bluuebunny/crossref_metadata_2025_split huggingface dataset:
B7H1/B7.1 Interaction Alleviate Acute Gvhd in a PD-1 Dependent Manner
Authors: Deng Ruishu , Cassady Kaniel , Li Xiaofan , Yao Sheng , Zhang Mingfeng , Racine Jeremy , Lin Jeffrey , Chen Lieping , Zeng Defu 
Published: November 2019

Abstract
Interactions of B7H1 (PD-L1) on antigen presenting cells with its two ligands, PD-1 and B7.1, on T cells play a pivotal role in controlling T cell activation, proliferation, anergy, and apoptosis. However, the interactions between the two pathways remain unknown. Using a graft-versus-host disease (GVHD) model of C57BL/6 donor to MHC-mismatched BALB/c recipient, we report here that:1) Wild-type (WT) conventional alloreactive CD4+ T (Tcon) cells induces more severe acute GVHD in B7H1 deficient recipients due to lack of both B7H1/PD-1 and B7H1/B7.1 interaction, resulting in reduced Tcon proliferation and apoptosis and therefore increased expansion of Tcon cells. In contrast, PD-1 deficient Tcon cells induced less severe GVHD in B7H1 deficient recipients compared to WT recipients due to lack of B7H1/B7.1 interaction and reduced Tcon cell proliferation. 2) Specific blockade of the B7H1/B7.1 axis by use of an anti-B7H1 mAb exacerbate acute GVHD. This is because anti-B7H1 mAb reduces donor Tcon cell proliferation, IL-2 production, expression of PD-1, and activation-induced apoptosis, resulting in expansion of alloreactive Tcon cells. In contrast, in the absence of PD-1 on donor T cells, specific blockade of B7H1/B7.1 interaction ameliorates acute GVHD due to reduction of donor Tcon proliferation without impact on Tcon cell apoptosis. 3) B7H1 fused to an immunoglobulin Fc domain (B7H1-Ig), when produced in vivo by hydrodynamic injection of B7H1 plasmid, ameliorates GVHD by augmenting both proliferation and apoptosis of WT- alloreactive Tcon cells. Conversely, in the absence of PD-1 on donor T cells, B7H1-Ig treatment has no impact on apoptosis but augments donor T cell proliferation and worsens GVHD. These results indicate that interaction of B7.1 with B7H1 augments Tcon cell proliferation, IL-2 production, and expression of PD-1. Higher expression of PD-1 leads to increased donor Tcon cell apoptosis mediated by the B7H1/PD1 pathway therefore ameliorates GVHD. But in the absence of PD-1, B7H1/B7.1 interaction only augments T cell proliferation and causes more severe GVHD. Additionally, by engaging both PD-1 and B7.1, B7H1-Ig can be a powerful therapeutic reagent for down-regulating the T cell immune response (This work was supported by NIH R01 AI066008).

Disclosures
No relevant conflicts of interest to declare.
The BRAF Pseudogene Is a Proto-Oncogenic Competitive Endogenous RNA
Authors: Karreth Florian , Reschke Markus , Chapuy Bjoern , Shipp Margaret A. , Chiarle Roberto , Pandolfi Pier Paolo 
Published: November 2019

Abstract
Non-coding RNAs have long been viewed as non-functional genomic relicts of evolution, but recetn findings have implicated their importance in physiology and disease. Recently, in vitro experiments demonstrated that the pseudogenes of PTEN and KRAS operate as natural miRNA decoys (competitive endogenous RNAs or ceRNAs) that regulate the expression of their parental genes. However, in vivo evidence for a causal role of pseudogenes in cancer development is lacking. To investigate whether the BRAF pseudogene (BRAFps) possesses oncogenic properties we generated transgenic mice carrying a Tet-inducible BRAF pseudogene allele. Global BRAFps overexpression resulted in the development of aggressive B-cell lymphoma after 6-12 months. These tumors were characterized by a profound expansion of B-lymphocytes in the spleen, as well as splenomegaly, lymphadenopathy and infiltration of the kidneys, lungs, and liver by neoplastic cells. The BRAFps-induced lymphoma was polyclonal, transplantable, dependent on continuous BRAFps expression, and cooperated with heterozygous loss of PTEN to accelerate disease onset. Mechanistically, we propose that BRAFps functions as a ceRNA that sequesters miRNAs from BRAF and possibly other targets. Indeed, overexpression of BRAFps results in elevated levels of BRAF in a Dicer-dependent manner. This, in turn, increased BRAF-dependent MAPK signaling and proliferation. To further validate the ceRNA activity of BRAFps, we engineered mice to express only the 3’UTR or CDS of BRAFps as each portion of the pseudogene may individually engage in miRNA-mediated crosstalk with BRAF. Notably, both BRAFps-CDS and BRAFps-3’UTR increased spleen and lymph node weights 6 months after induction. Interestingly, BRAFps-3’UTR elicited a lymphoma phenotype similar to full length BRAFps, while mice expressing BRAFps-CDS developed a more indolent form of this phenotype, suggesting that lymphomagenesis is primarily mediated by the BRAFps 3’UTR. BRAFps transcript was undetectable in primary human B-cells, but was aberrantly expressed in primary human DLBCL and human DLBCL cell lines. Expression of BRAF and BRAFps was positively correlated in human primary DLBCL and human DLBCL cell lines. In addition, gains or amplifications of the genomic locus containing BRAFps were found in various human cancer types. Overexpression of BRAFps in human lymphoma cells elevated BRAF levels, MAPK activation, proliferation and growth in xenografts. Our results demonstrate for the first time the oncogenic potential of a pseudogene in an engineered mouse model and indicate that ceRNA- mediated regulation is an important regulatory mechanism of gene expression in vivo.

Disclosures
No relevant conflicts of interest to declare.
High Ferritin Level Is High Risk Factor of II-IV Acute Gvhd or Severe Cytopenia Post-Transplant in Thalassemia HSCT
Authors: Liao Jianyun , Wen Jianyun , Li Chunfu 
Published: November 2019

Abstract
Purpose: To find out causes of II-IV acute graft-versus-host disease (GVHD) or severe cytopenia after hematopoietic stem cell transplantation (HSCT).
Methods: Sixty-four children with thalassemia major underwent HSCT from fifty-seven 7/8 (at allele of A, B, C, or DRB1) matched donors and seven 7/8 match associated with a DQB1 or DPB1 allele mismatched donors from January 2009 to September 2013. Thirty-three patients suffered from II-IV acute GVHD or severe cytopenia beyond day 28 post-transplant (SCB-28PT, WBC < 2G/L for 4 sequence weeks after HSCT, Group 1). The remains (n=31) associated with well status (Group 2). Current study focus on effect of ferritin, recipient age and donor age on the Group 1.
Results: Ferritin was significant higher in Group1 than Group 2 (4178.3 vs.3033.8 ng/ml, p=0.042). Ferritin was also proved as the dangerous cause by multi-factor binary regression analysis (p=0.017). There were insignificant in comparison of Group 1 and Group 2 in donor’s age (30.2 vs.30.4, p=0.939) and receptor’s age (6.3 vs. 6.4,p=0.889), respectively.
Conclusions: The current study showed high ferritin level is a high risk factor of II-IV acute GVHD or SCB-28PT.

Disclosures
No relevant conflicts of interest to declare.
In Human Beta-Globin Gene Locus, ERV-9 LTR Retrotransposon Interacts with and Activates Beta- but Not Gamma-Globin Gene
Authors: Tuan Dorothy , Pi Wenhu 
Published: November 2019

Abstract
Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) comprise over 40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions was not known. In the human b-globin gene locus, an ERV-9 LTR retrtransposon is located near the 5’ end of the locus control region (LCR) at 40-70 kb upstream of the human fetal g- and adult b-globin genes. To address the functional significance of the intergenic ERV-9 LTR, we generated transgenic (Tg) mice carrying a 100 Kb BAC clone spanning the entire human b-globin gene locus from the ERV-9 LTR to b-globin gene and showed that the LTR retrotransposon serves long-range, beneficial host function (Pi et al., PNAS 2010): The ERV-9 LTR containing multiple CCAAT and GATA motifs competitively recruits high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. Deletion of the ERV-9 LTR by Cre-loxP mediated in situ recombination in the BAC Tg mice suppresses transcription of b-globin gene but activates transcription of g-globin gene. The results indicate that the ERV-9 LTR activates transcription of b-globin gene in erythroid cells during development. Therefore, LTR deletion drastically suppressed b-globin gene and re-activated g-globin gene through a competitive mechanism of globin gene switching.
Alternatively, the primary effect of the ERV-9 LTR could be to suppress g-globin gene during development. Therefore, LTR deletion re-activated g-globin gene, which then suppressed transcription of b-globin gene. To differentiate between these two possibilities, we utilized Mx1-Cre mice to conditionally delete the ERV-9 LTR in the erythroid cells of the adult BAC transgene mice, in which g-globin gene was already silenced and b-globin gene fully activated. If the primary target of the ERV-9 LTR enhancer complex was g-globin gene, deletion of the ERV-9 LTR should not be able to activate the already silenced g-globin gene nor to suppress the fully active b-globin gene. However, the same effect on transcriptional suppression of b-globin gene and re-activation of g-globin gene was observed. These results indicate that the primary target of the ERV-9 LTR is the b-globin gene and not the g-globin gene. The molecular factors underlying the preferential interaction between the ERV-9 LTR and the b-globin gene are under investigation and will be presented

Disclosures
No relevant conflicts of interest to declare.
Pericardial Effusion Is a Marker of Increased Mortality in Thalassemia Major Patients
Authors: Meloni Antonella , Toia Patrizia , Sardella Leonardo , Serra Giuseppe , Chiari Roberta , D'Ambrosio Alfonso , De Franceschi Lucia , Neri Maria Giovanna , Resta Maria Chiara , Preziosi Paolo , Positano Vincenzo , Benni Monica , Pepe Alessia 
Published: November 2019

Abstract
Introduction. In different types of not-hematological diseases the presence of a small pericardial effusion (PE) was associated with worse survival even after adjustment for patient characteristics, suggesting that it is a marker of underlying disease.In thalassemia major (TM) pericardial effusion was shown to be one of the manifestations of heart disease but its potential prognostic importance has never been investigated in the modern era. Cardiovascular Magnetic Resonance (CMR) by cine SSFP sequences was demonstrated to be extremely sensitive to even a small amount of PE.
This is the first prospective study evaluating if the presence of pericardial effusion is associated with increased mortality in TM.
Methods. 1259 patients (648 females, mean age 31.02 ± 8.64 years) enrolled in the Myocardial Iron Overload in Thalassemia (MIOT) were prospectively followed from their first Magnetic Resonance Imaging (MRI) scan. CMR was used to quantify myocardial iron (MIO) overload by a multislice multiecho T2* approach and to assess biventricular function parameters and to detect PE by cine SSFP sequences.
Results. PE was present in 25 (2.0%) patients.Patients with and without PE were comparable for age and ratio of men/women. At the baseline, the percentage of patients with MIO (global heart T2* value < 20 ms) was comparable between patients with and without PE (12.0 % vs 28.7%; P=0.074) and left ventricular and right ventricular ejection fractions were not significantly different between the two groups.
Mean follow-up (FU) time was 44.55 ± 20.35 months and there were 15 deaths.
Mortality was greater for patients with PE compared to those without an effusion (8.0% vs 1.1%, P=0.034).
PE was a significant predictive factor for death (hazard ratio-HR=12.64, 95%CI=2.78-57.42, P=0.001). PE remained a significant prognosticator for death also in a multivariate model including MIO ms (PE: HR=17.36, 95%CI=3.65-82.62, P<0.0001and global heart T2* < 20 ms: HR=3.07, 95%CI=1.07-8.75, P=0.036).
Conclusions. PE is quite rare in TM patients and it is not related to myocardial iron overload. An important role in the development of PE could be played by the 'iron-induced' pericardial siderosis but, due to the limitations of the current non-invasive CMR techniques, we were not able to address this issue.
PE was found to be a strong predictor for death, independently by the presence of myocardial iron overload. The non-invasive diagnosis of pericardial effusion is important for a more complete definition of the cardiac involvement of TM patients. The increased risk of death associated with PE may be used along with other clinical characteristics when estimating a patient's prognosis and monitoring.

Disclosures
Pepe: Chiesi: Speakers Bureau; ApoPharma Inc.: Speakers Bureau; Novartis: Speakers Bureau.
Antibody Mediated Depletion of iNKT Cells Protects Against Hypoxia-Induced Pulmonary Injury in a Murine Model of Sickle Cell Disease
Authors: Pei Hong , Schaub Robert , Scheuplein Felix , Mashal Robert , Linden Joel 
Published: November 2019

Abstract
Background: Painful vaso-occlusive crises (pVOC) are caused by the polymerization of sickle deoxyhemoglobin, red cell sickling and vaso-occlusion. Vaso-occlusion is exacerbated by an inflammatory cascade that is initiated by iNKT cell activation. Townes sickle cell mice have mouse globins replaced by human globins, including the mutant human sickle b-globin gene (SS). NKT-14 is a monoclonal antibody directed specifically to the mouse invariant T cell receptor (iTCR). The antibody is a chimeric mouse IgG2a that, when bound to the mouse iNKT cell, promotes a rapid, specific and long lasting (> 14 days) depletion.
Aims: Our primary goal was to determine if selective iNKT cell depletion with NKT-14 reduces lung inflammation and injury in response to hypoxia-induced RBC sickling in Townes SS mice.
Methods: Hypoxic pulmonary injury was induced by placing mice in 8% O2 (Coy Lab products)followed by reoxygenation. Some mice were subject to a second period of hypoxia and continuously monitored to determine carotid Hb-saturation (Starr MouseOx). Total cell numbers and activation state of iNKT cells and neutrophils in lung were determined by FACS. Pulmonary vascular leak was quantified with Evans Blue dye.
Results: Twelve hours of 8% hypoxia followed 4 hours of reoxygenation (H/R) was found to produce non-lethal lung inflammation and injury in SS mice. Compared to isotype Ab controls, injection of four mice with 100µg/mouse of NKT-14 Ab three days prior to HR was found to decrease the number of pulmonary iNKT cells by 91% (P < 0.01). Following H/R, NKT-14 Ab treatment resulted in a significant 65% decrease in lung neutrophil accumulation (Fig. A, P < 0.01), and a 54% decrease in pulmonary vascular leak (Fig. B, P < 0.05). In addition, arterial Hb-Sat during a second exposure of SS mice to hypoxia was improved (Fig. C).
Figure 1 Figure 1.
Conclusions: Selective depletion of mouse iNKT cells by injection of NKT-14 Ab three days prior to H/R results in substantial reductions in pulmonary inflammation, vascular leak and injury as measured by Hb-Sat in SS mice. The results suggest that prophylactic Ab-mediated depletion of iNKT cells in humans with SCD may be useful for reducing the incidence and severity of pVOC and acute chest syndrome.

Disclosures
Schaub: NKT Therapeutics Inc: Employment, Equity Ownership. Scheuplein:NKT Therapeutics Inc: Employment, Equity Ownership. Mashal:NKT Therapeutics Inc: Employment, Equity Ownership.
KLF1 Null Neonates Display Hydrops Fetalis and a Deranged Erythroid Transcriptome
Authors: Perkins Andrew C , Magor Graham , Tallack Michael R , Gillinder Kevin R , Bell Charles , Williams Bronwyn 
Published: November 2019

Abstract
We describe a case of severe neonatal anemia with kernicterus due to compound heterozygosity for null mutations in KLF1, each inherited from asymptomatic parents. One of the mutations is novel. This is the first described case of a KLF1 null human. The phenotype of severe DAT-negative non-spherocytic hemolytic anaemia (NSHA), jaundice, hepato-splenomegaly, and marked erythroblastosis is more severe than that present in CDA type IV due to dominant mutations in the second zinc-finger of KLF1. There was a very high level of HbF expression into childhood (>70%), consistent with a key role for KLF1 in human hemoglobin switching. We performed RNA-seq on circulating erythroblasts and found human KLF1 acts like mouse Klf1 to coordinate expression of >500 genes required to build a red cell including those encoding globins, cytoskeletal components, AHSP, heme synthesis enzymes, cell cycle regulators, and blood group antigens. We interrogated recently published KLF1 ChIP-seq to determine directly regulated KLF1 genes in man. We identify novel KLF1 target genes including KIF23 and KIF11 which are required for proper cytokinesis. KIF23 mutations result in CDA type III so our finding provides a direct link btween CDA IV adn CDA III. We also identify new roles for KLF1 in autophagy, global transcriptional control and RNA splicing. We suggest loss of KLF1 should be considered in otherwise unexplained cases of severe neonatal NSHA or hydrops fetalis.

Disclosures
No relevant conflicts of interest to declare.
Expression of Core-Binding Factor-β Is Regulated By Mir-143 and Mir-223 during Granulopoiesis
Authors: Larsen Maria Torp , Borgkilde Anette , Clemmensen Stine Novrup , Borregaard Niels , Cowland Jack B. 
Published: November 2019

Abstract
Granulopoiesis is a strictly regulated process governed to a large extent by an ordered temporal expression of transcription factors. One of the earliest expressed transcription factors, obligatory for correct granulopoiesis, is the Runx1:CBF-beta heterodimer that induces cell division and transcription of genes encoding azurophile granule proteins. The expression level of Runx1:CBF-beta peaks in myeloblasts (MBs) and promyelocytes (PMs) and declines when cells enter the myelocyte (MC) stage, where cell proliferation ceases and terminal differentiation commences. Runx1 binds DNA directly and provides the transcriptional activation domain whereas CBF-beta binds to Runx1 and increases its DNA-binding affinity. CBF-beta is in most cases required for Runx1-mediated transcriptional activity.
microRNAs (miRNAs) post-transcriptionally regulate protein expression and are important for proper granulopoiesis. Using a combined density gradient and immunomagnetic purification protocol, we have isolated neutrophil precursors from human bone marrow and identified 135 differentially regulated miRNAs. Two of these, miR-143 and miR-223, were predicted to target CBFB mRNA that encodes the CBF-beta subunit. Both miRNAs are expressed at low levels in MBs and PMs and increases 5-fold when the cells mature to MCs and remain high in the following stages of neutrophil differentiation. This expression pattern supports a role for miR-143 and miR-223 as translational inhibitors of CBF-beta that diminish the transcriptional capacity of Runx1 and thus aid in terminating cell proliferation in MCs.
Transient transfection of HEK293 cells, that express CBF-beta endogenously, with pre-miR-143 and pre-miR-223 demonstrated a strong suppression of CBF-beta synthesis when both miRNAs were present. A luciferase reporter assay where the 3’-UTR of CBFB mRNA is inserted in the 3’-UTR of the firefly luciferase transcript confirmed that both miRNAs affect translation. Mutation of either the miR-143 or miR-223 miRNA recognition element (MRE) partly relieved repression of the luciferase mRNA and mutation of both MREs completely abolished translational repression. shRNA against CBFB mRNA was used to detect transcriptional targets affected by downregulation of CBF-beta in a murine promyelocyte cell line (MPRO) and in the myeloblastic cell line 32Dcl3 and expression of a number of genes encoding azurophilic granule proteins were found to be significantly diminished.
Together these data demonstrate that miR-143 and miR-223 affect CBF-beta expression and indicate that correct timing of expression of these two miRNAs is a prerequisite for proper granulopoiesis.
Grant acknowledgments: The Danish Medical Research Council, Novo Nordisk Foundation, Brøchner Mortensen Foundation, and the Danish Cancer Society.
MTL and AB contributed equally to the work

Disclosures
No relevant conflicts of interest to declare.
An Fc-Engineered CD133 Antibody for Induction of NK Cell Reactivity Against Cancer Cells
Authors: Rothfelder Kathrin , Leibold Julia , Grosse-Hovest Ludger , Buehring Hans-Joerg , Jung Gundram , Kanz Lothar , Salih Helmut R 
Published: November 2019

Abstract
CD133 is expressed in various types of solid tumors where it predicts an unfavourable clinical outcome (e.g. Linuma et al., J Clin Oncol. 2011). Moreover, CD133 has been shown to be preferentially expressed by cancer stem cells (CSC), as e.g. demonstrated by its usability to enrich CSCs containing populations within cancer cell preparations (e.g. Zeppernick et al., Nature 2008). As CSC display marked resistance to chemo- and radiation therapy, great efforts are presently made to develop immunotherapeutic approaches to specifically target CSC without harming healthy tissue. We here report on the preclinical characterization of a monoclonal antibody (mAb) directed towards CD133 for induction of NK cell reactivity against cancer cells. Two different anti-CD133 antibody clones (W6B3 and 293C3) were used to study expression levels of CD133 in different cell lines derived from solid tumors (A172 and U87MG, glioblastoma; CaCo-2, Colo-205, HCT-8, HT-29 and SW48, colorectal cancer; MDA-MB-231, MDA-MB-468 and BT474, breast cancer; WERI-Rb1, retinoblastoma). CD133 was recognized by W6B3 and 293C3 in 4 and 5 out of the 11 cell lines, respectively. The latter mAb was then chosen for the development of chimeric mAbs with either a wildtype human Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance its affinity to the activating Fc receptor CD16 (293C3-SDIE) and thereby its capacity to mediate antibody dependent cellular cytotoxicity (ADCC), an important antibody property that largely contributes to the clinical success of antitumor mAbs. After confirming the binding specificity of both 293C3-WT and 293C3-SDIE we employed cytotoxicity assays to comparatively analyze their ability to induce NK cell ADCC against different solid tumor cell lines. In all cases, profound induction of tumor cell lysis upon application of 293C3-SDIE was observed, and the therapeutic effect of the Fc-optimized mAb clearly exceeded that of its counterpart containing a wild type Fc part. Notably, colony forming unit assays did not reveal any toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells. Thus, treatment with 293C3-SDIE constitutes a promising novel strategy for immunotherapy of cancer, in particular considering its ability to preferentially target CSC which is presently under study.

Disclosures
No relevant conflicts of interest to declare.