. These data suggest that the expression of Notch1 should be regulated at post-transcriptional level by its 3′UTR at the HSC stage and our data provide the first evidence that the stage-specific translational regulation can play an important role in organization of hematopoietic development.
Human Cytomegalovirus: Degranulation Is a Prominent Feature of Functionally Impaired pp65- and IE-1-Specific T-Cells in Patients after Allogeneic Stem Cell Transplantation
Authors: Fuhrmann Stephan , Ganepola Susanne , Uharek Lutz , Thiel Eckhard , Ludwig Wolf-Dieter , Volk Hans-Dieter , Kern Florian 
Published: April 2020

Abstract
Human cytomegalovirus (CMV) reactivation and disease is still a frequent complication after allogeneic stem cell transplantation (allo SCT). It is well accepted that T-cell immunity is mandatory to control CMV infection and disease and much effort has been put into the development of cell-based monitoring assays. Nevertheless, no reliable marker for protective immunity has been established to date. Most studies use one CMV model antigen (pp65) to compare the frequencies of cytokine producers (mainly IFNg) or multimer-specific T-cells. Methods: In total, we recruited 16 patients after allo SCT, (7 high risk, 9 standard risk pts.). We used 8-colour flow cytometry to detect degranulation (mobilized CD107a/b), intracellular IFNg, TNFa, IL-2 production and CD28-expression in peptide pool stimulated pp65 and IE-1 specific CD8 T-cells. Results were compared to 7 healthy CMV exposed donors. Results: Degranulation identifies the highest percentage of CMV-specific T-cells in allo-transplanted patients (pp65: 0,94% degranulation and 0,31% IFNg; IE-1: 1,44% degranulation and 0,87% IFNg, mean frequency). These T-cells are relatively cytokine deficient compared to those in healthy donors (cytokine-production/degranulation ratio: SCT=0,42, healthy=0,72 for pp65, p=0,048; SCT=0,61, healthy= 1,00 for IE-1, p=0,133, U-test). The cytokine expression pattern differs between antigens used for stimulation, for example more IL-2-producers could be detected in the pp65 specific compartment (12,5% for pp65 and 4,5% for IE-1 of all activated CD8 T-cells, p=0,015).
Conclusion: This study demonstrates that degranulation is the most prominent marker of CMV-specific T-cells (pp65 and IE-1) in allo SCT patients. Looking at IFN-g producers only may underestimate the frequencies of CMV specific T-cells in this setting. Furthermore, these subsets have a divergent functionality in transplant recipients compared to healthy individuals. Our data challenge the concept of enumerating CMV specific T-cells to estimate immunity. We rather propose measuring functional differences in the T-cell response may help to identify patients with a high risk of CMV reactivation. A careful dissection of these differences is a prerequisite for the development of monitoring tools and adoptive T-cell transfer.
FTY720 Abrogates the Therapeutic Potential of Adoptively Transferred Treg Via Inhibition of IL-2 Induced in Vivo Expansion
Authors: Wolf Anna Maria , Hochegger Kathrin , Zeiser Robert , Duerr Christoph , Sixt Michael , Rosenkranz Alexander , Gastl Gunther , Wolf Dominik 
Published: April 2020

Abstract
CD4+CD25+ T cells (Treg) entry into secondary lymphoid organs (SLO) and local expansion after activation is at least in part responsible for their immunosuppressive action. Thus we hypothesized that trapping of adoptively transferred Treg in SLO would be an effective means to tip the balance towards a more immunosuppressive milieu within the LN microenvironment. Systemic application of the sphingosine-phosphate receptor agonist FTY720 has been proven to trap harmful effector T cells in SLO, thereby inhibiting their migration and destruction of target tissue. Here we provide first evidence that selective entrapment of adoptively transferred Treg in inflammatory LN can be achieved by blockade of SP-receptors upon ex vivo exposure of Treg to FTY720 before adoptive transfer. FTY720 exposure did not interfere with proper Treg localization within the T-cell areas of SLO as determined by immunofluorescent microscopy after co-transfer of either FTY720- or solvent exposed and subsequently differentially labelled Treg. However, despite the fact that the in vitro phenotype (including expression of adhesion and chemokine receptors), function (including anergy and suppressive activity) and survival (determined by Annexin/PI staining) of Treg remained unaltered by FTY720, it abrogated their protective effect after adoptive transfer in a murine model of acute experimental glomerulonephritis (determined by quantification of proteinuria and histological analysis) as well as in an acute GvHD model (determined by survival analysis and quantification of the in vivo expansion of luciferase-transgenic effector T cells by bioluminiscence technology). Notably, adoptive transfer of CFSE-labelled Treg revealed a markedly impaired proliferation of Treg in inflammatory SLO when pre-exposed to FTY720 ex vivo. Accordingly, FTY720 blocked Treg-proliferation induced by TCR-stimulation in combination with IL-2 in vitro. In line with this observation, FTY720 completely abolishes IL-2 induced phosphorylation of STAT-5. Thus, SP-1P receptors induce Treg trapping in inflammatory SLO but abrogate their in vivo immunosuppressive potential by inhibition of local Treg expansion.
A Prospectively Randomized Placebo-Controlled Trial of Epoetin-α in Patients with Advanced-Stage Hodgkin Lymphoma: Final Analysis of the GHSG HD15-EPO Trial
Authors: Engert Andreas , Borchmann Peter , Fuchs Michael , Josting Andreas , Haverkamp Heinz , Diehl Volker 
Published: April 2020

Abstract
Anemia is frequent among cancer patients and impacts organ function and quality of life of those affected. Anemia has been shown to be a negative prognostic factor in several malignancies including Hodgkin Lymphoma (HL). In addition, the advent of more aggressive treatment regimen such as BEACOPP escalated has resulted in more HL patients needing red-blood-cell transfusion. Thus, apart from the chemotherapy-related question of the GHSG HD15 trial, ie 8 cycles of BEACOPP escalated or 6 cycles of BEACOPP escalated or 8 cycles of BEACOPP14, patients in HD15 were randomized between Epoetin-α and placebo. Study drug was given at 40.000 I.E. weekly with a hemoglobin target level of 12–13g/dl. Primary endpoint was fatigue after the end of chemotherapy (CT) and 6 months later measured with the EORTC QLQ-C30 questionnaire (0–100 scale). With respect to the more recent safety discussion related to erythropoetins (EPOs) in general, we also analyzed efficacy and safety endpoints although the trial was not sufficiently powered for time-to-event analyses. Between1/2003 and 12/2006 1.379 patients were randomized for the EPO question of whom 1.329 were eligible. Of these 1.286 (96.8%) are fully evaluable. Patient characteristics were balanced between the placebo and the EPO arm. In those 688 patients who were randomized before 7/2005 and eligible for analysis, the CR/CRu rate was 90, 6% with a median follow-up of 28 months, 58 progressions/relapses (8.4%) and 33 deaths (4.8%) without differences between study arms. There was also no difference in terms of freedom from treatment failure (hazard ratio (HR) 0.9, 95%-confidence interval (CI) 0.6–1.4) and overall survival (HR 1.1, 95% CI 0.6–2.3). 51 serious adverse (7.4%) and 35 thrombovascular events (5.1%) were observed with no significant difference between arms. The median number of red blood cell units given significantly favored patients receiving Epoetin-α (2 vs 4; test for trend p&lt;0.001). The interim analysis for fatigue gave an average value of 61+/−25.8 (mean +/−SD) at the end of treatment and a reduced fatigue score of 33+/−25.3 at six months after CT.
Conclusion: This is the largest prospectively randomized, placebo-controlled trial with EPO in cancer patients. So far we found no difference in terms of fatigue, response, relapse, or side effects between those advanced-stage HL patients receiving Epoetin-α or placebo. The number of RBC units given was significantly reduced in the EPO group. However, all cases are needed for a final conclusion and will be presented.
PKC412, a Small Molecule Tyrosine Kinase Inhibitor, Regulates Survival and Proliferation Pathways in Waldenström Macroglobulinemia
Authors: Melhem Molly R. , Roccaro Aldo M. , Azab Abdelkareem , Ngo Hai T. , Sacco Antonio , Jia Xiaoying , Azab Feda , Runnels Judith , Burwick Nicolas , Quang Phong , Husu Emanuel N. , Leleu Xavier , Anderson Kenneth C. , Ghobrial Irene M. 
Published: April 2020

Abstract
Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase C (PKC) is a multigene family that that plays an important role in the regulation of cell growth and tumor progression. PKC412, a derivate of staurosporine, is a competitive inhibitor of ATP binding to PKC subtypes. This PKC inhibitor was used to examine its role in WM.
Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay. Cell signaling of HSP response, VEGF signaling and AKT pathways were determined by Western Blot analysis. Cell cycle and apoptosis analysis were obtained through flow cytometry.
Results: We showed that PKC412 induced cytotoxicity in WM cell lines tested (IC50 of 1uM) with no cytotoxic effect on healthy donor peripheral blood mononuclear cells at concentrations as high as 5uM. Cell proliferation and apoptosis analysis showed similar results, in a time independent manner. A G2/M arrest was observed upon cell cycle analysis, suggesting a possible mechanism for apoptosis. Similarly, the Akt pathway was investigated and was found to show a decrease in its activity in a dose dependent manner. Finally, PKC412 regulated VEGF levels in WM samples indicating an anti-angiogenic activity.
Conclusion: PKC412 has a significant effect on the survival and proliferation of WM cells, including regulation of angiogenesis. These studies provide the framework for clinical evaluation of this novel PKC inhibitor in Waldenström Macroglobulinemia.
Regulation of Histone Deacetylase in Waldenström Macroglobulinemia
Authors: Jia Xiaoying , Roccaro Aldo M. , Azab Abdel Kareem , Ngo Hai T. , Sacco Antonio , Melhem Molly R. , Azab Feda , Runnels Judith , Burwick Nicolas , Quang Phong , Husu Emanuel N. , Leleu Xavier , Anderson Kenneth C. , Bradner James , Ghobrial Irene M. 
Published: April 2020

Abstract
Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM.
Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence.
Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells.
Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.
5-Azacytosine (Azacytidine) and 2′-Deoxy-5-Azacytidine (Decitabine) Mediate Opposite Effects on NK Cell Anti-Tumor Reactivity
Authors: Schmiedel Benjamin J , Arélin Viktor , Krusch Matthias , Kanz Lothar , Salih Helmut R 
Published: April 2020

Abstract
The cytosine analogues 5-azacytosine (azacytidine) and 2′-deoxy-5-azacytidine (decitabine) display substantial therapeutic potential in patients with AML and MDS. Besides causing DNA demethylation, azanucleosides also mediate cytotoxic effects, and it appears that clinical responses are influenced by both epigenetic alterations and by apoptosis induction. However, the molecular changes induced by these drugs are still poorly understood. NK cells play an important role in tumor-immunosurveillance by confining development and progression of hematopoietic malignancies and are also important after therapeutic intervention like e.g. haploidentical stem cell transplantation. Thus it is important to define how a given therapeutic agent influences NK cell reactivity. Here we studied the effect of pharmacological concentrations of azacytidine and decitabine on NK cell effector functions. After preincubation with the azanucleosides (12h or more), NK cell cytotoxicity was found to be significantly enhanced by decitabine while, in contrast, azacytidine nearly completely abolished NK cell lysis of K562 and Raji target cells (up to 50% increase and 88% reduction, respectively; E:T ratio 10:1). Of note, modulation of NK cell cytotoxicity was also observed when the compounds were, after NK cell pretreatment, absent during the cytotoxicity assays. In contrast, neither the presence of either agent in cytotoxicity assays without preincubation nor pretreatment of target cells with either agent for 4h (corresponding to the time of the cytotoxicity assay) altered NK cell reactivity. These results indicate that azanucleosides are capable to modulate NK cell responsiveness to activating stimuli. In line, after pretreatment with the compounds NK cell IFN-γ production upon stimulation with IL-2 and IL-15 or in cocultures with target cells was found to be enhanced by decitabine but inhibited by azacytidine (up to 46% increase and 85% reduction, respectively). NK cell effector functions were not affected by deoxycytidine and cytidine, the physiological counterparts of the azanucleosides. While azacytidine treatment substantially induced NK cell apoptosis (about 30% after 24h) which may explain its inhibitory effect, no induction of apoptosis by decitabine was observed. Our data demonstrate that azacytidine and decitabine differentially affect NK cell anti-tumor reactivity and suggest that, while azacytidine causes NK cell apoptosis, decitabine enhances NK cell responsiveness via a yet unknown mechanism which is presently under study.
WP-1130 Induces Apoptosis in Multiple Myeloma Cells by Inactivation of Jak2 and Syk Tyrosine Kinases Via a Novel Mechanism of Selective Protein Trafficking
Authors: Lebovic Daniel , Kapuria Vaibhav , Hari Malathi , Talpaz Moshe , Jakubowiak Andrzej , Donato Nicholas 
Published: April 2020

Abstract
Multiple myeloma is a plasma cell malignancy affecting more than 50,000 people in the United States. The disease remains incurable despite significant recent advances in treatment, including the immunomodulatory agents, thalidomide and lenalidomide, and the proteasome inhibitor, bortezomib. Thus the search for additional agents is warranted. We have previously shown that WP1130, a tyrphostin-like compound, has activity in a number of hematological malignancies including CML, mantle cell lymphoma and multiple myeloma. In this study, we investigated the activity and mechanism of action of WP1130 on multiple myeloma cells and compared these activities to bortezomib. Both WP1130 and bortezomib demonstrated significant cytotoxicity in MM1S cells with IC50s of 1 mM and 2 nM, respectively and both compounds induced similar levels of apoptosis (67% vs 73% cell death with WP1130 and bortezomib, respectively). Both WP1130 and bortezomib increased cellular ubiquitination levels in distinct cellular compartments but we were unable to detect 20S proteasome inhibition by WP1130. The effect of WP1130 on signaling modules in MM cells was also investigated. WP1130 altered the subcellular distribution of a subset of tyrosine kinases, including Jak2 and Syk which were irreversibly displaced from the cytoplasm to detergent insoluble complexes within the cell. Although Jak2 remained stable in this fraction, Syk was subject to proteolysis and destruction in this compartment. WP1130-induced translocation of these kinases through association with the cytoskeletal network and was associated with loss of cytokine and B-cell receptor signal transduction. WP1130 did not alter the cellular distribution of other tyrosine kinase or phosphoproteins including Lyn, STAT3, STAT5, PI3K, AKT, ERK, suggesting some selective protein translocation and signal modulation by WP1130. These results support a novel mechanism of protein trafficking, selective kinase destruction and apoptosis by WP1130 in MM cells.