bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hematopoietic Defects in Cited2 Mutant Fetal Liver.
Authors: Chen Yu , Yang Yu-Chung 
Published: April 2020

Abstract
Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is a newly identified transcriptional modulator. Knockout of Cited2 gene is embryonic lethal because of heart and neural tube defects. Cited2 binds directly to CBP and p300, which have been shown to be crucial for hematopoietic stem cell self-renewal and proper hematopoietic differentiation, respectively. Cited2 also induces the expression of a polycomb-group gene, Bmi-1, which is essential for self-renewal of adult hematopoietic stem cells. These connections provided rationale to study the potential role of Cited2 in hematopoiesis. Mouse fetal liver is the major hematopoietic organ from day 10 postcoitus until right before birth. The smaller sized Cited2−/− fetal liver and significantly decreased fetal liver cellularity strongly suggest the potential defect in hematopoiesis. In vitro colony formation assay in methycellulose-based medium was used to characterize the hematopoietic progenitors. We found that fetal liver cells from E13.5, 14.5 and E15.5 Cited2−/− embryos gave rise to much less colonies, which reflects the decreased number and proliferative ability of hematopoietic progenitors due to Cited2 deficiency. Immunostaining of lineage-specific cell surface markers followed by flow cytometry was performed to characterize different hematopoietic populations in E14.5 and E15.5 fetal liver of wild type and Cited2−/− embryos. Cited2−/− fetal liver cells displayed a significant reduction in numbers throughout the hematopoietic hierarchy including hematopoietic stem cells (Lin− c-Kit+ Sca-1+), progenitor cells (Lin− c-Kit+), and differentiated cells of different lineages (CD45+, Ter119+, Mac-1+, Gr-1+), thus revealing a multi-level hematopoietic deficiency of Cited2−/− embryos. Long-term reconstitution experiment was then carried out to measure the ability of hematopoietic stem cells from Cited2−/− fetal liver cells to engraft and reconstitute hematopoietic system of congenic recipient mice. Mice transplanted with Cited2−/− fetal liver cells showed reconstitution of T cells whereas a 2-fold decrease in the reconstitution of B cell and myeloid lineages was observed, indicating a compromised ability of Cited2−/− fetal liver hematopoietic stem cells to maintain hematopoiesis. The results suggest an important role of Cited2 in hematopoietic differentiation and a selective function of Cited2 in B lymphoid &myeloid induction. The underlying mechanisms responsible for these defects will be pursued by microarray analysis of gene expression profile of Cited2−/− fetal liver cells, followed by more detailed phenotypic analyses of B and myeloid lineage markers plus in vitro and in vivo functional assays.
The Vent-Like Homeobox Gene Ventx2 Is a Novel Candidate for a Human Hematopoietic Regulatory Protein.
Authors: Arseni Natalia , Ahmed Farid , Sadlon Timothy J. , d’Andrea Richard J. , Hiddemann Wolfgang , Buske Christian , Feuring-Buske Michaela 
Published: April 2020

Abstract
Identification of the genes which are critically involved in normal and leukemic hematopoiesis is a major goal for experimental and clinical hematology. Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus it is of particular interest to examine the expression profile and function of the human homologue Ventx2 in hematopoietic development. We first analysed Ventx2 expression by RT-PCR in CD3, CD19 and CD33 cells highly purified by FACS sort from peripheral blood of healthy donors. Expression of Ventx2 was detected in T- and B- as well as differentiated myeloid cells indicating that Ventx2 is expressed in multiple hematopoietic lineages. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by RT-PCR (n=6). In an attempt to characterize the functional relevance of Ventx2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced a 3fold increase in the number of CFU-G (n=5; p&lt;0.001) compared to the GFP control (62 versus 25 CFU-G, respectively, per 1000 initially plated cells) without increasing the total number of colonies, indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays confirmed the effect of the homeobox gene with an over 9fold increase in the number of secondary CFU-G (511 vs. 54, respectively, per 1000 initially plated cells). When the effect of VENTX-2 on the frequency of LTC-IC was determined by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm (453 LTC-IC vs. 801 LTC-IC per 1x106 cells, respectively, p = n.s.). Furthermore, the number of colonies generated per LTC-IC did not significantly differ between the two arms (17 colonies for VENTX2 and 26 colonies for the control). In NOD/SCID mice VENTX2 induced a 2.9fold increase in the proportion of CD15+ mature myeloid cells within the GFP-positive compartment (n=7) compared to the control (n=9)(6.4 % vs. 2.2 %, respectively; p&lt;0.02), translating into 4 x 106 (± 1 x 106) human CD15+ /GFP+ cells per mouse in the VENTX2 group compared to 2x106 cells (± 6 x 105) in the control. These findings characterize VENTX2 as a novel regulatory protein in human hematopoiesis and add information about the role of non-clustered homeobox genes in early blood development.
Identification of Two Src Recruitment Sites in the Juxtamembrane Region of Flt3 with Opposing Effects on Flt3-Ligand-Induced Signaling.
Authors: Ronnstrand Lars , Heiss Elke , Sundberg Christina , Masson Kristina , Pedersen Malin , Sun Jianmin , Bengtsson Susanne 
Published: April 2020

Abstract
Early signal relay steps upon ligand-binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3-autophosphorylation and subsequent docking partners, are mainly unresolved. Here we demonstrate for the first time identification of ligand-induced in vivo phosphorylation sites in Flt3. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3-ligand-mediated responses in WT-Flt3, Y589F-Flt3 and Y599F-Flt3 expressing 32D cells. Compared to WT-Flt3-32D cells, 32D-Y589F-Flt3 showed upon ligand-stimulation enhanced Erk activation as well as proliferation/survival whereas 32D-Y599F-Flt3 cells displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for multiple signal relay molecules including Src family kinases. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered Src activation, impaired phosphorylation of the adapter molecules Cbl and ShcA and deficient receptor ubiquitination and degradation. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. pY599 was additionally found to interact with the protein tyrosine phosphatase Shp2. As Y599F-Flt3-32D lacked ligand-induced Shp2 phosphorylation and since silencing of Shp2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that recruitment of Shp2 to pY599 contributes to FL-mediated Erk activation and proliferation.
To summarize, our work presents novel insights in Flt3-mediated signal transduction. We have identified the in vivo autophosphorylation sites of the juxtamembrane region of Flt3, revealed Src family kinases and Shp2 as binding partners of pY589 and/or pY599, respectively, as well as their potential impact on FL-mediated signaling in Flt3-32D cells. Future work will now focus on elucidation of additional and possibly novel interaction partners of the found phosphorylation sites by employing an unbiased proteomics approach. With this gained knowledge it will be of interest to see whether ITDs differing in the nature of the duplicated tyrosines also confer distinct signaling behavior. If so, these tyrosines might serve as a diagnostic marker and point towards a successful combinatorial therapy consisting of a receptor tyrosine kinase inhibitor and an inhibitor for the specifically affected signal transduction pathway.
CrkL Mediates SDF-1-Induced Chemotaxis through Ras, Rac, and Erk Activation in Hematopoietic Cells.
Authors: Arai Ayako , Yan Weihua , Aoki Mae , Mizuchi Daisuke , Kurosu Tetsuya , Miki Tohru , Miura Osamu 
Published: April 2020

Abstract
The chemokine SDF-1 was originally identified as a growth promoting factor for pre-B cells and has been recognized to be indispensable for hematopoiesis from the findings on both its knock-out mice and those of its receptor CXCR4. SDF-1 is secreted by the bone marrow stromal cells and plays a crucial role for retention and homing of hematopoietic cells in the bone marrow through chemotaxis. In addition, SDF-1 regulates not only motility but also survival, proliferation, differentiation, and adhesion of the cells. Furthermore it was recently reported that leukemic cells show different responses to SDF-1 stimulation from normal hematopoietic cells. Despite its important roles in hematopoiesis, however, intracellular signaling pathways from CXCR4, especially those regulating chemotaxis of the hematopoietic cells, have largely remained to be clarified. We investigated the molecular mechanisms regulating chemotaxis induced by SDF-1 in hematopoietic cells and found that the enforced expression of adaptor molecule CrkL enhanced SDF-1-induced chemotaxis of murine hematopoietic cell lines, BAF3 and 32Dcl3, expressing CXCR4 on the cell surface. To examine the pathways up- and downstream of CrkL, we performed transient and stable transfection of CrkL and its mutants into BAF3 cell. Overexpression of CrkL enhanced SDF-1-induced Erk activation through Raf-1 and Mek. CrkL also enhanced SDF-1-induced activation of the Ras family GTPases Ras and Rap1 as well as the Rho family GTPase Rac. Among the dominant negative mutants of these small GTPases, those of Ras and Rac significantly suppressed CrkL-enhanced Erk activation. In addition, we also performed in vitro kinase assays using GST-CrkL as a substrate and found that SDF-1 induced phosphorylation of CrkL through the Src family tyrosine kinase Lyn. However, pretreatment with PP1, an inhibitor of Lyn, did not suppress activation of Ras, Rap1, Rac, and Erk at the concentration that suppressed the phosphorylation of CrkL. In addition, overexpression of a CrkL mutant that lacks the tyrosine targeted by Lyn did not reduce SDF-1-induced Erk activation. These results show that the phosphorylation of CrkL was not necessary for activation of the small GTPases and Erk. On the other hand, overexpression of a CrkL mutant lacking the N-terminal SH3 domain (CrkLdSH3N) required for the constitutive association with C3G, the guanine nucleotide exchange factor for Ras family GTPases, significantly reduced SDF-1-induced Erk activation. Therefore, this domain was required for the enhancement of SDF-1-induced Erk activation by CrkL through Ras. Finally we performed chemotaxis assays with transiently expressed mutants of these molecules. CrkLdSH3N as well as dominant negative forms of Ras and Rac reduced SDF-1-induced chemotaxis. Together, these results indicate that CrkL mediates SDF-1-induced chemotaxis of hematopoietic cells through activation of Ras, Rac, and the Raf/MEK/Erk pathway.
Novel Mechanism of Activated FLT3-Associated Leukemogenisis: FLT3 Regulates Wnt Signaling by Phosphorylating β-Catenin and Promoting β-Catenin Nuclear Localization.
Authors: Kajiguchi Tomohiro , Chung Eun Joo , Kiyoi Hitoshi , Naoe Tomoki , Neckers Len , Trepel Jane 
Published: April 2020

Abstract
Mutations of the gene encoding the FLT3 tyrosine kinase are the most common mutation in AML. Recently Wnt-dependent signaling has been suggested to cooperate with FLT3 in myeloid transformation. In the work presented here we have examined the mechanism of the interaction of FLT3 and the Wnt effector β-catenin in FLT3 leukemia cells. In comparison to cells with wild-type FLT3, cells with activating mutations of FLT3 including internal tandem duplication (ITD) of the juxtamembrane domain of FLT3 and the FLT3 D835Y mutation had elevated levels of tyrosine-phosphorylated β-catenin. Although β-catenin was located mainly in the cytoplasm in WT-FLT3 cell lines, it was primarily nuclear in cells with FLT-3 activating mutations. Treatment of activated FLT3 cells with the FLT3 kinase inhibitor AG1296 decreased tyrosine-phoshorylation of β-catenin and downregulated the level of nuclear β-catenin. Treatment of WT-FLT3 cell lines with FLT3 ligand increased tyrosine-phosphorylation of β-catenin and upregulated its nuclear accumulation. Immunoprecipitation experiments demonstrated that FLT3-ITD binds to β-catenin and treatment with AG1296 decreased interaction between FLT3-ITD and β-catenin. In vitro kinase assays using recombinant FLT3 and β-catenin showed that an active FLT3 kinase phosphorylates tyrosine residues of β-catenin directly and AG1296 decreased the activated FLT3-induced tyrosine-phosphorylation of β-catenin. The data demonstrate that activated FLT3 induces tyrosine phosphorylation of β-catenin and accumulation of nuclear β-catenin. We propose that increased β-catenin nuclear signaling is a potential mechanism of activated FLT-3-associated malignant transformation.
YM477, a Novel Orally-Active Thrombopoietin Receptor Agonist.
Authors: Suzuki Ken-ichi , Abe Masaki , Fukushima-Shintani Mari , Sugasawa Keizo , Hirayama Fukushi , Kawasaki Tomihisa , Hiramatsu Hidefumi , Nakahata Tatsutoshi 
Published: April 2020

Abstract
Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. The search for an orally-active nonpeptidyl small molecule TPO receptor agonist has resulted in the discovery of YM477. YM477 acted specifically on the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as TPO does. YM477, however, was shown to have high species specificity, effective in only humans and chimpanzees. Recently, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were characterized as an efficient engraftment model for human hematopoietic stem cells, as this model results in the production of human platelets. In this way, we examined the in vivo platelet-increasing effect of YM477 in human platelet-producing NOD/SCID mice in which human hematopoietic stem cells were transplanted. In this study, we used commercially available cryopreserved human fetal liver CD34+ cells as a source of human hematopoietic stem cells. The cells were transplanted into sublethally irradiated (240 cGy) NOD/SCID mice. Human platelets started to appear in peripheral blood of these mice 4 weeks after transplantation. The production of human platelets continued up to one year post-transplant. Various doses of YM477 (0, 0.3, 1, and 3 mg/kg/day) were orally administered for 14 days to NOD/SCID mice that had been confirmed to produce human platelets stably. Oral administration of YM477 dose-dependently increased the number of human platelets produced by these mice, with significance at 1 mg/kg/day and above. The increase in the human platelet count reached about 2.7-fold at 1 mg/kg/day and about 3.0-fold at 3 mg/kg/day on day 14. Withdrawal of YM477 administration caused the human platelet count to return to the pretreatment level. The number of murine platelets did not change during the study period. Next, to evaluate the function of human platelets produced in peripheral blood of these mice, the expression of activation-dependent marker CD62P (P-selectin) on human platelets stimulated with thrombin receptor agonist peptide (TRAP) were examined. CD62P expression on human platelets was induced by the stimulation of blood from transplanted mice with TRAP, suggesting that human platelets produced in NOD/SCID mice were functional. Furthermore, the maximum response of CD62P expression on human platelets induced by TRAP was evaluated before and after administration of YM477 at 3 mg/kg/day for 14 days. CD62P expression was not changed by administration of YM477, which was similar to the results obtained with a vehicle group. These results suggest that YM477 is an orally active TPO receptor agonist useful for treating patients with thrombocytopenia.
Expression of the Human Germinal Center Associated Lymphoma (HGAL) Protein Identifies a Subset of Classical Hodgkin Lymphoma of Germinal Center Derivation and Improved Outcome.
Authors: Natkunam Yasodha , His Eric D. , Zhao Shuchun , Elson Paul , Pohlman Brad , Levy Ronald , Lossos Izidore S. 
Published: April 2020

Abstract
Classical Hodgkin lymphoma (CHL) exhibit crippled somatic mutations in rearranged Ig genes and an immunophenotype unlike any other hematolymphoid malignancy. Expression profiles of CHL cell lines show that CHL is most closely related to EBV-transformed peripheral blood B-cells with marked loss of B lineage-specific gene expression. The Human Germinal center-Associated Lymphoma (HGAL) gene was identified as a candidate molecule that predicted improved outcome in diffuse large B-cell lymphoma. Previously, we showed that HGAL mRNA and protein are expressed specifically in germinal centers (GC), GC-derived lymphomas and a substantial proportion of CHL. Here, we sought to investigate whether HGAL protein expression could serve to distinguish biologically distinct subgroups of CHL. Ninety informative cases of CHL on a tissue microarray of 1.0 mm representative cores of paraffin embedded tissue were used. Staining was performed and the percent of stained Hodgkin cells was scored on a 3-tiered scale as negative (0%), weak (&lt;30%), and strong (&gt;30%), as previously described (Natkunam et al. Blood 2005, 105:3979–86). These cutoffs were chosen before results were correlated with clinical endpoints. Twenty-five patients (28%) showed no staining for HGAL and 25 patients (28%) had weak staining; HGAL was strongly expressed in 40 patients (44%). Thirteen of the 90 patients had died and 19 had failed (i.e. relapsed or died). The overall 5-year survival (OS) and failure-free survival (FFS) rates were 87% + 4% and 79% + 4%, respectively. HGAL expression was associated with improved OS in univariate analysis (p=0.04, no vs weak/strong staining), as were age &lt; 45 years (p&lt;0.001), stage I or II (p=0.02), and possibly histology (nodular sclerosing vs other, p=0.09). However, in multivariate analysis with age, stage and histology, HGAL expression no longer had an impact (p=0.93). HGAL did not impact FFS in either univariate (p=0.13) or multivariate analysis (p=0.87). The expression of the GC-specific marker HGAL in a subset of CHL suggests that a substantial proportion of CHL retain characteristics of lymphomas of GC derivation. The association with improved OS in univariate but not multivariate analysis suggests HGAL may be a biologic marker related to known clinical parameters of improved OS and FFS. Comparative studies with well-characterized GC markers, BCL6 and CD10, and with BCL2, MUM1 and Blimp-1 are underway to further define biologically distinct subsets of CHL. Analysis of additional cases of CHL is also on-going to substantiate these findings in multivariate analysis.
Potential Role of Immunosuppression in Therapeutic Effects Conferred by Cord Blood- Derived Mesenchymal Stem Cells.
Authors: El-Badri (Dajani) Nagwa S. , English Denis , Hakki Amal , Mudhusoodanan Sriram , Sanberg Cyndy D. , Sanberg Paul R. 
Published: April 2020

Abstract
Our previous studies consistently demonstrate enhanced neural protective effects of cord blood in comparison to stem cells from adult marrow. Similarly, cord blood possesses diminished immuno-stimulatory activity, and the basis of this effect has not been defined. Since neural damage may be potentiated by immune activation of inflammatory cascades, we examined the effects of cord blood-derived MSCs (mesenchymal stem cells) on immune responses. We isolated and characterized a population of cord blood MSCs. These cells maintained their pluripotency in culture. Progeny generated in the absence of differentiation were strongly adherent, did not express CD34, CD45, CD3, CD19 antigens, and did not generate hematopoietic colonies in methylcellulose. However, cultured cord blood MSCs possessed a remarkable ability to support the proliferation as well as the differentiation of hematopoietic cells in vitro. In addition, supernatants from cultured cord blood MSCs promoted survival of peripheral blood mononuclear cells cultured under conditions designed to induce cell stress and limit protein synthesis. We examined immune modulation by cord blood MSCs after co-culture with murine splenocytes. While spleen cells from normal C57Bl/6 mice exhibited a prominent IgM response after immunization with the T-cell dependent antigen, SRBCs, this response was significantly decreased after incubation with cord blood MSCs. Consistently, cord blood MSCs mitigated the enhanced mixed lymphocyte proliferative response C57Bl/6 T-cells exhibit when exposed to lymphocytes from non-related animals. To investigate whether these immune suppressive properties could be therapeutically useful in a transplantation model for autoimmune disease, cord blood MSCs were transplanted into sublethally irradiated BXSB mouse model for systemic lupus. Thirty days after infusion, defective IgM humoral immune responses of splenocytes of these mice normalized. This normalization paralleled normalization of the disrupted lymphoid cellularity observed in the spleens of diseased animals. Our results are consistent with the hypothesis that immune regulation is involved in the therapeutic utility of MSCs.