bluuebunny/crossref_metadata_2025_split huggingface dataset:
Iron Does Not “Jiggle Free” in Mitochondria: Is Mitoferrin the Only Answer?.
Authors: Paw Barry H. 
Published: October 2019

The developing erythron requires tremendous amounts of iron (Fe) for the synthesis of heme for hemoproteins, such as hemoglobin, and iron-sulfur (Fe/S) clusters of proteins, which are required to catalyze redox reactions and regulate Fe uptake and storage. The uptake of Fe from transferrin (Tf) involves the binding of Tf to its cognate receptor (TfR), followed by the endocytosis of the Tf-TfR complex.1,2 In the late endosome, the release of Fe3+ from TfR is achieved by acidification of the vesicle by the v-ATPase H+-pump. Steap3 reduces the liberated Fe3+ prior to its transport out of the endosome by the DMT1 transporter. In contrast to previous conventional models for a cytosolic intermediate state, new data have emerged showing the direct interorganellar transfer of Fe from the endosome to the mitochondria.3 Although it is assumed that the exported Fe is targeted to the mitochondria for eventual incorporation into heme and Fe/S clusters, our understanding of the precise mechanism of how Fe traverses the outer and inner mitochondrial membranes remains poorly understood. Work in yeast mutants have implicated the role of solute carriers, Mrs3/4p, in mitochondrial iron homeostasis and revealed that it is the reduced form of iron, Fe2+, that is imported into the mitochondria. Subsequent studies of the zebrafish mutant, frascati, led to the discovery of mitoferrin 1 (Mfrn1, slc25a37), the vertebrate ortholog of Mrs3/4, as the major iron importer across the inner mitochondrial membrane in developing erythroblasts.4 A structurally related paralog, mitoferrin 2 (Mfrn2, slc25a28), plays the analogous role of Fe importer in non-erythroid cells. Loss-of-function studies of Mfrn1 in the mouse have confirmed its requirement in mammalian primitive and definitive erythropoiesis and its essential role in heme and Fe/S biosynthesis. Several questions remain unanswered in mitochondrial Fe metabolism:
Do the two Mfrn importers account for all Fe imported into the mitochondria? How does Fe get across the outer mitochondrial membrane to reach the Mfrn importers? How does the translocated Fe in the matrix ultimately reach ferrochelatase to form heme? How is Fe translocated across the inner mitochondrial membrane?
Inducing the Tryptophan Catabolic Pathway, Indoleamine 2,3-Dioxygenase (IDO), for Suppression of Graft-Versus-Host Disease (GVHD) Lethality.
Authors: Jasperson Lisa , Bucher Christoph , Panoskaltsis-Mortari Angela , Mellor Andrew , Munn David H. , Blazar Bruce R. 
Published: October 2019

Abstract
Abstract 3547
Poster Board III-484
During graft-versus-host disease (GVHD), donor T cells become activated and migrate to tissue sites. Previously, we demonstrated a crucial role for the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in GVHD regulation. Here, we show that upon arrival in the colon, activated donor T cells produced IFN-γ that upregulated IDO, causing T cell anergy and apoptosis. IDO induces GCN2 kinase, upregulating a T cell stress response implicated in IDO immunosuppression. Donor T cells did not require GCN2 kinase to respond to IDO, suggesting toxic IDO metabolites, and not tryptophan depletion, were responsible for suppression. When exogenous metabolites were administered, GVHD lethality was reduced. To determine if IDO could be induced pre-transplantation for enhanced GVHD suppression, we first determined whether antigen presenting cells (APCs) or epithelial cells were primarily responsible for IDO expression and subsequent GVHD suppression. Recipients with wild-type vs. IDO−/− APCs had increased survival, regardless of epithelial cell expression of IDO, suggesting that APCs were suitable targets for inducing IDO. Administration of an agonist to toll-like receptor (TLR)-7/8, a receptor expressed primarily on APCs, induced IDO and reduced injury in the colon, and ameliorated lethality. We conclude that IDO upregulation may have therapeutic potential for preventing GVHD in the clinic.

Disclosures:
Mellor: NewLink Genetics, Inc.: Consultancy, Intellectual property interests in the therapeutic use of IDO and IDO inhibitors and receive consulting income and research support from NewLink Genetics, Inc., Research Funding. Munn:NewLink Genetics, Inc: Consultancy, Intellectual property interests in the therapeutic use of IDO and IDO inhibitors and receive consulting income and research support from NewLink Genetics, Inc, Patents & Royalties, Research Funding.
Tissue Damage and Lymphoma Derived Chemoattractive Signals Impact Regulatory T Cell Trafficking After Allogeneic Hematopoietic Cell Transplantation.
Authors: Dürr Christoph , Pfeifer Dietmar , Schmitt-Gräff Annette , Claus Rainer , Gerlach Ulrike , Graeser Ralph , Gerbitz Armin , Negrin Robert , Finke Jürgen , Zeiser Robert 
Published: October 2019

Abstract
Abstract 3564
Poster Board III-501
Regulatory T cells (Treg) have been shown previously to reduce graft-versus-host disease (GvHD) but allow for graft-versus-leukemia (GvL) effects. It is still unclear why the adoptive Treg transfer does not paralyze every immune response to the same extent. In that context Treg recruitment and localization may account for differential immune regulation. Therefore, we studied the impact of lymphoma and inflammation derived chemoattractive signals on Treg recruitment. Luciferase transgenic Treg accumulated in B cell lymphoma (BCL) tissue after allogeneic hematopoietic cell transplantation (alloHCT) when no conventional T cells were given. Microarray based analysis of the BCL tissue revealed increased expression of the CXC chemokine ligands (CXCL) 9, 10, 12 and CCL22. In vivo antibody based neutralization or interference with receptor signaling identified CCL22 and CXCL12 as critical chemokines for Treg attraction towards BCL tissue after alloHCT. Conversely, the CXCL9/10-CXCR3 axis was not critical for Treg recruitment. Depletion of antigen presenting cells (APC) from BCL tissue abrogated CCL22/CXCL12 secretion. CD11c-DTR recipients displayed reduced Treg recruitment towards BCL, indicative for a major role of host APC in the process of Treg recruitment. Strong local inflammatory stimuli caused by subcutaneous complete Freund's adjuvant injection caused local Treg accumulation and reduced recruitment to the BCL. Infusion of conventional T cells that caused subacute GvHD related inflammation redirected Treg towards secondary lymphoid organs and abdominal GvHD target organs.
In conclusion our study demonstrates the following novel findings: First Treg are recruited towards BCL infiltrated tissues after alloHCT and this process is dependent on CCL22 and CXCL12 production. Secondly, BCL infiltrating host type APC are the major source for CCL22 and CXCL12 and their targeted deletion impacts Treg recruitment towards BCL tissue. Thirdly, strong local or systemic inflammation can redirect Treg trafficking in the presence of BCL and affect the accumulation of this cell population at the tumor site which may explain why Treg preferentially regulate GvHD reactions post allografting.

Disclosures:
No relevant conflicts of interest to declare.
Update On the Risk of Leukemia in Genetic Subgroups of Congenital Neutropenia (CN): Comparison of Patients with Known Gene Mutations (ELA2, HAX1, WASP, G6PC3, p14).
Authors: Zeidler Cornelia , Donadieu Jean , Bolyard Audrey Anna , Vandenberghe Peter , Pracht Gusal , Beaupain Blandine , Hoy Ludwig , Zimmermann Martin , Bellanne-Chantelot Christine , Link Daniel C , Klein Christoph , Germeshausen Manuela , Dale David C. , Welte Karl 
Published: October 2019

Abstract
Abstract 3597
Poster Board III-534
An increased risk for malignant transformation (MDS or leukemia) is well documented in patients with congenital neutropenia (CN). In this study we assessed the incidence of leukemic transformation and potential risk factors for leukemic transformation in CN patients with known gene mutations, e.g. ELA2, HAX1, G6PC3, p14, WAS or no identified mutation, respectively, by combining all available data from the European and US Branches of the Severe Chronic Neutropenia Registry (SCNIR) and the French Neutropenia Registry (FR).
Data from mutational analysis were available for 407 patients. Mutations were identified in 259 CN patients, of whom 209 patients revealed ELA2 mutations, 20 HAX1 mutations, 18 WAS, 8 G6PC3 and 4 p14 mutations. In addition, in 57 patients neither ELA2 nor HAX 1 mutation were detectable and in another 91 patients ELA2 mutations could be excluded, but further genetic evaluation is not yet completed.
Secondary malignancies occurred in 50 of the 407 CN patients. The distribution by genetic subtype is shown in the table below:
CN subtype by gene mutation Total patient number (n) MDS/Leukemia (n/%) ELA2-CN 209 31 (14. 8%) HAX1-CN 20 4 (20%) ELA2 neg + HAX1 neg 57 6 (10.5%) ELA2 neg/ HAX1 not tested 91 5 (5.5%) WAS 18 4 (22.2%) G6PC3 8 0 p14 4 0 Total 407 50 (12.3%)
All subgroups benefit from G-CSF treatment. Median G-CSF maintenance doses required during the years prior to leukemic transformation compared by genetic subtype is shown in the following table 2:
CN subtype by gene mutation Without leukemia (n) Median G-CSF dose (μg/kg/d) With leukemia (n) Median G-CSF dose (μg/kg/d) ELA2-CN 75 5.0 15 15 HAX1-CN 15 5.0 4 13.4 ELA2-/HAX1- 36 7.2 4 10.5 WAS 15 2.6 4 3.0 G6PC3 8 3.9 0 na p14 4 5.1 0 na

Conclusion
Patients with severe congenital neutropenia who have mutations in ELA2, HAX1, or WAS and also those with no recognized mutation are at risk of secondary leukemia. So far, progression to MDS leukemia has not yet been described in the small number G6PC3 or p14 CN cases in our database. ELA2-CN or HAX1-CN patients requiring higher doses of G-CSF are at greater risk. Mutational analysis is helpful to identify the genetic cause of severe congenital neutropenia but does not serve to identify patients at risk of leukemic transformation.

Disclosures:
No relevant conflicts of interest to declare.
Expression Cloning of Genes Enabling Erythropoietin -Independent Erythropoiesis in Vitro.
Authors: Yokoo Tomoko , Kurita Ryo , Takahashi Atsushi , Okada Michiyo , Kawano Hirotaka , Miura Yoshie , Hiramoto Takafumi , Inoue Hiroyuki , Suehiro Youko , Sugiyama Daisuke , Lu Hua , Yoshimura Akihiko , Komatsu Norio , Tani Kenzaburo 
Published: October 2019

Abstract
Abstract 3608
Poster Board III-544
Erythropoietin (EPO) is essential during both ontogeny and the course of erythropoiesis. In primitive (yolk sac) erythropoiesis, however, the role of EPO is not fully understood. Elucidation of such role in primitive erythropoiesis would be very helpful for the development of ex vivo red blood cell expansion system from embryonic stem (ES) cells. Recently, we reported the establishment of the ex vivo induction of hematopoietic stem cells from common marmoset ES cells using lentiviral-Tal1/Scl gene transfer in the absence of any stromal cells (Stem Cells 24: 2014-2022, 2006). This method should also be applicable to both of human ES cells and induced pluripotential stem (iPS) cells, but the efficiency will be very critical for its clinical application. In the present study, we proceed further to find out unknown factors which accelerate ex vivo proliferation and differentiation of erythroid cells, constructed the human fetal liver cDNA expression lentiviral library (Mol Cell Biochem 319: 181-187, 2008) and screened for cDNAs which confer EPO independency to an EPO-dependent cell line, UT-7/Epo ( Blood 82: 456-464, 1993). Among twenty-two candidate genes cloned after screening of 6×10∧5 cDNA, we particularly focused on two full-length genes, ribosomal protein L11 (RPL11) and retinol dehydrogenase 11 (RDH11). Two candidate gene-transduced-UT-7/Epo cells, respectively named LV-RPL11 and LV-RDH11, showed complete EPO-independent survival and proliferation, increased expression of fetal γ-globin, and decreased expression of adult β-globin compared with parental UT-7/Epo cells in the presence of EPO. Cell cycle and apoptosis analyses showed decreased apoptotic cell death and increased S/G2/M cells in LV-RPL11 and LV-RDH11 cells compared with UT-7/Epo cells in the absence of Epo. Moreover, STAT5 phosphorylation and upregulation of its target genes, c-Myc, cyclin D and Pim, were observed in LV-RPL11, LV-RDH11 cells in the absence of EPO. In conclusion, the findings suggest that RPL11 and RDH11 play a role in EPO-independent erythropoiesis and might be applicable to ex vivo expansion of red blood cells from ES/iPS cells.

Disclosures:
No relevant conflicts of interest to declare.
Mesenchymal Stem Cells Inhibit Th17 Differentiation through PGE2 Production.
Authors: Tatara Raine , Ozaki Katsutoshi , Oh Lekuni , Hatanaka Keiko , Meguro Akiko , Matsu Haruko , Sato Kazuya , Nagai Tadashi , Muroi Kazuo , Ozawa Keiya 
Published: October 2019

Abstract
Abstract 3633
Poster Board III-569
Mesenchymal stem cells (MSCs) possess an immunomodulatory function and show promise as a cell therapy for graft-versus-host disease (GVHD). In a phase II study in Europe, injections of MSCs caused 60-70% overall response rate, with longer survival of complete responder. In contrast to its clinical efficacy, the molecular mechanism(s) underlying immunomodulation by MSCs has not been fully established. Prostaglandin E2 (PGE2), tumor growth factor-b1 (TGF-b1), and indoleamine-2,3-dioxygenase have been reported to mediate the immunomodulatory function of MSCs, and we reported evidence that nitric oxide is also a mediator (Blood 2007, 109, 228). Th17 is a recently recognized differentiation category, in which CD4 cells produce IL-17. It has been reported that Th17 is crucial for experimental autoimmune encephalomyelitis (a model of the human disease, multiple sclerosis) and is also thought to be important in other autoimmune diseases. Regulatory T cells (Treg) are another newly recognized differentiation category, in which CD4 T cells have high levels of Foxp3 expression and suppress T cell proliferation. It has been reported that Th17 and Treg can be induced by incubation with TGF-b1 and IL-6 or IL-21, and TGF-b1 and IL-2, respectively, and that these two differentiations are in a reciprocal relationship. Whereas the role of Th17 in GVHD is still controversial, Treg has been reported to suppress GVHD in a mouse model.
To elucidate the molecular mechanism(s) of the immunomodulatory function of MSCs, we herein sought to identify the effects of MSCs on these relatively new differentiations. MSCs inhibit Th17 differentiation even in conditions in which growth is not completely inhibited. Interestingly, an inhibitor of prostaglandin production, indomethacin, and an inhibitor of indoleamine 2,3-dioxygenase, 1-methyltryptophan, partially restore Th17 differentiation, whereas inhibitors of nitric oxide synthase do not. These results suggest that PGE2 and depletion of tryptophan, but not nitric oxide, mediate inhibitory effects of MSCs on Th17. Additionally, we found that MSCs produced PGE2 when co-cultured with CD4 T cells in Th17 differentiation condition and PGE2 per se suppresses Th17 differentiation. Thus, our results suggest that MSCs block Th17 differentiation through PGE2 prodction. In contrast to Th17 differentiation, Treg differentiation was not significantly inhibited by MSCs. However, MSCs still inhibited proliferation of T cells under these conditions, and T cell proliferation was restored by the addition of indomethacin. These results suggest that MSCs inhibit proliferation but not Treg differentiation through PGE2 production. The mechanism by which PGE2 differentially regulates these differentiations is unknown and remains an area for further investigation.

Disclosures:
Ozawa: Alexion: Research Funding.
Role of STAT3 in Immunoregulatory Function of Human Bone Marrow-Derived Mesenchymal Stem Cells.
Authors: Yoon Jinsun , Kim Seoju , Kim Eun Shil , Kim Byoung Kook , Lee Young 
Published: October 2019

Abstract
Abstract 3637
Poster Board III-573
The one of the best curative treatment modality in hematologic malignancies is an allogeneic hematopoietic stem cell transplantation (HSCT). However, graft-versus-host disease (GVHD) is a major obstacle of allogeneic HSCT. BM derived human MSCs are known to have immunoregulatory effect in vitro and in vivo via inhibiting alloreactive T lymphocytes, leading to their clinical use for the prevention of GVHD in HSCT. However, the molecular mechanism of immunoregulatory effect of human MSCs is not fully understood. In this study, the signaling of immunoregulatory effect was investigated by co-culture of human MSCs with lymphocytes. The proliferation of allogeneic T cells was inhibited by MSCs. Among the STATs, STAT3 was a key molecule in MLR co-cultured with MSCs. STAT3 siRNA treated MSCs did not inhibit the lymphocyte proliferation. After MSCs were trasnsfected with STAT3 plasmid, the fraction of CD4+CD25+FOXP3+ cells (Treg cells) were increased, while the fraction of CD4+, CD8+, CD25+ was decreased. In addition, Th1-related cytokines (IL-2, IL-12 and INF-γ) and Th17-related cytokines (IL-6, IL-17 and IL-21) were down-regulated, and Th2-related cytokines (GATA-3, IL-4 and IL-10) were up-regulated in MLR co-cultured with STAT3-ablated MSCs, while vice versa in MLR co-cultured with STAT3-transfected MSCs. Furthermore, ELISA showed that concentration of Th1-related cytokine (IL-2) in the supernatant of MLR co-cultured with STAT3-ablated MSCs was higher than that of control; while concentration of Th2-related cytokine (IL-4) was lower than that of control. These results suggested that induction of Th1 to Th2 shift by MSCs might be mediated via STAT3 molecule. In summary, STAT3 may be an indispensable molecule in the immunoregulatory effect in human MSCs via modulation of regulatory T cells.

Disclosures:
No relevant conflicts of interest to declare.
In Vivo Deficiency of Both Transcription Factors, C/EBPbeta and C/EBPepsilon, Results in Lack of Cytokine Response and a High Susceptibility for Overwhelming Infections.
Authors: Thoennissen Nils Heinrich , Akagi Tadayuki , Song Jee Hoon , Iwanski Gabriela B. , Koeffler H. Phillip 
Published: October 2019

Abstract
Abstract 3641
Poster Board III-577
Transcription factors including the CCAAT/enhancer binding protein (C/EBP) family are essential for hematopoietic cell development including myelopoiesis. In response to cytokines and/or infection, C/EBPbeta is important for emergent neutrophil production, whereas C/EBPepsilon induces terminal differentiation of granulocytes. We recently generated C/EBPbeta and C/EBPepsilon double-knockout (KO) mice and demonstrated the dependence of the hematopoietic system on both transcription factors. In order to study inflammatory response of this transgenic in vivo model, bone marrow-derived macrophages from wild-type (WT), single- as well as double-KO mice were cultured either with or without 100 ng/mL LPS and 100 ng/mL mIFNgamma as activating stimuli. After 24 hours of exposure, RNA was extracted, and gene expression difference was interrogated by whole genome microarray (Affymetrix®, mouse genome 430 2.0). The probes were normalized with the MAS 5.0 software for processing Affymetrix oligonucleotide array data, and genes were considered to be differentially expressed with a fold change level of at least 2. Remarkably, we found more than 75 genes which were up to 20-fold downregulated in the stimulated macrophages of the double-KO mice compared to either single KO or WT mice. A pathway-based analysis indicated that these deregulated genes formed networks associated with development and function of the hematopoietic system, inflammatory response and cell-cell signaling. We confirmed the expression for some of the most highly decreased genes by RT-PCR; for example, interleukin-(IL)1beta, IL-12beta, CD38, macrophage mannose receptor (Mrc-1), toll-like receptor-2 (TLR-2), C-type lectin receptor Mincle, and insulin-like growth factor (IGF-1) were almost not detectable in the activated macrophages of the double-KO mice compared to either single-deficient or WT mice. Lacking these and other important regulators of appropriate immune responses and hematopoiesis, the double KO died between 2 - 3 months of age due to systemic infections including formation of hepatic, peritoneal and lung abscesses with Streptococcus viridians, Enterococcus species and Escherichia coli. In conclusion, phentotype as well as expression pattern of mediators of inflammatory response and hematopoiesis of the double-KO mice was distinct from either the C/EBPbeta- or C/EBPepsilon-KO mice, demonstrating that each transcription factor is important in cytokine expression and host defense, but can in part compensate for each other in the single knockout genotype.

Disclosures:
No relevant conflicts of interest to declare.
Hypomethylation of Tumor Suppressor Genes in Acute Myeloid Leukemia: Characteristic of Cell Lines with MLL Abnormalities?.
Authors: Quentmeier Hilmar , Röhrs Sonja , Dirks Wilhelm G , Meyer Claus , Marschalek Rolf , Scherr Michaela , Slany Robert K. , Wallace Andrew , Drexler Hans G. 
Published: October 2019

Abstract
Abstract 365

Background:
Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemia (AML) and in mixed phenotype acute leukemia in infancy, a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type MLL carries histone methyltransferase activity and affects specific target genes, such us HOXA cluster genes. While the more than three dozen MLL fusion proteins known today exert different specific functions, they finally induce transcription of individual target genes. Consequently, acute lymphoblastic leukemias (ALL) with MLL mutations (MLLmu) exhibit typical gene expression profiles including high-level expression of HOXA cluster genes. Aim of this study was to find a correlation between the MLL mutational status and tumor suppressor gene methylation/expression in acute leukemia cell lines.

Results:
Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLLmu and MLLwt acute leukemia cell lines. 1.8/24 TSG were methylated in MLLmu AML cells, 6.2/24 TSG were methylated in MLLwt AML cells. Hypomethylation and expression of the tumor suppressor genes (TSG) BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLLmu acute myeloid leukemia (AML) cell lines. MLL wild-type (MLLwt) AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3 confirming epigenetic silencing of these genes in MLLwt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLLmu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene.

Conclusion:
These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLLmu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

Disclosures:
No relevant conflicts of interest to declare.