bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Transgenic Mouse Model for Congenital Dyserythropoietic Anemia Type I
Authors: Renella Raffaele , Roberts Nigel A , Sharpe Jacqueline A , Li Pik-Shan , Brown Jill M , Babbs Christian , Fugazza Cristina , Goardon Nicolas , Higgs Douglas R , Wood William G 
Published: October 2019

Abstract
The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown.
We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis.
These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.
High Levels of Growth Differentiation Factor 15 in Patients with Congenital Dyserythtopoietic Anemia Type I
Authors: Tamary Hannah , Shalev Hanna , Avraham Galit , Zoldan Meira , Levi Itai , Swinkels Dorine W. , Tanno Toshihiko , Miller Jeffery L 
Published: October 2019

Abstract
Growth differentiation factor 15 (GDF15) is a member of the TGF-beta superfamily of cytokines previously found to suppress hepcidin in primary human hepatocytes. GDF15 is secreted from human erythroblasts, and extremely high serum levels are present in β-thalassemia patients (Tanno et al., Nat. Med. 2007, 13, 1096–1101). To determine if elevated GDF15 levels are unique for thalassemia or more generally associated with iron-loading related to ineffective erythropoiesis, we determined the GDF15 levels, as well as, serum hepcidin (Swinkels DW et al, PLoS ONE PLoS ONE. 2008;3:e2706), ferritin, erythropoietin (EPO) and soluble transferrin receptor (sTfR) in patients with the congenital dyserythropoietic anemia type I (CDA I). Seventeen Israeli Bedouins with CDA I were studied, all homozygous for the founder R1040W mutation in the CDAN1 gene. All of the patients studied were young adults with a mean age of 29 years. Two patients previously underwent splenectomy, and one patient is currently transfusion-dependent. For comparison, ten healthy volunteers (HV) were studied. The mean level of GDF15 in CDA I patients was significantly elevated [10,239 ± 3,049 pg/ml (range 5,530–17,008) compared to 269 ± 238 pg/ml in healthy controls; p = 1.5×10−10]. Consistent with a previous study of dyserythropoietic anemia patients, significantly higher levels of soluble transferrin receptor were detected among the CDA I population (sTfR; CDA I, 86.4 ± 14.0 nmol/L; HV, 21.4 ± 6.2 nmol/L, p = 7.4×10−15). Serum EPO levels were also elevated (EPO; CDA I, 118 ± 59 IU/dL; HV, 2.0 ± 1.5 IU/dL, p = 2.3×10−7). For iron analyses, three patients with extensive transfusion histories were excluded. Among the remaining 14 patients, iron overload was demonstrated by elevated serum ferritin (CDA I, 916 ± 507 ng/ml; HV, 72 ± 60 ng/ml, p = 1.4×10−5). Despite the significant elevation in iron stores, significantly elevated levels of hepcidin 25 (Hep25) were not detected in the CDA I patients. Instead, a minor decrease in serum Hep25 levels were detected (Hep25; CDA I, 3.3 ± 2.8 nM; HV, 4.1 ± 3.0 nM, p = 0.27). Correlation analyses were performed between the iron parameters (Ferritin and Hep25) and GDF15, sTfR, or EPO levels. Only GDF15 demonstrated a significant positive correlation with ferritin and significant inverse correlations with Hep25 and the Hep25/Ferritin ratio. Weaker correlations with EPO were identified. Unexpectedly, the correlation trends for sTfR were opposite those of GDF15 in this group. These results demonstrate that GDF15 is immensely over-expressed in CDA I, and further suggest this cytokine contributes to hepcidin dysregulation and secondary hemochromatosis in humans with ineffective erythropoiesis.
Hsp90 regulates Ulk1-mediated autophagic clearance of mitochondria
Authors: Kundu Mondira , Yang Chia-Ying , McCastlain Kelly , Wu Junmin , Zhang Ji , Lindsten Tullia , Ney Paul , Thompson Craig B. 
Published: October 2019

Abstract
Recent studies have suggested that the serine-threonine kinase Ulk1, a mammalian homologue of the yeast autophagy regulator, Atg1, plays an important role in mitochondrial clearance during reticulocyte maturation. In order to gain insight into the regulation and activity of Ulk1, we used an unbiased proteomics approach to identify Ulk1-interacting proteins. Here, we demonstrate that Ulk1 interacts with heat shock protein 90 (Hsp90) and Cdc37, and is among a growing list of proteins whose steady state levels are regulated by this kinase-specific chaperone complex. Treatment of murine embryonic fibroblasts (MEFs) with the HSP90 inhibitor, 17AAG, inhibits the AKT/Tor pathway and induces autophagy despite the decreased steady state levels of Ulk1. By contrast, Ulk1-deficient MEFs are defective in autophagy induction, as measured by LC3 puncta formation (but not LC3 conversion) and ultrastructural analysis. Exposure of terminally differentiating erythroid cells to HSP90 inhibitors prevents the normal accumulation of Ulk1 protein and results in impaired autophagic clearance of organelles, similar to the effect of Ulk1 gene deletion. These findings highlight the importance of Ulk1 protein levels in terminal erythroid maturation and induction of autophagy and its regulation by the Hsp90/Cdc37 chaperone complex.
Disruption of Gamma Interferon Signaling through the STAT1 Pathway Enhances Alloreactivity While Abrogating GvHD
Authors: Capitini Christian M. , Larabee Shannon M. , Mackall Crystal L. , Fry Terry J. 
Published: October 2019

Abstract
BACKGROUND: Strategies that allow for sufficient alloreactivity to mediate graft-versus-tumor (GVT) effects after allogeneic bone marrow transplant (BMT) while minimizing graft-versus-host-disease (GVHD) remain elusive. Historically, gamma interferon (IFNγ) has been implicated as a critical mediator of both GVT and GVHD pathophysiology, and high levels of IFNγ have been implicated in immunosuppression. Given that immunosuppression is also a hallmark of GVHD, we hypothesized that modulation of IFNγ signaling could have important effects on both GVHD and GVT following BMT. We therefore compared immunocompetence to tumor challenge and tumor vaccination, in the presence or absence of IFNγ receptor (R1) and STAT1 signaling (a primary downstream mediator of IFNγ signaling) on donor bone marrow.
METHODS: We utilized a clinically relevant, minor histocompatibility antigen-mismatched, T cell-depleted BMT model, with delayed donor lymphocyte infusions (DLIs) into thymectomized mice to ensure that T cell recovery occurred exclusively from the DLI, as occurs in most clinical settings during the first 6 months post-BMT. Lethally irradiated female C57BL/6 (B6) × C3H.SW recipients were engrafted with normal B6, B6 IFNγR1-deficient, normal B6129, or B6129 STAT1-deficient bone marrow on day 0 and reconstituted with donor T cells on days 14 and 28 at a dose that induced sublethal GVHD, evidenced by weight loss, histology, and abrogated quantitative immune responses to an HY-directed tumor vaccine. Recipients were then challenged on day 42 with an HY-expressing tumor and followed for survival. Immune reconstitution was assessed by flow cytometry both prior to and after DLI administration.
RESULTS: Mild GVHD prevents vaccine-mediated immune responses and HY-expressing tumor protection in thymectomized recipients. Both IFNγR1 and STAT1-deficient marrow abrogate GVHD. STAT1-deficient marrow increased absolute numbers of splenic plasmacytoid dendritic cells (CD11cint/Class II+/Gr-1+/B220+) prior to DLI administration. In addition, an increase in the percentage and absolute number of splenic regulatory T cells was observed at the time of tumor challenge. Surprisingly, despite the immunosuppressive milieu, recipients of IFNγR1-deficient marrow had enhanced vaccine-mediated tumor protection as measured by overall survival, similar to syngeneic BMT controls.
CONCLUSION: IFNγ signaling is a critical mediator of GVHD pathophysiology, including the immunosuppression associated with this condition. Disrupting IFNγ signaling through the JAK1/STAT1 pathway is a novel strategy to selectively modulate alloreactivity, and results in enhanced anti-tumor immune responses following allogeneic BMT without exacerbating GVHD.
Active Immune Gene Therapy Using ISF35: Responses Associated with Priming for Death Receptor-Induced Apoptosis and Sensitivity to Fludarabine in Patients with CLL and Del 17p
Authors: Wierda William G , Castro Januario , Aguillon Robier A , Sampath Deepa , Jalayer Annette , Prussak Charles , Keating Michael J , Kipps Thomas J 
Published: October 2019

Abstract
CLL cells can be transduced with adenovirus vector to express CD40-ligand (CD154). Ad-ISF35 is the Ad-vector that encodes a novel molecule capable of ligating CD40 on CLL cells. We conducted a phase I clinical trial to evaluate tolerability, toxicities, and clinical activity of a single infusion of autologous ISF35-transduced CLL cells. Doses of 1×108, 3×108, and 1×109 transduced cells were administered. Three patients (pts) were treated at each dose level. All pts had active disease with an NCI-WG ‘96 indication for treatment, 2 were previously untreated and 7 were previously treated. Four of the previously treated pts had del 17p by FISH prior to ISF35. Pts experienced Grade 1–2 flu-like symptoms that resolved within 2–3 days. Infusions were associated with reduction in leukemia counts and lymph node size at all dose levels; there was no dose-response. These responses were associated with in vivo apoptosis of CLL cells demonstrated by Annexin V staining, including cells collected form pts with del 17p. In vivo phenotypic and biochemical changes occurred in bystander leukemia cells of treated pts, again including cells collected from pts with del 17p. These changes included increased expression of CD95 (FAS) and death receptor 5 (DR5). A notable biochemical change included de novo expression of bid, including in cells from pts with del 17p, which lasted several weeks. Bid links the intrinsic and extrinsic apoptosis pathways and facilitates death-receptor-induced killing. Leukemia cell sensitivity to purine analogue-based therapy can be increased following expression of p73 in CLL cells with del 17p or mutated TP53. We demonstrated that ISF35 induced in vivo expression of p73 following infusion of transduced cells. Treatment of patients with del 17p with purine-analogue based regimens resulted in responses, including complete remissions. Increases in T cell counts were also observed at all dose levels; there was no dose-response relationship. T-cell increases peaked at 1–4 wks post-infusion. These results demonstrate that Ad-ISF35-transduced autologous leukemia cells can be given safely up to 1×109 transduced cells, without dose limiting toxicities, and resulting in phenotypic and biochemical changes in bystander cells in vivo that render them able to present antigen and primes them for death-receptor induced apoptosis.
Generating of Human CD4+ and CD8+ T Lymphocytes toward NY-ESO-1 by T Cell Receptor (TCR) Modification and Transduction for Adoptive TCR Gene-Transfer
Authors: Krönig Holger , Hofer Kathrin , Sommermeyer Daniel , Peschel Christian , Uckert Wolfgang , Bernhard Helga 
Published: October 2019

Abstract
The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.
Efficient Gene Marking with a Clinical Gammaretrovirus Vector Expressing MGMTP140K in Baboons and Macaques
Authors: Beard Brian C , Welsh Megan L , Trobridge Grant D , Kiem Hans-Peter 
Published: October 2019

Abstract
Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.
MicroRNA Expression in Macrophages-Derived Microvesicles May Contribute to Cellular Survival and Differentiation
Authors: Ismail Noura , Marsh Clay B. , Hunter Melissa 
Published: October 2019

Abstract
Microvesicles (MV) (also know as exosomes) are small membrane-bound vesicles released by numerous cell types that contain proteins, mRNA and microRNA. We found that MV from activated monocytes drove survival and differentiation in naïve cells. We therefore were interested in understanding the content of MV produced by activated mononuclear phagocytes. Purified peripheral blood monocytes were treated in vitro for 24 h with or without the monocyte survival factors, GM-CSF or M-CSF, respectively. Examination of monocytes and macrophages by electron microscopy or culture supernatants by flow cytometry demonstrated that monocytes produced MV, which quantitatively increased upon differentiation. Treatment with GM-CSF resulted in more MV production than M-CSF-treated monocytes. To examine whether MV from differentiated cells induced myeloid maturation, the MV were collected and added to fresh monocytes; only MV derived from GM-CSF treated cells induced differentiation of naïve monocytes into macrophages. We next hypothesized that expression of microRNA contained in the MV modulated differentiation of monocytes. Profiling of MV from GM-CSF and M-CSF derived macrophages revealed only two significantly expressed microRNAs. We found that mir-155 was significantly elevated by two-fold in MV from GM-CSF-treated cells, while mir-340 was significantly increased seven-fold in M-CSF-derived MV. Notably, mir- 223 was the highest expressed microRNA in MV from both GM-CSF and M-CSF-treated cells. Recent data suggest that expression of mir-223 regulates myeloid, granulocytic and osteoclasts differentiation, and has a role in hematopoietic stem cell proliferation. While mir-223 is present in MV from both GM-CSF and M-CSF treated cells, it is possible that the low abundance of MV produce from M-CSF-treated cells resulted in less effective concentration to induce differentiation. In this model, it is also possible that regulation of proteins targeted by the increase in mir-155 and decrease mir-340 in the GM-CSF-derived MV are responsible for myeloid differentiation. Since changes in microRNA expression including mir-223 has been reported in AML, our data suggest that myeloid-derived MV in the peripheral blood containing mir-223 may be altered contributing to leukemogenesis.