bluuebunny/crossref_metadata_2025_split huggingface dataset:
Defective Production, Turnover, and Secretion of the Platelet α-Granule Protein P-Selectin In Mice with Disrupted FOG1-NuRD Interaction
Authors: Wang Yuhuan , Meng Ronghua , Blobel Gerd , Marks Michael S. , Poncz Mortimer 
Published: October 2019

Abstract
Abstract 547
We reported recently that point mutations in the GATA-1 co-factor FOG-1 that disrupt binding to the NuRD chromatin remodeling complex display defects in the erythroid and megakaryocyte (MK) lineages. Homozygous FOG-1 mutant (ki/ki) mice display a Gray Platelet Syndrome (GPS)-like macrothrombocytopenia. Specifically, ki/ki platelets exhibit a paucity of α-granules and increased numbers of lysosomal-like vacuoles, reminiscent of the platelet morphology observed in human patients with GPS due to GATA-1 mutations. To understand the molecular basis for the phenotype in ki/ki platelets, we compared mRNA and protein expression of α-granule proteins in wildtype (wt) and ki/ki MK. P-selectin mRNA in ki/ki MKs was 3–5 fold reduced when compared to controls, while platelet factor 4 (Pf4), platelet basic protein (PBP) and von Willebrand factor (Vwf), were only mildly reduced. Strikingly, P-selectin protein levels were more severely reduced in ki/ki MK and platelets being nearly undetectable by Western blot and immunohistochemistry. In contrast PF4, PBP, and vWF protein levels were normal. P-selectin loss was MK-specific as it was expressed at normal levels in endothelial cells. The dramatic decrease in P-selectin protein in ki/ki MK relative to the more moderate reduction in mRNA was due to a >5-fold decrease in protein half-life, as measured by metabolic pulse-chase and immunoprecipitation experiments. This suggests that P-selectin in ki/ki MKs is largely mistargeted to lysosomes. Nevertheless, immunofluorescence microscopy and volume de-convolution analyses of ki/ki platelets showed that the remaining P-selectin resided in vesicular compartments that were distinct from Lamp1 (a lysosomal marker)-containing granules, indicating that a fraction of P-selectin remains within α-like granules. Interestingly, the reduction in P-selectin levels in ki/ki platelets was accompanied by a defect in agonist-stimulated degranulation. While stimulation of wt platelets with AYP, thrombin or phrobol myristate acetate induced α-granule, dense granule and lysosome degranulation, ki/ki platelets failed to do so or undergo platelet aggregation. In summary, the GPS-like phenotype in FOG-1/NuRD interaction-defective MKs involves reduced P-selectin mRNA production, accelerated turnover of P-selectin, abnormal granule formation, and defective degranulation in response to diverse agonists. These findings may partially explain patients having platelet α-granule deficiency associated with decreased P-selectin and selective impairment of thrombin-induced activation with GPS. Future work will determine whether similar defects occur in human GPS.

Disclosures:
No relevant conflicts of interest to declare.
Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype
Authors: Woan Karrune , Cheng Fengdong , Wang Hongwei , Rock-Klotz Jennifer , Wang Zi , Yu Yu , Seto Edward , Luetteke Noreen , Yu Xue-Zhong , Villagra Alejandro , Sotomayor Eduardo M. 
Published: October 2019

Abstract
Abstract 588
We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models.
1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100.
2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25.

Disclosures:
No relevant conflicts of interest to declare.
Long-Term Prophylaxis In Children, Adolescents and Young Adults with Von Willebrand Disease – First Results of a Cohort Study
Authors: Halimeh Susan , Kruempel Anne , Rott Hannelore , Bogdanova Nadja , Budde Ulrich , Manner Daniela , Faeser Britta , Mesters Rolf M. , Nowak-Gottl Ulrike 
Published: October 2019

Abstract
Abstract 714
In children and young adults with von Willebrand disease [vWD] replacement therapy with von Willebrand factor (vWF)/factor VIII concentrates is increasingly performed as “prophylactic” therapy. Between 2000 and 2008, 82 consecutively enrolled children, adolescents and young adults with clinically meaningful bleeding episodes [spontaneous, peri- or postoperative] were diagnosed with vWD [type 1: 42/82; type 2: 24/82; type 3: 13/82; acquired: 3/82]. Here we report results of prophylaxic VWF replacement therapy in 32 patients [female: n=15; >Tanner 2: 9/15] with VWD type 1 [n=4], 2 [n=15] and 3 [n=13] in which a prophylactic treatment regimen was performed. Decision on prophylaxis was based on i) a pre-diagnostic bleeding score, and ii) on recurrent bleeds associated with microcytic anemia after diagnosis in patients with on-demand VWD therapy. Patients were treated with Humate P® (n=8; once switched to Wilfact®) or Wilate® (n=24). Median [min-max] dose was 40 (20-47) IU/kg, 23 patients were substituted twice weekly, 7 were treated three times, and two children 4 times per week. Within a six-months-period hemoglobin concentrations returned to normal values. Median [min-max] duration of prophylaxis was 3 years [1.0-9.0]. Recurrent bleeding episodes stopped in 31/32 patients, inhibitor development occurred in one. Following a 12-months observation period the bleeding frequency/bleeding score was significantly reduced, compared to pre-prophylaxis/pre-diagnostic values. The use of prophylactic VWF replacement therapy is an effective and well tolerated treatment modality, highly beneficial for children and young adults with vWD with recurrent bleeding events on on-demand therapy.

Disclosures:
No relevant conflicts of interest to declare.
Protective and Predisposing HLA Alleles In Dutch Classical Hodgkin Lymphoma Patients
Authors: Diepstra Arjan , Huang Xin , Nolte Ilja , Hepkema Bouke , Kooistra Wierd , Visser Lydia , Poppema Sibrand , Van Den Berg Anke 
Published: October 2019

Abstract
Abstract 749
Classical Hodgkin lymphoma (cHL) is characterized by an overwhelming reactive infiltrate and the involvement of Epstein Barr virus (EBV) as a causative agent in a proportion of patients. We previously did a population-based genetic screening study of the Human Leukocyte Antigen (HLA) region in Dutch cHL patients and found a strong association between HLA class I (HLA-A1) and susceptibility to EBV+ cHL. This association might be explained by the known lack of HLA-A1 restricted T cell responses to EBV latent antigens. We now performed a case-control study to determine allele frequencies of all major HLA class I and class II genes in this cHL population. HLA genotyping of 294 cHL patients was performed by a polymerase chain reaction-based sequence-specific oligonucleotide probe hybridization (PCR-SSOP) approach in Luminex assays. The resulting single nucleotide polymorphism (SNP) data were converted into two-digit HLA typing for the HLA class I and class II genes. Phenotype frequencies of serologically defined HLA-A, HLA-B and HLA-DR blood donors were retrieved from the database of the blood bank of the University Medical Center Groningen. Allele frequency differences between these controls and cHL patients (total group and EBV+ and EBV- subgroups separately) were analyzed by Chi-square tests. In addition, we analyzed the HLA genotyping data for every PCR-SSOP probe to assess potential differences between the EBV+ and EBV- cHL group using PLINK software. Frequencies of HLA-DR4 and HLA-DR7 were significantly decreased and HLA-B5 and HLA-B37 were significantly increased in cHL as compared to the controls. In EBV+ cHL, HLA-A1, HLA-B37 and HLA-DR10 frequencies were significantly increased and HLA-A2 was decreased. An increase in HLA-B8, an allele that can present EBV peptides and that usually is in strong linkage disequilibrium with HLA-A1, was not present in these patients. In EBV- cHL, HLA-DR5 was significantly more common and HLA-DR4 was underrepresented. The SNP analysis revealed significant differences for 16 HLA-A probes between EBV+ and EBV- cHL. Eight probes that represented a high susceptibility for EBV+ cHL were specific for HLA-A1, whereas the other 8 probes correlated with a low risk for EBV+ cHL and were specific for HLA-A2. Most of the corresponding SNPs had a presumed antigenic peptide or T cell receptor binding function. In conclusion, the current study demonstrates that certain HLA alleles are protective or predisposing for cHL with specific associations for the EBV+ and EBV- cHL subgroups. The genotype analysis indicates that the complete HLA-A1 and HLA-A2 alleles are more important than individual T-cell receptor or antigen binding polymorphisms for the association with EBV+ cHL.

Disclosures:
No relevant conflicts of interest to declare.
Mitotic Spindle Dysfunction Promotes Genomic Instability In Marrow Failure
Authors: Lombardo Katie , Stumpff Jason , Parkhurst Susan , Wordeman Linda , Shimamura Akiko 
Published: October 2019

Abstract
Abstract 880
Shwachman-Diamond syndrome (SDS) is an autosomal recessively inherited disorder associated with bone marrow failure and leukemia predisposition. The majority of patients harbor biallelic mutations in the SBDS gene. The SBDS protein has been implicated in several cellular functions including ribosome biogenesis and microtubule stabilization during mitosis. We have previously found that SBDS deficiency results in multipolar spindles, centrosome amplification and aneuploidy, implicating a role for SBDS in cell division. The mechanism by which SBDS functions to ensure proper spindle assembly and DNA segregation during mitosis remains unknown. Here we present evidence that SBDS functions to promote mitotic spindle stability both by directly modifying microtubule dynamics and through a microtubule crosslinking activity. Importantly, the microtubule stabilizing effects of SBDS appear to be essential for the growth and differentiation of hematopoietic progenitor cells. Specifically, we found that SBDS deficiency resulted in shortened mitotic spindle length and decreased spindle acetylation, a marker of microtubule stability. The loss of microtubule stability in the absence of SBDS function may be due to changes in microtubule dynamics or reduction in microtubule crosslinking activity, as we found that addition of recombinant purified wild-type SBDS to polymerized microtubules in vitro increases their polymerization rate and strongly promotes microtubule bundling. Interestingly, recombinant patient-derived missense mutant SBDS proteins showed a marked decrease in their microtubule bundling ability. To assess whether spindle destabilization contributes to marrow failure, we modeled hematopoiesis in the absence of SBDS in vitro. When SBDS expression was knocked down in human CD34+ cells, proliferation, differentiation, and hematopoietic progenitor colony formation were impaired, consistent with published data on primary marrow from SDS patients. The addition of taxol at concentrations that significantly impaired hematopoiesis in control CD34+ cells resulted in stable to improved hematopoiesis in the SBDS-deficient CD34+ cells. Based on these data, we hypothesize that spindle destabilization by SBDS loss promotes genomic instability, which in turn, contributes to marrow failure and leukemia predisposition.

Disclosures:
No relevant conflicts of interest to declare.
Cytokines Instruct Hematopoietic Lineage Choice
Authors: Schroeder Timm 
Published: October 2019

Abstract
Abstract SCI-13
The molecular control of multipotent cell lineage choice is one of the central questions in stem cell biology. Since decades, it is controversial whether cell extrinsic factors can actively influence lineage decisions of hematopoietic cells (instructive model). Alternatively, they may only allow the survival and proliferation of already unilineage-restricted cells, which have chosen one lineage by cell-autonomous mechanisms (selective / permissive model). One major reason for this longstanding controversy is the fact that hematopoiesis is usually followed by analyzing populations of cells - rather than individual cells - at very few time points of an experiment and without knowing their individual identities. However, the alternative lineage choice models can only be distinguished by constantly observing individual cell fates throughout differentiation.We therefore applied novel computer aided culture, imaging and cell tracking approaches to follow the fate of individual cells over many days. Analysis of primary mouse multipotent hematopoietic cell differentiation finally provided proof for the lineage instructive action of hematopoietic cytokines. This finding is of fundamental importance for our understanding of the molecular control of lineage choice. It shows that cell extrinsic signaling influences the - currently unknown - cell intrinsic molecular lineage choice and commitment machinery. Our novel approaches now allow identification of the signaling pathways mediating lineage instruction and their targets with the required precision. Moreover, we are developing technology for the quantification of lineage specific transcription factor proteins in individual living cells. This novel kind of continuous, live and quantitative molecular and cellular data is used for modeling the control of cell fates through integration of cell extrinsic signals with cell intrinsic molecular states.

Disclosures:
No relevant conflicts of interest to declare.
Origin of the Myeloma Stem Cell
Authors: Matsui William 
Published: October 2019

Abstract
Abstract SCI-4
Increasing evidence suggest that most human cancers consist of phenotypically and functionally heterogeneous cell types. Moreover, in some tumors, phenotypic-functional relationships exist and the capacity for clonogenic growth and self-renewal may be restricted to specific cell types. In multiple myeloma, malignant plasma cells make up the vast majority of tumor cells, phenotypically characterize the disease, and are responsible for clinical symptoms. However, myeloma plasma cells also appear terminally differentiated similar to their normal counterparts and incapable of long-term proliferation. Similar to other laboratories, we have identified minor populations of B cells that share the identical immunoglobulin gene rearrangements as the malignant plasma cells in most myeloma patients. We have further studied the functional capacity of these clonotypic B cells and found that they have ability to produce ectopic tumor growth both in vitro and in immunodeficient mice that can recapitulate human disease.1,2 Moreover, these cells are relatively resistant to several agents currently used to treat multiple myeloma suggesting that they have the capacity to persist following treatment and are responsible for tumor regrowth and disease relapse. In order to inhibit these drug resistant cancer stem cells, we have studied cellular pathways that regulate the self-renewal of normal adult stem cells and found that several, including the developmental pathway Hedgehog, are potential therapeutic targets.3 Finally, as these concepts are translated to the clinic, several challenges have emerged including the ability to detect activity against myeloma stem cells that make up only a minority of the overall tumor burden.4 To this end, we have begun to develop biomarker strategies to serially quantify myeloma stem cells in patients undergoing treatment and will examine their utility as clinical endpoints.
1. Matsui W, Huff CA, Wang Q, et al. Characterization of clonogenic multiple myeloma cells. Blood. 2004;103:2332–2336.
2. Matsui W, Wang Q, Barber JP, et al. Clonogenic Multiple Myeloma Progenitors, Stem Cell Properties, and Drug Resistance. Cancer Research. 2008;68:190–197.
3. Peacock CD, Wang Q, Gesell GS, et al. Hedgehog signaling maintains a tumor stem cell compartment in multiple myeloma. Proceedings of the National Academy of Sciences, USA. 2007;104:4048–4053.
4. Huff CA, Matsui W, Smith BD, Jones RJ. The paradox of response and survival in cancer therapeutics. Blood. 2006;107:431–434.

Disclosures:
Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding. Off Label Use: Rituximab in multiple myeloma.
Stability of Self-Renewal in Hematopoietic Stem Cells
Authors: Iscove Norman N. 
Published: October 2019

Abstract
Abstract SCI-42
For many years a distinction was drawn between prospectively separable murine HSC populations with long-term, essentially permanent reconstituting potential (LT-HSC), versus HSC populations yielding short-term engraftment lasting only 4 – 6 weeks after transplantation (ST-HSC). Recent work based on transplantation of single cells shows that highly purified populations of LT-HSC prepared by standard sorting parameters consist in fact predominantly of a distinct, newly recognized class of intermediate- term reconstituting cells (IT-HSC) whose grafts endure longer than short-term HSC but also eventually fail (1). IT-HSC are separable from long-term reconstituting cells on the basis of expression of more alpha2 integrin and less SLAM150. Crucial to recognition of the distinction between LT- and IT-HSC are the endpoints used to evaluate reconstitution. If blood erythroid or myeloid reconstitution is measured, IT reconstitution is readily distinguished by the disappearance of these elements by 16 wk post-transplant. If instead reconstitution is measured simply by presence of blood leukocytes of donor origin, which in the mouse are almost entirely lymphocytes, the distinction is not made because lymphoid elements persist even in fading IT clones to 24 wk or beyond. The observations imply a need for reinterpretation of most of the published descriptions of the biology and gene expression profiles previously attributed to LT-HSC but in fact derived from analysis of populations that consisted mainly of IT-HSC. The capacity now to separate LT- from IT-HSC creates new opportunities for probing the mechanisms that specify and sustain long term function in the former but not the latter. 1. Benveniste P, Frelin C, Janmohamed S, Barbara M, Herrington R, Hyam D, Iscove NN. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. 2010;6:48–58

Disclosures:
No relevant conflicts of interest to declare.
Chemotherapy-Resistant T Cells
Authors: Turtle Cameron J. 
Published: October 2019

Abstract
Abstract SCI-51
The mechanisms that maintain human T cell memory during normal and perturbed homeostasis are not fully understood. Patients undergoing multiple cycles of cytotoxic chemotherapy infrequently develop infections with viruses that are controlled by memory CD8+ T cells either during or after the period of profound lymphocytopenia, suggesting that some memory cells survive chemotherapy and provide protective immunity. We recently identified a subset of quiescent human CD161hi CD8+ T cells characterized by the capacity to rapidly efflux cytotoxic drugs and the fluorescent dye, rhodamine 123 (Rh123), which endowed them with the ability to survive chemotherapy in vitro and in vivo and to contribute to the recovery of lymphocyte numbers in patients receiving chemotherapy. Rh123 effluxing CD161hi memory CD8+ cells were identified in both the CD62L+ central memory (CM) and CD62L- effector memory (EM) pools, and were characterized by uniform expression of CD28, high levels of bcl-2, and low levels of granzyme B and perforin. CD161 and CD62L expression allowed partitioning of the CD8+ memory compartment into four phenotypic subsets with distinct transcriptional profiles and functional properties. Gene expression profiling of Rh123 effluxing CD161hi memory CD8+ cells in both CM and EM pools revealed a type 17 differentiation program with high levels of RORC, RORA, IL23R, CCR6, KLRB1, and CCL20, compared with CD161lo CM and EM cells. Transcriptional profiling also revealed alterations in the T cell receptor (TCR) signaling pathway in CD161hi cells that limits their capacity to proliferate and secrete cytokines, including IL-17, after TCR stimulation. Despite stringent regulation of TCR signaling in type 17-programmed CD161hi cells, proliferation and cytokine secretion could be induced by ligating CD28 to provide costimulation, and by IL-1β, IL-12, IL-18 or IL-23. The nature of the second and third signals provided to CD161hi T cells revealed plasticity in this subset of cells and determined whether the CD161hi progeny derived after expansion had the capacity to secrete IL-17, or had differentiated to Tc1-like cells with restored capacity to proliferate to TCR ligation alone. Thus, a surprisingly large fraction of the human CD8+ T cell memory pool is comprised of CD161hi CD8+ T cells that express a type 17 transcriptional program and possess drug efflux capacity that allows them to survive cytotoxic chemotherapy and contribute to recovery of lymphocyte numbers. The stringent regulation of CD8+CD161+ memory T cells in normal physiology also suggests that this subset may have a potential role in the pathogenesis of IL-17-mediated inflammatory diseases.

Disclosures:
No relevant conflicts of interest to declare.