bluuebunny/crossref_metadata_2025 huggingface dataset:
Matriptase-2, A Novel Suppressor of Hepcidin.
Authors: Camaschella Clara , Nai Antonella , Pagani Alessia , Silvestri Laura
Published: October 2019

Abstract SCI-26

Hepcidin inhibition is a process essential to increase iron release from duodenal cells and macrophages to plasma in conditions of high iron requests such as iron deficiency, hypoxia and erythropoietic expansion. Among the reported potential inhibitors of hepcidin the serine protease matriptase-2 has a strong effect in vivo both in mice and in humans. Matriptase-2 is a member of the transmembrane serine protease (TTPS) family, encoded by TMPRSS6 gene, whose expression is tissue restricted, mainly to the liver and to a lesser extent to kidney and olfactory epithelium. The role of matriptase-2 in iron metabolism was first defined in the Mask mouse, which after birth develops a type of iron deficiency anemia refractory to oral iron and a peculiar pattern of hair loss, because of inappropriate overexpression of hepcidin and impaired iron absorption, a phenotype due to the deletion of the matriptase-2 serine protease domain. An identical phenotype is reported in Tmprss6 -/- mice and a correspondent phenotype is observed in iron-refractory iron-deficiency anemia (IRIDA) patients. We have shown that matriptase-2 cleaves through a proteolytic processing the bone morphogenetic protein (BMP) co-receptor hemojuvelin from plasma membrane in vitro, indicating that the suppression of the BMP signaling is essential for hepcidin inhibition. The finding also suggests that the BMP pathway requiring hemojuvelin as coreceptor is the main iron-dependent pathway of hepcidin regulation. The hepcidin inhibitory effect is observed also in zebrafish and is abolished in the human homologue of Mask. We have investigated several missense TMPRSS6 mutants affecting different domains of the protein, reported in IRIDA patients, in comparison with wild type matriptase-2 and Mask. Transient overexpression in hepatoma cells shows that all mutants are deficient in the ability to inhibit the hepcidin promoter in a classic luciferase-based assay and that the decreased hepcidin inactivation broadly corresponds to the inability to cleave hemojuvelin from plasma membrane. Studies of the in vitro processing of the mutants indicate that determinants of the pathogenic effect others than the protease activity are the intracellular processing and the ability of self-activation of matriptase-2. These results have implications for the molecular pathogenesis of IRIDA.

Disclosures

No relevant conflicts of interest to declare.
Development of Hepcidin Agonists and Antagonists.
Authors: Nemeth Elizabeta
Published: October 2019

Abstract SCI-27

Hepcidin binding to its receptor, the iron exporter ferroportin, leads to degradation of the ligand-receptor complex and decreased flow of iron into plasma from duodenum, macrophages and hepatocytes. Dysregulated production of hepcidin or its aberrant interaction with ferroportin causes a number of iron disorders. Hepcidin deficiency, either relative or absolute, underlies excessive iron loading in hereditary hemochromatosis, beta-thalassemia and hepatitis C. A rare type of hereditary hemochromatosis is also caused by ferroportin resistance to hepcidin. On the other side of the spectrum, hepcidin excess causes iron restriction and anemia, as is observed in patients with iron-refractory iron deficiency anemia. Hepcidin also contributes to development of anemia in different inflammatory disorders and chronic kidney disease, and may cause erythropoietin resistance. Hepcidin agonist and antagonists thus may be useful for treatment of different iron disorders. Antagonist strategies for lowering hepcidin levels have been reported, and include hepcidin-neutralizing monoclonal antibodies or agents interfering with the BMP-related hepcidin production (soluble hemojuvelin and dorsomorphin). To undertake the rational design of drug leads, we analyzed the critical structural features that determine the interaction of hepcidin and Fpn. We showed that the thiol form of Fpn residue Cys326 is essential for hepcidin binding, as is the presence of a disulfide bond on hepcidin. The mechanism of binding appears to involve disulfide exchange between the two molecules. Treatment of ferroportin-expressing cells with thiol-reactive agents prevented hepcidin binding and allowed continuous export of iron from cells. Systematic mutagenesis of additional residues on hepcidin showed that His3, Phe4 and Phe9 are also important for binding to ferroportin. To provide leads for hepcidin agonists, we then synthesized a mini-hepcidin containing only the 9 N-terminal hepcidin residues (DTHFPICIF), and showed that it displayed significant activity in vitro. Proteolysis-resistant analogs of this peptide were also active in vivo after intraperitoneal injection in mice. The small size of these peptides may allow oral bioavailability. Understanding the mechanisms of hepcidin regulation and its interaction with ferroportin will facilitate the development of new agents for the treatment of iron overload disorders and iron-restricted anemias.

Disclosures

Nemeth: Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Patient-Specific Pluripotent Stem Cells for Hematologic Disease.
Authors: Daley George Q.
Published: October 2019

Abstract SCI-40

Pluripotent stem cells can be isolated from embryos (embryonic stem cells; ES cells) or generated by direct reprogramming of somatic cells (induced pluripotent stem cells; iPS cells). Both types can be differentiated into a multitude of cell lineages to serve disease research and cell replacement therapies. Additionally, genetically matched pluripotent stem cells generated via nuclear transfer (ntES cells), parthenogenesis (pES cells), or direct reprogramming (iPS cells) are a possible source of histocompatible cells and tissues for transplantation. We have used customized ntES cells to repair genetic immunodeficiency in mice (Rideout et al., Cell 2002); however, generation of ES cells by nuclear transfer remains inefficient, and to date has not been achieved with human cells. We have also generated ES cells with defined histocompatibility loci by direct parthenogenetic activation of the unfertilized oocyte (Kim et al., Science 2007). Compared to ES cell lines from fertilized embryos, pES cells display comparable in vitro hematopoietic activity, but appear compromised in repopulating hematopoiesis in irradiated adult mouse recipients. We are currently comparing the performance of ntES, pES, and iPS cells in murine models of thalassemia. We have generated human iPS cells by direct reprogramming of human somatic cells with OCT4, SOX2, MYC, and KLF4 (Park et al., Nature 2008), and have generated disease-specific iPS cells from patients with a number of hematologic conditions (Park et al., Cell 2008; Agarwal et al., submitted). Applications of disease-specific cells for investigating the mechanisms of reprogramming and for probing aspects of human bone marrow disorders will be discussed.

Disclosures

Daley: iPierian: Consultancy, Equity Ownership; Epizyme: Consultancy; Solasia: Consultancy; MPM Capital: Consultancy.
Genetic Modifiers of Thrombosis in Mice.
Authors: Ginsburg David
Published: October 2019

Abstract SCI-44

The genetic factors responsible for the highly variable clinical course of inherited bleeding disorders including von Willebrand disease and hemophilia are largely unknown. Similar factors are also likely to contribute to the variability of common thrombotic disorders, including factor V Leiden. Studies by our lab over the past 10 years have used the power of mouse genetics to identify genes contributing to this variability (referred to as ‘modifier‘ genes). By performing genetic crosses between inbred strains of mice with elevated plasma levels of von Willebrand Factor (VWF) and other strains with low levels, we have mapped a total of 6 genetic factors contributing to the control of murine plasma VWF levels. Similar studies in ADAMTS13-deficient mice are in progress aimed at characterizing genes modifying susceptibility thrombotic thrombocytopenic purpura. We have also conducted large scale mutagenesis studies in the mouse in an effort to identify larger numbers of genes contributing to thrombosis risk in the setting of Factor V Leiden, and most recently are extending this approach to similar genetic screens in zebrafish. Finally, recent advances in human genetics are expanding the potential opportunities for directly identifying bleeding and thrombosis modifier genes in humans.

Disclosures

No relevant conflicts of interest to declare.
The Lymph Node Niche.
Authors: Mempel Thorsten R.
Published: October 2019

Abstract SCI-51

Lymph nodes provide specialized stromal environments that support the maintenance and homeostasis of T and B lymphocyte populations and are also staging grounds for lymphocyte effector responses against pathogens and transformed cells. They serve as immune information hotspots by collecting lymph fluid from peripheral tissues, especially our external and internal epithelial body surfaces, thus displaying a condensed representation of foreign and self-antigens at these sites in addition to integrating innate alarm signals that report tissue damage or pathogen invasion. Naïve B and T cells constantly traffic through these environments via the blood stream and efferent lymphatic vessels, which allows for efficient matching of their antigen receptor repertoires with the regional antigenic landscape. Depending on the absence or presence of signs of a potential threat to the organism, the result may be either tolerance or immunity towards the origin of these antigens. The architecture of lymph nodes is optimized to facilitate the presentation of lymph-borne antigen in various forms and to guide naïve lymphocytes in their search for 'their' cognate antigen in the form in which they are able to 'see' it. It also facilitates the cellular crosstalk with other immune cell populations that shape and regulate an ensuing adaptive response if cognate antigen is encountered in an immunogenic context. Our conception of how these various tasks are accomplished has recently been enriched through new methodological approaches that include the dynamic in situ or in vivo visualization of cellular and molecular processes using modern microscopy technology. We will review some recent insights into the function of lymph nodes derived from these studies.

Disclosures

No relevant conflicts of interest to declare.
A Key Role for Tumor Suppressor BTG1 In the Regulation of Metabolic Stress Responses: Implications for Asparaginase Resistance In Pediatric Acute Lymphoblastic Leukemia.
Authors: van der Meer Laurens , Levers Marloes , van Emst Liesbeth , van Galen Joost C. , van Kessel Ad Geurts , Kuiper Roland P. , Hoogerbrugge Peter , van Leeuwen Frank N.
Published: October 2019

Abstract 1028

Background:

L-Asparaginase (ASNase) is a key component of multi-agent chemotherapy regimens used in the treatment of pediatric Acute Lymphoblastic Leukemia (ALL). This bacterially-derived enzyme lowers blood asparagine levels by catalyzing the hydrolysis of L-asparagine, leading to specific killing of leukemic blasts. However, relapses and associated therapy resistance occur in about 20% of the patients. Although the molecular mechanisms that contribute to ASNase resistance are not well understood, increased expression of asparagine synthethase (ASNS), the enzyme responsible for asparagine synthesis, has been linked to ASNase resistance both in ALL cell lines and in primary leukemias. We recently reported that micro-deletions affecting the B-cell translocation gene 1 (BTG1) occur in about 10% of pediatric pre-B ALL cases. Here we show that BTG1 loss contributes to ASNase resistance by up-regulation of ASNS.

Results:

Using RNA interference, we show that loss of BTG1 expression promotes cell growth and renders pre-B ALL cells completely refractory to ASNase induced cell death. Resistance to ASNase in the BTG1 knockdown cells is accompanied by increased expression of ASNS, while knockdown of this metabolic enzyme is sufficient to reverse therapy resistance, indicating that upregulation of ASNS is required for the observed resistance phenotype. We further show that BTG1 associates with and regulates the activity of the transcription factor ATF4, a key regulator of metabolic stress responses and a central component in the regulatory network that controls ASNS expression.

Conclusion:

Together, our experiments identify BTG1 as an important regulator of ASNS expression which acts by modulating ATF4 function. We expect that a detailed molecular understanding of how BTG1 loss contributes to ASNase resistance, will lead to the identification of pharmacological targets that can be used to improve treatment responses in therapy-resistant ALL.

Disclosures:

No relevant conflicts of interest to declare.
Heterogeneous Interactions of Leukemic and Control Bone Marrow Stroma Cells with an AML Cell Line and AML Leukemic Blasts.
Authors: Buttkereit Ulrike , Mohamad Sana , Lindemann Monika , Goethert Joachim R. , Opalka Bertram , Duerig Jan , Duehrsen Ulrich
Published: October 2019

Abstract 1047

Tumor-stroma interaction plays a pivotal role for malignant cell survival, proliferation, immune escape, and drug resistance. We had previously shown that bone marrow (BM) stroma from leukemia patients had different gene expression patterns and support of normal CD34+ cells compared to controls (Ctr). Aim of this study was to evaluate growth, survival, and colony-forming potential issues of M-07e AML cells and AML blasts as indicator cells (IC) upon contact with non-leukemic and leukemic stroma. Plastic-adherent BM cells from leukemic or Ctr non-leukemic donors were cultured for several weeks. Polyclonal as well as isolated single fibroblast colony (F-CFU) cultures were set up. Short-term and long-term co-cultures of BM stroma used M-07e AML cells and AML blasts as IC. After long-term co-culture colony forming units (CFU) were determined in the adherent and non-adherent fraction of the IC. Proliferation of IC was determined by cell counting and 3H-TdR incorporation assays. The human HS-5 stroma cell line was used in selected experiments. The F-CFU frequency was determined from 39 Ctr samples, 6 MDS samples, 59 leukemic samples, and 12 lymphoma samples with BM infiltration. Succession of F-CFU numbers in respective samples was AML / MDS < Ctr < MM / FL II << 2°AML. Polyclonal stroma cells from AML BM supported M-07e IC survival slightly but not significantly better than Ctr stroma. No noteworthy support for primary AML blasts was observed with either stroma. Stroma from both AML and Ctr donors stimulated colony formation of M-07e IC without marked differences in the frequency of compact and diffuse colonies. In 3H-TdR assays M-07e IC were stimulated by IL-3 and certain but not all stroma samples tested. Notably, IL-3-induced proliferation of M-07e IC was decreased in the presence of stroma and almost completely suppressed by HS-5 cells. When single F-CFU were isolated, expanded, and tested for their capacity to support M-07e IC stimulating as well as non-stimulating F-CFU were found. Support competence of stroma cells decreased with passage number. Primary BM polyclonal stroma cells and single F-CFU from leukemic and non-leukemic donors showed a broad heterogeneity with respect to support of cell growth or colony-forming potential in favour of the AML M-07e IC line. Thus, the interaction of stroma and normal or leukemic hematopoietic cells seems to be a complex system awaiting further investigations.

Disclosures:

No relevant conflicts of interest to declare.
Micafungin Versus Voriconazole for Empirical Antifungal Therapy In Febrile Neutropenic Patients with Acute Myeloid Leukemia: A Randomized, Controlled Trial.
Authors: Oyake Tatsuo , Kowata Shugo , Murai Kazunori , Ito Shigeki , Akagi Tomoaki , Kubo Koumei , Sawada Kenichi , Ishida Yoji
Published: October 2019

Abstract 1062

Background:

Invasive fungal infections (IFIs) incur significant morbidity and mortality among neutropenic patients after chemotherapy. The risk for these infections is related to the intensity and duration of neutropenia, and varies from 2% to 40%. Mortality rates associated with documented IFIs are considerable, reportedly ranging from 30% to 60%. Empirical antifungal therapy is the standard care for neutropenic patients with hematological malignancies who remain febrile despite broad-spectrum antibacterial treatment. Several antifungal agents including voriconazole (VRCZ) or liposomal amphotericin B (L-AMB) have been studied as empirical therapy for febrile neutropenia (FN). However, limited data are available concerning the efficacy of micafungin (MCFG) in FN patients with acute myeloid leukemia (AML).

Methods:

We conducted a randomized, cooperative group, open-label trial comparing MCFG (150 mg once daily) with VRCZ (6 mg/kg twice on day 1 followed by 4 mg/kg twice daily) as first-line empirical antifungal treatment for 95 hospitalized FN patients with AML during induction or consolidation chemotherapy (MCFG, 49; VRCZ, 46). The efficacy end point was a favorable overall response, as determined by a five-component end point according to the criteria of Walsh et al (N Engl J Med 2004; 351: 1391).

Results:

At the time of enrolment, there were no significant differences in the demographics or baseline characteristics between the two groups. The mean treatment duration for MCFG and VRCZ was 10 and 9 days, respectively. The efficacy rates of MCFG and VRCZ were not significantly different (37.8% vs. 32.4%). The rates of breakthrough fungal infections (proven, probable and possible IFIs), successful treatment of baseline fungal infections, survival 7 days after end of therapy, and resolution of fever during neutropenia were similar in the two groups. However, premature discontinuation of therapy occurred less often in the MCFG group than in the VRCZ group (32.4% vs. 55.9%, P=0.0457*). In safety evaluation, there were fewer adverse events in the MCFG group than in the VRCZ group (27.0% vs. 64.7%, P=0.0013*). *: Chi square test

Conclusions:

MCFG was as effective as VRCZ, and better tolerated than VRCZ as empirical antifungal therapy in FN patients with AML.

Disclosures:

No relevant conflicts of interest to declare.
An Open Label, Non Randomized, Propspective Phase IV Clinical Trial Evaluating the Immunogenicity of Branded Enoxaparin Versus Biosimilars In Healthy Volunteers.
Authors: Gomes Marise , Ramacciotti Eduardo , Hoppensteadt Debra , Walenga Jeanine M. , Lewis Bruce E , Jeske Walter , Fareed Jawed
Published: October 2019

Abstract 1086

Biosimilar enoxaparin preparations are in use outside the U.S. Due to compositional variations, their interaction with platelet factor 4 (PF4) differs leading to differential immunogenic responses between branded and biosimilar agents. To compare their immunogenic response, branded enoxaparin (Clexane®, Sanofi-Aventis) and a biosimilar version (Cutenox®, Gland-Pharma) were administered to healthy volunteers (n=110/drug) at a dose of 40 mg SQ for10 days. Blood samples drawn on days 1 and 10 were analyzed for anti-heparin/PF4 antibody (A-HPF4-Ab) titers and subtypes by ELISA (GTI, Brookfield, WI). Treatment with each LMWH resulted in comparable A-HPF4-Ab generation as compared by using the total absorbance for each population (p<0.05). However in the thrombin generation assays clexane group showed a stronger inhibition (45+12% vs 32+9%). None of these antibodies activated platelets as determined by the serotonin release assay. The two groups alos showed comparable AXa and AIIa responses (p<0.05). Antibody subtyping demonstrated different profiles between LMWHs. For IgG (Clexane1=0.15±0.04, Clexane10=0.21±0.06, Cutenox1=0.17±0.04, Cutenox10=0.28±0.10) with a significant time effect (p<0.0001), a significant drug effect (p<0.0001), and a significant time by drug interaction (p=.0009). Post hoc comparisons showed a difference between the drugs at time 0 (p=0.03), a difference between the drugs at time 10 (p<0.0001) and a significant time effect for each drug (p<0.0001). For IgA (Clexane1=0.12±0.02, Clexane10=0.15±0.02, Cutenox1=0.12±0.03, Cutenox10=0.13±0.02) with significant effects for time (p<0.0001), drug (p=0.0078) and for the drug × time interaction (p<0.0001). The post hoc comparisons showed a significant drug effect at time 10 (p<0.0001). There was a significant time effect for Clexane (P<0.0001) but not for Cutenox. For IgM (Clexane1=0.11±0.01, Clexane10=0.13±0.02, Cutenox1=0.11±0.03, Cutenox10=0.13±0.02), there was only a time effect (p<0.0001). The post hoc comparisons showed no difference between drugs at either time, but significant time effects for each drug (p<0.0001). The immunogenic potential of LMWHs varies in terms of ability to generate A-HPF4-Ab, the antibody subtypes generated, and their cross-reactivity with pre-formed A-HPF4-Ab. Such parameters may be useful in defining the bioequivalence of generic LMWHs. Future studies evaluating the immunogenicity of different compounds in patients exposed to biosimilar drugs are warranted.

Disclosures:

Jeske: PolyMedix, Inc.: Research Funding.
What Is the Best Time to Initiate Peri-Operative Heparin Therapy In General Surgery Patients? A Risk-Benefit Dilemma.
Authors: Rostambeigi Nassir , Greenlee Susan , Huebner Marianne , Farley David
Published: October 2019

Abstract 1088

Background:

While many studies support the use of prophylactic heparin therapy around the time of operation in cancer patients, little data shows the optimal timing of such therapy in general surgery (GS) patients.

Methods:

From 2001–2008, 95 patients identified with thromboembolism reported in our GI/General Surgery Division and were compared with a matched control group for: age, gender, type of operation, date of operation, diagnosis of malignancy, and BMI. Timing of heparin therapy, characteristics of TE or bleeding events, driving distance from our institution, history of smoking or OCP use, anticoagulation use after operation, length of hospital stay, and mortality were collected. Peri-operative heparin therapy was defined as heparin given within a 24 hr window of incision. The McNemar's test for proportions was performed. Conditional logistic regression was used for the analysis of risk factors and survival curves were implemented for mortality comparisons.

Results:

mean age and BMI were similar (57 yrs, 33 kgM-2), 45% were female and 53% had malignancy in both groups. Duration of operation was similar (204 vs. 191 minutes, P=NS). peri-operative heparin administration was similar in the two groups (56%, 64%, p=0.05). But, pre-operative therapy was seen more in the control group (77% vs. 51%, p=0.01). Regression model showed a protective effect for heparin if it was given pre-operatively (OR=0.37, p=0.047) with no effect if it was given beyond 10 hours. Mean blood transfusion units (97 and 106 mL) and hemorrhagic events (4.5% and 5%) were similar (p=NS). Median (range) length of hospital stay was longer (19 days (0-201) vs. 6 (0-66), p=0.001) and two-year survival lower in the TE group (p=0.03).

Conclusions:

data from our institutional case-control study shows that heparin administration before GS is associated with more than 2-fold reduction in the risk of thromboembolism. The optimal period appears to be between 1–10 hours prior to the time of incision. Large prospective trials need to be desgined to more accurately determine the time of heparin therapy.

Disclosures:

No relevant conflicts of interest to declare.
Prothrombin Complex Concentrate Reverses the Anticoagulant Effect of Rivaroxaban In Healthy Volunteers.
Authors: Eerenberg Elise S , Sijpkens Meertien K , Kamphuisen Pieter W , Meijers Joost CM , Levi Marcel
Published: October 2019

Abstract 1094

Background:

Rivaroxaban is a novel oral antithrombotic agent in the class of specific factor Xa inhibitors, and is licensed in Europe and Canada for the prevention of venous thromboembolism after elective orthopedic surgery. Despite its stable pharmacokinetic and pharmacodynamic profile, immediate reversal may be required in case of a major bleeding or emergency surgery. No specific antidote is currently available, although Prothrombin Complex Concentrate (PCC) seems effective in animal studies. This study is the first to investigate the ability of PCC to reverse the antithrombotic effect of Rivaroxaban in humans.

Methods:

In a randomized, double-blind, placebo-controlled trial, twelve healthy male subjects received Rivaroxaban 20mg twice daily for two and a half days. One group (n=6) was then randomized to receive a single bolus of 50 IU/kg PCC (Co-fact®, Sanquin, the Netherlands) while the other group (n=6) was given a similar volume of saline.

Results:

The prothrombin time (PT) was significantly prolonged by Rivaroxaban (15.8 sec ± 1.3 versus 12.3 ± 0.7 at baseline; p< 0.001). Immediately after the infusion of PCC, the PT normalised almost completely (12.8 ± 1.0; p< 0.001), which was sustained for 24 hours. Saline did not reverse the PT prolongation (16.2 ± 0.8; p= 0.4). Furthermore, Rivaroxaban inhibited the endogenous thrombin potential (ETP) (51% ± 22, baseline 92 ± 22; p= 0.002), with normalisation after administration of PCC (114 ± 26; p< 0.001), but not after saline (41 ± 6; p= 0.2).

Conclusions:

This study provides the first data that Prothrombin Complex Concentrate reverses the anticoagulant effect of Rivaroxaban in humans and may serve as an antidote for the new oral factor Xa inhibitors.

Disclosures:

Eerenberg: Sanquin Netherlands: Research Funding, make available of Prothrombin Complex Concentrate for this study. Kamphuisen:Sanquin Netherlands: Research Funding, make available of Prothrombin Complex Concentrate for this study. Levi:Sanquin Netherlands: Research Funding, make available of Prothrombin Complex Concentrate for this study.
Predictors of the Indefinite-Duration Anticoagulation Treatment Strategy In Patients with Cancer-Associated Thrombosis.
Authors: Spirk David , Korte Wolfgang , Husmann Marc , Frauchiger Beat , Banyai Martin , Aujesky Drahomir , Baumgartner Iris , Kucher Nils
Published: October 2019

Abstract 1097

Background:

In patients with cancer and acute venous thromboembolism (VTE), current consensus guidelines recommend anticoagulation therapy for an indefinite duration or until the cancer is resolved.

Methods and results:

Among 1’247 patients with acute VTE enrolled in the Swiss Venous Thromboembolism Registry (SWIVTER) from 18 hospitals, 315 (25%) had cancer of whom 179 (57%) had metastatic disease, 159 (50%) ongoing or recent chemotherapy, and 83 (26%) tumor surgery within 6 months. Patients with cancer were older (66±14 vs. 60±19 years, p<0.001), more often hospitalized at the time of VTE diagnosis (46% vs. 36%, p=0.001), immobile for >3 days (25% vs. 16%, p<0.001), and more often had thrombocytopenia (6% vs. 1%, p<0.001) than patients without cancer. The 30-day rate of VTE-related death or recurrent VTE was 9% in cancer patients vs. 4% in patients without cancer (p<0.001), and the rates of bleeding requiring medical attention were 5% in both groups (p=0.57). Cancer patients received indefinite-duration anticoagulation treatment more often than patients without cancer (47% vs. 19%, p<0.001), and LMWH mono-therapy during the initial 3 months was prescribed to 45% vs. 8%, p<0.001, respectively. Among patients with cancer, prior VTE (OR 4.0, 95%CI 2.0–8.0), metastatic disease (OR 3.0, 95%CI 1.7–5.2), outpatient status at the time of VTE diagnosis (OR 3.8, 95%CI 1.9–7.6), and inpatient treatment (OR 4.4, 95%CI 2.1–9.2) were independently associated with the prescription of indefinite-duration anticoagulation treatment.

Conclusions:

Less than half of the cancer patients with acute VTE received a prescription for indefinite-duration anticoagulation treatment. Recurrent VTE, metastatic cancer, outpatient VTE diagnosis, and VTE requiring hospitalization were associated with an increased use of this strategy.

Disclosures:

Spirk: sanofi-aventis (suisse) sa: Employment.
Thrombin Activation of Protease-Activated Receptor-2 on Neutrophils Primes the Respiratory Burst.
Authors: Bock Ashley , Kelher Marguerite , Khan Samina , Hansen Kirk , Dzieciatkowska Monika , Silliman Christopher C
Published: October 2019

Abstract 1119

Thrombin is a multifunctional serine protease involved in hemostasis, fibrinolysis, and pro-inflammatory activation of innate immunity through stimulation of protease-activated receptors (PARs). Thrombin is considered the prototypical protease to activate PARs through cleavage of the N-terminal exodomain of the receptor, thereby unmasking a tethered ligand which activates it. Neutrophils (PMNs) are reported to express PAR-2; however, little is known about the changes in PMN physiology following protease activation of PAR-2. In addition, proteomic analyses (two dimensional gel electrophoresis and mass spectroscopy (MALDI-TOF)) of injured patients (blunt trauma) who developed acute lung injury (ALI) and multiple organ failure (MOF) have identified a number of serine proteases which accumulate post-injury. Moreover, these identical proteases were also found via proteomics in stored, but not fresh, packed red blood cells, which are used to resuscitate these injured patients. We hypothesize that thrombin primes PMNs through activation of PAR-2. Methods: PMNs were isolated from whole blood drawn from healthy human donors using dextran sedimentation, ficoll-hypaque gradient centrifugation and hypotonic lysis of red blood cells. The proteins from whole cell lysates were separated by SDS-polyacrylamide electrophoresis, transferred to nitrocellulose and immunoblotted with a monoclonal antibody to PAR-2. The PMNs were also incubated for 3–30 minutes at 37°C with thrombin [1-10 U/ml] followed by fMLP [1 μM] activation of the NADPH oxidase. Oxidase activity was measured by the superoxide dismutase-inhibitable reduction of cytochrome c at 550 nm. Results: PMNs express PAR-2 as detected by western blotting of whole cell lysates. Thrombin [1 U/ml] for 30 min, significantly primed the fMLP-activated respiratory burst: buffer control: 1.6 +/− 0.5 vs. 1 U/ml thrombin: 2.2 +/− 0.3* (*p<.05, n=5). Higher concentrations of thrombin or shorter incubation times did not induce PMN priming. Furthermore, pre-incubation with leupeptin [50 μM] and 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) [250 μM], two effective protease inhibitors, abrogated the thrombin-induced PMN priming by 100±18%. We conclude that thrombin primes the PMN respiratory burst, which is dependent upon its protease activity. Because PMNs are the effector cell in ALI/MOF and priming agents have been linked to the development of clinical ALI and MOF, proteases that accumulate in the patient plasma post-injury or are transfused with stored PRBCs may be etiologic in post-injury ALI/MOF.

Disclosures:

No relevant conflicts of interest to declare.
Is Transfusion of Rh Positive Red Blood Cells to ccDee Patients From a Cosmopolitan Population Safe?.
Authors: Vandeput Marie-Helene , Latinne Dominique , Gervais Thibaut , Eeckhoudt Stephane L
Published: October 2019

Abstract 1121

Introduction:

D variant is a common phenotype in Africans. However, the allele multiplicity challenges the serological methods which often fail to detect D variant patients in such populations. Recently, two cases of strong anti-D allo-immunization occurred in our blood bank in ccDee patients who received transfusions with Rh positive red blood cells. The D phenotype of these patients was determined using two different monoclonal and one polyclonal anti-D antibodies, with normal agglutination (4+) observed. Considering these results, we decided to investigate the prevalence of D variant for the ccDee patients of our cosmopolitan population. The aim of this study was to determine whether ccDee blood recipients should systematically undergo molecular screening for D variant prior to transfusion in order to avoid anti-D allo-immunization.

Materiel and methods:

Fresh EDTA blood from 35 ccDee patients (22 Africans and 13 Europeans) was analyzed using both serological and molecular methods. Serological characterization was performed using two monoclonal and one human polyclonal anti-D antibodies. In addition, each sample was tested using an extended partial RhD typing set from Diamed (consisting of 12 monoclonal antisera). Molecular determination was performed using two polymerase chain reactions with sequence-specific priming (PCR-SSP) kits intended to diagnose either partial D or weak D phenotypes (BAGene, BAG, Lich, Germany).

Result:

Among the 35 patients screened, discordant results between serological and molecular methods were found in 12. While serological tests revealed a normal D phenotype for these patients with all anti-D antibodies tested, molecular tests diagnosed them as D variant (nine were DAU; two were weak D types 4.0 and 4.1; one was DAR). As expected, the vast majority (9/12) of these patients were of African descent. No discordance between serological and molecular screening was observed for the rest of the screened population (23 patients).

Conclusion:

Our study reveals that approximately one-third (12/35) of our ccDee patients present a D variant genotype despite normal serological results. It emphasizes the fact that transfusion of Rh positive red blood cells to ccDee patients (mainly of African origin) requires precautionary measures. Molecular D variant screening in ccDee patients should be performed in order to improve the handling of transfusions in ccDee patients.

Disclosures:

No relevant conflicts of interest to declare.
Carbon Monoxide-Releasing Molecule-Derived CO Regulates Tissue Factor, Plasminogen Activator Inhibitor Type 1, and Thrombomodulin Production by Human Endothelial Cells.
Authors: Maruyama Keiko , Morishita Eriko , Ohtake Shigeki , Yachie Akihiro , Nakao Shinji
Published: October 2019

Abstract 1146

Background and purpose:

Heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). HO-1 is rapidly induced by various oxidative stresses and inflammation, thereby playing an important role in the self defence system. Recently, Yachie, et al. reported the first human case of HO-1 deficiency. This patient showed prominent signs of intravascular hemolysis, endothelial cell injury, and abnormalities in the coagulation / fibrinolysis system, suggesting the involvement of HO-1 or HO-1 products, such as CO, regulation of coagulation / fibrinolysis system. The current study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberated CO in the presence of dimethyl sulfoxide (DMSO), modulates the expression of tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1) and thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs).

Methods:

HUVECs were pretreated with CORM-2 at the concentration of 50 μM for 3h, washed and stimulated with tumor necrosis factor-α(TNF-α, 10 ng/ml) for additional 2 or 5h. The mRNA and protein levels of TF, PAI-1 and TM in the cultured HUVECs were determined by real-time reverse transcriptase polymerase chain reaction and Western blotting. To determine whether CORM-2 affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) in the HUVECs were analyzed with Western blotting.

Results:

After TNF-α stimulation, TF mRNA levels were approximately 6-fold and PAI-1 mRNA levels were approximately 8-fold increased, and TM mRNA levels were decreased by 20% compared to the control. Similarly to the mRNA expression, TF and PAI-1 protein levels were increased while the TM protein level was decreased. On the other hand, pretreatment of HUVECs with CORM-2 significantly decreased TF mRNA levels (approximately 80% suppression), and PAI-1 mRNA levels (approximately 90% suppression) while increased TM mRNA levels by 3-fold as compared to the TNF-α-stimulated group (p<0.05; Figure 1). Similarly, the pretreatment with CORM-2 inhibited TNF-α-induced TF and PAI-1 protein up-regulation and TM protein down-regulation. CORM-2 inhibited TNF-α-induced activation of p38MAPK and ERK1/2 by 50% compared to the control.

Conclusions:

These results indicate that CO liberated by CORM-2 suppresses the TNF-α-induced TF and PAI-1 up-regulation and prevents the TNF-α-induced TM down-regulation in HUVECs via MAPK signaling pathways. Thus, the modulation of endothelial function by CO may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation.

Disclosures:

No relevant conflicts of interest to declare.
Serum Sickness and Plasma Cytokine Profiles After Treatment with Antithymocyte Globulin In Severe Aplastic Anemia Patients.
Authors: Feng Xingmin , Scheinberg Phillip , Nunez Olga , Biancotto Angelique , Takaku Tomoiku , McCoy J. Philip , Young Neal S.
Published: October 2019

Abstract 1162

Antithymocyte globulin (ATG) is the standard therapy for severe aplastic anemia (SAA). Currently, two different preparations are commercially available, produced in horse and rabbit. As foreign proteins, ATGs elicit immune response in humans. Foreign antigen and responding antibody form circulating immune complexes that may deposit in tissues and cause serum sickness (SS), usually around day 10 after initiation of ATG therapy. The relationship of human anti-ATG antibodies with SS and the cytokine profiles of ATG-treated patients are not defined. We studied human anti-ATG antibody titers and 27 cytokines in the plasma of 27 patients who received horse ATG (hATG) and 25 patients who received rabbit ATG (rATG) for SAA. Cyclosporine was administered with both ATGs to achieve trough levels of 200–400 ng/mL. Plasma samples were obtained at: baseline pre-treatment, weekly in the first month, and at 3 and 6 months after ATG treatment. Nine of 25 (36%) rATG recipients and 5 of 27 (19%) hATG recipients developed SS at about week two. When measured by enzyme-linked immunosorbent assay, human anti-hATG or rATG antibody titers peaked during weeks 2–3, and then declined at month 2, reducing to baseline levels at month 6. All 9 rATG-treated patients who developed SS had significantly higher antibody titers at week 2 than did 16 rATG-treated patients without SS (O.D. 1.704 ± 0.173 vs 0.664 ± 0.076, p < 0.0001), a difference also presented at month 3 (p = 0.002). In hATG-treated patients, human anti-hATG antibody titers did not appear to correlate with the occurrence of SS. We measured cytokines using a 27-plex cytokine magnetic bead-based Luminex assay. Both hATG and rATG-treated patients without SS showed similar cytokine profiles: a peak in MIP-1β, IL-8, IL-10, IL-13, G-CSF and IP-10 at day 2, which rapidly declined to baseline; this early-phase response may be the direct effect of ATG infusion. In rATG-treated patients, SS patients had significantly higher plasma levels of IFNγ, IL-1ra, IL-12 (p < 0.0001), IL-4, IL-1β (p = 0.0001), IL-6 (p = 0.0002), IL-10 (p = 0.0003), IL-17, GM-CSF, FGFb (p = 0.0015), IL-15, MIP-1α (p = 0.0046), PDGF (p = 0.0052), IL-13 (p = 0.0066), and IL-2 (p = 0.0077) than did non-SS patients at weeks 2 and 3; this late-phase response may be secondary to heterologous antibodies to rATG. In conclusion, human anti-ATG antibodies were frequently detected after ATG administration; antibody titers associated with SS in rATG-treated patients; and SS patients and non-SS patients showed broadly distinct cytokine profiles.

Disclosures:

No relevant conflicts of interest to declare.
p53 Dependent and Dose Dependent Effects of Rps29 Mutation In the Zebrafish Embryo.
Authors: Taylor Alison M. , Humphries Jessica M. , White Richard M. , Murphey Ryan D. , Burns Caroline E. , Zon Leonard I.
Published: October 2019

Abstract 1170

Diamond Blackfan anemia (DBA) is a rare congenital disease characterized by red cell aplasia and craniofacial abnormalities. Ribosomal protein genes are often mutated in patients with this disease, but the mechanism of action is still being investigated. To elucidate the effect of mutations in ribosomal proteins, we are studying a zebrafish rps29 mutant with hematopoietic and endothelial defects. Hematopoietic stem cells (HSCs) in rps29-/- embryos are significantly decreased, as assayed by runx1 and cmyb expression. Although the aorta and posterior cardinal vein form in the mutant, intersomitic vessel formation is affected. To test whether decreased p53 levels can rescue these defects, we crossed fish with mutated p53 into the rps29 background. In rps29-/-;p53-/- embryos, the vascular and HSC phenotypes are rescued, demonstrating that p53 may be required for these effects of rps29 knockdown. We performed a microarray comparing rps29-/- embryos and their siblings to identify genes that are differentially expressed in the mutant. Using gene set enrichment analysis (GSEA), we determined that the list of genes up-regulated in the rps29 mutant is enriched for genes up-regulated by p53 in response to irradiation. Many of the genes identified have known roles in apoptosis and stress response. We have also identified genes whose expression correlates with the number of wildtype copies of rps29. Orthopedia homolog a (otpa), which is specifically expressed in forebrain and hindbrain tissues at 24 hours post fertilization (hpf), is decreased in heterozygous siblings and further decreased in homozygous siblings. In addition, p53 knockdown partially increases otpa levels in the mutant. These data support a model where p53 activation is one of the critical downstream mediators of rps29 knockdown in several tissues, but the mechanism of tissue specificity remains unclear. The otpa phenotype suggests that regulation of some genes is dependent on rps29 levels. The zebrafish rps29 mutant will be a useful model for understanding how a decrease in ribosomal protein levels can cause specific defects in hematopoietic and neural tissues.

Disclosures:

Zon: FATE, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Analysis of Cell Aggregation In Thawed Hematopoietic Stem Cell Products Using Micro-Flow Imaging.
Authors: Wu Luke , Martin Teresa , Halpenny Michael , Giulivi Antonio , Allan David S.
Published: October 2019

Abstract 1187

Background:

Cell aggregation can be observed after thawing cryopreserved hematopoietic stem cell products used in transplantation. Micro-flow imaging (MFI) is a novel method of imaging biologic fluids and provides a means of quantifying cell aggregation.

Method:

Umbilical cord blood (UCB) was collected following informed consent and cryopreserved in 5% DMSO and 37% (w/v) pentastarch in liquid nitrogen. Aliquots of cryopreserved peripheral blood progenitor cell (PBPC) products were cryopreserved under similar conditions. Cells were thawed rapidly and filtered before analysis using MFI.

Result:

Large cellular aggregates were identified in all thawed PBPC samples analyzed (n=4) with a mean reduction in total cells of 19.7 ± 9.8% at 1 hour and 49.8 ± 15.9% at 24 hours. Cellular aggregates ≤ 170 um were visualized, sized and digitally imaged with MFI. Analysis of larger clumps was limited due to size of the analysis chamber. Cell transparency and circularity were unchanged over 24h after thawing and similar to freshly isolated PBPCs. Mononuclear cell yields did not diminish over 24 hours in freshly isolated PBPC (n=3, p>0.05) or in fresh UCB (n=3, p>0.05). Thawed UCB samples did not demonstrate significant clumping and maintained higher yields of total cells compared with PBPCs (p=0.02). Filtering of thawed PBPC products using standard blood transfusion filters (Baxter, 170 um) did not change the qualitative nature of observed cellular aggregates. The loss of cells in PBPC products correlated with early apoptotic mononuclear cells (7AAD-AnnV+). Treatment with DNase I eliminated the presence of observable cell clumping in thawed PBPCs at 24h with a marked improvement in cell yield at 24h (p<0.0001).

Conclusion:

Micro-flow imaging provides novel insight regarding cellular aggregation of blood stem cell products. Significant cell clumping occurs in cryopreserved PBPC after thawing and aggregates are not removed by standard transfusion filters. Aggregation increases with time after thawing and may influence the final dose of cells available for engraftment after transplantation. Fresh PBPC collections contain few aggregates and both fresh and thawed cryopreserved UCB demonstrated minimal cell clumping. Apoptosis induced by cryopreservation and thawing may contribute to increased aggregation in PBPCs and DNase I appears to preserve higher yields of total cells. New cryoprotectants that limit apoptosis are needed and methods to reduce cell clumping appear warranted.

Disclosures:

Martin: Brightwell Technologies: Employment.
Involvement of a B1 Progenitor In a Murine Model of BCR-FGFR1 Induced Leukemogenesis.
Authors: Tidwell Josephine A , Ren Mingqiang , Cowell John K
Published: October 2019

Abstract 1221

Constitutive activation of the FGFR1 kinase domain through rearrangement with dimerization domains from various proteins leads to an atypical myeloproliferative disease (MPD). Although the majority of these rearrangements result in development of T-cell leukemia/lymphoma, in the case of the BCR-FGFR1 rearrangement the phenotype is predominantly a myeloid and B cell MPD. To investigate the etiology of this disease, a murine model of BCR-FGFR1 was created using a bone marrow transduction/transplantation approach. Consistent with the human disease, recipient mice developed clear myeloproliferations marked by CML-like basophilia as well as extramedullary leukemic transformation containing myelo- or lymphoblasts, with neoplasms of myeloid and B cell lineages. Flow analysis demonstrated a CD43+ phenotype in leukemic B cells suggesting a block in the differentiation of pro-B progenitor cells. A cell line (BBC1) has now been established with this immature B cell immunophenotype albeit B220-, consistent with a recently described B1 progenitor. These cells can differentiate in vitro with characteristics of both macrophage and dendritic lineages. When BBC1 cells are injected into normal mice, leukemogenesis marked by a hypercellular bone marrow and splenomegaly is induced. Although BBC cells show the same progenitor B cell immunophenotype seen in the parent cells throughout the hematopoietic system, tumor cells in the peritoneum lose CD43 expression, down-regulate CD19 expression and upregulate CD11b and F4/80 demonstrating a capacity to differentiate in this environment. In this model, therefore, the etiology of BCR-FGFR1 disease closely mimics that seen in humans and we have identified a likely B cell subtype involved in leukemic transformation.

Disclosures:

No relevant conflicts of interest to declare.