bluuebunny/crossref_metadata_2025_split huggingface dataset:
Analyzing the Stepwise Developmental Pathway From ES/IPS Cells to Functional Mature Erythrocytes.
Authors: Niwa Akira , Fukatsu Tomoki , Umeda Katsutsugu , Kato Itaru , Sakai Hiromi , Morishima Tatsuya , Okita Keisuke , Yamanaka Shinya , Heike Toshio , Nakahata Tatsutoshi 
Published: October 2019

Abstract
Abstract 2534
Poster Board II-511
Induced pluripotent stem (iPS) cells, reprogrammed somatic cells with embryonic stem (ES) cell–like characteristics, are generated by the introduction of combinations of specific transcription factors. Despite the controversy surrounding the gene manipulation, it is expected that iPS cells should contribute to regenerative medicine, disease investigation, drug screening, toxicology, and drug development in future. In the fields of hematology, iPS cells could become used as a new feasible source for transplantation therapy without immunological barrier and for the investigation of various kinds of hematological defects. Previous studies on ES / iPS cells have already demonstrated that they can develop into various lineages of hematopoietic cells including erythrocytes following the similar processes occurred in embryo and fetus. However, it is important to establish the more effective system for developing functional blood cells. Here we present the methods for selectively inducing mature red blood cells from ES / iPS cells in vitro, and show the functional equality of them to natural blood cells. First, Flk1+ mesodermal progenitors were derived from ES / iPS cells on OP9 stromal cells at an efficacy of more than 50% and collected by fluorescence activated cell sorter. Then, those sorted cells were cultured in the presence of exogenous erythropoietin and stem cell factor. They highly selectively developed into erythroid lineages including enucleated red blood cells. Sequential FACS analysis using the antibodies against transferrin receptor CD71 and erythroid specific antigen Ter119 in combination with DNA staining dye Hoechst 33342 demonstrated that ES / iPS cell-derived erythropoiesis in our system follow the normal erythroid developmental pathway occurred in vivo. RT-PCR and Western blot analyses proved the expression of heme biosynthesis enzymes on the produced erythrocytes. Finally, the oxygen dissociation curve showed that ES / iPS cell-derived erythroid cells are functionally virtually equivalent to natural red blood cells as oxygen carriers. Taken together, our system can present the effective methods of investigating the mechanisms of normal erythropoiesis and the deficits in syndromes with disrupted red blood cell production.

Disclosures:
No relevant conflicts of interest to declare.
Interferon-Gamma Is a Critical Regulator of the Hematopoietic Stem Cell Response to Chronic Infection.
Authors: Baldridge Megan T , King Katherine Y , Weksberg David C , Boles Nathan C , Goodell Margaret A 
Published: October 2019

Abstract
Abstract 2549
Poster Board II-526
Hematopoietic stem cells (HSCs) are the progenitors of all blood cells including the effector cells of the immune system. HSCs remain dormant during most of the life of an organism, but can be activated by stress conditions to replenish peripheral blood cells. Infection is a common stress that leads to the consumption of immune cells, and the bone marrow must compensate for increased immune system demands by increasing production of downstream progeny during chronic infection. How this compensation is accomplished is poorly understood.
We investigated the HSC response to chronic infection using an in vivo mouse model of Mycobacterium avium (M. avium) infection. We found that infection stimulates changes in multiple hematopoietic progenitor compartments to maintain peripheral blood homeostasis. Infection activates the HSC itself to differentiate and proliferate, as measured by BrdU incorporation. Since interferon-gamma (IFNg) levels are high during M. avium infection, we hypothesized that this cytokine may be an important mediator of the HSC response. We confirmed that IFNg levels in the bone marrow, where the HSC resides, are elevated in infected mice. Consistent with our hypothesis, we found that the proliferative response to M. avium infection is abrogated in Stat1-deficient and IFNg-receptor-deficient mice, but is unaffected in IFNa-receptor-deficient mice, indicating that the HSC response to M. avium infection is critically mediated by IFNg.
To determine if IFNg alone is sufficient to stimulate the HSC, we tested and confirmed that HSCs express the IFNg receptor, and are thus capable of responding directly to IFNg signaling. We next established that IFNg stimulates expression changes in known IFN regulatory molecules such as Stat1 and Irf9 in the HSC. In agreement with our findings using M. avium, IFNg alone causes HSCs to proliferate both in vitro and in vivo, as assessed by BrdU incorporation. Furthermore, we found that HSCs from an IFNg-deficient mouse model exhibit decreased baseline proliferation and superior engraftment in a competitive transplantation assay. Thus, IFNg participates in maintaining balance between quiescence and proliferation of HSCs even in the absence of infection.
Despite historical data that IFNs are suppressors of bone marrow function, this report signifies the first in vivo evidence that IFNg has a powerful and direct effect on HSC proliferation. Thus it appears that IFN regulation of the bone marrow is a complex, and perhaps context-dependent, phenomenon. These findings inform our understanding of HSC biology as well as our ability to treat patients affected by chronic infections, such as HIV and tuberculosis, illnesses that can be complicated by bone marrow suppression.

Disclosures:
No relevant conflicts of interest to declare.
Short-Term Hematopoietic Stem Cells (ST-HSC) Have Full Long-Term Capacity with Sustained but Reduced Potential Compared with LT-HSC.
Authors: Colvin Gerald A. , Berz David , Quesenberry Peter J. , Papa Elaine , Liu Liansheng 
Published: October 2019

Abstract
Abstract 2550
Poster Board II-527
We evaluated the hypothesis that there was a homing defect between long-term (LT) hematopoietic stem cell (HSC) (KLS-Flk2-) and ST-HSC (KLS-Flk2+) that explained differences in engraftment potential and duration. Short-term HSC by definition have limited self-renewal capacity, generally described as giving rise to lymphohematopoiesis for 4–12 weeks before senescence. We performed three large animal engraftment studies into lethally ablated mice (950cGy split dose) looking at engraftment of both ST and LT-HSC cells delivered via intravenous, intraperitoneal and intra-femoral route. Two-hundred or 500 cells derived from B6/SJL mice were administered to each animal along with 300,000 recipient (C57/BLK) whole bone marrow cells for radioprotection following prior published studied [PNAS:98;14541, Stem Cells 24:1087] with optimization of flourochromes for better discrimination with our Cytopeia sorter. The animals were serially transplanted after eight months or one year to secondary recipients. In our hands, the ST-HSC engrafted animals did not lose chimerism over time. Review of the literature revealed that there were not confirmatory studies from those published from the initial one publication describing the ST-HSC. We found the ST-HSC were not short-term and persisted for one year in primary recipients and at least 3 months in secondary recipients. Engraftment kinetics favored LT-HSC over ST-HSC with engraftment examples at one year of 62% compared with 30% respectively when administered intravenously, 10% verses 4% given intra-femoral and 0.5% verse 0.3% given intraperitoneal. Chimerism was on average 50% better for the LT-HSC when compared with the ST-HSC and was irrespective of route proving that the differences seen are not due to homing deficiency but rather intrinsic differences in the two stem cell pools. Prior studies gave a maximum of 100 cells. Cell number was purposely increased for better differentiate of subtle differences in engraftment kinetics for statistical reasons. To avoid contamination of Flk2+ cells in the Flk2- cohort and vise-versa, discrimination of the gates were enhanced from that which was published prior. Double sorting of the cells confirmed that there was no appreciable cross contamination but obviously we cannot totally rule that out as a potentially confounding factor. In conclusion we found that ST-HSC as described have long-term capacity with intrinsic differences in engraftment potential that is not driven by a homing defect.

Disclosures:
No relevant conflicts of interest to declare.
Myeloid Translocation Gene (MTG)-16 Binds Intracellular Domains of Notch Receptors to Coordinate Notch-Dependent Cell Fate Specification.
Authors: Engel Michael E , Nguyen Hong , Mariotti Jolene , Hunt Aubrey A , Hiebert Scott 
Published: October 2019

Abstract
Abstract 257
The Notch signaling pathway regulates gene expression programs to control the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of Notch receptors is cleaved by the γ-secretase complex and then translocates to the nucleus. There it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional co-repressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors. The MTG16 NHR3 domain contributes to CSL binding, while N-ICD binding sites lie within the PST1 and PST2 domains. The Notch intracellular domain disrupts the MTG16—CSL interaction, suggesting a pivotal role in regulating the Notch transcription complex. Using co-culture of Lin-/Sca-1+/c-Kit+ (LSK) cells on OP9-DL1 stromal cells, we show that ex vivo fate specification in response to Notch signal activation is altered in Mtg16 (−/−) hematopoietic progenitors. While Notch signal activation specifies lymphoid fate in Mtg16 (+/+) LSK cells, Mtg16 (−/−) LSK cells display cell surface marker expression reminiscent of myeloid differentiation. We used this lineage allocation assay to assess the contribution of MTG16 to cell fate determination. Lymphoid fate specification is restored by MTG16WT expression in Mtg16 (−/−) LSK cells. However, an MTG16 mutant deficient in N1-ICD binding is defective in this assay, suggesting this region is important to Notch-dependent lineage allocation. These data suggest that MTG family proteins interface with critical components of the Notch transcription complex and intimate a functional relationship between MTG proteins and Notch signaling in normal and malignant hematopoiesis.

Disclosures:
No relevant conflicts of interest to declare.
Measurement of Urine Pyridinoline and Deoxypyridinoline as Markers of Increased Bone Degradation in Sickle Cell Disease.
Authors: Nur Erfan , Mairuhu Willem , Brandjes Dees P. , van Zanten Ton , Biemond Bart J. , Otten Hans-Martin 
Published: October 2019

Abstract
Abstract 2572
Poster Board II-549

Introduction:
Sickle cell disease (SCD) is commonly manifested through skeletal involvement. Besides the characteristic acute musculoskeletal pain, SCD is also associated with chronic skeletal complications such as osteopenia and osteoporosis. During bone resorption, the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are released into circulation with subsequent urinary excretion. Measurements of urinary PYD and DPD could serve as valuable tools in detecting osteoporosis in the follow-up of SCD patients but perhaps also in determining the severity of bone infarction during painful crises. Therefore we compared urinary concentrations of PYD and DPD of SCD patients during asymptomatic state and painful crisis with those of race- and age-matched healthy controls.

Methods:
Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in SCD patients both during asymptomatic state (n=38) and painful crisis (n=27) and healthy controls with normal HbA hemoglobin (n=25) using high performance liquid chromatography (HPLC).

Results:
PYD and DPD concentrations were higher in asymptomatic SCD patients compared to controls ((54.8 (41.5–68.6) vs. 44.1 (37.7–49.9),P=0.005 and 11.6 (9.3–15.2) vs. 8.5 (6.8–10.4),P=0.004 respectively), with further increments during painful crisis (63.3 (51.8–76.0),P=0.041 and 15.3(13.0–21.5),P=0.003 respectively). In the asymptomatic patients levels of PYD and DPD were significantly correlated to the degree of hemolysis.

Conclusion:
In sickle cell patients bone resorption is increased and significantly correlated to the degree of hemolysis, compatible with their susceptibility to osteopenia and osteoporosis. Measurement of pyridinoline and deoxypyridinoline could have additional value as biomarkers of osteoporosis in SCD. During painful crises a further increment in bone degradation was observed.

Disclosures:
No relevant conflicts of interest to declare.
Plasma Protein Oxidation and Triglyceride Levels in Sickle Cell Anemia.
Authors: Oztas Yesim , Unal Selma , Ozgunes Nuriman 
Published: October 2019

Abstract
Abstract 2578
Poster Board II-555

Introduction:
A mutant and unstable hemoglobin is characteristic in sickle cell anemia (SCA) with an increased formation of methemoglobin, hemichrome and haemin. Increased oxidative stress and altered lipid balance in the plasmas of SCA patients have been documented previously. Lower total plasma cholesterol (TC) and higher-than-avarage triglyceride (TG) levels have also been reported in SCA patients compared to healthy controls. Plasma phospholipids (PL) were normal or increased according to various studies. However, to our knowledge only a few reports documented the plasma protein oxidation in SCA patients. In this study protein oxidation, lipid levels, bilurubin levels and their probable correlation were investigated in the plasmas of SCA patients, carriers and healthy controls.

Patient and Methods:
15 SCA patients, 10 carriers and 10 healthy controls were included in the study. The patients had not been transfused and hospitalized and had no crisis for the last three months. The oxidation status of plasma and its proteins were detirmined by total sulfhydryl (T-SH) and protein carbonly levels respectively. TC, TG and bilurubin levels were detirmined in the plasma and PL levels were detirmined in the lipid extract from plasma.

Results:
SCA patients had lower T-SH levels compared to controls (p=0.003) and higher protein carbonyl levels compared to carriers (p=0.001) and healthy children (p=0.032). T-SH values were 417.41±77.90 μM, 457.46±106.26 μM, 522.59±77.18 μM and protein carbonyl levels were 0.93±0.15 nmol/mg protein, 0.74±0.08 nmol/mg protein , 0.82±0.05 nmol/mg protein for patients, carriers and controls respectively. Significantly lower mean TC levels than controls (p<0.0001) and higher TG levels than carriers (p=0.001) were observed in SCA patients. TC levels were 116.67±23.60, 127.53±22.40, 151.62±16.53 and TG levels were 89.31±23.50 mg/dl, 60.94±9.18 mg/dl, 82.96±21.06 mg/dl for patients, carriers and controls respectively. Phospholipid levels were not different between the groups. TG levels were positively correlated to protein carbonyl levels (p=0.002) and to bilirubin levels (p=0.033). Bilurubin levels and protein carbonyl levels are also positively correlated (p<0,0001).

Conclusions:
Alteration of lipid balance besides protein oxidation and hemolysis were observed in the plasmas of SCA patients compared to the carriers and healthy group. The positive correlation between total bilurubin, TG and carbonyl levels indicates that oxidative stress may affect the SCA plasma extensively, ie. lipids, proteins and erythrocytes are all affected.

Disclosures:
No relevant conflicts of interest to declare.
Evidence for a Pathological Ratio of CD44s/CD44v6 in Chronic Lymphoctic Leukemia (CLL) Cells.
Authors: Hertweck Magdalena , Erdfelder Felix , Filipovich Alexandra , Uhrmacher Sabrina , Gandhirajan Rajesh Kumar , Gehrke Iris , Paesler Julian , Razavi Regina , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl Anton 
Published: October 2019

Abstract
Abstract 2628
Poster Board II-604
Chronic Lymphocytic Leukemia (CLL) is the most common adult-onset leukemia in the western world. It is characterized by an accumulation of functionally incompetent monoclonal CD5+ B-lymphocytes and an increased resistance to apoptosis. CD44 is a cell surface transmembrane glycoprotein which has more than ten isoforms generated by alternative splicing. It is expressed on different cells as e.g. cells of lymphohematopoietic origin, epithelial cells and keratinocytes. CD44 is involved in lymphocyte activation, recirculation and homing. It acts as an adhesion molecule and plays an important role in angiogenesis, cell proliferation, differentiation and migration. One of these variants, CD44 variant 6 (CD44v6), is shown to be responsible for an increased apoptotic resistance in Jurkat cells. Our aim was to show that i) CD44 is overexpressed in CLL cells, ii) that this overexpression is regulated by the WNT pathway, which is known to be constitutively active in CLL cells, and iii) that this overexpression is a possible cause for the increased resistance to apoptosis in CLL cells. Peripheral blood of CLL patients was incubated with specific antibodies directed against CD5, CD19, CD44 standard (CD44s) and CD44v6 and measured by flow cytometry. We measured the mean fluorescence intensity of CD44s and CD44v6 on CD5/CD19 double positive CLL cells. The patient pool included patients with poor prognosis ( ZAP70+, CD38+) as well as patients having a good prognosis (ZAP70-, CD38-). CD19 positive B-cells from healthy volunteers, aged from 18 to 60, were analyzed for their expression of CD44s and CD44v6 as well. Expression levels of CD44s in 37 CLL samples and of CD44v6 in 29 CLL samples were measured and compared those of 22 healthy volunteers. We found a high expression of CD44s in CLL-cells (mean: 368.02) but an even higher expression in healthy B-cells (mean: 538.8; p<0.05). In contrast, the CD44 variant 6 was expressed five- to six-fold higher in CLL cells compared to healthy B cells (mean CLL cells: 20.0; mean healthy B cells: 3.6; p<0.001). Taken together, we found that there is a significantly altered ratio of CD44s/CD44v6 expression in CLL cells when compared to normal B cells. As CD44v6 is known to be oncogenic this phenomenon may contribute to the malignant phenotype of these cells. Currently, we are investigating the effect of WNT3a, a WNT signaling initiator, on the expression of CD44v6 in CLL cells and the effect of an anti human CD44v6 antibody on the apoptosis rate in CLL-cells.

Disclosures:
Hallek: BayerScheringAG: Honoraria, Research Funding.
Clinical Implementation of a Novel High-Throughput Screen of Primary Leukemia Cells to Customize Therapy for Relapsed/Refractory Acute Myeloid Leukemia (AML).
Authors: O'Dwyer Kristen M , Shum David , Heaney Mark , Brentjens Renier J. , Maslak Peter , Jurcic Joseph G , Djaballah Hakim , Frattini Mark G. 
Published: October 2019

Abstract
Abstract 2657
Poster Board II-633
For most patients with relapsed and refractory acute myeloid leukemia, therapeutic success is unpredictable with current cytotoxic chemotherapeutic regimens. Moreover, multiple courses of therapy have been shown to result in co-morbid conditions, which can often preclude the patient from being able to receive the potentially curative therapy of allogeneic bone marrow transplantation. With the aim of identifying a successful chemotherapy regimen for these patients, based on patient-specific myeloid blast cell chemo-sensitivities, we have developed a high-throughput screening assay that utilizes primary (patient-derived) leukemia cells and tested for chemo-sensitivity against a panel of established chemotherapeutic agents as well as novel agents. In our laboratory, we were able to identify in vitro resistance against the various drugs already clinically administered, including cytarabine, etoposide, and anthracyclines. Importantly, we were able to show in vitro sensitivity to chemotherapy agents that had not been previously administered. The index patient was a 32-year-old woman with primary refractory acute myeloid leukemia who had received six different therapeutic regimens prior to our testing, all of which demonstrated in vitro resistance using our assay. The blasts were sensitive, however, to 6-thioguanine with an inhibitory concentration (IC50) of 70 nM. Based on the in vitro data, the patient began a combination treatment regimen containing oral 6-thioguanine as her white blood cell (WBC) count had increased to greater then 50,000 cells/ml on her previous therapy. Following a maintenance regimen with these agents, her circulating blast count decreased to less then 10,000 cells/ml, and she had partial recovery of the neutrophil count, resulting in a decrease in the overall leukemia burden. This result was not seen with her previous treatment regimens. Importantly, the majority of this therapy was administered in the outpatient setting. Subsequently, we have collected specimens from more than ten patients with AML and used this screening assay to predict their response to various chemotherapeutics. In each case, we have accurately predicted the in vivo clinical response (both sensitivity and resistance) to different chemotherapeutic agents. This demonstrates that the technology currently being used to screen drug therapy for patients with relapsed and refractory AML is clinically useful and has a potential role in designing individualized drug treatment regimens.

Disclosures:
No relevant conflicts of interest to declare.
BACH2 Regulates Foxp3 Expression in Umbilical Cord Blood CD4+/45RA+ T-Cells.
Authors: Lesniewski Mathew Lawrence , Haviernik Peter , Weitzel R. Patrick , Greco Nicholas J , Paul Phil , Laughlin Mary L 
Published: October 2019

Abstract
Abstract 2677
Poster Board II-653
There are clear differences in the response of umbilical cord blood (UCB) T-cells with regards to improved immune tolerance after allogeneic stem cell transplant and lower incidence of acute graft versus host disease (aGvHD) which have not been determined at a cellular level. Activated, UCB CD4+ T-cells express less TNF-α and IFN-γ then their adult complement. At the protein level, NFAT1, a transcription factor known to regulate the expression of FoxP3, TNF-α, and IFN-γ is attenuated in resting UCB CD4+ T-cells. In addition, activated NFAT1 temporal response to stimulation is delayed by ∼6 hours. However our analysis by gene array did not see any attenuation of FoxP3 mRNA compared to normal naïve adult peripherial CD4+/CD45RA+ T-cells. The absence of a key transcription factor activity having little or no effect on subsequent downstream gene expression is normally a strong indicator of compensatory regulatory factor(s). Previous observation demonstrated that the transcription factor BACH2 plays a compensatory regulatory role in the up-regulation of IL-2 in UCB CD4+/CD45RA+ T-cells. Studies of the effect of BACH2 inhibition on FoxP3 expression in UCB CD4+/CD45RA+ T-cells were conducted. Loss of BACH2 expression correlated with the loss of FoxP3 mRNA and protein expression in transient siRNA knockdown experiments as observed by qRT-PCR, and validated by Western Blot. Using cross-linking chromatin immunoprecipitation (ChIP), we show that BACH2 binds to the proximal promoter and within intron 1 of FoxP3 gene in naïve UCB CD4+/CD45RA+ T-cells and within the intron 1 of adult CD4+/CD45RA+ T-cells. This report summarizes studies demonstrating a functional role for BACH2 in UCB CD4+/CD45RA+ T-cells. Transient knockdown of BACH2 by siRNA results in decreased FoxP3 expression at both at mRNA and protein level prior to CD3/CD28 stimulation. There were several putative BACH2 DNA binds sites both associated and unassociated with NFAT1 sites. ChIP analysis demonstrated that BACH2 co-precipitated with 2 separate sites surrounding exon 1 of the FoxP3 gene in unstimulated UCB CD4+/CD45RA+ T-cells, but with only one site in unstimulated AB CD4+/CD45RA+ T-cells. In addition, the bZip protein sMafK was observed co-immunoprecipitated with the same DNA regions in UCB and AB CD4+/CD45RA+ T-cells.

Disclosures:
No relevant conflicts of interest to declare.