bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effective Depletion of Alloreactive T Cells by CD95l-Transduced Cells with Preservation of FoxP3 Regulatory T-Cells.
Authors: Jacobsen Eva-Maria , Wenger Till , Herr Ingrid , Strauss Gudrun , Debatin Klaus-Michael , Friedrich Wilhelm , Muller Susanna M. 
Published: April 2020

Abstract
Adoptive transfer of donor T-cells after stem cell transplantation across HLA barriers could bridge the period of prolonged immunodeficiency until new thymic derived T-cells develop. In such an approach, however, donor T-cells reactive to alloantigens of the recipient induce GvHD, necessitating their selective elimination, inactivation or modulation. Depletion of alloreactive cells has been attempted by monoclonal antibodies, but prevention of GvHD was insufficient in clinical application so far. We developed a system allowing efficient selective depletion of alloreactive cells by CD95L-mediated apoptosis, which in addition has the advantage of preserving FoxP3 regulatory T-cells. An apoptosis-resistant B-cell line (C1RA1) was transduced with lentiviral CD95L (C1RA1-CD95L). CD95L was stably expressed by 65–80% of the cells and was fully functional to induce apoptosis in Jurkat cells. MNC of donor A were stimulated with APC of donor B, and, after 5 days of culture, viable responder cells were enriched and cultured with C1RA1-CD95L for another 3 days. T cells obtained by this culture system had lost proliferative activity towards cells of donor B, while proliferation against 3rd-party cells was fully preserved. Likewise, cytotoxic activity against donor B was markedly reduced. Since CD4+CD25+ T-cells were still detectable at levels of 7 to 10% of MNC at the end of the culture despite effective depletion of alloactivated cells, we analyzed for the presence of FoxP3+ regulatory T-cells (Treg). While gene expression of FoxP3 was almost undetectable in MNC before culture, it was present after activation of MNC and was strongly expressed after induction of apoptosis with C1RA1-CD95L at the end of our culture system. Suppression of proliferation of about 50% by these cells was noted in a suppressor assay using fresh MNC from donor A stimulated with cells of donor B. When suppression was analyzed in a stimulation system with 3rd-party stimulatory cells, reduction of proliferation was less prominent, indicating the possibility that the suppressive activity of Treg preserved in our culture system could be alloantigen-specific. In summary, we have developed a culture system, allowing alloantigen-specific depletion of T cells while preserving FoxP3+ regulatory T-cells. This sequential system of alloantigen-specific stimulation followed by CD95L-mediated apoptosis of activated cells by an unrelated, modified cell line offers the advantage of broad applicability in clinical transplantation settings.
Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.
Authors: Yoshida Shuro , Ishikawa Fumihiko , Yasukawa Masaki , Miyamoto Toshihiro , Yoshimoto Goichi , Kawano Noriaki , Shimoda Kazuya , Akashi Koichi , Shultz Leonard D. , Harada Mine 
Published: April 2020

Abstract
Transplantation of human leukemic cells into severe combined immunodeficiency (SCID) mice has been used to analyze developmental mechanisms of human leukemogenesis. Previous models, however, were limited in efficient or long-term engraftment of leukemia initiating cells. Here we report a new SCID model that supports highly efficient long-term engraftment of primary human acute myelogenous leukemia (AML) cells. We have established a novel immune-compromised mouse by backcrossing a complete null mutation of the common cytokine receptor g chain onto NOD-scid mice (NOD/SCID/IL2rgnull mice), and reported that normal human cord blood-derived hematopoietic stem cells efficiently engrafted in newborn NOD/SCID/IL2rgnull mice as compared to NOD/SCID/b2mnull mice (Ishikawa et al, Blood in press). Injection of 5x106 total bone marrow mononuclear cells from primary AML patients (FAB subtypes: M1, M2, M3, M4 and M7) into sublethally-irradiated newborn NOD/SCID/IL2rgnull mice, however, did not result in efficient engraftment of AML cells, while predominant proliferation of human CD4+ and CD8+ T cells was seen. These human T cells expressed CD45RO, and levels of human IFN-g in sera of the recipients significantly elevated, suggesting that human T cells were activated and inhibited the engraftment of human AML cells in the xenogeneic setting. We thus transplanted AML cells after T cell depletion. Strikingly, transplantation of 4x106 T cell-depleted AML bone marrow cells into neonatal NOD/SCID/IL2rgnull mice resulted in the efficient AML engraftment, whose levels were significantly higher than those in transplantation of the same number of T cell-depleted AML cells into NOD/SCID/b2mnull newborns or NOD/SCID/IL2rgnull adults. We also transplanted 103–104 hCD34+hCD38− bone marrow cells purified from AML patients. These low-doses of hCD34+hCD38− cells also successfully engrafted, progressively giving rise to hCD34+hCD38+ and hCD34− leukemic cells over 16 weeks. hCD34+hCD38− cells purified from the bone marrow of primary NOD/SCID/IL2rgnull recipients again reconstituted AML in secondary recipients, indicating that this system supports self-renewal capacity of AML stem cells within the hCD34+hCD38− fraction. Thus, the NOD/SCID/IL2rgnull newborn system provides a powerful model to study human leukemogenesis as well as the interaction between human T cells and AML cells in vivo.
Divergent Roles of Histone Deacetylases in Human Adult Eythropoiesis.
Authors: Ohishi Kohshi , Katayama Naoyuki , Yamamura Kentaro , Heike Yuji , Takaue Yoichi , Shiku Hiroshi 
Published: April 2020

Abstract
Histone deacetylases (HDACs) plays a regulatory role in the expression and activity of transcription factors. Several studies suggested that HDACs influence the proliferation and differentiation of hematopoietic progenitors. Here, we studied the role of HDACs in human adult erythropoiesis, using a HDAC inhibitor FK228 [depsipeptide]. We first examined the effect of FK228 on the growth of erythroid progenitors by incubating G-CSF-mobilized peripheral blood CD34+ cells (500 cells/well) in semisolid serum-free cultures containing IL-3, SCF, or IL-3+SCF in the presence or absence of FK228. Addition of FK228 (0.5 ng/ml) to the cultures with IL-3 and SCF+IL-3 resulted in 32-fold and 12-fold increases, respectively, in the number of erythroid colonies that consist of CD36+ glycophorin A (GPA)low/negative cells, compared with the cultures without FK228. FK228 also increased the number of cells comprising erythroid colonies in the cultures with IL-3 and SCF+IL-3. No colony growth was detected with SCF and SCF+FK228. We next examined the effect of FK228 on BFU-E colony formation by incubating CD34+ cells with EPO or SCF+EPO in the presence or absence of FK228. While BFU-E colony formation was observed with SCF+EPO but not with EPO alone, addition of FK228 to the cultures containing SCF+EPO almost completely inhibited BFU-E colony formation. To further elucidate the effect of FK228 on BFU-E progenitors that grow in an EPO-dependent fashion, CD34+ cells were cultured for 7 days with SCF, Flt3L, TPO, and IL-3, and CD36+ cells were purified. Although BFU-E colony formation was induced by incubation of the CD36+ cells with EPO, it was strongly repressed by the presence of FK228. Suspension cultures of CD36+ cells revealed that viable cell numbers in the cultures with EPO+FK228 rapidly declined from 2 days of cultures, in contrast to an increase in the number of erythroid cells in the cultures with EPO. Annexin V/propidium iodide assay showed that, upon exposure to FK228, a remarkably higher fraction of the CD36+ erythroid progenitors expressed an annexin V+PI− phenotype, indicative of an apoptotic cell death, compared with the control cultures (71 ± 8% vs 4 ± 1%). On the other hand, even though FK228 blocked the BFU-E colony formation from CD34+ cells in the presence of SCF+EPO, FK228 did not significantly increase the population of cells that undergo an apoptotic cell death in CD34+ cells. These findings suggest that HDACs, while playing an essential role in SCF/EPO- and EPO-dependent erythropoiesis, negatively regulate the action of IL-3 and IL-3+SCF on the growth of early erythroid progenitors. Our results indicate that HDACs have diverse functions in human adult erythropoiesis.
Conversion of Alloantigen-Specific CD8+ T-Cell Anergy to T-Cell Priming through In Vivo Ligation of Glucocorticoid-Induced Tumor Necrosis Factor Receptor.
Authors: Kim Hawk , Kwon Byungsuk , Kim Juyang , Choi Woon S. , Kim Hye J. , Suh Jae-Hee , Sakaguchi Shimon 
Published: April 2020

Abstract
In this study, we investigated the effect of an agonistic mAb (DTA-1) against glucocorticoid-induced tumor necrosis factor receptor (GITR) in a murine model of systemic lupus erythematosus (SLE)-like chronic graft-versus-host disease (cGVHD). A single dose of DTA-1 inhibited the production of anti-DNA IgG1 autoantibody and the development of glomerulonephritis, typical symptoms of cGVHD. DTA-1-treated mice showed clinical and pathological signs of acute GVHD (aGVHD), such as lymphopenia, loss of body weight, increase of donor cell engraftment, and intestinal damage, indicating that DTA-1 shifted cGVHD towards aGVHD. The conversion of cGVHD to aGVHD occurred because DTA-1 prevented donor CD8+ T cells from being anergic. Functionally active donor CD8+ T cells produced high levels of IFN-γ and had an elevated CTL activity against host antigens. In in vitro MLR, anergic responder CD8+ T cells were generated and DTA-1 stimulated the activation of responder CD8+ T cells that were fated to be anergic. We further confirmed in vivo that donor CD8+ T cells, but not donor CD4+ T cells, are responsible for the DTA-1-mediated conversion of cGVHD to aGVHD in a CD4+CD25+ regulatory T cell-independent manner. These results indicate that donor CD8+ T-cell anergy is a restriction factor in the development of aGVHD and that GITR stimulation prevent CD8+ T-cell anergy by activating CD8+ T cells prone to anergy. In sum, our data suggest GITR as an important costimulatory molecule regulating cGVHD versus aGVHD and as a target for therapeutic intervention in a variety of related diseases.
Toll like Receptor 4 Is Involved in In VIvo Antiphospholipid-Mediated Thrombosis and Endothelial Cell Activation.
Authors: Pierangeli Silvia S. , Vega-Ostertag Mariano E. , Raschi Elena , Liu Xiaowei , Borghi Maria O. , Meroni Pier Luigi 
Published: April 2020

Abstract
Background: Antiphospholipid antibodies (aPL) are associated with thrombosis and pregnancy loss in patients with Antiphospholipid Syndrome (APS). aPL and bacterial lipopolysaccharide (LPS) bind to and activate endothelial cells (EC) through NFκB and p38 MAPK pathways. Recent studies suggest that aPL might interact with toll-like receptor-4 (TLR-4), the receptor for LPS.
Aim: to investigate the role of TLR-4 in antiphospholipid syndrome (APS).
Methods: we examined: i) the aPL effects on thrombosis and EC activation in LPS non-responsive (LPS−/−) mice that display a spontaneous mutation of TLR-4 vs LPS-responsive (LPS+/+) mice displaying wild type TLR-4, ii) the prevalence of TLR-4 Asp299gly and Thr399Ile polymorphisms - both associated with decreased response to LPS - in 110 APS patients (with arterial and/or venous thrombosis) vs 220 controls (of same ethnic background). IgGs were purified from a patient with APS (IgG-APS) and from control serum (IgG-NHS). LPS −/− and LPS +/+ mice, in groups of nine, were treated with IgG-APS or with IgG-NHS twice intraperitoneally. Size of induced thrombi and # of leukocyte (WBC) adhering to endothelial cells in the microcirculation of endothelium of the cremaster muscle of mice (as a means to measure endothelial cell activation) were determined in vivo, seventy-two hours after the first injection. TLR-4 Asp299gly & Thr399Ile polymorphisms were evaluated by Allele-Specific PCR.
Results: LPS +/+ mice treated with IgG-APS produced significantly larger thrombi when compared to mice treated with IgG-NHS (2166 ± 1419 μm2 vs 1176 ± 841 μm2) and significantly larger number of WBC adherence to ECs (4.5 ± 1.9 vs 2.2 ±1.1). Thrombus size and number of adhering WBC to ECs were significantly lower in LPS −/− mice treated with IgG-APS compared to LPS +/+ mice treated with IgG-APS [thrombus size: 779 ± 628 μm2 vs 2166 ± 1419 μm2 (p&lt;0.05) and number of adherent WBC to EC: 1.0 ± 0.5 vs 4.5 1.9 (p&lt;0.05)], respectively. The titer of anticardiolipin antibodies in the sera of mice injected with aPL was 48.2 ± 17.1 GPL (for LPS −/− mice) and 50.8 ± 11.2 GPL (for LPS +/+ mice), respectively (NS). A significant reduction in TLR-4 Asp 299gly & Thr399Ile polymorphisms was observed in APS patients (5%) compared to controls (11.4%) (p&lt;0.05).
Conclusions: These findings strongly suggest that TLR-4 is involved in aPL interaction with endothelial cells and mediates their pathogenic effects.
A t(8;21) Fusion Protein Disrupts the Spindle Checkpoint and Promotes Aneuploidy.
Authors: Boyapati Anita , Peterson Luke F. , Yan Ming , Le Beau Michelle M. , Zhang Dong-Er 
Published: April 2020

Abstract
The 8;21 translocation occurs in approximately 8% of patients with acute myeloid leukemia. This chromosomal lesion results in the fusion of the AML1 (RUNX1) gene on chromosome 21 to the ETO (MTG8) gene on chromosome 8 and generates the AML1-ETO fusion protein. We identified a truncated form of AML1-ETO which lacks approximately 200 amino acids at the C-terminus of full length AML1-ETO and strongly induces leukemia development (Yan et al, PNAS101(49): 17186–91, 2004). This protein is almost identical to a natural splice form of AML1-ETO designated AML1-ETO9a, which is expressed in t(8;21) patients. To study the leukemogenic properties of the truncated form of AML1-ETO (AEtr), we examined cell cycle profiles to compare it to full length AML1-ETO. In contrast to full length AML1-ETO which induced growth arrest of K562 cells, AEtr did not inhibit cell proliferation. Interestingly, AEtr expressing cells continued to proliferate in the presence of microtubule toxins that normally activate the spindle checkpoint and arrest cells in prometaphase of mitosis. Evaluation of critical components of the spindle checkpoint revealed a significant decrease in BubR1 protein in AEtr cells compared to vector control cells. BubR1 is a potent inhibitor of the anaphase promoting complex and reduction of BubR1 by either siRNA approaches or gene deletion in mice results in aneuploidy. Cytogenetic analyses of leukemic splenocytes from mice transplanted with AEtr cells identified multiple chromosomal abnormalities, including chromosome loss and gain as well as translocations. Observations of chromosomal alterations in t(8;21) patients have been well documented and include loss of either sex chromosome in 70% of patients, deletions of chromosome 9, and trisomy 8. These results indicate that inactivation of the spindle checkpoint through BubR1 reduction may be an important event in the development of t(8;21) leukemias.
Inactivation of NIPA Results in Premature Mitotic Entry and Subsequent Mitotic Catastrophe.
Authors: Bassermann Florian C. , Muench Silvia , von Klitzing Christine , Morris Stephan W. , Peschel Christian , Duyster Justus 
Published: April 2020

Abstract
Ubiquitin-mediated destruction of regulatory proteins marks a vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process as they determine specificity of the ubiquitination process and thus regulate proteasomal degradation of target proteins. Recently, a class of E3 ligases referred to as SCF complexes has been identified. The substrate binding specifity within this class of E3 ligases is mediated by a class of molecules termed F-box proteins. We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that defines an oscillating ubiquitin ligase (SCF-NIPA) which targets nuclear cyclin B1 in interphase. We have now determined the consequence of inactivating NIPA with regard to cell cycle regulation using an RNAi approach. Kinetic analysis of cell cycle phase transition times revealed a premature onset of Cdk1/cyclin B1 kinase activity and early mitotic entry in cells treated with NIPA siRNA. Cyclin B1 was shown to accumulate within the nucleus in these cells correlative to a reduced ubiquitination activity of the SCF-NIPA complex. Subsequent to premature mitotic entry, NIPA inactivated cells arrested in prometaphase and mitotic catastrophe was observed thereafter. We searched for relevant proteins involved in this process and found Survivin, a member of the inhibitor of apoptosis proteins (IAP) to be strongly downregulated in NIPA siRNA treated cells. Survivin has been shown to function as a mitotic checkpoint molecule that induces mitotic catastrophe subsequent to aberant mitosis when inactivated or downregulated. While the precise functional relationship between Survivin and NIPA is still under investigation, the current data distinguishes NIPA as a central molecule in timing mitotic entry. Given this function, NIPA directly influences the fidelity of DNA replication and segregation. Interference with NIPA function may therefore be an oncogenic principle that favours genomic instability in tumor cells.
Elevation of Kinase Interacting with Stathmin (KIS) and Promotion of Cellcycle Progression by KIS in Leukemia Cells.
Authors: Nakamura Satoki , Ono Takaaki , Sugimoto Yuya , Kobayashi Miki , Sahara Naohi , Shinjo Kaori , Shigeno Kazuyuki , Ohnishi Kazunori 
Published: April 2020

Abstract
Purpose: The protein p27 is an important regulator of cell cycle. An increase in p27 causes proliferation of cells, while a decrease in p27 induces quiescence of cells. p27 is regulated by transcriptional, translational and proteolytic mechanisms. Among them, an importnant mechanism in the regulation of p27 is proteolysis. Kinase interacting with stathmin, KIS, is a serine / threonine kinase, and regulates cell cycle progression through the phosphorylation of p27 on serine 10. The S10 phosphorylation on p27 plays an important role in p27 degradation. KIS that phosphorylates p27 on S10 and its role in the regulation of cell cycle progression have not been defined in leukemia cells. In this study, we investigated the role of KIS in leukemia cells.
Materials and Methods: We examined the biological significance of KIS expression in K562, NB4, U937, CEM, MOLT4, and SUP-B15 leukemia cells and relationship with the G1 regulaters, such as p27. Moreover, we generated the lentivirus vector inserted with the dsDNA of KIS small interfering RNA (siRNA), and effects of down-regulation of KIS by siRNA transfection were investigated in leukemia cells.
Results: RT-PCR and western blot analysis showed high KIS expression in all leukemia cells. The p27 phosphorylation on S10 was significantly lower in the leukemia cells transduced with KIS siRNA and depletion of KIS enhanced growth arrest. The down-regulation of KIS induced G1 arrest in cell cycle, but not apoptosis. In cell cycle analysis, control leukemia cells showed 42.3 ± 1.8 %, but leukemia cells transfected with KIS siRNA showed 67,1 ± 2.1 % in G1 fraction. Moreover, these cells significantly showed small population in S ansd G2 fractions compared compared with controls.
Conclusion: These findings suggest KIS expression promotes cell cycle progression in leukemia cells. Depletion of KIS using siRNA in leukemia cells induced cell cycle arrest in G1 phase compared with control cells. In this study, we showed that KIS might be the target for the molecular therapy.
Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.
Authors: Wunderlich Mark , Krejci Ondrej , Wei Junping , Lu Thomas X. , Mulloy James C. 
Published: April 2020

Abstract
The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.