bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Myeloproliferative Disorder in the Tc1 Mouse Model of Down Syndrome
Authors: Alford Kate A. , Vanes Lesley , Li Zhe , Orkin Stuart H. , Fisher Elizabeth M. C. , Tybulewicz Victor L. J. 
Published: April 2020

Abstract
Down syndrome (DS) children have a one in ten chance of being diagnosed with leukemia within the first ten years of life. Acute megakaryoblastic leukemia (AMKL) is a subtype of acute myeloid leukemia (AML) that accounts for nearly 50% of these leukemias. AMKL is associated with a self-regressing neoplasia found almost exclusively in DS newborns called Transient Myeloproliferative Disorder (TMD). In all cases of TMD and DS-AMKL, leukemic blast cells show mutations in the gene encoding the hematopoietic transcription factor GATA1, resulting in production of a truncated form of the protein called GATA1s. Mutations in GATA1 are not seen in non-DS-AMKL or other DS leukemias and it is clear both trisomy of human chromosome 21 (HSA21) and a mutation in GATA1 are required for the development of both TMD and AMKL. However, it is unknown which genes on HSA21 need to be trisomic in order to predispose an individual with DS to AMKL. Our group has generated mice (termed the Tc1 mice) that contain an almost complete, freely segregating copy of HSA21. These mice display phenotypic features of DS. We have examined adult hematopoiesis in these mice. Blood samples taken from a cohort of Tc1 mice were examined from 4 weeks until 60 weeks of age. Complete blood cell counts show that whilst the mice do not develop leukemia they displayed persistent macrocytosis and had reduced erythrocyte numbers. Crossing the Tc1 mice with mice that express GATA1s protein did not perturb or exacerbate this phenotype. Over the age of 15 months more than 50% of Tc1 mice examined were found to have developed splenomegaly. These mice displayed megakaryocyte hyperplasia and had increased numbers of cells of the erythroid lineage. In vitro colony forming assays demonstrated an increase in the frequency of megakaryocytic and granulocyte-macrophage progenitors in the spleen, consistent with extramedullary hematopoiesis. In the bone marrow, no abnormalities were seen in the lineage-, c-Kit+, Sca1+ (LSK) compartment, however there was a significant increase in the percentage of common myeloid progenitors (CMP) and a corresponding decrease in megakaryocyte-erythrocyte progenitors (MEP). This suggests a possible block in development from CMP to MEP. These data demonstrate defects in hematopoietic development in a proportion of adult Tc1 mice. However, preliminary data suggest that these mice do not develop a neonatal myeloproliferative disorder that is comparable with human TMD. It may be that the phenotype seen in the adult Tc1 mice is due to defects in hematopoietic progenitors that are different to those responsible for development of TMD and DS-AMKL. This mouse model may therefore provide a useful tool to examine the role of HSA21 genes in adult hematopoietic disorders.
Outcome of Patients with Myeloproliferative Disorders Treated with Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Donato Michele L. , Prann Angela , Ullrich Anna , Feldman Tatyana , Goldberg Stuart L. , Goy Andre , Masood Aisha , Pecora Andrew L. , Siegel David S. , Rowley Scott D. 
Published: April 2020

Abstract
Myeloproliferative disorders (MPD) are characterized by marrow clonal proliferation leading to ineffective hematopoiesis. Between 1998 and 2007, 27 patients (pts) with myeloproliferative disorders were treated with hematopoietic stem cell transplantation. Patients included 9 males (33%) and 18 females (67%) with a median age of 52 years (range 30–68). Diseases included Philadelphia chromosome-negative chronic myelogenous leukemia (CML) (n=2, 7%), myelofibrosis (n=21, 78%), polycythemia vera (n=2, 7%) and essential thrombocythemia (n=2, 7%). Twelve patients (44%) had abnormal cytogenetics, 24 pts (89%) had splenomegaly and 9 pts (33%) had a prior splenectomy. Preparative regimens included busulfan and cyclophosphamide (n=11, 41%), cyclophosphamide and total body irradiation (n=3, 11%), fludarabine and busulfan (n=3, 11%), fludarabine and cyclophosphamide (n=1, 4%), fludarabine and melphalan (n=7, 26%), and fludarabine with low dose total body irradiation (n=2, 7%). Patients with unrelated donors also received ATG. Twelve patients received an unrelated donor product, 7 pts received bone marrow and 20 pts received peripheral blood stem cells.
The 5-year overall Kaplan-Meier product-limit survival estimates (figure 1.) was 60.9%. The day 100 transplant-related mortality was 18.5%, all in patients receiving either cyclophosphamide and total body irradiation or cyclophosphamide and busulfan. Overall, there were twice as many deaths in patients receiving a non-fludarabine-based regimen (n=7, 25.9%) compared with patients receiving a fludarabine-based regimen (n=3, 11.1%) though the difference was not statistically significant (p=0.15). The overall incidence of grade II–IV acute GVHD was 44% and chronic GVHD was 64%. The overall relapse rate was 16%. No significant differences in relapse rates by acute (p=0.40) and chronic GVHD (p=0.53) or by fludarabive vs non-fludarabine based regimens (p=0.24) were observed. In univariate Cox proportional hazard models, chronic GVHD was the only significant factor associated with worse survival (p=0.035).
In conclusion, myeloproliferative disorders can be successfully treated with allogeneic hematopoietic stem cell transplantation. Chronic GVHD significantly reduces overall survival. The optimal preparative regimen still remains to be determined.
Figure 1. Kaplan-Meier product-limit survival estimates of MPD treated with allogeneic hepatopoietic stem cell transplantation (n=27). Figure 1. Kaplan-Meier product-limit survival estimates of MPD treated with allogeneic hepatopoietic stem cell transplantation (n=27).
Previously Unknown Interactions Between AML1 and MLL Provide Epigenetic Regulation of Gene Expression in Normal Hematopoiesis and in Leukemia
Authors: Huang Gang , Elf Shannon , Yan Xiaomei , Wang Lan , Liu Yan , Sashida Goro , Gural Alex , Menendez Silvia , Lee Jennifer , Yang Youyang , Zhao Xinyang , Nimer Stephen D. 
Published: April 2020

Abstract
In all organisms, the fundamental process of transcriptional regulation requires transcription factors, which bind to DNA in response to extra-cellular signals and regulate transcription of target genes. In eukaryotes, this process also involves epigenetic regulation, which includes DNA and histone modifications. Hematopoiesis and leukemia are excellent model systems for studying the higher eukaryotic regulations of gene expression and for identifying important molecules involved in genetic and epigenetic transcriptional regulation. The Mixed-Lineage Leukemia (MLL) protein, a Set1-like H3K4 methyltransferase, and the heterodimeric transcriptional factor AML1/CBFβ are critical for definitive and adult hematopoiesis. They are required for the generation of all hematopoietic lineages and act as tumor suppressors in human leukemia. We have previously shown that the regulation of PU.1 by AML1 is mediated by 3 AML1 binding sites in the PU.1 upstream regulatory element (URE), located −14 kb relative to the transcription start site in mice (Huang et al. Nat Genet. 2008). To understand whether AML1 plays a critical role in regulating the PU.1 locus at the chromatin level, we have utilized this PU.1 regulation system as a model to study the potential interplay between AML1/CBFβ and MLL. We found that MLL binds to the evolutionarily conserved Runt-domain of AML1, enhances the formation of the AML1/CBFβ heterodimer, and blocks the ubiquitin-proteasome mediated degradation of AML1. We also found that AML1/CBFβ is required for maintenance of the H3K4-me3 histone mark at the PU.1 upstream regulatory element (URE) and promoter region, and that MLL is required for AML1 to regulate PU.1 expression. In contrast, we found that several leukemia-associated MLL fusion proteins, including MLL-AF4, MLL-AF9, MLL-ENL, and MLL-AF10, no longer stabilize AML1 but rather induce AML1 degradation and downregulate PU.1 expression. Taken together, our data indicate that MLL and some of its leukemia-associated fusion proteins physically and functionally interact with AML1. Their effects on target genes, particularly PU.1, may be critical for the normal regulation of hematopoiesis and for the aberrant regulation that underlies acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).
Aberrant Splicing of the E-Cadherin Gene Is a Novel Mechanism of Gene Silencing in Chronic Lymphocytic Leukemia Cells
Authors: Sharma Sanjai , Lichtenstein Alan 
Published: April 2020

Abstract
Premature termination codons (PTCs) in tumor suppressor genes can contribute to oncogenesis. PTCs prematurely terminate transcription, leading to expression of a truncated, mRNA. The nonsense-mediated degradation (NMD) pathway degrades the aberrant transcripts resulting in loss of expression. To identify RNA transcripts with PTCs in chronic lymphocytic leukemia specimens (CLL), we employed a microarray technique to screen for genes which upregulate their RNA signal upon NMD blockade. In the list of genes with highest upregulation, we identified the E-cadherin gene, a known tumor suppressor gene. On further investigation, we found that the PTC was due to stabilization of an alternatively spliced E-cadherin transcript. This transcript completely lacks exon 11 (exon skipping), resulting in a frameshift and a PTC which is recognized by the NMD pathway and degraded. RT-PCR analysis demonstrated that the exon 11 skipping occurs in normal B cells as well but at a much lower frequency. A real time PCR based strategy to quantify the relative amount of spliced transcript confirmed a 10–30 fold increase in exon 11 skipping in CLL cells (n=29) as compared to normal B cells (n=4) and the degree of skipping increased with increased Rai stage. In addition, real time PCR also demonstrated a significant decrease in total wild type E-cadherin RNA expression in 58% of CLL specimens compared to normal B cells. As prior work demonstrates an upregulated wnt/β-catenin pathway in CLL and, and that E-cadherin can be a physiologic regulator of this pathway, we tested wnt pathway reporter expression in CLL specimens. High expression was identified and ectopic expression of E-cadherin in some samples with E-cadherin loss was sufficient to inhibit the wnt-reporter activity. This suggests that E-cadherin loss, possibly due to exon 11 alternative splicing and exon skipping plays a role in upregulated wnt signaling. As there are no mutations in exon 11 or its flanking intronic regions in CLL cells, our results suggest a novel mechanism of E-cadherin gene inactivation in which the trans-factors/splicing factors in malignant B lymphocytes induce an increased nonproductive splicing of a tumor suppressor gene.
MicroRNA-181b Inhibits ATRA Induced Differentiation in the APL Cell Line NB-4 Via Negative Regulation of the Tumorsupressor RASSF1A
Authors: Braeuer Daniela , Hartmann Jens-Uwe , Christopeit Maximilian , Behre Gerhard 
Published: April 2020

Abstract
MicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in AML and granulopoiesis. In this study, we analysed the expression of miRNAs in the acute promyelocytic leukemia cell line NB-4 during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarray platform (Exiqon) and quantitative PCR analysis. We identified several ATRA regulated microRNAs. Among them we found microRNA-181b as negatively regulated by ATRA. MicroRNA-181b is known to be overexpressed in several cancers such as thyroid papillary carcinoma, colorectal cancer, acute lymphocytic leukemia (ALL) and also acute promyelocytic leukemia (APL). To determine the role of microRNA-181b in the formation and maintenance of the APL-blast phenotype, we performed LNA knock down and transient overexpression experiments. Here we could show in quantitative PCR analysis an inhibitory effect of microRNA-181b on the transcription of the ATRA induced granulocytic differentiation markers C/EBPα and C/EBPβ. Furthermore in FACS analysis we could observe a reduction of the ATRA stimulated formation of the granulocytic surface marker CD11b under microRNA-181b overexpression conditions. To identify the mechanism how microRNA-181b affects ATRA induced differentiation we performed data base analysis to identify putative targets of microRNA-181b. One promising candidate is the tumorsuppressor RASSF1A. RASSF1A was already shown to be involved in proliferation and differentiation by regulation of cell cycle processes. As first group we were able to show in this study the ATRA induced upregulation of RASSF1A in NB-4 cells by quantitative PCR analysis. To demonstrate the negative effect of microRNA-181b on RASSF1A we analysed RASSF1A protein level under LNA knock down and overexpression conditions of microRNA-181b. Here we could observe an upregulation of RASSF1A protein level when microRNA-181b was downregulated. And in addition, a higher amount of microRNA-181b elicits the reduction of RASSF1A protein level. The regulatory effect of RASSF1A in the ATRA induced differentiation of NB-4 cells we confirmed by using siRNA against RASSF1A. In this experiment the FACS analysis showed a reduction of ATRA induced CD11b expression under siRNA mediated knock down of RASS1A. In conclusion, these findings support our hypothesis that microRNA-181b can act as oncogenic microRNA and thereby promotes the leukemic phenotype of APL-blasts by negative regulation of the tumorsuppressor RASSF1A.
NK Cell Anti-Tumor Reactivity Is Impaired by Platelet-Derived TGF-B through NKG2D Downregulation
Authors: Kopp Hans-Georg , Placke Theresa , Schmiedel Benjamin J , Kanz Lothar , Krusch Matthias , Salih Helmut R 
Published: April 2020

Abstract
NK cells can recognize and eliminate malignant cells thereby preventing both local tumor progression and metastatic spread. Besides by the direct interaction with their target cells, NK cell activity is influenced by the interplay with other hematopoietic cells influencing the activity of both involved cell types. Recently, we and others demonstrated that thrombocytopenia inhibits metastasis in mice, which is dependent on the presence of NK cells as shown by NK cell depletion experiments (Jin et al., Nat Med2006, 12: 557; Nieswandt et al., Cancer Res1999, 59: 1295). These data suggest that thrombocytopenia indirectly inhibits tumor dissemination by allowing NK cells to exert their anti-tumor effector functions. However, yet nothing is known regarding the molecular mechanisms underlying platelet-NK cell interaction, especially in humans. Here we report that human platelets express high levels of MHC class I, and incubation of MHC class I-negative tumor cells with platelets induced formation of tumor cell-platelet aggregates resulting in tumor MHC class I “pseudoexpression” which can inhibit NK cell reactivity via KIR triggering. Furthermore, tumor cell-induced platelet aggregation, alike stimulation with classical platelet agonists such as thrombin or collagen, resulted in secretion of platelet-derived soluble factors (releasate) containing, among others, high levels of the immunoregulatory cytokine TGF-b. Presence of releasate in cocultures of NK cells and tumor target cells was found to mediate a substantial inhibition of NK cell cytotoxicity and IFN-g production. Of note, platelet releasate caused a marked downregulation of the activating immunoreceptor NKG2D expressed on NK cells, which was partially restored by a neutralizing TGF-b antibody. Confirming the functional relevance of this finding, neutralization of TGF-b in platelet releasate by the same antibody also partially restored both the impaired NK cell cytotoxicity and IFN-g production in cocultures with tumor cells. Taken together, our data demonstrate that platelets may substantially influence NK cell anti-tumor reactivity by various mechanisms including inhibition of NKG2D-mediated NK cell immunosurveillance. Further elucidation of platelet ability to confer expression of immunregulatory surface molecules to tumor cells and the various soluble factors carrying NK cell-modulatory activity may reveal novel targets to prevent tumor dissemination.
Down Regulation of Hepcidin and Interleukin 1-Alpha in Pbmc from Patients with Beta thalassemia
Authors: Lai Maria-Eliana , Vacquer Stefania , Carta Maria Paola , Mulas Claudia , Cocco Pierluigi , Dessi Sandra 
Published: April 2020

Abstract
Thalassemia is an inherited blood disease characterized by low levels or absence of normal globin chains. Severe forms of thalassemia, thalassemia major (TM), require regular blood transfusions, the main cause of secondary hemosyderosis. However, iron overload is a potential complication, even in patients (pts) with thalassemia intermedia (TI), who do not require transfusions. Among them, iron overload is mainly the result of an excessive absorption of dietary iron, caused by a down-regulation of hepcidin, an hepatic hormone that acts as a major regulator of systemic iron homeostasis. Very low mRNA levels of hepcidin have been reported in urine as well as in liver biopsies from patients with TI. It has been also reported that, interleukin-1 alpha (IL-1α) stimulates hepcidin transcription, raising the question as to whether a down-regulation of this cytokine may be responsible, at least partially, for the excessive iron absorption occurring in patients with TI. Here, we assessed whether IL-1α was involved in hepcidin regulation in β-thalassemia. Peripheral blood mononuclear cells (PBMCs) were isolated from 6 TM pts, 8 TI pts and 6 controls. mRNA was obtained from PBMCs by RT-PCR. Hepcidin mRNA levels were reduced in both TM and TI pts when compared to the age matched normal controls. Neverthless, hepcidin mRNA levels were significantly lower in TI pts, as compared to TM pts receiving regular blood transfusions. Among TI pts lower hepcidin mRNA level was associated with lower level of IL-α. Tumor necrosis factor alpha (TNF-α) does not seem to be involved in the regulation of hepcidin transcription. Interestingly, the levels of mRNA expression of acyl-CoA-cholesterol acyltransferase (ACAT), the enzyme responsible for intracellular cholesterol ester accumulation, and thus, for atherosclerotic plaque formation were strongly induced in PBMCs in TI pts. These results suggest that PBMCs hepcidin and IL-1-α measurements could possibly be used in the future as simple, ease and sensible diagnostic tools for the detection of iron overload in patients with thalassemia. ACAT expression may even be used as therapeutic target in preventing atherosclerotic complications such as pulmonary thromboembolism, cerebral thrombosis, and leg ulcers. frequently occurring in such patients.
Long-Term Efficacy of the Terminal Complement Inhibitor Eculizumab in a Patient with Cold Agglutinin Disease (CAD)
Authors: Roeth Alexander , Huettmann Andreas , Duehrsen Ulrich , Philipp Thomas 
Published: April 2020

Abstract
Cold agglutinin disease (CAD) is a rare form of autoimmune hemolysis in which monoclonal or polyclonal IgM autoantibodies can activate the complement system in colder areas of the body (e.g. extremities), resulting in red blood cell destruction. Typically prednisone is ineffective and, although chemotherapy is often used, only on rare occasions does it suppress autoantibody production. Although CAD is often mild and self-limited, some cases require hospitalization and can be life-threatening. Supportive transfusions of red blood cells may also be necessary. Based on what is known about the pathophysiology of CAD, blockade of the terminal complement cascade by eculizumab could be a possible therapeutic approach. Eculizumab, a monoclonal antibody targeting complement factor C5, has shown high efficacy in patients with hemolytic paroxysmal nocturnal hemoglobinuria (PNH) through inhibition of the terminal complement system. We tested the efficacy of eculizumab in a transfusion-dependent patient with CAD refractory to extensive previous treatment. Eculizumab was dosed as follows: 600 mg IV every 7±2 days × 4; 900 mg 7±2 days later; and then 900 mg every 14±2 days. The patient has continued dosing for approximately one year. As eculizumab treatment may increase the risk for meningococcal disease, a standard vaccination scheme was administered prior to treatment. Transfusion requirements as well as clinical and biochemical indicators of hemolysis were monitored. Our results showed that intravascular hemolysis, measured by lactate dehydrogenase (LDH), was reduced by 46% from 780±50.5 U/L (mean±SE) during the 1 year period prior to treatment to 446±35.5 U/L during 1 year of eculizumab treatment. Reduction in hemolysis resulted in improvement of anemia (from a mean hemoglobin level of 10.0 g/dL 1 year prior to treatment to 11.8 g/dL during 1 year of eculizumab treatment) with a significant reduction in the number of PRBC units transfused (from a mean of 18 units during the pre-treatment period to 0 units during eculizumab treatment). Ferritin levels also decreased from 666±70.8 μg/L (mean±SE) before treatment to 313±8.9 μg/L during treatment. The patient reported an improvement of fatigue and overall quality of life. There were no adverse events observed and the ongoing therapy with eculizumab continues to be safe and well tolerated in this patient. In summary, this is the first report of eculizumab efficacy in a patient with CAD. Chronic blockade of terminal complement with eculizumab resulted in reductions in hemolysis and transfusion requirements and improvements in anemia and fatigue. As no alternative effective treatment options are available, these promising results provide a rationale for a clinical trial for the use of eculizumab in patients with CAD.