bluuebunny/crossref_metadata_2025_split huggingface dataset:
Impact of Cardiac Stage and Hematologic Response on AL Amyloidosis Patients with Renal Involvement
Authors: Wong Sandy W. , Toskic Denis , Warner Melissa , Moreno-koehler Alejandro , Fein Daniel , Fogaren Terry , Lee Lisa , Oliver Colin , Guthrie Spencer D. , Comenzo Raymond L. 
Published: October 2019

Abstract
Cardiac stage and depth of hematologic remission are major predictors of survival for AL amyloidosis patients (Wechalekar et al., Blood, 2013; Dispenzieri et al., JCO, 2004; Palladini et al., JCO, 2012). Renal staging in AL amyloidosis (AL) has been studied in the context of renal survival (Palladini et al., Blood 2014). Influences on survival for renal patients have yet to be fully defined. We performed a retrospective study of all AL patients with renal involvement diagnosed at our center between 7/1/08 and 6/30/15. In this cohort of consecutive patients (n=80) median age was 63 (IQR 55-70) and 56% were men. Eighty-eight percent had lambda plasma cell disease and median involved FLC was 140mg/L (69-485). Thirty-nine percent were renal stage 1, 44% stage 2, and 16% stage 3. Median 24-hour proteinuria and serum creatinine were 6.23 g (3.47-10.70) and 1.03 mg/dL (0.80-1.80) respectively, and median eGFR was 72 mL/min (41-90). Fifty-eight percent had cardiac involvement, of whom 11% were cardiac stage 1, 54% stage 2, and 34% stage 3, while 18% had GI and 9% peripheral nerve involvement.
As first-line therapy, 70% received bortezomib-based regimens and 25% melphalan-based autologous stem cell transplant. By intention-to-treat, at 6 months after beginning therapy, 54% of patients had a hematologic response of PR or better, and renal and cardiac responses occurred in 13% and 14% of patients respectively, while renal progression occurred in 6%. Median overall survival (OS) for this cohort (n=80) was 67 months. Those with cardiac involvement (n=45) had a median OS of 41 months and, while median OS was not reached for cardiac stage ≤ 2, it was 31 months for those who were stage 3 (P<0.05) (Figure). Median OS was also not reached for patients achieving hematologic response ≥ VGPR with a median follow-up of 19 months.
In conclusion, for AL patients with renal involvement, both cardiac stage and depth of hematologic response are important contributors to overall survival. Furthermore, as this real world intention-to-treat analysis demonstrates, there is a continuing need for better therapies for both the hematologic disease and the organ damage associated with AL.
Figure. Figure.

Disclosures
Oliver: Prothena Biosciences, Inc.: Employment, Equity Ownership. Guthrie:Prothena: Employment, Equity Ownership, Other: Leadership. Comenzo:Takeda: Consultancy, Research Funding; Prothena: Consultancy, Research Funding; Karyopharm: Research Funding; Janssen: Consultancy, Research Funding.
Beta-Globin Gene Targeting in Human Hematopoietic Stem Cells Using Cas9 Ribonucleoprotein and rAAV6
Authors: Dever Daniel Patrick , Porteus Matthew 
Published: October 2019

Abstract
The b -hemoglobinopathies including sickle cell disease (SCD) and b -thalassemia ( b -thal) affect millions of people worldwide . SCD and b -thal are caused by mutations in the b -globin gene (HBB) resulting in either abnormal sickling or severely reduced protein production, respectively. A curative strategy for the b -hemoglobinopathies would be ex vivo HBB gene correction in patient-derived hematopoietic stem and progenitor cells (HSPCs) followed by autologous hematopoietic stem cell transplantation (auto-HSCT). We report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in long-term repopulating HSCs derived from mobilized peripheral blood. We combine electroporation of Cas9 protein complexed with chemically modified sgRNAs and delivery of a HR donor by recombinant adeno-associated viral vectors, serotype 6 (rAAV6). Notably, by including a reporter gene in the HR donor, we are able to identify and purify a population of HSPCs with >90% of cells having targeted integration at the HBB gene. These cells can be identified because HR-mediated integration causes the reporter gene to be expressed at log-fold higher levels than the non-integrated reporter. When transplanted into immunodeficient mice, the purified population gives rise to engraftment of HBB-edited human cells in primary and secondary recipients, confirming the presence of long-term repopulating hematopoietic stem cells (LT-HSCs). Importantly, we show efficient correction of the SCD-causing E6V mutation in SCD patient-derived CD34+ HSPCs by either editing the nucleotide mutation or knocking in an anti-sickling b -globin cDNA. Edited SCD CD34+ cells were shown to express adult b -globin (HbA) mRNA after HSPCs were differentiated into erythrocytes in vitro, confirming intact transcriptional regulation of the edited HBB allele. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs for HR at the HBB locus to advance the development of next generation therapies for b -hemoglobinopathies.

Disclosures
Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership.
Specific Deletion of Keap1 in Endothelial Cells or/and Macrophages of SCD Mice Induces Nrf2 to Protect Tissues Against Oxidative Insults
Authors: Keleku-Lukwete Nadine , Harit Panda , Naganuma Eriko , Suzuki Mikiko , Yamamoto Masayuki 
Published: October 2019

Abstract
Sickle cell disease (SCD) model mice frequently suffer from hemolysis. Endothelial cells face hemolysis, and they activate inflammatory cells following their own activation. Heme and iron discharge into blood stream enhanced by the following ischemia reperfusion injury as consequence of intermittent vaso-occlusion, jeopardize functions of SCD mouse organs. Transcription factor Nrf2 regulates expression of the genes involved in antioxidant cell defense. The roles that Nrf2 plays in pathological conditions in association with oxidative insults make the Nrf2 pathway attractive for developing new drugs for SCD treatment. Recently, we demonstrated that systemic Nrf2 induction significantly relieves inflammation and organ damage of SCD mice, while hemolysis is still unaffected (PNAS 2015). To examine the mechanisms underlying the contribution of Nrf2, we analyzed SCD mice by means of cell type-specific activation of Nrf2 through conditional loss of its repressor Keap1. We generated compound SCD mice with Keap1 floxed and cell-specific Cre expression alleles (SCD::Keap1F/F::Tie1-Cre and SCD::Keap1F/F::LysM-Cre mice targeting endothelial cells and macrophages, respectively). In lungs of SCD::Keap1F/F::Tie1-Cre mice, expression of Nrf2 and Nrf2-target genes were upregulated, while expression of inflammatory cytokines were significantly repressed. In both genotypes, necrosis and inflammation of liver and lungs were significant improvement, compared to littermates control mice. Plasma leucocytes counts, plasma heme level, total bilirubin and indirect bilirubin levels, were significantly reduced in both genotypes compared to controls, and these levels were slightly higher in the SCD::Keap1F/F::LysM-Cre mice compared to SCD::Keap1F/F::Tie1-Cre mice. 8-OHdG staining of lungs showed smaller number of positive cells in SCD::Keap1F/F::Tie1-Cre mice. These results indicate that Nrf2 activation in both endothelial cells and macrophages contributes to relieve chronic complications of SCD.

Disclosures
No relevant conflicts of interest to declare.
Hydrogen Exchange Mass Spectrometry Reveals Allosteric Interactions Between the Distal and the Proximal Domains of Human ADAMTS13
Authors: Zander Catherine B. , Mayne Leland , Englander S. Walter , Zheng X. Long 
Published: October 2019

Abstract
Background: Deletion of the distal C-terminal domains, or a decrease in pH, or the binding of a monoclonal antibody to the C-terminal domains of ADAMTS13 increases its proteolytic activity towards VWF peptide substrate, VWF73, by approximately 2-fold; this suggests an allosteric interaction between the distal and proximal domains of ADAMTS13. However, neither the underlying mechanism of the allosteric interaction nor the exact sites of interaction between the proximal domains and the distal C-terminal domain are fully understood.
Objective: To determine the binding sites and kinetic interactions between the proximal and distal domains of ADAMTS13 and to gain insight into the mechanism of allosteric regulation of ADAMTS13 function.
Methods: Hydrogen deuterium exchange plus LC-mass spectrometry (HX-MS) and surface plasmon resonance (SPR) were employed to determine conformational changes and direct binding interactions between the proximal and distal domains of ADAMTS13.
Results: Recombinant full-length (rFL-A13) and an ADAMTS13 variant truncated after the spacer domain (rMDTCS) were purified to homogeneity. After incubation with deuterium for various times (0-6 hours), both rFL-A13 and rMDTCS were digested with pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Overall, 188 of the total 338 peptides in rFL-A13 were identified, spanning the metalloprotease, cysteine-rich, and spacer domain. Only 16 peptides were found to be less accessible (or protected from deuterium-hydrogen exchange) in rFL-A13 than in rMDTCS. Two of these regions reside in the metalloprotease domain (residue 78-127 and 282-304). The next region is at a loop on the cysteine-rich domain (residue 446-473 and 495-501). The next peptides are in the adjacent loops of the spacer domain with one peptide 596-603, and five overlapping peptides spanning from 632-642, and then finally one peptide from the C-terminal of the MDTCS variant (residue 676 to 688). These results indicate that the distal C-terminal domains interact with multiple discrete sites in the proximal domains of ADAMTS13, resulting in protection from hydrogen-deuterium exchange. Such an interaction was further demonstrated by direct binding experiments with SPR. The distal domains including CUB, T2-CUB, and T5-CUB bound directly to M, MDT, and MDTCS under flow with high affinity.
Conclusion: Our findings demonstrate the allosteric interactions between the distal domains and the proximal domains at multiple discrete sites. Such an interaction may regulate catalytic efficiency and substrate specificity of the metalloprotease domain of ADAMTS13 under various conditions.

Disclosures
Zheng: Ablynx: Consultancy; Alexion: Research Funding.
Platelet Alpha-Storage Pool Defect Combined with Non-Classic Delta-Storage Pool Deficiency - with a Severe Bleeding Tendency
Authors: Bergmann Frauke , Schwierczek Kathrin , Sollfrank Stefanie , Czwalinna Andreas , Erkel Joseph , Lackner Karl , Walter Ulrich , Rossmann Heidi , Jurk Kerstin 
Published: October 2019

Abstract
Aims :We report about a 35 year old male patient with severe and frequent epistaxis and hematoma since infancy. He presented with mild thrombocytopenia and increased mean platelet volume. Von Willebrand's disease and subhemophilia had been excluded. Previously, he was diagnosed with immune thrombocytopenic purpura. He never underwent elective surgery. His parents were asymptomatic. However, his young daughter also suffers from severe bleeding symptoms and was referred to our coagulation outpatient clinic to investigate the bleeding disorder.
Methods:Platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. Lysates of gel-filtered platelets were analyzed for total granule P-selectin, CD63 and von Willebrand factor (VWF) content by Western blotting and for serotonin levels by ELISA, respectively. Molecular genetic analysis was performed using whole exome sequencing and pyrosequencing.
Results: Platelet aggregation in response to ADP was not inducible. Platelet aggregation in response to epinephrine resulted in monophasic curves. Platelet aggregation in response to TRAP-6 or collagen was inducible but impaired and accompanied by disaggregation. In contrast, arachidonic acid- and U46619-induced platelet aggregation as well as ristocetin-induced platelet agglutination/aggregation was within the normal range. Also platelet surface expression of GPIIb/IIIa, GPIb/V/IX, GPVI and CD29 was normal. Activation of GPIIb/IIIa in response to ADP, convulxin or TRAP-6 was inducible but diminished and in response to epinephrine or arachidonic acid normal. P-selectin (alpha-granule marker) and CD63 (dense body/lysosome marker) surface expression was not inducible by thrombin or convulxin and markedly reduced in response to TRAP-6 and arachidonic acid. Immunoblot analysis of isolated platelets demonstrated pronouncedly decreased content of total P-selectin, VWF and CD63. Fluorescent staining of adenine nucleotides by mepacrine in patient's platelet dense bodies and secretion of ATP induced by arachidonic acid (normal aggregation) were absent. However, patient's platelet serotonin levels were within the normal range. The molecular genetic diagnostic showed an undescribed mutation in the GATA1 gene (c.886A>C hemizygot, p.T296P). The bioinformatic check confirmed a pathogenetic importance of this mutation located in the domain I of the C-terminal zinc finger.
Conclusion: We describe the first case of a combined platelet alpha- and partial delta-storage pool disorder where the dense granules lack adenine nucleotides but contain normal levels of serotonin - causing a severe bleeding disorder.

Disclosures
No relevant conflicts of interest to declare.
Natural IgM Against Oxidation-Specific Epitopes Inhibit Microvesicle-Driven Coagulation
Authors: Obermayer Georg , Afonyushkin Taras , Puhm Florian , Schwameis Michael , Jilma Bernd , Binder Christoph J 
Published: October 2019

Abstract
Increasing evidence demonstrates an important role for immune functions in coagulation and thrombosis, and both microbial and sterile triggers have been implicated in these responses. Oxidation-specific epitopes (OSE) are products of lipid peroxidation that are present on oxidized low-density lipoproteins and dying cells, and represent important sterile triggers of inflammation in cardiovascular diseases. We have previously shown that a large part of naturally occurring IgM antibodies have specificity for OSEs and that 50% of circulating procoagulant microvesicles (MV) carry OSEs, such as malondialdehyde-adducts (MDA), on their surface. Because OSE-specific natural IgM bind MVs and increased levels of circulating MVs have been implicated in an increased thrombotic risk, we studied the capacity of these antibodies in directly modulating the procoagulant potential of MVs in vitro and in vivo. We first showed that increasing concentrations of three different monoclonal IgM antibodies with specificity for MDA (LR04, NA17, E014) significantly delayed MV-induced fibrin formation in plasma, while an MDA-specific IgG antibody or F(ab')2 fragments of LR04 had no effect. Moreover, peak thrombin generation of normal, factor VII- or factor XII- deficient microvesicle poor plasma (MVPP) substituted with tissue factor positive MVs was significantly reduced in the presence of LR04 but not a control IgM antibody, indicating that it exerts its effect on the common pathway of the coagulation cascade. A similar anticoagulatory effect of LR04 was observed in freshly drawn whole blood analyzed by rotational thrombelastometry. Addition of LR04, compared to a control IgM antibody, increased clotting time, clot formation time, and the time to reach maximum clot firmness, and decreased maximum clot firmness. Finally, in a murine pulmonary embolism model, we could show that 30-minute survival of mice that received intravenous injections of epinephrine together with pro-coagulant MVs derived from the pancreatic cancer cell line HPAFII was significantly increased in mice co-injected with LR04 compared to controls. Our study identifies an inhibitory role of MDA-specific IgM antibodies in MV-induced coagulation. These effects are exerted on the common pathway of the coagulation cascade and require the intact structure of IgM antibodies. The findings may offer novel diagnostic and therapeutic approaches in the prevention and treatment of thrombosis.

Disclosures
No relevant conflicts of interest to declare.
Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Acute Myeloid Leukemia Progression and Drug Response
Authors: Passaro Diana , Di Tullio Alessandro , Abarrategi Ander , Rouault-Pierre Kevin , Foster Katie , McNaughton Linda Ariza , Lassailly Francois , Bonnet Dominique 
Published: October 2019

Abstract
The biological and clinical behavior of hematological malignancies are not only determined by the properties of the leukemic cells themselves, but are also highly affected by interaction with the microenvironment, pointing to the existence of an active crosstalk between the two compartments. Previous studies showed that acute myeloid leukemia (AML) cells actively modify endothelial cells ex vivovia several pathways, mainly mediated by VEGF. However, as anti-VEGF therapies haven't produced successful results in clinical trials, an extensive study of the crosstalk between AML and the vascular niche in the bone marrow (BM) is required to provide new therapeutic strategies. In the present study we combined the use of mouse models of AML, human AML patient-derived xenografts (PDX) and direct analysis on patient-derived BM biopsies to provide a global, reliable picture of the bone marrow vasculature in AML disease. We found several abnormalities in the vascular architecture and function in PDX, such as increased number of endothelial cells, increased microvascular density (MVD), decreased vascular mean diameter and increased hypoxia. Furthermore, using two-photon confocal intravital imaging we witnessed increased vascular permeability upon AML engraftment, observed homogeneously among different PDX. Interestingly, induction chemotherapy failed to normalize the vascular permeability in the BM, despite significant reduction in AML engraftment. We identified increased nitric oxide (NO) as a major mediator of the AML-induced vascular leakiness in the BM. Increased levels of NO and activated NOS3 were found in PDX and in an independent cohort of patient-derived BM biopsies. Strikingly, inhibition of NO production using genetic and pharmacological approaches reduced the vascular permeability, potentiated the normal HSC function and significantly improved treatment response in PDX. These results strongly support the notion of a primary function of the vascular permeability in AML progression, drug response and in affecting normal stem cell function, and they call for clinical trials incorporating NOS inhibitors during the remission phase to target the abnormal vascular niche and improve the treatment response.

Disclosures
No relevant conflicts of interest to declare.
Identification of New Multiple Myeloma-Specific Cell Surface Antigens As Immunotherapeutic Targets
Authors: Hosen Naoki , Hasegawa Kana , Aoyama Yasutaka , Ichihara Hiroyoshi , Mugitani Atsuko , Nakao Takafumi , Manabe Masahiro , Oji Yusuke , Sugiyama Haruo 
Published: October 2019

Abstract
Cancer-specific cell surface antigens are ideal targets for therapies using monoclonal antibodies (mAbs) and their derivatives, such as chimeric antigen receptor (CAR)-T cells. However, such antigens are not likely to remain unidentified following extensive searching by transcriptome or proteome analyses. However, we hypothesized that cancer-specific antigens formed by post-translational events, such as glycosylation, complex formation, or conformational changes, might have been missed in previous screens. Such antigens could be discovered by thoroughly searching for cancer-specific mAbs and characterizing the antigens recognized by these mAbs. To test our hypothesis, we applied this strategy to identify novel therapeutic targets specific for multiple myeloma (MM), a major hematological cancer. We first identified two MM-specific mAbs designated as MMG49 or R8H283 after screening more than 10,000 anti-MM mAb clones. Then, we identified the antigens recognized by these mAbs by an expression cloning method. Interestingly, genes identified as antigens for both mAbs were not specific to MM cells, suggesting that both mAbs recognize MM-specific epitopes formed by post-translational events. Finally, we showed that these mAbs or CAR-T cells derived from them could reduce tumor burden in MM-xenograft models, but did not damage normal hematopoietic cells. These results not only demonstrate that these mAb or CAR-T cell therapy is promising for MM, but also suggest that MM-specific immunotherapetic target antigens formed by post translational events may be still missed.

Disclosures
Aoyama: Alexion: Honoraria.
Gene Disruption of Zebrafish Sbds Phenocopies Human Shwachman-Diamond Syndrome but Suggests More Global and Lineage Defects
Authors: Oyarbide Usua , Kell Margaret J , Farinas Javier , Topczewski Jacek , Corey Seth 
Published: October 2019

Abstract
Introduction: Shwachman-Diamond Syndrome (SDS) is a complex disorder resulting in short stature, neutropenia, bony dysplasia, and pancreatic malabsorption. Individuals with SDS are also at increased risk for developing myelodysplastic syndrome and/or acute myeloid leukemia (AML). Its estimated incidence of 1 in 77,000 births makes SDS difficult to study and develop newer therapies. SDS results from mutations in the SBDS gene. Genetic ablation of Sbds results in early embryonic lethality (ED 7.5) in mice. Zebrafish provide an attractive, alternative model organism to study human diseases. Genome analysis of zebrafish (Danio rerio) revealed the presence of a single sbds gene, encoding a protein 90% identical to the human orthologue.
Methods: We used CRISPR/Cas9 genome editing technique to generate indel mutations in sbds andestablished multiple zebrafish lines. We used zebrafish sbds mutants to analyze the function of this gene during vertebrate development and in the pathophysiology of SDS.
Results: Western blotting showed progressive decrease of Sbds expression with no detectable protein at 8 days post fertilization (dpf). These results suggest that the presence of Sbds during early stages of the development is due to maternal deposition, which was confirmed by qPCR and immunoblot demonstration of sbds transcript and proteins in in all stages of the development including the 1 cell-stage. Unlike Sbds-/- mice, homozygous mutant sbdsnu132live up to 6 weeks. They display marked growth retardation. We also observed a significantly low number of neutrophils at 5 dpf and 15 dpf. No differences were found in macrophages and hemoglobinization. Interestingly, multiple organs, especially the pancreas, liver, digestive tract and eye showed histologic evidence of atrophy at 21 dpf. Differences were also observed in fat deposition and bone formation. These defects were not seen at 15 dpf, suggesting a critical stage in development dependent on functional sbds.
Conclusions: To our knowledge, we established the first zebrafish model of SDS, making it a highly relevant model to investigate its pathophysiology and develop new small molecule therapies. Comparison of null states between mice and zebrafish suggest that loss of Sbds results in two phenotypes: an early embryonic stage required for viability and development and a post-embryonic (larval) stage critical for granulocyte production and normal size. Data will be presented on bone formation, neutrophil chemotaxis, metabolomics, and RNAseq in the zebrafish model.
This work was supported by grants from the NIH R21CA15920, Shwachman-Diamond Syndrome Foundation and the Alex Turnquist Memorial Research fund of Shwachman-Diamond America.

Disclosures
No relevant conflicts of interest to declare.