bluuebunny/crossref_metadata_2025_split huggingface dataset:
Manipulating Higher Order Chromatin Structure of the β-Globin Locus by Targeted Tethering of a “looping” Factor
Authors: Deng Wulan , Gregory Philip D , Reik Andreas , Blobel Gerd 
Published: October 2019

Abstract
Abstract 647

The mammalian β-globin locus is under the coordinated control of multiple transcription factors to ensure the correct expression of the globin genes during development. The distal β-globin locus control region (LCR) physically interacts with β-like globin promoters to form developmentally dynamic chromatin loops. The hematopoietic transcription factor GATA-1 and its associated cofactor Ldb1 bind to the LCR and the β-globin promoter and are essential for loop formation and β-globin expression. However, the molecular basis of chromatin looping and its cause-effect relationship with transcriptional activation are unclear. Here, we examined whether Ldb1 is an effector of GATA-1 during loop formation. Specifically, we tested whether artificial tethering of Ldb1 to the endogenous β-globin promoter and LCR can substitute for GATA-1 function. Ldb1 was fused to artificial zinc finger proteins (ZFP) designed to bind to the LCR and β-globin promoter. Ldb1-ZFPs were introduced pairwise into murine GATA-1 null erythroid cells in which the β-globin locus is relaxed and transcriptionally silent. In vivo binding of the Ldb1-ZFPs to their targets was verified by ChIP assay. Strikingly, expression of Ldb1-ZFPs but not Ldb1 or ZFPs alone led to substantial activation of β-globin transcription in the absence of GATA-1. Moreover, chromosome conformation capture experiments showed that Ldb1-ZFPs triggered physical association between the LCR and the β-globin promoter. Recruitment of RNA polymerase II (Pol II) and its phosphorylation at serine 5 are critical LCR-dependent regulatory steps in β-globin transcription. We found that Ldb1-ZFP expression facilitated Pol II recruitment at the β-globin promoter and serine 5 phosphorylation to the same level as GATA-1-expressing erythroid cells. This is consistent with an Ldb1-ZFP-induced LCR-β-globin promoter chromatin loop. In concert, these results indicate that Ldb1 is a critical effector for GATA-1 by mediating enhancer-promoter loops. In broader terms, our results suggest that chromatin loop formation can be sufficient for gene activation in the absence of an essential transcription factor. We are currently in the process of examining whether targeting of the LCR to embryonic and fetal globin genes can be used to activate them in adult cells. Targeted chromatin loop formation may provide a method to activate fetal or adult hemoglobin expression in individuals with β-thalassemia or sickle cell anemia.

Disclosures:
No relevant conflicts of interest to declare.
Transfusion of Fresh but Not Stored Blood Reduces Infarct Size and Improves Cardiac Function Following Acute Myocardial Infarction In Anemic Rats
Authors: Xenocostas Anargyros , Hu Houxiang , Chin-Yee Nicolas J , Chin-Yee Ian , Feng Qingping 
Published: October 2019

Abstract
Abstract 661

Background:
Anemia is a predictor of poor outcomes in acute myocardial infarction (MI) and may increase the degree of ischemia, resulting in greater tissue injury. The only existing therapeutic intervention used to acutely correct anemia is red blood cell (RBC) transfusion, although its effectiveness at improving outcomes in anemic subjects following MI remains controversial. We have previously shown that transfusion of fresh blood in anemic rats following MI decreased infarct size, and improved cardiac function and survival (Hu et al. Transfusion 2010; 50:243-251). However, it was not known if the transfusion of stored blood, which develops a series of chemical and corpuscular changes collectively known as the “storage lesion”, would be equally effective in this model.

Experimental Design:
Acute MI was induced by coronary artery ligation in 49 male Sprague-Dawley rats, 38 of which were made anemic (Hb = 80–90 g/L) using a combination of dietary iron deprivation and phlebotomy, and 11 with normal Hb levels. Rat blood was stored for 7 days in CPDA-1 to induce a storage lesion similar to the one seen in human blood stored under identical conditions for 28 days (d'Almeida et al. Transfusion Medicine 2000; 10:291-303). Anemic animals were randomly assigned to one of three groups: i) fresh blood transfusion (stored for <4 hours) to increase the Hb to 100 g/L, ii) transfused with stored blood to increase the Hb to 100 g/L, or iii) no transfusion. At 24 hours post-MI, infarct size, cardiac function and survival rates were determined.

Results:
A similar degree of left ventricular (LV) myocardial ischemia (area at risk) was induced in all groups (P=NS). In agreement with previous results, the infarct size to area at risk ratios were significantly increased in the anemic (Hb 80–90 g/L) animals compared to non-anemic MI controls, and transfusion of fresh blood to a target Hb level of 100 g/L after MI decreased infarct size (P<0.05) and improved both systolic (+dP/dtmax) and diastolic (−dP/dtmin) LV function compared to anemic animals (P<0.05). However, the transfusion of stored blood into anemic animals post-MI did not have these effects when compared to the group receiving fresh blood (P<0.05). Furthermore, survival was increased in anemic animals receiving fresh blood (9/11; P<0.05) but not stored blood (7/14; P=NS) compared to the non-transfused group (5/13).

Conclusions:
The prolonged storage of blood negates the beneficial effects of fresh blood transfusion, which include reductions in infarct size, and improvements in cardiac function and short-term survival following acute MI in this animal model.
(This study was supported by the Canadian Institutes of Health Research, Canadian Blood Services and the Bayer Partnership Fund).

Disclosures:
No relevant conflicts of interest to declare.
Dendritic Cell Activation by PF4/H Multimolecular Complexes
Authors: Afonina Galyna , Joglekar Manali , Qi Rui , Arepally Gowthami M. 
Published: October 2019

Abstract
Abstract 722
Heparin-Induced Thrombocytopenia (HIT) is caused by antibodies to multimolecular complexes of Platelet Factor 4 (PF4) and heparin (H). Little is known about the cellular mechanisms underlying the PF4/H immune response. Our previous studies have shown that mice injected with murine (m) PF4/H complexes develop a de novo immune response to mPF4/H, but do not respond to injections of mPF4 alone or H alone. In other studies using this model, we have shown that the HIT immune response is T-cell dependent, requires the presence of PF4/H multimolecular complexes and does not engage TLRs via MyD88. To examine the cellular basis of the HIT immune response, we performed studies addressing requirements for antigen presentation. We first isolated splenocytes from non-immunized C57Bl/6 mice and incubated 4×105 splenocytes with mPF4 alone (10mg/ml, final concentration.), heparin alone (0.4U/ml, final), mPF4/H complexes (10mg/ml and 0.4U/ml, final), buffer or LPS (1 mg/ml, final, positive control). We noted significant levels of IL-12 in wells incubated 24hrs with mPF4/H (109 ± 7 pg/mL) or LPS (256 ± 22 pg/mL) but not wells containing mPF4 (33 ± 7 pg/mL), H (5 ± 7 pg/mL), or buffer (9 ± 13 pg/mL). In other studies, we noted that splenic dendritic cells (DCs) were primarily activated by mPF4/H complexes and that cellular activation, as gauged by IL-12 (Figure 1A) or IFN-g (data not shown) occurred in a heparin-dependent manner. DCs activation by mPF4/H complexes was not dependent on CXCR3 or pattern recognition receptors, such as receptor for dectin, mannose or complement receptor 3 (CR3 or CD11b antibody). To determine the effect of DC activation on T-cell responses, we performed mixed lymphocyte reaction assays using DCs isolated from C57Bl/6 mice (3×105 cell/well) and naïve T-cells from Balb/c mice (1:5 cellular ratio). Pre-incubation of Bl/6 DCs with mPF4/H complexes, but not mPF4, H or buffer alone resulted in Balb/c T cell activation and release of cytokines indicative of T helper (Th) 1 immune response (Figure 1B). In summary, we show that DC s are directly activated by PF4/H multimolecular complexes, and that cellular activation by complexes results in a predominant Th1 polarization. Additional studies are underway to identify the relevant receptor for DC activation, and additional pathways of antigen processing and presentation that are necessary for the initiation of a PF4/H specific adaptive immune response.

Disclosures:
No relevant conflicts of interest to declare.
Deficiency In Stress Response Gadd45a and Gadd45b Alters MAPK Kinase Signaling Leading to Functional Defects In Innate Immune Responses of Neutrophils and Macrophages In Vitro and In Vivo
Authors: Salerno Dominic M. , Tront Jennifer , Hoffman Barbara , Liebermann Dan 
Published: October 2019

Abstract
Abstract 927
Stress sensor GADD45 proteins modulate p38-NF-Kb and JNK signaling; which play major roles in leukocyte activation and innate immunity. We have previously documented that GADD45a-deficient and GADD45b-deficient mice exhibit impaired responses to hematological stress, including acute stimulation with inflammatory cytokines (Gupta et. al, Oncogene. 25:5537, 2006). Whether GADD45 genes modulate myeloid compartment function, notably the response of macrophages & granulocytes to inflammatory stress, remains largely unexplored. Data obtained thus far indicate that bone marrow (BM) derived neutrophils and macrophages lacking GADD45 family members exhibit defects in chemotaxis towards inflammatory stimuli. BM derived neutrophils and macrophages lacking GADD45 family members were also observed to exhibit defects in the production of reactive oxygen species (ROS) upon treatment with LPS (500ng/mL) in vitro. Furthermore, BM derived macrophages from mice lacking GADD45a and GADD45b were found to be defective in phagocytosis and cellular adhesion upon stimulation with LPS (500ng/mL) in vitro. Additionally, it was observed that BM derived neutrophils and macrophages lacking GADD45 family members exhibit defects in inflammatory cytokine secretion in response to activation with LPS. Notably, GADD45a, GADD45b & GADD45g null mice injected intraperitoneally with sublethal doses of LPS were significantly more susceptible to septic shock compared to wt mice, showing significantly increased morbidity. Moreover at 18 hrs. post-injection, the spleens of KO mice harbored apoptotic foci in the white pulp, identified as tingible body macrophages ingesting dying cells. To dissect the signaling pathways involved, MAPK specific chemical inhibitors were used to correlate MAPK signaling alteration to defects in innate immune responses of neutrophils and macrophages lacking GADD45a or GADD45b. LPS stimulated wt neutrophils treated with MAPK inhibitors exhibited defects in signaling and innate immune responses mimicking neutrophils and macrophages lacking GADD45a or GADD45b. Collectively, these novel data provide evidence to show that deficiency in GADD45a and GADD45b leads to functional defects in the myeloid innate immune responses of neutrophils and macrophages in vitro and in vivo. These findings increase our understanding of innate immunity and have the potential to contribute towards development of novel approaches to clinical treatment of sepsis.

Disclosures:
No relevant conflicts of interest to declare.
Calorie Restriction and Aging
Authors: de Cabo Rafael 
Published: October 2019

Abstract
Abstract SCI-2
A prominent manifestation of aging is a reduced ability to respond to environmental stressors, including heat and oxidative stress. Reduced stress tolerance and decreased ability to maintain homeostasis are at least partially responsible for the increased morbidity and mortality that occurs with advancing age. The age-related attenuation of stress pathways and increased expression of stress-response genes with aging are examples of the growing body of evidence linking reduced stress responsiveness to aging. In 1935, McCay and colleagues first reported that reducing the caloric intake of rodents could significantly lengthen their mean and maximal life span, slowing down basic aging processes. The effect of calorie restriction (CR) on delaying aging has been replicated in many animal species including nonhuman primates, although in these, potential life span alterations cannot be ascertained for several more years due to their longevity CR causes a reduction in body weight, tissue growth, blood glucose, insulin levels and body temperature. In addition, CR prevents the age-related decline in tolerance to different stressors such as oxidative and heat, and the age-related reduction in expression of protective heat shock and oxidative stress proteins. While CR is the only intervention that has consistently been shown to increase maximum life span and prevent or delay the onset of age-associated pathophysiological changes in laboratory rodents, the underlying mechanisms remain elusive. Using calorie restriction (CR) as their benchmark research tool, gerontologists are making progress in identifying dietary and pharmacologic interventions that may be applicable to retarding aging processes in humans.

Disclosures:
No relevant conflicts of interest to declare.
Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT) for Patients with Severe Sickle Cell Disease (SCD)
Authors: Hsieh Matthew M , Fitzhugh Courtney D , Weitzel R. Patrick , Coles Wynona , Link M Beth , Tisdale John F. 
Published: October 2019

Abstract
Abstract 10
Allo-HSCT remains the only curative approach for patients with SCD, yet a high risk of procedural toxicities and graft-versus-host disease (GvHD) limit this approach in adult patients with end organ damage from SCD. We chose a low-dose radiation approach utilizing sirolimus based upon its unique ability to promote T cell tolerance. Entry criteria include irreversible SCD-related complications (stroke, nephropathy, or tricuspid regurgitant jet velocity >2.5 m/s), or reversible complications not ameliorated by a 6-month course of hydroxyurea (frequent vaso-occlusive crises or acute chest syndrome). Conditioning was achieved with 1mg/kg of alemtuzumab divided over 5 days, a single total body irradiation dose of 300cGy, and oral sirolimus targeting trough levels between 10–15 ng/ml. Donor chimerism was measured by microsatellite PCR among CD3 and CD14/15 positive white cells. Twenty-three patients have been transplanted to date, and their ages ranged from 17 to 64 years (median 28). All are alive at 2 months to 7 years post allo-HSCT. Conditioning was well-tolerated. All received unmanipulated G-CSF mobilized peripheral blood progenitors obtained from 8/8 HLA-matched siblings. CD34+ cell doses ranged from 5.5 to 31 × 10e6/kg (median 14.4), and CD3+ cell doses ranged from 1.6 to 5.4 × 10e8/kg (median 3.4). Three patients engrafted temporarily but lost their grafts between the 2nd and 3rd months post transplant and had recurrent SCD. Twenty patients engrafted with mean myeloid chimerism of 97.5% (median 89%) and CD3 chimerism of 42% (median 49%). Improvements in laboratory parameters of engrafted patients are as follows: pre vs post HSCT hemoglobin, 8.7 vs 12.1 g/dL; reticulocyte count, 181 vs 74 k/uL; LDH, 328 vs 202 units/L; total bilirubin, 2.87 vs 1.0 mg/dL. Hemoglobin electrophoresis revealed replacement by donor type hemoglobin in 19 patients by 1 year, with amelioration of the SCD phenotype allowing for therapeutic phlebotomy. In 17 patients at 1 year or more post allo-HSCT, 5 had CD3 chimerism >50% which allowed complete withdrawal of immunosuppression; they have maintained stable mixed chimerism. Two patients received preemptive treatment with ganciclovir or foscarnet for presumed CMV reactivation (blood PCR >300 genome/mL) before day 30. Treatment in both patients was discontinued 1 week later with negative blood PCR and prophylaxis with acyclovir was resumed. There were 2 patients with zoster at 2 and 3 years post allo-HSCT. Finally, no engrafted patient to date has developed any evidence of acute or chronic GVHD. Our results demonstrate that this relatively simple conditioning regimen is well-tolerated across a broad age range of adults with severe SCD, is effective in achieving stable mixed chimerism without the development of GVHD, and is sufficient to induce functional tolerance.

Disclosures:
No relevant conflicts of interest to declare.
B Cell Receptor-ERK1/2 Signal Cancels PAX5-Dependent Repression of BLIMP1 Probably Through PAX5 Phosphorylation: A Mechanism of Antigen-Triggering Plasma Cell Differentiation
Authors: Yasuda Takahiko , Hayakawa Fumihiko , Kurahashi Shingo , Naoe Tomoki 
Published: October 2019

Abstract
Abstract 1109
Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C).

Disclosures:
Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.
KSHV Latency Locus Augments B Cell Response In Vivo
Authors: Sin Sang-Hoon , Dittmer Dirk P 
Published: October 2019

Abstract
Abstract 1115
A small subset of genes is transcribed during latency in Kaposi sarcoma-associated herpesvirus (KSHV) lymphomas. To better understand viral lymphomagenesis we generated a transgenic mouse expressing all viral latent genes including LANA, vFLIP, vCyc, kaposin and all viral miRNAs. All viral miRNAs were expressed in B cells. The complete latency locus induced activation of mature B cells more robustly than a single LANA protein transgenic mouse. It led to the specific expansion of marginal zone (MZ) B cells. The transgenic mice showed an augmented primary response to T-dependent antigen leading to acute MZ activation and germinal center (GC) activation. Furthermore, they exhibited an augmented response to TLR4 stimulation. Our data indicates that the KSHV latency locus fuels transformation processes as a chronic antigenic stimulator. These data supports a model of lymphomagenesis, in which virally infected B cells become hyperresponsive to B cell stimulation, thus providing an expanded pool for secondary genetic alterations.

Disclosures:
No relevant conflicts of interest to declare.
FAK Regulates Progenitor B Cell Growth, Localization and Retention in Bone Marrow Microenvironments
Authors: Park Shin-Young , Wolfram Peter , Manis John , Beggs Hilary , Nombela-Arrieta Cesar , Pivarnik Gregory , Silberstein Leslie E 
Published: October 2019

Abstract
Abstract 1119
Progenitor B cells are thought to reside in complex bone marrow microenvironments, e.g. niches, where they receive signals for growth and maturation. Here we have conditionally targeted in B cells the non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK), because of its function as an integrator of cell extrinsic signals including CXCL12 and VCAM-1. The number of progenitor (pro-, pre- and immature) B cells is reduced by 30–40% in CD19-Cre Fak fl/fl mice compared to wild type mice, and ex vivo cultured, Fak deleted pro-B cells exhibit impaired IL-7 ± CXCL12 mediated proliferation and survival. A novel quantitative laser scanning cytometry approach was used to demonstrate that Fak deletion also disrupts the non-random gradient distribution in femoral bone marrow of progenitor B cells, which normally are preferentially localized in endosteal regions of the metaphyses and to a lesser degree of the diaphysis. Increased numbers of pro-B cells are detected in the periphery of Fak deleted mice, and mobilization of pro-B cells induced by the inflammatory antigen NP-CGG-alum is increased 3-fold in Fak-deleted versus wild type mice. In addition, intravenously transferred Fak deleted pro-B cells have defective homing specifically to the extra-vascular compartment of the bone marrow. Collectively, these studies illustrate the importance of FAK in regulating progenitor B cell homeostasis and maintenance of their spatial distribution in bone marrow niches.

Disclosures:
No relevant conflicts of interest to declare.
Abnormalities of the Hematopoietic Stem Cell Compartment in Children After in Utero Exposure to AZT
Authors: Andre-Schmutz Isabelle , Cortivo Liliane Dal , Six Emmanuelle M. , Kaltenbach Sophie , Radford-Weiss Isabelle , Cagnard Nicolas , Cocchiarella Fabienne , Benachi Alexandra , Mandelbrot Laurent , Mavilio Fulvio , Cavazzana-Calvo Marina , Blanche Stéphane 
Published: October 2019

Abstract
Abstract 1123
Mother to child HIV-1 transmission prophylaxy with antiretroviral (ARV) during pregnancy is remarkably effective. Although now based on various ARV-combinations, azydothymidine (AZT) remains the cornerstone drug in all guidelines. AZT hematologic toxicity after in utero exposure consisted in a reversible anemia but also in a persistant slight decrease on platelet, neutrophils and lymphocytes count up to 2 years of age. This long lasting biological effect evokes an alteration of the hematopoietic stem cell compartment. The very long term impact is unknown.
Twenty six neonates from HIV-1 infected mothers treated with an AZT based combination during pregnancy in Paris area were included in the study and compared to 20 neonates from healthy mothers. Umbilical cord blood was sampled after obtaining written informed consent from the mother. The experimental scheme included phenotypical and functional analysis of the hematopoietic compartment as well as transcriptome analysis of hematopoietic progenitors. Phenotypic analysis revealed a tendancy to higher numbers of CD34+ cells in the AZT group as compared to the control group. A similar finding was observed for hematopoietic stem cells defined on the basis of a Lin-CD34+ CD38loCD90+CD45RA- phenotype. Multipotent progenitors Lin-CD34+ CD38loCD90-CD45RA- were also increased at a statistically significant level. Affymetrix analysis of gene expression profiles was performed on CD34+ cells sorted from 8 AZT and 6 controls. Ingenuity analysis revealed significant differences in genes involved in hematopoiesis and cell cycle. Among hematopoietic genes for which differences of expression were noted between the two groups, 14 have a known role in the maintenance and differentiation of hematopoietic stem cells. Quantitative RT-PCR are actually ongoing to further characterize the gene expression profile of AZT CD34+ cells.
These results support the hypothesis of an alteration of the hematopoietic stem cell compartment in children after in utero exposure to AZT. Potential hematotoxicity of other ARV drugs must be evaluated to delineate the safest as possible mother to child HIV-1 transmission prophylaxy.

Disclosures:
No relevant conflicts of interest to declare.