bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of ATRA Inducible RARα2 Isoform Is Deregulated in AML.
Authors: Glasow Annegret , Barrett Angela , Gupta Rajeev , Grimwade David , von Lindern Marieke , Enver Tariq , Zelent Arthur 
Published: April 2020

Abstract
Retinoids exert a variety of effects on both normal and malignant hematopoietic cells. To date, three different retinoic acid receptor (RAR) and retinoid X receptor (RXR) genes have been characterized, each encoding multiple N-terminal protein isoforms. RXRs serve as co-regulators for RARs, and many other nuclear receptors integrating different signalling pathways. All-trans-retinoic acid (ATRA) signaling pathway is of critical importance for optimal myelomonocytic differentiation and its disruption by translocations of the RARα gene leads to acute promyelocytic leukemia (APL). APL associated fusion oncoproteins, such as PML-RARα and PLZF-RARα, function through recruitment of histone deacetylases (HDACs) and DNA methyltransferases (DNMTs), thus promoting an inactive chromatin state and leading to repression of RARα target genes. Recently, we demonstrated that up-regulation of RARα2 expression by ATRA directly correlates with differentiation of APL and non-APL AML cells and that RARα2 transcription is silenced by DNA methylation in AML cell lines. Using primary AML samples as well as normal cord and peripheral blood derived cells representing different stages of myelomonocytic development we now show that expression of RARα2 increases with maturation of hematopietic cells. Expression of RARα1 on the other hand, which is transcribed from a distinct promoter, remains relatively constant throughout the different stages of myelomonocytic development. The levels of RARα1 expression in various primary AML cell types appear to be similar to those found in normal hematopietic cells. Consistent with data derived from AML cell lines, however, the RARα2 isoform is poorly expressed in all samples. Compared with CD34+/CD133+ or CD34+ progenitors, and more mature CD33+ myeloid cells, RARα2 is expressed at much lower levels in a variety of primary AML cells and its expression is not effectively induced by myelomonocytic growth factors and/or ATRA. Negatively acting epigenetic changes, such as DNA methylation, appear to be responsible for deregulated expression of RARα2 in AML cells, although their pattern and extent differs significantly between AML cell lines and primary AML samples. Taken together our data suggest that agents, which revert negatively acting epigenetic changes may restore expression of the RARα2 isoform in AML cells and render them more responsive to ATRA as well as other differentiation inducers.
MHC Class-II Expression Reveals Differential Regulation of BLIMP-1 Target Genes.
Authors: Tooze Reuben , Stephenson Sophie , Doody Gina , Rawstron Andy 
Published: April 2020

Abstract
BLIMP-1 initiates plasma cell differentiation by inhibiting the expression of a limited set of transcription factor genes. BLIMP-1 mediates repression of its targets by promoter occupancy and recruitment of the Groucho corepressor, histone deacetylase and the G9a lysine methyltransferase. The relative contribution of these mechanisms to the stable silencing of BLIMP-1 target genes in plasma cells is not known. Epigenetic changes initiated by BLIMP-1 could be maintained by independent factors or alternatively may be dependent on continuous occupancy of the promoter by BLIMP-1. The aim of this study was to investigate the mechanisms operating at two well-defined BLIMP-1 target genes, MHC2TA and PAX-5. Repression of PAX-5 is essential for extinction of the B-cell phenotype, while repression of MHC2TA mediates the loss of MHC class-II expression characteristic of plasma cell differentiation. PAX-5 is silenced in virtually all cases of myeloma, whereas MHC class-II continues to be expressed in a subset of primary myeloma cells and many myeloma cell lines. Using chromatin immunoprecipitation we demonstrate that BLIMP-1 constitutively binds MHC2TA promoter-3 and is present equally in MHC2TA expressing and non-expressing myeloma cell lines. In expressing cells MHC2TA promoter-3 is associated with acetylation of histone H3 lysine-9, a marker of open chromatin, whereas histone H3 lysine-9 is neither acetylated nor methylated in non-expressing cells. In contrast the PAX-5 promoter is associated with trimethylation of histone H3 lysine-9, a marker of repressed heterochromatin, in all the cell lines examined, but constitutive BLIMP-1 occupancy is not detectable. Although BLIMP-1 does not silence MHC2TA expression in the MHC class-II positive U266 cell line, it is mediating transcriptional repression, since siRNA knockdown of BLIMP-1 is associated with elevated MHC2TA mRNA and MHC class-II surface expression. In contrast PAX-5 mRNA and its target CD19 continue to be repressed. MHC class-II re-expression was associated with loss of CD138 and the entry of the majority of cells into apoptosis. Our data demonstrate that MHC class-II expression in myeloma cell lines is associated with defective silencing of MHC2TA despite BLIMP-1 occupancy, and reveal differences in the contribution of epigenetic modifications and promoter occupancy in the maintenance of target gene silencing by BLIMP-1. Loss of BLIMP-1 results in a partial reversion of plasma cell phenotype that is associated with induction of apoptosis. As continual high levels of BLIMP-1 expression appear to be critical for plasma cell survival, suppression of BLIMP-1 represents a potential therapeutic pathway.
Detection of the Acquired Tyrosine Kinase JAK2 Gene Mutation by Real Time PCR as Minimal Residual Disease Marker for Patients after Allogeneic Hematopoetic Stem Cell Transplantation.
Authors: Elmaagacli Ahmet H. , Steckel Nina K. , Koldehoff Michael , Ditschkowski Markus , Trenschel Rudolf , Beelen Dietrich W. 
Published: April 2020

Abstract
Recently, a single point mutation (VAL617Phe) in the tyrosine kinase gene JAK2 was identified in some patients with polycythaemia vera, essential thrombocythaemia and idiopathic myelofibrosis by Baxter and coworker. Here, we assessed the frequency of these acquired JAK2 gene mutations by real-time PCR in patients with myelofibrosis with myeloid metaplasia (MMM) (n=25), atypical bcr-abl negative CML (n=8), MDS (n=53), AML M6 (erythroleukaemia) (n=8) and AML megakaryocytic leukaemia) M7 (n=2) prior to a scheduled allogeneic transplant. Further, we evaluated the use of the detection of JAK2 gene mutation as a MRD marker after transplant. A JAK2 gene mutation was found in 13 of 25 patients with MMM, 1 of 8 patients with atypical CML, 2 of 2 patients with AML M7, but none in patients with MDS (n=53) or patients with AML M6 (n=8). All tested 24 healthy donors had no detectable JAK2 mutation by PCR. In 15 patients with a detectable JAK2 gene mutation prior to transplant, we evaluated the chimerism status and the detection of the JAK2 mutation by PCR simultaneously. Four of these 15 patients revealed a mixed chimerism after transplant at different time periods. In three of these four patients a JAK2 gene mutation was detectable contemporarily by PCR, whereas 11 patients with a complete chimerism detected by STR-PCR had no evidence for a JAK2 mutation at the same time point. Three patients with MMM in whom a JAK2 mutation could be detected again relapsed again of MMM. These data demonstrate that JAK2 gene mutation can be used as a MRD marker for MMM in patients who had this gene mutation prior to transplant.
BCR-ABL Decreases the Expression of CCN3 Leading to Increased Clonogenic Potential and Cell Growth.
Authors: McCallum Lynn M.R. , Lu Wanhua , Price Susan , Planque Nathalie , Perbal Bernard , Pierce Andrew , Whetton Anthony , Irvine Alexandra E. 
Published: April 2020

Abstract
Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we have identified downregulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity and detected reduced CCN3 expression in human CML cell lines and primary human CML cells. We now report a reciprocal relationship of BCR-ABL and CCN3 expression and the functional consequence of expressing CCN3 in BCR-ABL+ cells. Real-time PCR was used to examine the relationship between BCR-ABL and CCN3 expression in human K562 cells. Parental K562 cells showed high expression of BCR-ABL (4.68 x104 transcripts in 5 μL of cDNA) whilst CCN3 expression was not detected. Treatment with siRNA directed against BCR-ABL (0.5 μg per106 cells) for 72 hours resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (mean Ct change 1.9 ± 0.2 and 2.6 ± 0.5 for BCR-ABL and CCN3 respectively, n=3, p=0.001). Similarly, K562 cells treated with imatinib (1 micromolar) for 96 hours showed a 5.9 fold decrease in BCR-ABL expression and a 4.2 fold increase in CCN3 expression (mean Ct change 2.5 ± 0.1 and 2.1± 0.2 for BCR-ABL and CCN3 respectively, n=3, p=0.001). To investigate CCN3 function, we expressed CCN3 in BCR-ABL+ cells using Nucleofector technology (Amaxa, GmbH). K562 cells were transfected with either the pCb6+ vector (Invitrogen,UK) or pCb6+ vector containing the CCN3 construct. Cells were analysed 24 hours post-transfection by flow cytometry and also after 7 days in methyl cellulose culture to determine clonogenicity. Cell cycle analysis was performed on 20,000 events using the winMDI software. CCN3 expression in BCR-ABL+ cells resulted in an accumulation of cells in the subG0 phase of the cell cycle (mean for subG0 9.9% ± 4.6 and 21.8% ± 0.7 for the pCb6+ vector alone and pCb6+ vector containing CCN3 construct respectively). CCN3 expression significantly increased the number of cells within the subG0 area of the cell cycle (n=3, p=0.027). In addition, CCN3 expression reduced the clonogenic capacity of BCR-ABL+ cells. K562 cells transfected with the pCb6+ vector containing CCN3 construct formed significantly fewer colonies on methyl cellulose in comparison to cells that had been transfected with the pCb6+ vector alone (n=3, p=0.027).
This study demonstrates a reciprocal relationship between CCN3 and BCR-ABL expression. CCN3 is known to be a negative growth regulator and increased expression of CCN3 in BCR-ABL+ cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.
Activation of Phospholipase C γ 2 in Lymphoma Cells.
Authors: Huynh Minh Q. , Wuendisch Thomas , Ramaswamy Annette , Neubauer Andreas 
Published: April 2020

Abstract
The Nf-κB signaling pathway is important for lymphoma survival and cell proliferation. In recent immunohistological studies, the B-cell receptor (BCR), Src kinases and PLC γ 2 were shown to be highly expressed in primary mediastinal B-cell lymphoma and lymphocyte predominant Hodgkin lymphoma, but not in classical Hodgkin lymphoma. Activation of PLC γ 2 is a crucial step in regulating Nf-κB through protein kinase C (PKC). The PLC-PKC cascade also activates the Ras-Raf-Erk signaling pathway by recruiting GRP3 to the cell membrane. These signaling cascades are well studied in physiological B-cells, but there is little data about the role of PLC γ 2 in lymphomas. Our study compared the PLC γ 2 expression in low-grade gastric MALT lymphoma tissue and chronic gastritis tissue from the same patient by quantitative PCR. In 8 out of 10 cases PLC γ 2 was overexpressed in MALT lymphomas, but not in gastritis. We performed functional studies with the PLC-inhibitor U73122 and PLC γ 2 specific siRNA in two lymphoma cell lines (SSK41, Raji). Proliferation of lymphoma cells was significantly reduced by inhibition of PLC γ 2 on protein level and by knocking down PLC γ 2 mRNA by transfection with a specific siRNA. Our results indicate that proliferation and survival of lymphoma cells depend, at least in part, on the activated PLC-PKC signaling cascade and that it is possible to reduce lymphoma survival by the “small molecule inhibitor” U73122. PLC γ 2 could be a new target for lymphoma treatment.
3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.
Authors: Levantini Elena , Okuno Yutaka , Zhang Pu , Koschmieder Steffen , Radomska Hanna S. , Fenyus Maris , Guiney Maureen , Tenen Daniel G. 
Published: April 2020

Abstract
CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.
Immunomodulation of Transgene Responses Following Naked DNA Transfer of Factor VIII into Hemophilia A Mice.
Authors: Ye Peiqing , Rawlings David J. , Thompson Arthur R. , Ochs Hans D. , Miao Carol H. 
Published: April 2020

Abstract
Naked DNA transfer of liver-specific, high-expressing plasmid pBS-HCRHPI-FVIIIA in Rag2(−/ −) SCID mice produced persistent high-level gene expression of human factor VIII (hFVIII) (Miao, Hum. Gene Ther. 2003). However, in immunocompetent hemophilia A mice, a robust humoral immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity (Ye, Mol. Ther. 2004). Transient immunomodulation strategies were explored to prevent the formation of inhibitory antibody formation. Eight groups of mice (n=8) were treated by naked DNA transfer of plasmid pBS-HCRHPI-FVIIIA. Each group were subjected to treatment with single or combined immunosuppressive regimen: CyclosporineA (CSA) daily for 14 days; Rapamycin daily for 14 days; Mycophenylate mofetil (MMF) daily for 14 days; combination of CSA and MMF; combination of Rapamycin and MMF; a monoclonal antibody (MR1) against murine CD40 ligand on days -1, 1, 2, 7, & 14; recombinant murine Ctla4Ig on days 1 & 2; and combination of MR1 and Ctla4Ig. Combination regimens were given using the same combined schedule and dosages. All animals treated with immunosuppression had delayed or no immune responses against hFVIII except the group treated with CSA only. The most effective treatment was observed in animals treated with the combination of Ctla4Ig and MR1. Seven of 8 animals failed to develop detectable inhibitors. One animal developed transient low-titer antibodies. This group of animals produced persistent, therapeutic levels of hFVIII gene expression for over 6 months. Tolerized animals were subsequently challenged by the T dependent antigen, bacteriophage Φx174, and exhibited a normal primary and secondary response including amplification and isotype switch. These results strongly suggest that transient immunomodulation strategies to disrupt B- and T- cell interactions at the time of plasmid injection is effective to promote long-term immune tolerance that is specific for FVIII without altering subsequent immune responses to other T cell dependent antigens.
Antigen-Dependent Suppression of Graft Versus Host Disease by Foxp3-Induced Regulatory T Cells in Transplantation.
Authors: Albert Michael H. , Liu Yan , Anasetti Claudio , Yu Xue-Zhong 
Published: April 2020

Abstract
Adoptive transfer of polyclonal CD4+CD25+ regulatory T cells (Tregs) can tolerize transplantation alloresponses and prevent lethal acute graft-versus-host disease (GVHD). For optimal suppressive function, Tregs need to be activated via their T-cell receptors (TCR), but the antigen specificity of wild type Tregs remains elusive, and therefore controlling potency and duration of Treg activity in the transplantation setting remains not feasible. In this study, we used a murine lethal acute GVHD model system to test the hypothesis that specifically activated, antigen-specific Tregs induced by foxp3 transduction could suppress the response of T effector cells to alloantigens in vitro and prevent GVHD in vivo more effectively than polyclonal Tregs.
We found that the suppressive potential of TCR transgenic (Tg), antigen-specific CD4+CD25+ Tregs was much greater than that of polyclonal Tregs in vitro and in vivo. When activated by their specific peptide antigen, Tg Tregs protected 95% of recipients from lethal GVHD even at ten times lower doses than polyclonal Tregs. To facilitate the acquisition of larger numbers of antigen-specific Tregs, we transduced naive CD4+CD25- cells with foxp3, and observed that these foxp3-induced Tregs also suppressed alloresponses in vitro and prevented GVHD in vivo as effectively as naturally derived CD4+CD25+ Tregs. To enhance translational feasibility, we then used an antigen-specific CD4 Th1 T-cell clone as a source of Tregs after transduction with foxp3, and found those Tregs to effectively prevent GVHD in 90% of recipients. We further found that prevention of GVHD via foxp3-induced Tregs was also dependent on their activation by either a specific alloantigen expressed on recipient cells or by immunization with a specific peptide antigen.
The findings of this study provide a basis for the concept that the onset and potency of alloresponse suppression in GVHD can be regulated by using Tregs with known antigen specificity. The novel evidence that antigen-specific T cell clones can be used as the cell source for foxp3-induced Tregs further improves the feasibility of using Tregs for modulating immune responses in vivo. These data suggest a novel approach to control induction of tolerance using Tregs as an adoptive immunotherapy in allogeneic transplantation.
Early Treatment with CD4+CD25+ Regulatory T Cells Provides Prolonged Suppressive Effects Which Control Evolving but Not Established Graft-Versus-Host-Disease.
Authors: Nguyen Vu H. , Zeiser Robert , Dasilva Daniel , Contag Christopher N. , Negrin Robert S. 
Published: April 2020

Abstract
CD4+CD25+ Regulatory T cells (Treg) reduce the incidence and severity of acute graft-versus-host disease (GvHD) in murine models of allogeneic bone marrow transplantation (BMT) when given at the time of transplantation at a 1:1 ratio with donor effector T cells (Tcon). In our previous study with Treg trafficking, bioluminescene imaging (BLI) indicated a persistence of signal consistent with a prolonged survival of Treg in vivo following allogeneic BMT. In the current study, we evaluated the duration of Treg suppression and the impact of Treg on evolving and established GvHD. Lethally irradiated Balb/c (H2d) hosts received 5x106 T-cell depleted bone marrow (TCD-BM) cells from wild-type FVB mice (H2q) on day 0. On day 2, 3x106 splenocytes, or 1x106 purified CD4+/CD8+ T cells (Tcon) from luciferase+ transgenic mice (FVB) were infused to induce GvHD. Treg, purified from wt-FVB mice, in a 1:1 dose ratio with Tcon, were infused either on day 0, 2, 9, or 23 post-transplantation. Bioluminescence imaging was used to localize and quantitate the proliferation of Tcon in the absence or presence of Treg. Signal intensity, measured by photons/second/mouse, was significantly decreased in animals which received Treg at day 0, 2, or 9 (p-value &lt; 0.05). More importantly, the greatest reduction in signal intensity occurred when Treg were given prior to the induction of GvHD by Tcon. This reduction was associated with a significantly lower clinical score for GvHD. Studies in which Treg are given up to 10 days prior to the addition of Tcon show similar findings. At day 23, when clinical GvHD was fully established in mice which received Tcon, the addition of Treg did not alter the increasing BLI signal level or the clinical course such that all animals died of GvHD (p-value=0.38). Lymphoid reconstitution was not affected by the addition of Treg prior to the induction of GvHD. In dose titration studies whereby Treg are given two days prior to the induction of GvHD, a 10-fold dose reduction in Treg was sufficient to significantly reduce Tcon proliferation and suppress clinical GvHD. We next assessed the duration of Treg suppressive effect by inducing GvHD on day 7, 14, 19 with luc+ Tcon following the infusion of Treg on day 0 of allogeneic BMT. Treg provided protection from the Tcon challenge at all 3 time points, leading to improved survival (p-value &lt; 0.05). We conclude that Treg provide prolonged protection due to their ability to proliferate in vivo in an allogeneic setting. The capacity of Treg to proliferate in vivo permits a significant reduction in the number of Treg needed for adoptive transfer to induce a clinical response, a practical finding given the rarity of Treg. In addition, we conclude that Treg suppress the early proliferation of Tcon, allowing them to prevent and control evolving but not established GvHD.