bluuebunny/crossref_metadata_2025_split huggingface dataset:
Brain-Derived Neurotrophic Factor Is a Potential Osteoclast Stimulatory Factor In Multiple Myeloma
Authors: Sun Chun-Yan , Hu Yu , She Xiao-Mei , Qin You , Zhang Lu , Chen Lei , Ai Li-Sha , Fang Jun 
Published: October 2019

Abstract
Abstract 1917

Background and Objective:
Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In the present study, we investigated whether BDNF was present in marrow from patients with MM and possibly involved in MM cell-induced osteolysis.

Methods and Results:
Levels of bone marrow plasma BDNF was measured by ELISA in a cohort of individuals with MM and controls. The concentration of BDNF was found to be significantly elevated in patients with MM (879 ± 93) pg/ml when compared with bone marrow plasma derived from normal control subjects (186 ± 52) pg/ml (p < 0.001). Moreover, bone marrow plasma levels of BDNF positively correlated with plasma cell burden and extent of bone disease in MM patients. In osteoclast formation assay, bone marrow plasma from 31 of 37 patients with MM tested significantly stimulated the formation of osteoclast when compared to controls (61.8 ± 7 [mean ± SEM for the 31 patients] versus 25.2 ± 6 TRAP+ multinucleated cells/well [mean ± SEM for the 12 controls]; p < 0.01). The effect was significantly blocked by a neutralizing antibody to BDNF (p < 0.05), suggesting a critical role for BDNF in osteoclast activation. Furthermore, BDNF was found to dose-dependently increased the formation of multinucleated, TRAP+ osteoclast. The direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. Using reverse-transcriptase polymerase chain reaction analysis and western blotting assay, we demonstrated that BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF. These data suggested that TrkB is the functional receptor mediating BDNF's effect on osteoclast formation. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein (MIP)-1α (r = 0.45, p < 0.005) and receptor activator of nuclear factor-κB ligand (RANKL) (r = 0.68, p < 0.0001), two major osteoclast stimulatory factors in MM.

Conclusion:
Taken together, our results demonstrate the ability of MM cells to secret BDNF correlates with the severity of osteoclastic bone resorption, and provide evidence that BDNF play a causal role in the development of MM bone lesions through TrkB receptor.

Disclosures:
No relevant conflicts of interest to declare.
Combined Bendamustine, Prednisolone and Lenalidomide (RBP) In Refractory or Relapsed Multiple Myeloma. First Results of a Phase I Clinical Trial
Authors: Pönisch Wolfram , Heyn Simone , Wagner Ina , Mohren Martin , Hoffmann Franz-Albert , Schüler Frank , Schmalfeld Marion , Al-Ali Haifa K. , Jäkel Nadja , Niederwieser Dietger W 
Published: October 2019

Abstract
Abstract 1971

Introduction:
While the role of lenalidomide monotherapy in the treatment of relapsed/refractory patients with multiple myeloma (MM) is established, combination therapies with Lenalinomide are still under investigation. Bendamustine is a bi-functional alkylating agent with a purine-like benzimidazole ring effective in combination with steroids, thalidomide and bortezomib for the treatment of patients with MM. In the current trial, combination therapy of bendamustine, lenalinomide and prednisolone (RBP) was tested for feasibility and safety in patients with relapsed or refractory MM.

Patients and Methods:
This is a phase I trial examining dosing of lenalidomide in combination with bendamustine and prednisolone. The first cohort of patients received a starting dose of 10mg/d d1-21 lenalidomide, 60mg/m2/d d1-2 bendamustine and 100mg/d d1-4 prednisolone. Escalation steps in the next cohorts included 15, 20 and 25mg of lenalidomide followed by an escalation step of 75 mg/m2 bendamustine. Three patients were enrolled at each dose level and the first two cycles were evaluated for maximum tolerable dose. Patients received RBP in 4-week cycles for a maximum of 8 cycles in order to evaluate efficacy. Patients with stable or responding disease following 8 cycles of RBP received single-agent oral lenalidomide 10 mg once daily on days 1–21 of each 28-day cycle as maintenance.

Results:
: Nine patients (3 at each dose level of 10 mg, 15 mg or 20 mg lenalidomide) have been enrolled to date and 9 patients have completed at least 2 cycles. Response was assessed using modified EBMT criteria to include near complete remission (nCR) and very good partial remission (VGPR). 8 of 9 patients responded after at least 2 cycles with 2 VGPR, 4 PR, 1 MR and 1 stable disease. One patient experienced progressive disease. None of the 9 patients developed dose-limiting hematoxicity as defined by an ANC < 1,0 × 109/l with fever for > 3 days or an ANC <0,5 × 109/l for > 7 days or platelet count < 25 × 109/l for > 3 days. Neutropenia was reported in 4 patients (CTC grade ≥ 3) but no thrombocytopenia (CTC grade ≥ 3) was observed. No grade 3 or 4 non hematological toxicity was encountered and no dose modification was required.

Conclusions:
RBP with a dose of 20 mg lenalidomide d 1–21 and 60 mg/m2 bendamustine d 1–2 is well tolerated in patients with relapsed or refractory MM. Maximum tolerable dose was not reached. Further dose increase according to the protocol is in progress.

Disclosures:
Niederwieser: Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.
Filamin A Deficiency Leads to Premature Platelet Formation and Increased Clearance
Authors: Begonja Antonija Jurak , Weber Sarah E. , Hoffmeister Karin M. , Hartwig John H. , Falet Herve 
Published: October 2019

Abstract
Abstract 2027
Filamin A (FlnA) is a large cytoplasmic protein that crosslinks and stabilizes actin filament networks and links membrane glycoproteins and signaling proteins to the underlying cytoskeleton. FlnA loxP-PF4-Cre mice that specifically lack FlnA in their platelets and megakaryocytes (MKs) have a macrothrombocytopenia with increased tail bleeding time. FlnA KO platelets have decreased surface expression of the von Willebrand factor receptor (vWfR) and are cleared rapidly from the circulation of WT mice, indicating an inherent surface defect that leads to detection and removal. FlnA loxP-PF4-Cre mice have a marked increase in MK numbers in bone marrow and spleen. Detailed analysis of platelet production by FlnA KO MKs in vitro reveals an altered maturation program and defects in vWfR stability, compared to WT. Surprisingly, the surface expression of vWfR components (Ibα, Ibβ, IX and V) on FlnA KO MKs is normal or increased compared to WT MKs, although the total amount of GPIbα is decreased and GPIbα is not associated with the actin cytoskeleton. Analysis of the GPIbβ subunit shows increased degradation in FlnA KO MKs and platelets. Although FlnA KO and WT MK cultures contain comparable cell numbers, FlnA KO MKs more rapidly convert their proplatelets into large CD61+ platelet-sized particles. These findings suggest that aberrant platelet maturation by FlnA KO MKs results in enlarged platelets that are cleared rapidly because of altered vWfR expression.

Disclosures:
No relevant conflicts of interest to declare.
Role of the Krüppel-Type Zinc Finger Transcription Factor ZBP-89 In Human Globin Gene Regulation and Erythroid Development
Authors: Woo Andrew J. , Kim Jonghwan , Xu Jian , Huang Hui , Cantor Alan 
Published: October 2019

Abstract
Abstract 2067
The molecular mechanisms underlying developmental globin gene regulation remain incompletely understood. Prior studies have identified key cis-regulatory elements within the beta globin locus that contain core regions of closely spaced functional binding sites for GATA, NF-E2p45/maf and GT/GC box binding transcription factors. We recently identified the GT/GC-box binding transcription factor ZBP-89 as a novel GATA-1 interacting partner, and showed that it is involved in erythroid development in mice (Woo et al. 2008. Mol. Cell Bio. 28:2675-2689). Brand et al. independently isolated ZBP-89 in NF-E2p45/mafk complexes from induced mouse erythroid leukemia (MEL) cells (Brand et al. 2004. Nat. Struct. Mol Biol. 11:73-80). In the current study, we show that ZBP-89 protein levels increase during in vitro erythroid differentiation of human bone marrow derived CD34+ cells. This correlates with the onset of alpha and beta globin gene transcription. ChIP-chip studies using ENCODE v2.0 arrays demonstrate that ZBP-89 occupies key cis-regulatory elements within both the beta globin (locus control regions HS3, HS2; delta and beta proximal promoters; and an intergenic region between gamma1 and delta globin) and alpha globin (HS-48, HS-40, HS-10 and alpha globin proximal promoters) loci in primary human erythroid precursors. Comparative analysis across the entire ENCODE array reveals a strong positive correlation between ZBP-89 occupancy, RNA polymerase II occupancy, and the activating histone marks acetylated histone 3 (AcH3) and trimethylated histone 3 lysine 4 (H3K4me3); and a negative correlation with the repressive mark trimethylated histone 3 lysine 27 (H3K27me3). Motif analysis under the ZBP-89 occupancy peaks indicates a preference for GGGG(G/A)NGGGG in vivo binding sites. Lentiviral shRNA mediated knock down of ZBP-89 in the in vitro differentiated CD34+ cells results in 30–50% reduction of alpha-, gamma-, and beta-globin gene expression, as well as modestly decreased expression of a number of additional erythroid-specific genes. Co-immunoprecipitation experiments demonstrate physical association between ZBP-89 and the GCN5/Trapp histone acetyltransferase complex. Based on these findings, we propose that ZBP-89 participates with GATA-1 and NF-E2 in the final epigenetic changes required for high-level expression of globin and other erythroid genes in terminally differentiating human erythroid cells.

Disclosures:
No relevant conflicts of interest to declare.
Antigen Presenting Cell-Mediated Ex Vivo Expansion of Human Umbilical Cord Blood Cells Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity
Authors: Shah Nina , Xing Dongxia , Lee Dean A. , Cooper Laurence J.N. , Decker William , Li Sufang , Robinson Simon , Yang Hong , Parmar Simrit , Tung Shawndeep , Wang Michael , Champlin Richard , Bollard Catherine M , Shpall Elizabeth J. 
Published: October 2019

Abstract
Abstract 2100

Background:
Multiple myeloma (MM) is the second most common hematologic malignancy in adults and, to date, is incurable. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM. Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (UCB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. Here we demonstrate the anti-myeloma activity of UCB-NK cells expanded to clinical grade by a novel technique using artificial antigen presenting feeder cells (“K562 Clone 9” cells) modified to express IL-21 (“K562-cl9-mIL21”).

Methods:
Cord blood mononuclear cells (CBMCs) were cultured in 10% human serum albumin media with IL-2 (500 IU/ml) and K562-cl9-mIL21 feeder cells (2:1 feeder: CBMC ratio) for 21 days. Thereafter, cells were subjected to CD3-immunomagnetic depletion. CD3-negative cells were then used as effector cells in functional assays. Flow cytometry was used to confirm NK cell purity (C56+/CD3- cells) and a standard chromium-51 assay was performed to determine NK cell cytotoxicity. Targets included K562 cells, MM cell lines RPMI 8226, ARP-1 and U266, autologous non-neoplastic UCB cells (negative control) and bone marrow-derived CD138+ plasma cells from myeloma patients.

Results:
Expansion of CBMCs with K562-cl9-mIL21 yielded a >2000 fold expansion of NK cells, compared with 47 fold expansion of CD56-selected cells cultured with IL-2 alone (p <0.05). After CD3 depletion, UCB-NK cultures were comprised of 92% CD56+/CD3- cells. K562-cl9-mIL21-expanded UCB-NK cells demonstrated cytotoxicity against the classic NK cell target K562 as well as MM cell lines RPMI 8226, ARP-1 and U266 (Fig 1). In addition, these UCB-NK cells were also active against primary, bone marrow-derived CD138+ plasma cells from myeloma patients, an effect which was augmented by pre-incubation of UCB-NK cells with lenalidomide (Figure 2, NK=UCB-NK cells, NKR=UCB-NK cells+lenalidomide).

Conclusions:
UCB-NK cells can be expanded ex vivo to clinically relevant doses for allogeneic NK cell therapy via co-culture with K562-cl9-mIL21 feeder cells. Expanded UCB-NK cells are cytotoxic to myeloma cell lines and primary myeloma cells. Further development of UCB-NK cells as an adjunct therapy in stem cell transplantation for myeloma is warranted.

Disclosures:
Wang: Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding.
Genome-Wide Linkage Analysis Reveals Novel Loci Modifying Plasma Von Willebrand Factor Undetected by Genome-Wide Association
Authors: Desch Karl , Li Jun Z , Ozel Ayse Bilge , Thornburg Courtney , Siemieniak David , Ginsburg David 
Published: October 2019

Abstract
Abstract 2116

Plasma levels of von Willebrand Factor (VWF) are highly variable among healthy people and ~70% of this variability is heritable. Elevated levels of VWF are associated with thrombosis while low levels of VWF cause the common bleeding disorder, von Willebrand Disease (VWD). The genome-wide association study (GWAS) is a powerful tool for identifying common genetic variants contributing to complex traits, though less common variants are likely to be missed by this approach. We performed combined GWAS and linkage analysis in a cohort of healthy young college students. The study was approved by the University of Michigan IRB (IRBMED #2005-0080). 1189 healthy individuals (14-35 yrs of age) were recruited, comprising 507 sibships. VWF levels were determined by alphalisa assay (mean 109%, range 26 – 410%). VWF level heritability (h2) was 73% among siblings and 82% among monozygotic twins. Samples were de-identified and DNA was extracted from peripheral blood, quantified and genotyped using the Illumina Omni-1 quad SNP chip at the Broad Institute (MIT, Boston). 1182 individuals passed genotyping quality control with >97% call rates for 979,855 SNPs. Genotyping quality control was performed as part of the NIH's GENEVA program. GWAS on log transformed, age and sex adjusted VWF levels identified a major peak over the ABO locus (p<1×10-36), [Figure 1] confirming the known influence of ABO blood type on plasma VWF. Preliminary genome-wide linkage analysis (MERLIN) performed on plasma VWF levels in the 507 sibships identified 5 novel loci with peak LOD scores greater than 5 (chromosomes 2, 7, and 21), in addition to the ABO locus on chromosome 9 [Figure 2]. Other than ABO, none of these loci were detected in our GWAS analysis, or the previously reported CHARGE Consortium VWF GWAS (Smith, N et al. Circulation, 2010). Our analysis is now being extended to additional hemostasis and other related phenotypes using the same SNP data. In conclusion, linkage analysis identified novel loci contributing to the variation of VWF in healthy siblings that were undetected by GWAS. Though GWAS is an effective tool for the analysis of many complex traits, including plasma VWF variation, the majority of the heritable component of these traits remains unexplained. However, the missing heritability may be due, at least in part, to multiple rare variants at a limited number of loci. Our results suggest that genome-wide linkage analysis in multiple sibships may identify such loci and that this approach may be broadly applicable to hemostasis and other complex traits. Identification of the major loci regulating plasma VWF levels may lead to an improved prediction of bleeding and thrombotic risk and could suggest novel targets for therapeutic intervention.

Disclosures:
No relevant conflicts of interest to declare.
CD34+/CD38- Leukemia Stem Cells Aberrantly Express CD82 Adhesion Molecule
Authors: Ikezoe Takayuki , Nishioka Chie , Yang Jing , Serada Satoshi , Naka Tetsuji , Yokoyama Akihito , Udaka Keiko 
Published: October 2019

Abstract
Abstract 2168
To identify molecular targets in leukemia stem cells (LSCs), this study compared the protein expression profile of freshly isolated LSCs (CD34+/CD38- compartment) with that of non-LSC (CD34+/CD38+ compartment) counterparts from individuals with acute myelogenous leukemia (AML) using isobaric tags for relative and absolute quantitation (iTRAQ). A total of 98 proteins were overexpressed, while six proteins were underexpressed in LSCs compared with their non-LSC counterparts. Proteins overexpressed in LSCs included a number of proteins involved in DNA repair, cell cycle arrest, gland differentiation, anti-apoptosis, adhesion, and drug resistance. Aberrant expression of CD82, a family of adhesion molecules, in LSCs was noted in additional clinical samples (n=6) by flow cytometry. In addition, we found that imatinib-resistant chronic eosinophilic leukemi EOL-1R cells expressed a greater amount of CD82 and remained in a dormant state compared to the parental EOL-1 cells. Interestingly, down-regulation of CD82 in EOL-1R cells by a small interfering RNA stimulated their migration capacity, as assessed by the transwell assay. These observations suggested that the aberrant expression of CD82 probably played a role in adhesion of hematopoietic cells to bone marrow microenvironment. Targeting CD82 could detach LSCs from bone marrow niche and sensitized these cells to anti-leukemia agents.

Disclosures:
No relevant conflicts of interest to declare.
B-Cell Depletion Using IgG1 Anti-CD20 Spares MZ B Cells and Promotes Tolerance to Human FVIII In a Murine Model of Hemophilia A
Authors: Zhang Ai-Hong , Skupsky Jonathan , Scott David W. 
Published: October 2019

Abstract
Abstract 2206
Preventing and reversing inhibitor formation remains one of the major challenges for hemophilia A therapy. Anti-CD20 mAb (Rituximab) has been reported to be beneficial for hemophilia A patients who failed immune tolerance induction (ITI). However, the evaluation of anti-CD20 therapy often is complicated in the clinical setting by concomitant use of other immune modulating drugs, such as hydrocortisone and IVIG. In this study, we tested the effect of B-cell depletion per se on tolerance induction to FVIII in a mouse model of hemophilia A. Two subclasses of anti-mouse CD20 monoclonal antibodies with differential effects were used. We previously showed that IgG1 anti-CD20 selectively depleted follicular (FO) B cells and spared marginal zone (MZ) B cells, while IgG2a anti-CD20 efficiently depleted both. In FVIII primed mice (inhibitor titer = 30.7 ± 4.8 BU/ml), a single dose of IgG1 anti-CD20 pretreatment prevented the increase in inhibitor formation in the majority of treated mice given daily, high dose FVIII i.v. injection as a model for ITI. Surprisingly, only a marginal effect was achieved when we repeated the same protocol using IgG2a anti-CD20 for B-cell depletion, which efficiently depletes both FO and MZ B cells. To examine tolerance to FVIII, we re-challenged the treated mice with 2 μg FVIII intraperitoneally three months after the initiation of B cell depletion using IgG1 anti-CD20 when the number of peripheral B cells had recovered 60 % or more. The inhibitor titers remained significantly lower in the IgG1 anti-CD20 group after this FVIII boost injection (60.9 ± 33.2 versus 190.3 ± 33.5 BU/ml in control IgG1 group; p = 0.02). Importantly, after the mice were subcutaneously challenged with an unrelated antigen, OVA in CFA, there was no significant difference in anti-OVA IgG titers between the two groups. Taken together, these results suggested that selectively depletion of FO B cells by IgG1 subtype anti-CD20 mAb treatment may facilitate the tolerance induction to FVIII. (Supported by NIH R01 HL061883 and a fellowship from the American Heart Association)

Disclosures:
No relevant conflicts of interest to declare.
De-Immunization of Immunodominant HLA-DR Epitopes In the C2 Domain of Factor VIII
Authors: Song Chang , Moise Leonard , Tassone Ryan , McClaine Elizabeth , Martin William D. , DeGroot Annie S. , Scott David W. 
Published: October 2019

Abstract
Abstract 2207
Development of anti-factor VIII (FVIII) antibodies that neutralize FVIII activity is a major obstacle of replacement therapy in hemophilia A. To create a less immunogenic therapeutic protein, recombinant FVIII can be modified to reduce binding of FVIII sequences to HLA, based on predicted anchoring residues. This process is known as ‘de-immunization’. Previously, we found that immunogenic peptide epitopes in the C2 domain of FVIII could be modified to reduce MHC class II binding resulting in lower antigenicity in vitro in hemophilic E16 mice (H-2b). Here, we extended these studies to HLA-DR transgenic mice and identified immunodominant C2 peptides in E16, DR3 and DR4 mice as well as in DR3 mice crossed to E16 mice (DR3×E16). E16 and DR transgenic mice were immunized with the designated original high class II-binding peptides (ORG) or modified peptides (MOD) emulsified in CFA, and lymph node (LN) cells were tested in T-cell proliferation and ELISpot assays against the ORG and MOD peptides. We found that while p2191-2210 dominantly stimulates H-2b, p2271-89 is immunodominant in DR3 transgenic animals; DR3×E16 T cells recognized both epitopes as would be predicted. On the other hand, p2231-51 and p2310-30 were immunodominant in DR4 mice. We also found that modifications of dominant epitope peptides 2191, 2271, 2231, 2310 showed lower HLA class II binding and T-cell proliferation in corresponding mouse strains; stimulation of IL17-a and IFNγ cytokine production were reduced with modified 2191 in DR3xE16 mice. Direct immunization with rFVIII containing modified peptides is required to validate potential immunogenicity. These results will be utilized to design and produce a functional full-length modified rFVIII that can be used as a less immunogenic therapeutic protein for hemophilia A patients. (Supported by NIH grant R43 HL088834-01 to ADG.)

Disclosures:
Moise: EpiVax: Employment. Tassone: EpiVax: Employment. McClaine: EpiVax: Employment. Martin: EpiVax: Employment, Equity Ownership, Patents & Royalties. DeGroot: EpiVax: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Scott: EpiVax: Consultancy.