bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional and Biochemical Changes in Stored Human Red Blood Cells.
Authors: McMahon Timothy J. 
Published: October 2019

Abstract
Transfusion of red blood cells (RBCs) is associated with excess mortality and morbidity in some anemic patients with critical illness, and no benefit in others, but the underlying mechanisms are poorly understood. Specifically, little is known of how “storage lesions” of the RBC may contribute to adverse outcomes or lack of benefit from a transfusion. The recent discovery of an active role of the human RBC in regulating blood flow – a major determinant of its own function, O2 delivery – prompts a reexamination of storage-induced changes in this and related RBC functions, and motivates specific identification of the relevant mediators. RBCs release the vasodilators S-nitrosothiols (SNOs) and ATP upon physiological exposure to hypoxia, contributing to O2-dependent blood flow regulation. The result is hypoxic vasodilation peripherally and modulation of hypoxic vasoconstriction in the lung. We show that conventionally stored human RBCs become deficient in SNOs within three hours of acquisition and independent of exposure to storage solution or leukofiltration; both hemoglobin-bound SNO and membrane SNO are depressed. Early changes (hours) are also seen in the function of stored RBCs, namely in RBC regulation of vascular tone in vitro and in the lungs of intact mice in vivo, whereas declines in RBC deformability take place more gradually (from days to weeks). We also confirm storage-induced depletion of RBC ATP, a lesion which may contribute to the functional declines in both deformability and in RBC-dependent vasoactivity. Time courses, therefore, vary widely for several storage-induced functional and biochemical defects that may contribute to adverse clinical outcomes following RBC transfusion. Strategies that either prevent the loss of vasoactive mediators during RBC storage, or replete bioactive (S)NO after RBC storage, are reviewed and may inform the rational design of strategies to improve the risk-benefit balance associated with RBC transfusion in critically ill and other anemic patients.
Genetic Origin of Myeloma.
Authors: Chesi Marta , Bergsagel Peter Leif , Kuehl Michael 
Published: October 2019

Abstract
Multiple myeloma (MM) is a tumor of isotype-switched plasma cells that accumulate in the bone marrow, replacing normal plasma cells and leading to bone destruction and marrow failure. It is often preceded by a stable monoclonal gammopathy (MG) with limited (<10%) bone marrow plasmacytosis that progresses stochastically to MM at a rate of between 1–2% per year. Primary genetic events thought to be associated with disease initiation are present at similar frequencies in both MG and MM. In approximately 40% of cases, there are recurrent chromosome translocations involving the immunoglobulin heavy chain gene that have the hallmarks of being mediated by errors in switch recombination (90%) or somatic hypermutation (10%). These involve recurrent loci that fall into three categories: Cyclin D (15% CCND1 at 11q13, <1% CCND2 at 12p13, 2% CCND3 at 6p21), MAF (5% c-maf at 16q23, 2% MAFB at 20q12, <1% MAFA at 8q24.3), and MMSET (usually with FGFR3 at 4p16, 15%). Approximately one-half of cases are hyperdiploid (48–75 chromosomes) with trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19 and 21. A unifying feature of MM is dysregulated expression of a cyclin D gene, either directly by Ig gene translocation, by transactivation by MAF (CCND2), or by unknown mechanisms (MMSET – CCND2, Hyperdiploid – CCND1). Interestingly, about 2% of patients express no cyclin D, and one-half of these patients have bi-allelic deletion of RB, highlighting the critical role of the CCND/RB axis in the pathogenesis of MM. Deletion of chromosome 13 is seen in one-half of MM but is much more common in the tumors that express CCND2 (>80%) than in those that express CCND1 (~30%). Although with largely redundant functions, CCND1 has additional ability to antagonize RB not shared by CCND2, suggesting that CCND2 tumors may be more critically dependent on the absolute level of RB protein. A variety of secondary genetic events associated with disease progression occur with a similar frequency in hyperdiploid and non-hyperdiploid tumors (17p loss or p53 mutations, RAS mutations, secondary Ig translocations, MYC translocations, and inactivation of p18INK4c or RB1). In contrast, a promiscuous array of mutations that activate the NFKB pathway are less common in hyperdiploid MM, which, nonetheless, has high levels of presumably ligand-dependent NFKB activity and appears particularly dependent on the bone marrow microenvironment. Both some of the primary (MAF, MMSET) and secondary (17p deletion) genetic events are associated with a poor prognosis with melphalan-based regimens and appear to benefit from the early introduction of bortezomib.
Developing a Therapeutic Anti - Dendritic Cell Antibody to Prevent Graft Versus Host Disease.
Authors: Hart Derek , Seldon Therese , Jones Martina , Sheng Yonghua , Palkova Anna , Cullup Hannah , Munro Trent , Sinfield Laura , Rice Alison M , Wilson John , Gray Peter , Barnard Ross , Mahler Stephen , Munster David 
Published: October 2019

Abstract
Abstract 3554
Poster Board III-491

Introduction
Host and donor dendritic cells (DC) stimulate alloreactive donor T lymphocytes, and initiate GVHD. We have shown that polyclonal antibody to the DC surface activation marker human CD83 (anti hCD83), which depletes activated DC, can prevent human DC and T cell induced lethal xenogeneic GVHD in SCID mice without impairing T cell mediated anti-leukaemic and anti-viral (CMV and influenza) immunity (J Exp Med 2009; 206: 387). Therefore, we made and tested a polyclonal anti mouse CD83 (RAM83) antibody in murine HSCT models and developed a human mAb against hCD83 as a potential new therapeutic immunosuppressive agent.

Methods
RAM83 specificity and function was tested by flow cytometry and allogeneic MLRs. It was administered as a single dose on day -1 of murine syngeneic and allo HSCT. Human and mouse single chain variable fragment (scFv) phage libraries were panned on recombinant human CD83 extracellular domain. After screening for specificity and affinity, the clones were reformatted to human IgG1 and expressed by transfected CHO cells. The purified mAb were tested for their ability to block a mixed leucocyte reaction (MLR).

Results
Lin-,Sca1+,Kit+ murine stem or progenitor cells were CD83-ve. RAM83 treated mice receiving syngeneic HSCT survived >35days (normal day 14 WBC). Full MHC mismatched (2× 60mg/kg cyclophosphamide + 2× 500cGy conditioned), B6 [H-2b] into BALB/c [H-2d]) recipient mice treated with a single dose of RAM83 developed GVHD later and survived longer (mean 26.9 days ±3.3) compared to controls (RAneg 12.3±1.3days, p=0.001; Nil antibody 7.7±1.5 days, p=0.0008). Daily high dose (50mg/kg) cyclosporin A delayed GVHD by a mean 18.2±3.3 days. Four phage clones that bind to human CD83 have been reformatted. Of the two tested to date, one mouse/human chimeric mAb has lost binding affinity, probably as a result of immunoglobulin variable region glycosylation. The other reformatted fully human anti-CD83 (3C12) mAb blocked a MLR, by an ADCC mechanism.

Conclusions
mAb targeting of activated DC is a promising novel approach to immunosuppression. The 3C12 clone and others will be subjected to preclinical testing in the xenogeneic SCID mouse model. The human mAb, which prevents GVHD without impairing the desired graft versus leukaemia effect will be developed for phase 1 clinical trials in clinical alloHSCT.

Disclosures:
Hart: CRC-BT: Consultancy.
Efficient MGMTP140K-Mediated In Vivo Selection and Chemoprotection of Long-Term Repopulating Cells in Nonhuman Primates Following Reduced Intensity Conditioning.
Authors: Beard Brian C , Trobridge Grant D , McCune Jeannine S , Kiem Hans-Peter 
Published: October 2019

Abstract
Abstract 3572
Poster Board III-509
Strategies using gene-modified hematopoietic stem cells to treat various severe hematopoietic diseases, including but not limited to hemoglobinopathies, will likely require high levels of gene marking. Here we have established efficient and stable in vivo selection in nonhuman primates using methylguanine methyltransferase (MGMTP140K). In the macaque (Macaca nemestrina) we were able to increase pre-chemotherapy lentiviral gene marking levels of 11.3% in granulocytes and 15.3% in lymphocytes to a post-chemotherapy gene marking level of 76.9% in granulocytes and 49.0% in lymphocytes. Furthermore, stable increases in gene marking were also observed in red blood cells (RBCs) and platelets (PLTs) with a pre-chemotherapy gene marking level of 5.6% and 6.7%, respectively, and a post-chemotherapy gene marking level of 15.2% and 64.0%, respectively. Importantly, the chemotherapy regimen was well tolerated, and engraftment was polyclonal as determined by analyzing long-term repopulating clones by LAM-PCR. In order to minimize extra-hematopoietic toxicity we have began to test a more clinically applicable conditioning regimen in the macaque model. This reduced intensity conditioning regimen should allow treatment of patients with severe hematopoietic or infectious diseases, who may not tolerate a high dose conditioning regimen. We tested targeted busulfan for conditioning to provide sufficient myelosuppression and to facilitate engraftment of chemoprotected hematopoietic stem cells while minimizing extra-hematopoietic toxicity. Following conditioning with busulfan (4 mg/kg/day for 2 days) and infusion of gene modified cells (∼1.7 × 107 CD34-selected cells/kg), there was moderate cytopenia with ANC <500/mL for 7 days and thrombocytopenia with a nadir of 18,000/mL. Following stable hematopoietic recovery, we observed gene marking, determined by RT-PCR, in total white blood cells as a provirus copy number of 0.04 (∼4% gene marking) that, following a single cycle of O6BG (x2) and BCNU, rose to 0.16 (∼16% gene marking). Currently, gene marking has been stable for more than 9 months following chemotherapy. The treatment was well tolerated with only transient elevated liver enzymes following O6BG/BCNU treatment and no additional extra-hematopoietic toxicity has been observed. Clonality studies before and after in vivo selection is underway using a combination of LAM-PCR and a modified whole genome pyrosequencing approach. In summary, we have attained efficient and stable in vivo selection of long-term repopulating cells in nonhuman primates, and have extended this approach to use a reduced intensity conditioning regimen that should be well tolerated in patients with many hematopoietic diseases.

Disclosures:
No relevant conflicts of interest to declare.
The Transient Neutropenia Following G-CSF Administration Is Associated with An Accumulation of Neutrophils in the Pulmonary and Splenic Vasculature.
Authors: Dejesus Catherine E. , Egen Jackson G. , Alvarez Xavier , Metzger Mark E. , Combs Christian A. , Malide Daniela A. , Yu Zu Xi , Donahue Robert E. 
Published: October 2019

Abstract
Abstract 3586
Poster Board III-523
Shortly following G-CSF administration there is a dramatic yet transient disappearance of neutrophils from the circulation. To evaluate this disappearance, C57BL/6 LysM-EGFP knock-in male mice between 6-12 weeks of age were used. The LysM promoter/enhancer is specifically expressed in neutrophils and monocytes permitting evaluation of tissue distribution using EGFP marker localization by confocal microscopy. The ratio of EGFP+ leukocytes to background cellularity was determined in fixed volumes of tissue collected from the lung, liver, spleen, and kidney of mice either receiving saline (n=4) or at 15 minutes (n=6), 30 minutes (n=6), or 240 minutes (n=4) following 50ug/kg SQ rmG-CSF administration. No changes in ratio were observed in triplicate images of the liver or kidney of mice following G-CSF administration; however, there was a significant increase in EGFP+ leukocytes in the spleen and lung shortly following G-CSF administration. It increased from a control value of 6.3(2.6)% (n=12) to 15.9 (6.8)% (n=18) at 30 minutes in the lung and from 1.6 (0.7)% (n=12) to 8.3 (4.2)% (n=18) at 30 minutes in the spleen. While the value for the pulmonary images was less at 240 minutes (9.2 (5.1)%) the level of EGFP+ cells in the spleen continued to rise (12.3 (7.2)%). The increases were in neutrophils based on nuclear morphology. Thus the transient loss of neutrophils from the circulation following G-CSF administration appeared to be due to an accumulation of neutrophils within the pulmonary and splenic tissues. To confirm this observation, tissues were collected from three rhesus macaques 30 minutes following 10ug/kg SQ G-CSF. These tissues were compared to those of a control. Neutrophils were labeled with CD18 and a secondary antibody conjugated to FITC for quantification by confocal microscopy. As before, ratios of fluorescence were determined. The mean threshold intensity for CD18+ cells for the lung lobes for those animals receiving G-CSF (3.8 (3.0)%, n=54) was greater than control specimen lungs (1.5(1.1)%, n=18) and slightly increased for the spleen( from 4.1(3.7)% to 8.5 (6.3)%, n=9). Detailed confocal microscopy using a myeloid/histiocyte antigen clone Mac387 which labels neutrophils and monocytes, an endothelial marker anti-caveolin-1-Cy3 antibody, and a nuclear stain (To-Pro3) showed (see below, stained green, red, and blue, respectively) that the neutrophils were elongated, appeared activated, and were tightly adhered to the endothelium of the pulmonary vasculature. These results suggest that the transient neutropenia following G-CSF administration is associated with an accumulation of neutrophils within the pulmonary and splenic tissues.

Disclosures:
No relevant conflicts of interest to declare.
The Role of Beta 2 Integrins in Macrophage Migration During Resolution of Inflammation.
Authors: Kushchayeva Yevgeniya , Mishchuk Darya , Ugarova Tatiana 
Published: October 2019

Abstract
Abstract 3600
Poster Board III-537
The mobilization of blood monocytes and their differentiation into macrophages during the immune-inflammatory response helps to prepare the tissue for resolution. During the resolution phase of inflammation macrophages do not die locally: some cells emigrate by draining lymphatics whereas some remain at the site of inflammation. The major myelo-monocytic integrin αMβ2 (Mac-1, CD11b/CD18), together with two related integrins αDβ2 (CD11d/CD18) and αXβ2 (CD11c/CD18), mediate critical adhesive reactions of monocyte/macrophages. However, the roles of these adhesion receptors in control of macrophage retention at sites of inflammation and their emigration to lymph nodes are unclear. Using a mouse model of sterile peritonitis induced by thioglycollate injection, we examined the dynamics of macrophage β2 integrins during the resolution phase of inflammation. Macrophages were defined by FACS analyses as a population of cells expressing αMβ2high, αDβ2+ and CD115+. The initial population of resident β2, positive for βDβ2 and negative for αXβ2. The thioglycollate-challenged mice showed a ∼4-fold increase in macrophages on day 3 followed by a progressive decrease to normal resident cell numbers by day 13. Expression of αMβ2 on macrophages on day 3 decreased by 2.5-fold as a result of dilution of the initial population of αMβ2high resident macrophages by infiltrating blood monocytes expressing αMβ2low. However, after day 3, the density of αMβ2 on macrophages gradually increased and by day 13 returned to the high levels characteristic of resident macrophages. By contrast, expression of αDβ2 and αXβ2 on inflammatory macrophages increased by 2-fold by day 6-9 compared to that on resident macrophages and then returned to the resident levels by day 3. Thus, although the number of macrophages decreased from day 3 to day 9 by several fold, the population of macrophages which remained in the peritoneum was enriched in cells expressing the high levels of αMβ2 and αDα2. Tracking migration of fluorescently labeled peritoneal cells demonstrated that a population of macrophages which leaves the inflamed peritoneum and enters lymph nodes consists of cells expressing low levels of αMβ2 and αDβ2. These data suggested that upregulation of β2 integrins, especially αMβ2, may be responsible for the retention of macrophages in the peritoneum. Indeed, the rate of macrophage emigration from the peritoneum in the αMβ2-deficient mice was significantly higher than that in wild-type mice. The results indicate that macrophage emigration from the inflamed site is controlled by the level of integrin αMβ2 and αDβ2 with low expressors being migratory and high expressors remaining in the peritoneum. The data also highlight the importance of integrins αDβ2 and αXβ2 as specific markers of inflammatory macrophages.

Disclosures:
No relevant conflicts of interest to declare.
Rescue of Erythropoietin-Deficient Mice From Anemia by Complementation with BAC Transgene.
Authors: Yamazaki Shun , Suzuki Norio , Obara Naoshi , Pan Xiaoqing , Hirano Ikuo , Jishage Kou-ichi , Honda Kurisu , Minegishi Naoko , Yamamoto Masayuki 
Published: October 2019

Abstract
Abstract 3614
Poster Board III-550
To maintain tissue oxygen homeostasis, erythropoietin (Epo) regulates the erythropoiesis. During embryonic development Epo is produced in the liver, but after birth and throughout adulthood Epo is produced and secreted from the kidney and support definitive erythropoiesis in the bone marrow. Anemia or hypoxic stress provokes transcription of the Epo gene and the mechanisms how the Epo gene expression is activated in a tissue-specific and hypoxia-inducible manner are one of the research topics of the molecular biology or gene regulation study. Since Epo-null mice die in utero of definitive erythropoiesis failure in the fetal liver, physiological significance of the Epo production for the adult erythropoiesis remains to be clarified. We have found that transgenic reporter gene expression driven by a BAC clone containing 180-kb mouse Epo gene locus recapitulates the Epo gene expression in the liver and kidney. To examine whether the Epo gene transcriptional activity within the 180-kb BAC is sufficient to recapitulate physiological Epo production, we attempted to rescue Epo-null mice from embryonic lethality with a BAC transgene harboring a floxed allele of the Epo gene. We found that transgene-derived Epo recovered the erythropoiesis in the fetal liver of Epo-null mice and Epo-null mice with the BAC transgene were born showing a Mendelian ratio. The rescued mice showed no hematological abnormality and were fertile. In the rescued mice bleeding anemia transiently increased plasma Epo level and after recovery from anemia plasma Epo level declined similarly to wild-type mice. In an RT-PCR analysis Epo production in the liver and kidney of the rescued mice was practically equivalent to that in the wild-type counterpart, while other tissues produced only marginal amount of Epo. These results thus demonstrate that the BAC transgene contains the Epo gene regulatory domain region sufficient to fully recapitulate the endogenous Epo gene expression and to compensate for the Epo-deficiency. We also found that the Epo gene deletion from the BAC transgene by general deletor-Cre transgene provoked the Epo-null phenotype, demonstrating that conditional inactivation of the Epo gene is attained in the BAC-rescued mice. The latter system will be a precious tool to investigate adult stage-specific roles of Epo.

Disclosures:
Yamamoto: Chugai Pharmaceutical, Co., Ltd. : Research Funding.
Single Cell Phospho-Flow Analysis of Cytokine Stimulation in Human Hematopoietic Progenitors Reveals That G-CSF Acts Directly On Human Hematopoietic Stem Cells.
Authors: Gibbs Kenneth , Gilbert Penney , Weissman Irving L. , Blau Helen M. , Nolan Garry P. , Majeti Ravindra 
Published: October 2019

Abstract
Abstract 3617
Poster Board III-553
Hematopoietic stem and progenitor cells (HSPC) give rise to all blood cell lineages for the lifetime of an individual. However, their low frequency has precluded the use of traditional biochemical methods to study their signaling biology. We developed a phospho-specific flow cytometry approach that allows for the simultaneous interrogation of multiple elements of signaling networks in primary human HSPC at the single cell level without prospective purification by cell sorting. We found that HSPC contain biochemically distinct signaling subpopulations, and that, although considered quiescent, HSC from cord blood and adult bone marrow respond directly to several cytokine stimuli through multiple signaling nodes including JAK/Stat, Erk, and Akt. Surprisingly, we found that a subset of HSC respond rapidly and directly to in vitro G-CSF stimulation through Stat3 signaling. The addition of G-CSF to purified cultures of HSC significantly increased the percentage of cells that had entered the cell cycle. Moreover, when G-CSF was added to cultures of purified human HSC spatially segregated in hydrogel microwell arrays, we observed a significant increase in the frequency of HSC proliferation. These results demonstrate that contrary to the accepted paradigm, G-CSF acts directly on human HSC. Cytometric single cell characterization of the molecular behavior of HSPC in a wide variety of hematopoietic malignancies can now be applied to study the signaling behavior of other primary and rare stem cell populations.

Disclosures:
Weissman: U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties; Stem Cells Inc.: Cofounder and Director; Cellerant Inc.: Cofounder; Amgen: Equity Ownership. Nolan:BD Biosciences: Consultancy.
STAT3 Controls Neutrophil Progenitor Growth and Differentiation During Emergency Granulopoiesis.
Authors: Zhang Huiyuan , Nguyen-Jackson Hoainam , Panopoulos Athanasia , Li Haiyan S , Murray Peter J , Watowich Stephanie 
Published: October 2019

Abstract
Abstract 3619
Poster Board III-555
Granulocyte colony-stimulating factor (G-CSF) controls neutrophil production in the bone marrow under steady state conditions and during demand-driven hematopoiesis occurring in response to infection. STAT3 is a principal signaling molecule activated by the G-CSF receptor (G-CSFR). We previously reported that STAT3 has an important role in demand-driven granulopoiesis, although its cellular and molecular mechanisms have been unclear. To address this, we investigated STAT3 function in emergency granulopoiesis stimulated by G-CSF administration or infection with Listeria monocytogenes, which is restrained by the G-CSF response pathway in vivo. Our results show that STAT3-deficiency renders hematopoietic stem cells and myeloid progenitors refractory to the proliferation-inducing effects of G-CSF or Listeria monocytogenes infection. STAT3-deficient myeloid progenitors have a cell autonomous defect in G-CSF-responsive cell cycle progression and undergo delayed granulocyte maturation relative to wild type cells. To define STAT3 target pathways in granulocytic progenitors, we investigated the expression of CCAAT enhancer binding protein (C/EBP) beta, a transcription factor that is necessary for G-CSF-driven emergency granulopoiesis. We found that STAT3 directly regulates G-CSF-responsive C/EBPbeta expression by binding to Cebpb promoter. Moreover, we show that STAT3 and C/EBPbeta co-regulate c-Myc during emergency granulopoiesis. These results place STAT3 as a crucial G-CSF-responsive signal transducer during demand-driven granulopoiesis, through its regulation of critical transcription factors in developing granulocytes.

Disclosures:
Zhang: Amgen: Research Funding. Nguyen-Jackson:Amgen: Research Funding. Watowich:Amgen, Inc: Research Funding.