bluuebunny/crossref_metadata_2025_split huggingface dataset:
MicroRNAs 17-5p/20a/106a Function as a Master Gene Complex Controlling Monocytopoiesis through AML1 Targeting.
Authors: Fontana Laura , Pelosi Elvira , Greco Paolo , Testa Ugo , Liuzzi Francesca , Croce Carlo Maria , Brunetti Ercole , Peschle Cesare 
Published: April 2020

Abstract
MicroRNAs (miRs) are a novel regulatory class of non-coding, single-stranded RNAs of ~ 22 nucleotides, identified in plants and animals. MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3′ untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation. MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation. However, little is known on their role in hematopoiesis. Furthermore, in diverse types of cancer miRs may function as “oncomirs”.
We investigated the role of miR-17-5p, -20a and -106a, located in the miR-17-5p-92 and miR-106a-92 clusters respectively, in monocytic-macrophage (Mo) differentiation-maturation. In unilineage Mo culture generated by cord blood (CB) hematopoietic progenitor cells (HPC), these miRs are gradually and markedly downmodulated, whereas AML1 is upmodulated at protein but not at mRNA level. The acute myeloid leukaemia-1 protein (AML1, also known as Runt-related transcription factor 1, Runx1) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which plays a pivotal role in Mo differentiation-maturation. Luciferase assay shows that miR-17/20/106 bind the AML1 mRNA 3′UTR, implying that during monocytopoiesis their decline unblocks AML1 translation. Mo culture transfection with miR-17/20/106 stimulates blast cell proliferation and inhibits differentiation-maturation: this is coupled with AML1 translation blockade, leading in turn to inhibition of M-CSF receptor (M-CSFR) expression. Equivalent results were observed in Mo clonogenic culture of HPCs. Knockdown of endogenous miR-17/20/106 by anti-miR oligonucleotide transfection causes opposite effects. Altoghether, our studies indicate that miR-17/20/106 downmodulation during monocytopoiesis “unblocks” the translation of AML1, which in turn transactivates the M-CSFR, thereby allowing Mo differentiation and maturation. We hence propose that these miRs function as a master gene complex underlying the monocytopoiesis gene program, at least in part through the regulation of two pivotal proteins, AML1 and M-CSFR.
This study also sheds light on other relevant aspects:the regulatory action of miR-17/20/106 action on AML1 mRNA is restricted to the Mo lineage, implying a role for lineage-specific factor(s) to modulate the interaction of these miRs with their mRNA target;the subtle complexity of mechanisms controlling the transcription/processing of the miR-17-5p-92 cluster, implied by the differential expression patterns of the miRs comprised in the 17-5p-92 polycistron in Mo culture;the leukemogenesis mediated by AML1 fusion proteins.
EVI1 Interacts with and Represses RUNX1: A Novel Mechanism of Deregulated Hematopoiesis in EVI1-Positive Hematopoietic Cells.
Authors: Senyuk Vitalyi , Sinha Kislay K. , Yanamandra Sastry , Nucifora Giuseppina 
Published: April 2020

Abstract
Myelodysplastic syndrome (MDS) comprises a group of disorders with overlapping features. The majority of recurring chromosomal abnormalities associated with MDS are large deletions. There are two genes, RUNX1 and EVI1, whose deregulation is also known to be associated with MDS. RUNX1 is a master regulator of hematopoiesis that specifically binds to the DNA and regulates the expression of hematopoietic genes. Lately it has become clear that RUNX1 is frequently mutated in patients with MDS. Surprisingly, the mutations are limited to one allele, suggesting that a partial loss of function is sufficient to contribute to the disease. The second gene, EVI1, encodes a large nuclear protein characterized by two domains of zinc fingers. While the precise role of EVI1 is not known, this protein is not expressed in normal bone marrow and is inappropriately activated in very aggressive MDS by chromosomal translocations. Because alterations of both genes are not usually observed together, we have recently investigated whether there could be a potential link between the pathways regulated by RUNX1 and EVI1. Here, we report that in vivo and in vitro EVI1 physically interacts with RUNX1. We mapped the interaction domains to the eighth zinc finger motif of EVI1 and to the DNA-binding Runt domain of RUNX1. We found that this interaction alters the ability of RUNX1 to bind to its DNA consensus and represses the ability of RUNX1 to activate a reporter gene, suggesting that the interaction between EVI1 and RUNX1 could impair the hematopoietic programs regulated by RUNX1. These data suggest that inappropriate expression of EVI1 could contribute to hematopoietic transformation in part by a new mechanism that involves EVI1 association with key hematopoietic regulators leading to their functional impairment. Our results also indicate that the interaction region between RUNX1 and EVI1 could represent a novel target for therapeutic drugs for EVI1-associated leukemia.
Thalidomide Induces gamma-Globin Gene Expression in Adult Primary Erythroid Cells through Increased Intracellular Reactive Oxygen Species Mediated Activation of p38 MAPK Signal Pathway.
Authors: Aerbajinai Wulin , Zhu Jianqiong , Gao Peter , Chin Kyung , Rodgers Griffin P. 
Published: April 2020

Abstract
Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide’s therapeutic effect are still being defined. We hypothesize that thalidomide induce the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin gene expression in a dose-dependent manner. The averaged gamma/gamma+beta percentage ratio was 12.89% ± 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% ± 0.07% in Epo alone (P&lt;0.01). Interestingly, we found that intracellular ROS level was significantly increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner. Thalidomide also increased the histone H3 phospho-acetylation and histone H4 acetylation. In contrast, treatment with the anti-oxidant N-acetylcycteine (NAC) inhibited the thalidomide induced p38 activity and histone H3, H4 acetylation. These data indicate that thalidomide induced the gamma-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.
Computational Modeling of Nitric Oxide Bioavailability in Hemolytic Anemias.
Authors: Jeffers Anne , Gladwin Mark , Kim-Shapiro Daniel 
Published: April 2020

Abstract
Intravascular hemoglobin (Hb) scavenges nitric oxide (NO), which decreases the availability of endothelial-derived NO, thereby reducing smooth muscle relaxation. Under normal conditions, there are various mechanisms that limit consumption of NO by Hb in vivo includinga physical barrier to NO diffusion across the red blood cell (RBC) membrane,an unstirred layer creating a concentration gradient in NO outside the RBC, andand a cell-free zone between the endothelium where NO is made and the location of the RBCs.
However, in hemolytic conditions, these mechanisms can be compromised, leading to reduced levels of NO reaching the smooth muscle cells to cause vasodilation. Although it is generally accepted that cell-free Hb scavenges NO more effectively than Hb encapsulated in the red blood cell (RBC), some do not believe that the low concentrations of cell-free Hb that are present in hemolytic conditions, such as sickle cell disease, can impact NO bioavailability in the presence of the large amounts of RBC encapsulated Hb (about 10 mM in heme) present in vivo. We have performed computational modeling looking at the effects of cell-free Hb on NO bioavailability within blood vessels. We have found that concentrations of cell-free Hb as low as one micromolar significantly reduces the availability of NO. Thus, 0.01% hemolysis has a significant affect on steady state NO levels. Extravasation of cell-free Hb into the interstitial space of as low as one micromolar further reduces the bioavailability of NO. At low hematocrit values, cell-free Hb scavenging of NO was found to be more efficient than at high hematocrit values. We have also found that the effect of RBC membrane permeability diminishes at cell-free Hb concentrations of only four micromolar and above. These theoretical results support experimental evidence indicating that cell-free Hb contributes to the pathology of hemolytic diseases. Supported by NIH grant RG0489.
Additive Effect of Butyrate and Hemin on Fetal Hemoglobin Induction in Erythroid Cells from Sickle Cell Disease and Beta-Thalassemia.
Authors: Fathallah Hassana , Taher Ali , Bazarbachi Ali , Atweh George F. 
Published: April 2020

Abstract
High levels of fetal hemoglobin (HbF) are known to ameliorate the pathophysiology of β-globin disorders. The objective of this study is twofold: the first is to evaluate the efficacy of hemin as an inducer of HbF in erythroid cells from patients with sickle cell disease (SCD) and β-thalassemia (β-thal); the second is to determine if the combination of butyrate and hemin can induce higher levels of expression of HbF than either agent alone. BFU-E derived cells from the peripheral blood of two patients with homozygous SCD, three patients with β-thal, one patient with sickle β-thalassemia (S/β-thal) and one normal individual (AA) were cultured in the absence (control) or presence of butyrate (B), hemin (H) or butyrate and hemin (B+H). As expected, the levels of γ-globin mRNA [expressed as % γ/(β+γ)] increased upon butyrate exposure in progenitor-derived erythroid cells from SS and S/β-thal patients, and to a lesser extent in patients with β-thal (P = 0.01). In contrast, butyrate did not increase γ-globin expression in BFU-E derived colonies from the AA individual. Moreover, hemin exposure increased the γ/(β+γ) ratio in all subjects (P = 0.02). These findings confirm that hemin can be an effective HbF inducing agent in SCD and β-thal. Although the mechanism of induction of HbF by hemin is not known, unlike butyrate, hemin is clearly not a direct inhibitor of histone deacetylases and is likely to induce HbF by a different mechanism of action. Thus, we investigated the effect of the combination of hemin and butyrate on γ-globin gene expression. Interestingly, the combination of butyrate and hemin resulted in additive increases in the γ/(β+γ) ratios in all patients compared to butyrate alone (P = 0.03) or hemin alone (P = 0.01) (Table I). Just as importantly, exposure to both drugs resulted in a decrease in the α/(β+γ) mRNA imbalance in β-thal, which is the predominant pathophysiological feature of this disorder. In conclusion, combination therapy consisting of butyrate and hemin, which are two agents with different mechanisms of action and different toxicity profiles, may provide a more effective way of inducing HbF in patients with SCD and β-thal.
Table I mRNA SCD β-Thal S/β-Thal AA n 2 3 1 1 %γ/(β+γ) Control 36 42 26 7.1 B 45 50 41 6.9 H 55 55 52 15 B+H 60 61 59 13 α/(β+γ) Control 3.1 8.9 1.8 1.9 B 2.0 7.7 2.9 1.7 H 3.0 7.5 1.7 1.0 B+H 2.9 6.4 2.2 1.3
Effects of Sickle Cell Disease on Hematopoietic Stem Cell Function and the Bone Marrow Microenvironment.
Authors: Javazon Elisabeth H. , Kean Leslie S. , Perry Jennifer , Butler Jessica , Archer David R. 
Published: April 2020

Abstract
Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted &gt; hemi &gt; sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.
Endothelial Cell-Derived Microparticules (EMPs) Can Induce In Vitro Maturation of Both Plasmocytoid and Myeloid Dendritic Cells.
Authors: Garnache-Ottou Francine , Angelot Fanny , Biichle Sabeha , PLumas Joel , George Francoise Dignat , Saas Philippe , Tiberghien Pierre , Seilles Estelle 
Published: April 2020

Abstract
The implication of plasmocytoid dendritic cells (pDCs) in transplantation is more and more reported, especially because of their tolerogenic properties supported by the expansion of regulatory T cells. Endothelial microparticles (EMPs) are plasma membrane-derived vesicules shed from endothelial stimulation involved in physiological and pathological processes, including intercellular communication, hemostasis and immunity. However, the interaction between DC and EMP are not well-known, especially in the context of alloreactivity. Therefore, the aims of the current study were to explore the DC/EMP interactions, more specifically the capacity of EMPs to modulate the maturation of DC. Firstly, we validated an original co-culture model involving a tumoral pDC lineage (GEN 2.2) and EMPs generated in a standard fashion from an endothelial cell lineage (HMEC U608), stimulated or not with TNF-α . EMPs was analyzed by FACS (FITC labelled annexin V) and supernatant resulting from the last wash of EMPs was used as negative control. By incubating increasing amounts of EMPs we showed that EMPs can lead to GEN pDC maturation in a dose- effect manner as evidenced by a significative increase in the HLA-DR and CD83 expression, compared to the negative controls or, of importance, compared to the maturation with CPG-A (ligand of TLR9 mimicking bacterial DNA), CD40L or R848 (ligand of TLR7 mimicking viral RNA). To confirm and expand these findings, we then cocultured with EMPs (and similar controls)
circulating pDC obtained by an anti-BDCA-4 MoAb-mediated positive selection of healthy donors PBMC , and myeloid DC (mDC) obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4.
Maturation of both normal circulating pDC and mDC occurred in the presence of EMPs and once again, in a dose-dependant fashion. The cytokine secretion pattern of the EMPs- matured mDC and pDC, determined by the Luminex technology, was as expected for such cell populations. The EMPs matured pDC secreted IL-1, IL-6, IFN-α , IL-12 and IL-10 but not TGF-β . In contrast, the EMPs matured mDC secreted mainly proinflammatory cytokines : IL-1, IL-6 and IL-8 but not IL-10. Overall, our studies demonstrate that EMPs can induce the maturation of both pDC and mDC. Such endothelial microparticules, generated by inflammatory and hemostatic disorders, could play a role in post-transplant immune responses. Determining the factors resulting in EMPs-induced pDC vs mDC maturation may open the possibility for EMPs-related immuno-intervention.
ISG12 Is a Critical Modulator of Innate Immune Responses in Animal Models of Septis.
Authors: Schabbauer Gernot , Papac-Milicevic Nikolina , Uhrin Pavel , Binder Bernd 
Published: April 2020

Abstract
Sepsis is still a major burden for the society with a high incidence of morbidity and mortality each year. Molecular mechanisms underlying the systemic inflammatory response syndrome (SIRS) associated with sepsis are still ill defined and most therapies developed to target the acute inflammatory component of the disease are insufficent.
Recently the role of nuclear receptors (NRs) in transcriptional regulation of inflammatory processes became a major topic of interest. Nuclear receptors, such as the peroxisome proliferators-activated receptors (PPARs), have been found to exert anti-inflammatory properties by interfering with the NFkB pathway.
We are interested in the nuclear envelope protein, interferon stimulated gene 12 (ISG12), which directly interacts with NRs. ISG12 is a co-factor stimulating nuclear export of NRs, thereby reducing the anti-inflammatory potential of NRs such as PPARg or NR4A1. To examine the role of ISG12 in acute inflammation we generated ISG12 deficient mice.
We can demonstrate by reverse genetics in ISG12 deficient mice that lack of ISG12 is beneficial in experimental sepsis and endotoxemia. Furthermore we can show that several acute inflammatory parameters, such as systemic IL6 cytokine levels, are downregulated in septic ISG12-/- animals. Consistently, similar results could be obtained in in vitro experiments in peritoneal macrophages derived from ISG12 deficient mice.
In contrast, mice deficient for the nuclear receptor NR4A1 exhibited an exacerbated innate immune response and showed a significantly higher mortality after lethal septic challenge. This dramatic phenotype could be restored in ISG12/NR4A1 double deficient mice.
We conclude from our data in vitro and in vivo that ISG12 is a novel modulator of innate immune responses regulating anti-inflammatory nuclear receptors such as NR4A1.
Replication of Genetic Markers Associated with Clozapine-Induced Agranulocytosis.
Authors: Gerson Stanton L. , Dettling Michael , Dain Brad , Lachowicz Michael , Kane John , Malhotra Anil , Cascorbi Ingolf , Mosyagin Igor , Reed Carol , Athanasiou Maria 
Published: April 2020

Abstract
Clozapine is a highly efficacious drug for the treatment of schizophrenia, but its use is limited, in part due to the side effect of agranulocytosis. In order to reduce the incidence of clozapine-induced agranulocytosis (CIA), patients are required to submit to a blood monitoring program. A genetic component to CIA is suggested by published associations in a number of genes. A genetic test assessing the risk of CIA might allow safer and more broad use of clozapine. We previously conducted a candidate gene-based case-control study to discover genes associated with CIA. Cases were patients with an ANC&lt;500 during clozapine treatment, and controls were patients treated for at least one year without a significant reduction in WBC or ANC. For each case, we attempted to enroll 2 age-, gender- and ethnicity-matched controls. We collected blood, medical histories and informed consent from 33 CIA cases and 54 controls. Seventy-four candidate genes were sequenced in the exons, intron-exon boundaries, 5′ untranslated region and promoter region for each of the cases and controls, and a haplotype analysis was conducted using logistic regression. A total of 20 haplotype markers in HLA-DQB1, 1 in HLA-C, 1 in NTSR1, 1 in DRD1 and 6 in CSF2RB were significant with permutation test adjusted p-values under 0.05.
A replication analysis was performed on an independently collected cohort of 49 cases and 78 controls from Germany. Cases were patients who developed an ANC&lt;500 during clozapine treatment, and controls were patients treated for at least two years without an adverse effect on the WBC or ANC. We attempted to replicate 28 haplotype markers from 4 of the 5 genes. We did not attempt to replicate the findings from HLA-C. Permutation tests were performed for markers within genes, and the Benjamini-Hochberg FDR method was applied to the best permutation p-values from the four genes. Markers with p-values less than 0.05 after these adjustments were considered replicated. One genetic marker in HLA-DQB1 replicated further implicating this as the strongest genetic association with CIA. Markers in CSF2RB were not statistically significant, but warrant further study. The strength of the findings is sufficient to develop a diagnostic test for CIA risk. These findings may also be applicable to agranulocytosis and neutropenia induced by other drugs.