bluuebunny/crossref_metadata_2025_split huggingface dataset:
The ZZ Domain of Sequestosome-1/p62 Plays An Important Role In Stromal Cell Support of Myeloma Cell Growth and Osteoclast Formation
Authors: Teramachi Jumpei , Windle Jolene J. , Roodman David , Kurihara Noriyoshi 
Published: October 2019

Abstract
Abstract 128
The marrow microenvironment provides a critical supportive role in myeloma (MM) and enhances both tumor growth and bone destruction through activation of multiple signaling pathways in stromal cells. We reported that sequestosome 1 (p62) plays a key role in the formation of signaling complexes that result in NF-kB, p38 MAPK, and PI3K activation in the marrow microenvironment of patients with MM. These results suggest that p62 is a potential therapeutic target for blocking the supportive effects of the marrow microenvironment in MM. The goal of this study was to identify the domains of p62 responsible for increased MM cell growth and osteoclast (OCL) formation mediated by NF-kB and p38 MAPK signaling, as a means to develop inhibitory peptides/molecules as potential therapeutic agents for MM. To pursue this objective, we generated deletion constructs of p62 that lacked specific p62 domains: ΔSH2, ΔPB1, ΔZZ, Δp38, ΔTBS and ΔUBA domains. We then transfected these constructs into a p62-knockout (KO) stromal cell-line we established from p62-KO mice and examined their RANKL, IL-6, and VCAM-1 expression induced by TNF-a. GFP-labeled MM1.S myeloma cells or normal CFU-GM, a source of OCL precursors, were then co-cultured with the p62-KO cells transduced with the different p62 deletion constructs and compared to wild type (WT) stromal cells. IL-6 production and VCAM-1 expression induced by TNF-a was 50% lower in non-transduced p62-KO stromal cells compared to WT stromal cells. Further, in contrast to WT cells, RANKL was not induced by TNF-a in the p62-KO stromal cell-line, and OCL formation in co-cultures of p62-KO stromal cells with CFU-GM was very low. Transduction of p62-KO stromal cells with the ΔSH2, ΔPB1, Δp38 and ΔUBA constructs restored stromal cell support of MM growth, VCAM-1 and IL-6 production. However, p62-KO stromal cells transduced with the ΔZZ construct did not increase MM cell growth, or increase IL-6 and VCAM-1 expression, or fully restore the capacity of the p62-KO stromal cells to support OCL formation. These results demonstrate that the ZZ domain of p62 is required for stromal cell support of MM cell growth, increased IL-6 and VCAM-1 expression, and OCL formation. These results suggest that dominant negative constructs or small molecules that target the ZZ domain of p62 should block p62 function and inhibit support of MM cells and OCL formation by the marrow microenvironment.

Disclosures:
Roodman: Amgen, Celgene, Acceleron & Millennium: Consultancy.
Chronic Lymphocytic Leukemia (CLL) Signaling Profiling Identifies CLL-Specific Signaling Impairment That Discriminates CLL B-Cells From Normal Circulating B Cells
Authors: Palomba M. Lia , Piersanti Kelly , Zelenetz Andrew D , Brink Marcel R.M. van den , Altan-Bonnet Gregoire 
Published: October 2019

Abstract
Abstract 1374
CLL is the most common leukemia in the Western world. It is characterized by the clonal expansion of CD5+/CD19+ B cells, expressing surface immunoglobulins (sIg), most often of the IgM subset. Clinically, it can either manifest as an indolent disease with little impact on the lifespan of a subset of patients or as an aggressive, highly drug-resistant disease with lethal outcome but all possible scenarios in between these extremes are routinely observed. The degree of somatic mutations of the sIg has been shown to affect survival; patients expressing highly mutated sIg have a better prognosis than patients whose sIg are less than 2% different from germline sequences. Therefore, it is intuitive that signaling through the sIg, or B-cell receptor (BCR), must have a role in determining the fate of the CLL cells, veering the intracellular machinery towards proliferation or apoptosis.
We have systematically investigated the downstream events caused by BCR stimulation via crosslinking of the sIg in a series of about 60 heterogeneous CLL patients and 15 normal donor peripheral blood mononuclear cells (PBMCs). Using multiplexed phosphoflow cytometry we were able to assess the intensity of multiple signaling events at specific stages of the BCR signaling pathway. By combining phosphospecific staining and classic immunophenotyping we were also able to evaluate signaling intensity at the at the single cell level as well as correlating the signal intensity of CLL-specific surface markers to that of the phosphoproteins analyzed. We used principal component analysis to combine phosphospecific staining within individual cells and generate a variable that discriminates efficiently between CLL and PBMCs B-cells.
We found that signaling properties of CLL B-cells were impaired when compared to normal B cells from healthy donors, both at the level of upstream members of the BCR signaling pathway (pBLNK, pSYK, pZap70, pBTK, pPLCg2) and downstream components such as pp38 and pERK. However, within the cohort of CLL samples we identified a small subpopulation in which signaling proceeded as well or more efficiently than in normal B cells. We also used our principal component variable to quantify the heterogeneity of signaling responses within populations of CLL B-cells and correlate them with clinical parameters that are known prognostic indicators in CLL.
In conclusion, multiplexed phosphoflow cytometry of individual CLL cells provides a novel and highly specific method to determine the use of signaling pathways (such as BCR-mediated) in comparison to normal B cells. Our studies indicate a significant decrease in BCR signaling of CLL B cells, which involves both upstream and downstream components.

Disclosures:
van den Brink: Cytheris: Research Funding.
Toxico-Kinetics of Recombinant VWF In Non-Human Primates and Rabbits
Authors: Muchitsch Eva-Maria , Dietrich Barbara , Rottensteiner Hanspeter , Gritsch Herbert , Ehrlich Hartmut J. , Turecek Peter , Schwarz Hans Peter 
Published: October 2019

Abstract
Abstract 1414
Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. We showed previously by in vitro and ex vivo studies that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine and rat ADAMTS13, whereas ADAMTS13 from rabbits and cynomolgus monkeys is able to cleave human rVWF.
Here we tested the pharmacological behavior of human rVWF in rabbits and cynomolgus monkeys. The animals were infused once with different doses of human rVWF. VWF antigen rose sharply in a dose-dependent manner (∼25 IU/ml VWF:Ag for the highest dose, 15 min after injection) and then declined gradually (∼7 IU/ml VWF:Ag for the highest dose, 18 hours after injection). By contrast, the ADAMTS13 activity did not show relevant changes throughout the entire test period in the rabbit or in the monkey samples, indicating that an excess of intravenously administered rVWF as the substrate does not exhaust its enzyme ADAMTS13. Most importantly, neither rabbits nor cynomolgus monkeys showed any exaggerated pharmacological or toxic effects upon rVWF administration. Even the administration of 14 daily doses of rVWF to cynomolgus monkeys did not lead to any toxicological effect.
Our data indicate that rabbits and cynomolgus monkeys, two species with a sufficient rVWF cleavage capacity by endogenous ADAMTS13, are appropriate animal models for a meaningful preclinical evaluation of the rVWF product, which allows the therapeutic safety margins for human patients to be determined.

Disclosures:
Muchitsch: Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Gritsch:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment.
Cleavage of Recombinant and Plasma-Derived VWF by Human ADAMTS13
Authors: Rottensteiner Hanspeter , Varadi Katalin , Vejda Susanne , Ehrlich Hartmut J. , Scheiflinger Friedrich , Schwarz Hans Peter , Turecek Peter 
Published: October 2019

Abstract
Abstract 1415
A recombinant human CHO-expressed von Willebrand factor (rVWF) consisting of ultra-high molecular weight (UHMW) multimers resembles the VWF stored in Weibel-Palade bodies of endothelial cells. Once secreted into plasma, UHMW multimers are rapidly cleaved by ADAMTS13 and are usually missing in plasma-derived VWF (pdVWF).
Here we analyzed in vitro whether the kinetics of cleavage of rVWF by ADAMTS13 is similar to that of pdVWF. The kinetics of ADAMTS13-mediated proteolysis of rVWF were explored under denaturing conditions (1.5 M urea) or under shear stress so as to expose the ADAMTS13 cleavage site of VWF.
Multiple assays showed that rVWF was efficiently cleaved by ADAMTS13. UHMW multimers disappeared within seconds at physiological concentrations of ADAMTS13. Using lower concentrations of ADAMTS13 (10-30 mU/ml, equivalent to 1–3% of normal human plasma), UHMW were cleaved within 30 minutes. The typical satellite bands appeared very early in an ADAMTS13 dose-dependent manner. Virtually the same results were obtained when human plasma was used as a source for ADAMTS13. Although pdVWF differs from rVWF in its multimeric structure, the decrease in activity was similar for rVWF and pdVWF. Finally, the extent of ADAMTS13 cleavage was similar for rVWF and pdVWF when exposed to shear stress using a cone-plate viscometer.
Our data clearly indicate that rVWF is a good substrate for ADAMTS13. Ongoing phase 1 studies demonstrated that rVWF is indeed processed by the protease when administered in humans with severe VWD.

Disclosures:
Rottensteiner: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.
Identification of a VPS33B-Binding Protein That Facilitates Alpha Granule Formation In Human Megakaryocytes and Platelets.
Authors: Urban Denisa , Li Ling , Wasmuth James , Christensen Hilary , Parkinson John , Kahr Walter H. 
Published: October 2019

Abstract
Abstract 1444
Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are observed in isolation, in combination with α-granule deficiency, or as part of a syndrome in the Hermansky-Pudlak, Chediak-Higashi and Griscelli syndromes. The biogenesis of α-granules is poorly understood. Our laboratory has identified VPS33B as a central player in the formation of platelet α-granules. VPS33B has yet to be characterized in detail, however, its homolog VPS33A is known to be part of a multiprotein complex involved intracellular vesicle trafficking. Studies in our laboratory suggest that VPS33B is also part of a multiprotein complex. We performed a yeast two-hybrid library screen with VPS33B as bait and found another member of the complex: the unidentified gene product of chromosome 14 open reading frame 133 (C14orf133). Sequence analysis indicated this to be human VPS16B. Our studies show that VPS16B specifically binds to VPS33B but not its homologue, VPS33A. Furthermore, we show that VPS33B forms a distinct complex from that of its homologue VPS33A. VPS16B was found to co-localize with trans-Golgi, late endosome and α-granule markers in megakaryocytic Dami cells. Ongoing studies suggest that knockdown of VPS16B affects α-granule formation. We conclude that VPS16B, much like its binding partner VPS33B, plays a crucial role in megakaryocyte and platelet α-granule biogenesis.

Disclosures:
No relevant conflicts of interest to declare.
The Risk of Low Bone Mineral Density with Long-Term G-CSF Therapy for Severe Chronic Neutropenia.
Authors: DiMeglio L. A. , Bolyard Audrey Anna , Marrero Tracy M. , Alter Blanche P , Bonilla Mary Ann , Boxer Laurence A. , Link Dan , Newburger Peter E. , Rosenberg Philip S. , Shimamura Akiko , Dale David C. 
Published: October 2019

Abstract
Abstract 1484
Low bone mineral density (BMD) is a known risk factor for fractures. Low BMD has been reported in individuals with severe chronic neutropenia (SCN), and attributed both to the diseases causing neutropenia and to G-CSF therapy. However, given the rarity of SCN, few data exist regarding associations of BMD z-scores with disease characteristics such as type of neutropenia and duration of G-CSF therapy. We present data here obtained from BMD reports collected through the Severe Chronic Neutropenia International Registry (SCNIR).
We reviewed BMDs on 128 subjects [40 children (< 21 years of age), 87 females] having sufficient information about lumbar spine BMD by dual-xray absorptiometry (DXA) for evaluation. For subjects with multiple BMD measurements available, the most recent one was used for analysis. Mean age was 32.0 years (range 0.6–85 years).
57 subjects had idiopathic SCN (mean age 40 years), 40 had congenital (mean age 15 years), 28 were cyclic (mean age 41 years) and 3 were autoimmune (mean age 18 years). 122 subjects had received G-CSF at the time of the BMD assessment (mean 8.8 years, range 0.1–19.9 years). 11 of the adults were on bisphosphonate therapy for low BMD at the time of the BMD assessment; no children were on anti-resorptive therapy.
BMDs in these subjects were, on average, low. For the children, the BMD z-score (age matched mean ±1 standard deviation) was -1.0 ± 1.1, with 17.5% of children having BMDs that were low for age (Z-score < -2.0). For the adults the BMD t-score was -1.1 ± 1.4, with 46% of adults meeting t-score criteria for osteopenia (≤ -1.0) and 9% meeting criteria for osteoporosis (< -2.5). BMDs were lowest in those with congenital neutropenia, followed by those with cyclic neutropenia.
For children, BMDs were lower in those who had received longer G-CSF therapy (r= -0.506, p=0.002). This association was not seen in adults (r= 0.074, p= 0.5).
The low BMDs and the correlation of lower BMD with longer G-CSF treatment in children suggests there is an association of bone loss with the childhood diseases causing SCN. The data also suggest that regular assessments of bone health should be made in SCN patients, particularly those on long-term G-CSF therapy.

Disclosures:
Boxer: Amgen, Inc.: Equity Ownership. Dale:Amgen: Consultancy, Research Funding.
Leukocyte Integrin αXβ2 Is the Principal Receptor for Candida Albicans on Macrophages and Is Essential for Antifungal Host Defense.
Authors: Jawhara Samir , Soloviev Dmitry 
Published: October 2019

Abstract
Abstract 1493
The induction of cell-mediated immunity to pathogens is the prime task of cells of the innate immune system. Integrin αMβ2 (Mac-1, CR3, CD11b/CD18) is expressed on polymorphonuclear neutrophils (PMNs), monocytes, macrophages (MFC) and NK cells and has been implicated in diverse responses of these cells to infection, including phagocytosis, cell-mediated killing, chemotaxis and cellular activation. In PMNs integrin αMβ2 serves as the principal receptor involved in recognition of the common opportunistic fungal pathogen Candida albicans, and this integrin recognizes the fungal mannoprotein pH-regulated Antigen 1 (Pra1). Professional antigen-presenting cells of myeloid lineage MFC and dendritic cells express integrin αXβ2 (p150,95, CD11c/CD18) which is highly homologous to Integrin αMβ2, but the role of αXβ2 in the immune response to bacterial and fungal infections remains unclear. Employing αM - and αX-KO mice and mutated strains of C. albicans in a model of murine systemic candidiasis, we first demonstrate that mice deficient in αXβ2 exhibit significantly increased susceptibility to the invasive fungal infection. C. albicans induced lethality in αXβ2-KO mice more rapidly (2- to 3-fold faster) than the same inoculum in wild-type mice. MFC derived from mice deficient in α×β2 have a reduced ability to kill or phagocyte C. albicans. When the fungus was injected into mice, 6 hrs after peritoneal of thioglycollate, neutrophil recruitment to C. albicans showed a 20%-30% clearance of the fungus in the αMβ2-KO murine intraperitoneal lavages and a 50–60% rise in the αXβ2-KO lavages, which is consistent with our prior data showing αMβ2-dependency for PMNs-mediated antifungal defense activity. In contrast, when the fungus was injected into the peritoneal cavity 16 hrs after thioglycollate, when most recruited leukocytes are MFC, the C. albicans injected into αXβ2-KO mice showed a 23%-25% clearance of the fungus from the lavages while fungal clearance in αMβ2-KO lavages increased by only 30–40%. Thus, MFC antifungal activity is αXβ2-dependent. These results were further confirmed by in vitro fungicide assays employing isolated murine peritoneal PMNs and MFC. Fungal survival was greatest with PMN derived from αMβ2-KO mice and MFC derived from the αXβ2-KO mice-derived MFC. Disruption of the PRA1 gene of C. albicans, which is the primary ligand for αMβ2, had no effect on MFC fungicide activity, which suggests that αXβ2 recognizes a C. albicans ligand different from Pra1. Taken together, these results suggest that αXβ2 is a principal receptor for C. albicans on macrophages and plays critical role in host antifungal defense in a Pra1-independent mechanism.

Disclosures:
No relevant conflicts of interest to declare.
Overall Survival of Lymphoma Patients In Randomized Controlled Trials Comparing Primary G-CSF Chemotherapy Support to Control.
Authors: Lyman Gary H. , Crawford Jeffrey , Culakova Eva , Poniewierski Marek S. , Huang Mingyan , Kuderer Nicole M. , Dale David C. 
Published: October 2019

Abstract
Abstract 1502

Background
Randomized controlled trials (RCTs) have shown that granulocyte colony-stimulating factor (G-CSF) can reduce the risk of neutropenic complications as well as early mortality (Kuderer et al. JCO 2007). The impact of G-CSF support of chemotherapy on overall survival in patients with lymphoma is assessed here in a systematic review of RCTs of chemotherapy with versus without G-CSF in studies with at least 2 years of follow up.

Methods
A systematic review of RCTs of patients with malignant lymphoma receiving chemotherapy with versus without primary G-CSF support and reporting overall mortality by study arm identified 16 eligible studies. Data extraction was conducted by two independent reviewers. Heterogeneity was evaluated using Cochran's Q-statistic and the Inconsistency Index (I2). Weighted summary estimates of relative risk (RR) and absolute risk (AR) were based on the method of Mantel and Haenszel. Chemotherapy relative dose intensity (RDI) with G-CSF support compared to control was meta-regressed on the logarithm of RR for mortality. Egger's regression intercept was utilized to assess publication bias.

Results
Sixteen (16) eligible RCTs of patients receiving chemotherapy with (n=2,731) versus without (n=2,757) primary G-CSF support were identified. Deaths were reported in 927 patients randomized to receive primary G-CSF and in 1,064 controls. No significant heterogeneity was observed (Q=12.50, P=.641; I2=0%). Overall RR for mortality with versus without G-CSF support of chemotherapy was 0.876 [95% CI: 0.822 – 0.935; P<.001]. The AR reduction for mortality in G-CSF-supported patients versus control was 4.8% [95% CI: 2.4% - 7.15; P<.001]. An inverse trend was observed between the difference in RDI between treatment arms and RR for mortality [Slope = -0.39; 95% CI: -0.78 – 0.01; P=.057]. The greatest reduction in mortality was observed in the 5 RCTs of dose dense chemotherapy [RR=0.830; 95% CI:0.742-0.930; P=.001]. Significant treatment effect was observed in 7 studies limited to older patients [RR=0.910;95% CI: 0.847–0.978;P=.010] and 9 permitting all adult age groups [RR=0.813;95% CI: 0.715–0.926;P=.002]. No difference in treatment effect was observed based on study funding source and no evidence for publication bias was observed.

Conclusions
A highly significant reduction in overall mortality was associated with G-CSF support compared to control in 16 RCTs of lymphoma patients receiving chemotherapy. Patients receiving greater RDI with G-CSF support, especially those receiving dose dense regimens, experienced the greatest reduction in mortality.

Disclosures:
Lyman: Amgen: Research Funding. Crawford:Amgen: Consultancy. Dale:Amgen: Consultancy, Research Funding.
Isolation and Characterization of Renal Erythropoietin-Producing Cells: Contribution of HIF3α In Vivo.
Authors: Pan Xiaoqing , Suzuki Norio , Hirano Ikuo , Yamazaki Shun , Yamashita Toshiharu , Ohneda Osamu , Minegishi Naoko , Yamamoto Masayuki 
Published: October 2019

Abstract
Abstract 1541
Understanding the nature of renal erythropoietin-producing cells (REPs) remains a central challenge for understanding the mechanisms involved in hypoxia/anemia-induced erythropoietin (Epo) production in adult mammals. Previous studies have shown that REPs are renal peritubular cells, but further details are lacking. Here, we describe our approach used to isolate and characterize REPs. We bred mice bearing Epo/GFP combined with Epo-3’-enhancer deletion. These mice exhibit perinatal anemia (average Hct = 18% at P4–6). This enabled identification and purification of REPs based on GFP expression in the kidney. Light and confocal microscopy revealed that GFP immunostaining was confined to fibroblasts residing in the peritubular interstitium, which is consistent with our previous observation from the 180-Kb Epo/GFP reporter assay. Flow cytometry revealed that the GFP fraction constituted approximately 0.2% of the whole kidney cells; with most (63%) cells co-expressing CD73, a well-known marker for Epo-expressing cells. qRT-PCR confirmed that Epo expression was increased by approximately 100-fold in the purified REP population compared with that of the unsorted cells or CD73 fraction. Gene expression profiling showed Hif2α and Hif3α enrichment in the purified GFP/REP population. Moreover, in mouse hypoxia experiments, Epo expression was approximately 2.5-fold higher in Hif3α–/– kidney cells than in wild-type cells. The molecular strategy described here provides a means to isolate a pure population of REPs, allowing the analysis of gene expression profile of a defined population of cells essential for Epo production in the kidney. Our findings have led to a novel hypothesis that positive regulation by HIF2α and negative regulation by HIF3α might be necessary for correct renal Epo induction.

Disclosures:
Yamamoto: Chugai Pharmaceutical, Co., Ltd.: Research Funding.