bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.
Authors: Fuchs Tobias , Brill Alexander , Dürschmied Daniel , Schatzberg Daphne , Hartwig John H. , Myers Daniel , Wakefield Thomas W. , Wagner Denisa D. 
Published: October 2019

Abstract
Abstract 1345

Introduction
Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability.

Disclosures
No relevant conflicts of interest to declare.
The Second Signal in Antigen-Presenting Cells: Complementary JAK-1 Directs Pro- and Anti-Inflammatory Cytokine Production Induced by CD40L.
Authors: Conzelmann Michael , Wagner Andreas , Hildebrandt Anke , Rodionova Elena , Hess Michael , Zota Annika , Giese Thomas , Falk Christine , Breit Stephen , Hecker Markus , Ho Anthony D. , Dreger Peter , Luft Thomas 
Published: October 2019

Abstract
Abstract 1348
Poster Board I-371
Aberrant expression of CD40 ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. However, CD40 activation alone is unable to induce interleukin (IL)-12p70 secretion, whereas IL-6, IL-12p40, TNF-alpha and IL-10 are produced. We demonstrate that cytokine-induced Janus kinase (JAK)-1 is unique in mediating a complementary pro-inflammatory signal for the production of IL-12p70 whilst at the same time inhibiting IL-10 secretion in human APCs.
CD40- and JAK/STAT signals recruit complementary pathways: CD40 signals involve RelA and cRel for the induction of IL-12p70 and RelA for IL-10. In contrast, RelB inhibited IL-12p70 production. JAK1 complemented CD40-induced NF-kB signals by phosphorylating STAT-1 and inducing IRF-1 and IRF-8 expression resulting in IL-12p70 production. JAK-1-dependent inhibition of IL-10 transcription was mediated either by STAT-1 directly binding to the IL-10 promotor or by IRF-8 that in a complex with PU.1 (Spi-1) binds to inhibitory IFN-gamma-activated sequence (GAS)-sites of the IL-10 promotor. Our results demonstrate that specific inhibition of the “second signal” JAK1 will shift the balance of CD40-induced cytokine panels from IL-12p70 towards IL-10 in human APCs.

Disclosures
No relevant conflicts of interest to declare.
The Gene RUNX1 and Its Possible Relation with the Alteration of Granulocytes Cells and with the Progression of Chronic Myeloid Leukemia.
Authors: Mascarenhas Cintia C , Cunha Anderson Ferreira , Brugnerotto Ana Flavia , Gambero Sheley , Costa Fernando Ferreira , Pagnano Katia Borgia Barbosa , Souza Carmino Antonio De 
Published: October 2019

Abstract
Abstract 2215
Poster Board II-192
The exact action mechanism of the protein BCR-ABL is unknown, studies in vitro and in animal models showed that the activity of protein tyrosine kinase (TK), by itself, is sufficient to cause the development of CML. Thus, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. The use of methods to detect the difference in gene expression between CML and normal granulocytes could bring new insights in the understanding of these complex mechanisms, highlighting new therapeutic targets for the CML treatment. Evaluation of the differential gene expression between CML and normal granulocytes using the Subtractive Suppression Hybridization (SSH) and investigation of the involvement of a set of genes in modulating the development of CML. The SSH libraries were constructed using a pool of granulocytes RNA's extracted from CML patients and from healthy blood donors. Real-Time (RT-PCR) was used to validate the results and evaluation of gene expression in granulocytes and in mononuclear cells in the pool of patients and controls used for the construction of libraries. The expression of the RUNX1 gene, whose expression was the most differential between the libraries, was evaluated in mononuclear cells of patients with CML using RT-PCR. The comparison between granulocytes of CML patients and control granulocytes showed 39 genes exclusively expressed in CML and 169 genes in controls. For validation, we evaluated the expression of genes RUNX1, KPNA6, TOB1, SEPT5, CDC42SE, ITCH, MIER and GP1BA by RT-PCR in the pool of patients and controls used for the construction of libraries. Among these genes, the RUNX1 gene showed substantial difference, and was also studied in mononuclear cells from 20 patients with CML in different stages of the disease and using different types of treatment with TK inhibitors. The expression of this gene was highly altered in patients in advanced phase of disease when compared with patients who are responding to treatment. The genes identified in this study may be related to the development and progression of CML. Among them, the RUNX1 gene was shown to be one of the most promising, since the occurrence of chromosomal translocations in this gene has been associated with different types of acute leukemia – however its association with CML is not yet clear. The results showed a relationship between the expression of this gene and progression of the disease, beyond the identification of several genes that may lead to a better understanding of CML and help in identifying new therapeutic targets. This work was supported by FAPESP.

Disclosures:
Off Label Use: Development of TKI in the treatment of CML.
Treatment of Severe Acyclovir-Resistant Herpes Simplex Virus Infection with Continuous Infusion of High Dose Acyclovir Following Hematopoietic Cell Transplantation.
Authors: Kim Janet H. , Schaenman Joanna , Ho Dora Y. , Brown Janice (Wes) M. 
Published: October 2019

Abstract
Abstract 2224
Poster Board II-201

Although acyclovir (ACV)-resistant herpes simplex virus (HSV) occurs in healthy patients, higher rates have been reported in 7-27% of immunocompromised patients, particularly in the setting of acyclovir prophylaxis. Infection due to ACV-resistant HSV (ACV-R HSV) is frequently severe and poses challenges to treatment, particularly following hematopoietic cell transplantation (HCT). Unfortunately, currently available alternative therapies including foscarnet, cidofovir, and ganciclovir are frequently limited by their toxicity profile. We present a series of five HCT patients from 1997-2008 with severe infections due to ACV-R HSV who were successfully treated with continuous infusion of high dose acyclovir. A Pubmed search of published literature from 1948-2009 confirmed this as the first report of ACV-R HSV in HCT patients treated with this regimen.
The characteristics of the patients are summarized in the table. Patients ranged in age from 24 to 55 and had undergone an allogeneic HCT following a myeloablative preparative regimen. All grafts were composed of peripheral blood hematopoietic cells. Underlying diseases included acute myelogenous leukemia, myelodysplastic syndrome, and essential thrombocytosis. Four of the five patients were receiving treatment for GVHD of the skin (grades 2-4), with regimens that included varying doses of prednisone. Infection was due to HSV-1 in three patients and HSV-2 in two. In vitro testing revealed that high median inhibitory concentrations were necessary to inhibit viral replication by 50% (MIC50) for all five isolates consistent with resistance to acyclovir, ganciclovir, and foscarnet. Failed HSV treatment regimens included standard, intermittent doses of acyclovir (5-10 mg/kg IV every eight hours), foscarnet, cidofovir, and/or famciclovir. Adverse reactions to these agents included severe renal insufficiency, hyponatremia, and neutropenia. The doses of ACV administered via continuous infusion ranged from 30 to 50 mg/kg per day, and time to resolution of lesions ranged from 1 week to 2 months. No patients experienced toxicity associated with continuous infusion of high dose acyclovir and therapy could be continued as an outpatient when appropriate.

Conclusions:
Continuous infusion of high dose acyclovir for acyclovir resistant HSV is an attractive and more tolerable alternative for patients failing standard dose intermittent infusion acyclovir or intolerant of alternative standard therapies such as foscarnet, cidofovir, or ganciclovir. At this time it is not clear how the altered pharmacokinetics that results from continuous infusion of high doses of acyclovir overcomes viral resistance. We are currently investigating the mechanisms by which the sustained levels of ACV provide this therapeutic advantage.

Disclosures:
No relevant conflicts of interest to declare.
Different Clinical Results with the Use of Different Dosing Schedules of Azacitidine in Patients with Myelodysplastic Syndrome Managed in Community-Based Practice: Effectiveness and Safety Data From the Spanish Azacitidine Compassionate Use Registry.
Authors: Garcia Regina , de Miguel Dunia , Bailen Alicia , MD José Ramón González, , Sanz Guillermo , Falantes Jose F , Andreu Rafael , Tormo Mar , Duarte Rafael F , Figueredo Antonio 
Published: October 2019

Abstract
Abstract 2773
Poster Board II-749

Background:
Azacitidine (AZA), a hypomethylating agent recently approved in Europe for the treatment of myelodysplastic syndrome (MDS), prolongs the median survival time in patients enrolled in clinical trials (Fenaux et al 2009). AZA was available for clinical trial or compassionate use in Spain before receiving its marketing authorization from the Spanish Medicines Agency in May 2009. The dosing of AZA in community-based hematology clinics could differ from that approved by the health authorities.

Material and Methods:
We present the preliminary analysis of the clinical results of data from a longitudinal, multicenter Spanish patient registry, which retrospectively collected data from community-based hematology clinics on the disease course and management of patients with MDS treated under compassionate use conditions with AZA, in whom the dosing schedule administered was documented. AZA was administered to the patients in three different dosing schedules over 28-day cycles; Group A: days 1, 2, 3, 4 and 5 / Group B: days 1, 2, 3, 4, 5, 8 and 9 / Group C: days 1, 2, 3, 4, 5, 6, 7. Treatment assignation was based on patient status and the feasibility of weekend drug administration. As of August 1, 2009, data from 147 patients with MDS diagnosed according to the WHO criteria had been collected.

Results:
We evaluated 144 patients in whom the dosing schedule was documented at the time of this analysis. At baseline, the patient characteristics were comparable, except for ECOG performance status, with a higher prevalence of an ECOG ≥ 2 in the day 1-7 dosing schedule group (Group C) (Table 1). Group A accounted for 36% of the patients, group B for 32%, and group C for 32%. The mean initial dose of AZA was comparable between groups. Despite the different dosing schedules, the median number of cycles administered was similar between groups. The overall treatment response rates (International Working Group 2006 criteria) varied according to dosing schedule: 58% in group A, 65% in group B and 74% group C (Table 1). AZA was generally well tolerated, but the adverse events profile differed according to dosing schedule group (Table 1)

Conclusion:
Our results demonstrate that different treatment schemes are feasible for patients with MDS treated with AZA in the community-based setting, but they have different effectiveness and the safety profiles. These data show a better efficacy/safety profile for the dosing schedule approved by the EMEA. Further analyses are awaited.

Disclosures:
No relevant conflicts of interest to declare.
P52 Activation in Monomorphic B-Cell Post-Transplant Lymphoproliferative Disorders without BAFF-R Expression.
Authors: Proust Alexis , Rince Patricia , Creidy Rita , Lazure Thierry , Fabre Monique , Joab Irène , Guettier Catherine , Raphael Martine 
Published: October 2019

Abstract
Abstract 2932
Poster Board II-908
Post-transplant lymphoproliferative disorders (PTLD) are one of the worse prognostic complications after solid organ or bone marrow stem cells transplantation. Most of them are associated to EBV known to activate NF-kB pathway especially by constitutive p65 expression. BAFF cytokine, a B cell-activating factor belonging to the tumour necrosis factor (TNF) family, have been described to modulate cell growth and survival in non Hodgkin lymphomas. However, little is known on the association between EBV, BAFF/BAFF-R signalling and canonical and non canonical NF-kB pathways expression in PTLD. Thus, we intend to study the role of EBV, NF-κB and BAFF/BAFF-R expression in PTLD.Our study has been investigated in two different contexts of diffuse large B-cell lymphoma (DLBCL). 20 cases of DLCBL resulting from immunocompetent patients (DLBCL/IC) and 13 from post-transplant recipients (DLBCL/PTLD) were compared. Indeed, all cases were characterized by histology and immunohistochemistry (IHC) was used to detect B-cell markers and to identify their germinal center (GC) or non GC (NGC) origin. EBV was detected by in situ Hybridisation (ISH) using EBER probe. Latent proteins LMP1 and EBNA2 as well as replicative protein ZEBRA were also detected by IHC. In addition, p50 and p52 proteins expression was carried out to study NF-kB pathway activation.
We showed in DLBCL/IC, regardless of the ontogenic profile GC/NGC, that BAFF-R is expressed in 40% of cases, while in DLBCL/PTLD NGC pattern, BAFF-R is expressed in only one case out of 13 (7,7%) (p<0,05). Moreover, there was no significant difference in p50 expression between the categories of DLBCL studied. In contrast, we have shown a significant expression of p52 in DLBCL/PTLD (8 out of 13 cases) compared to DLBCL/IC (4 out of 20 cases) (p<0.005). In DLBCL/PTLD, there was no expression of BAFF-R ; this pattern could be related to the presence of EBV and LMP1 since p52 expression is mostly observed in EBV+ DLBCL/PTLD (5 out of 7 p52+ cases).
In conclusion, the activation profile of DLBCL/PTLD is mostly not associated with BAFF/BAFF-R expression while NF-kB2 pathway is activated.

Disclosures:
No relevant conflicts of interest to declare.
ALL-Associated JAK1 Mutations Confer Hypersensitivity to the Anti-Proliferative Effect of Type I Interferon.
Authors: Hornakova Tekla , Chiaretti Sabina , Lemaire Muriel , Foà Robin , Tartaglia Marco , Renauld Jean-Christophe , Knoops Laurent 
Published: October 2019

Abstract
Abstract 3066
Poster Board III-3
Recently, we and others reported activating mutations in JAK1 in acute lymphoblastic leukemia (ALL). These mutations are relatively common in adult patients with T cell ALL. JAK1 is a tyrosine kinase that associates to different cytokine receptors to mediate signal transduction. The associations of the mutant JAK1 with receptors like IL-2R or IL-9R are necessary to promote tumorigenicity by inducing constitutive signaling via the activation of the receptor complex. Because JAK1 mutations confer poor prognosis to the patients, there is a need for new therapies that could specifically target the leukemic blast.
Starting from patient samples, we show here that JAK1-mutant ALL blasts are characterized by a type-I interferon (IFN) transcriptional signature. This signature was recapitulated in vitro by the expression of JAK1 mutants in BW5147 and BaF3 hematopoietic cell lines. Binding of JAK1 to the IFN receptor was essential since mutations in the FERM domain abrogated this effect. Beside the constitutive activation of the type I IFN signaling cascade, JAK1 mutations also strongly potentiated the response to IFN in vitro. Typically, the proliferation of cell lines expressing JAK1A634D was abrogated by type I IFNs. Interestingly, we found that different JAK1 mutations differentially potentiate responses to type I IFNs or to IL-9, another cytokine using JAK1 to mediate its effects. This suggests that the type of mutation influences the specificity of the effect on distinct cytokine receptor signaling. Finally, we also showed in an in vivo leukemia model that cells expressing JAK1A634D are hypersensitive to the anti-proliferative and anti-tumorigenic effect of type I IFN, suggesting that type I IFNs should be considered as a potential therapy for ALL with JAK1 activating mutations.

Disclosures
No relevant conflicts of interest to declare.
CD44-Induced Differentiation of Acute Monoblastic Leukemia Cells Is Mediated by Prolonged Activation of ERK1/2 and by Cytokines.
Authors: Bourcier Sébastien , Sansonetti Arnaud , Durand Laetitia , Chomienne Christine , Robert-Lézènès Jacqueline , PhD Florence Smadja-Joffe Sr., 
Published: October 2019

Abstract
Abstract 3103
Poster Board III-40
We have previously reported that specific monoclonal antibodies (mAbs) to the cell adhesion molecule CD44, trigger monocytic differentiation of blasts from patients with acute monoblastic leukemia (1) and that cytokines contribute this differentiation (2). To further analyze the mechanisms mediating CD44-induced monocytic differentiation of leukemic cells, we have used as a model the THP-1 leukemia cell line which differentiation has been induced by the Hermes-3 anti-CD44 mAb. Monocytic differentiation has been checked at day 3 by measuring, using flow cytometry, the increase in the expression levels of CD11b (myeloid-specific) and CD14 (monocyte-specific) differentiation antigens. Control THP-1 cells (untreated or treated with isotypic IgG) display only a little CD11b and do not express CD14.
First, using western blot analysis, we show that CD44-ligation with Hermes-3 mAb rapidly triggers ERK1/2 MAP kinase phosphorylation, that persists for about 6-8 hours. This prolonged ERK1/2 phosphorylation is essential for CD44-induced differentiation, since its delayed inhibition (using the specific chemical inhibitor U0126) fully abrogates the differentiation process. Similar results have been obtained in Hermes-3 - treated primary blasts from patients with acute monoblastic leukemia. Interestingly, ERK1/2 signalling mediates normal monocytic differentiation induced by cytokines (3). Accordingly, our present results suggest that CD44-ligation might reactivate this physiological differentiation pathway persisting in monoblastic leukemia cells (THP-1 cells and primary leukemic monoblasts).
Second, using real-time RQ-PCR and ELISA assays, we show that ERK1/2 activation leads to synthesis and secretion of a burst of cytokines, including IL-1b, IL-6, IL-8, GM-CSF and TNF-alpha. Among these cytokines, TNF-alpha- and IL-6, acting through an autocrine-paracrine pathway, play an important part in the CD44-induced differentiation process, since their functional inhibition with specific mAbs dramatically decreases the extent of this differentiation. TNF-alpha has a critical role in the increase of CD11b expression level. In addition, it cooperates with IL-6 for inducing CD14 expression. However, TNF-alpha- and IL-6 inhibition does not totally abrogate CD44-induced differentiation. This suggests that another unidentified cytokine is perhaps involved in the differentiation process. Alternatively, CD44-activated ERK1/2 pathway may directly reprogram the expression of monocytic differentiation genes. Interestingly, addition of exogenous IL-6 enhanced the level of CD14, suggesting that IL-6 and CD44-ligation may cooperate for differentiation therapy of acute monoblastic leukemia.
1. Charrad et al. Nat. Med 5:669,1999 2. Delaunay et al. Leukemia 22:873, 2008

Disclosures
No relevant conflicts of interest to declare.
Prevention of Venous Thromboembolism (VTE) Associated with Pregnancy in Women with a Past History of VTE.
Authors: Rozanski Camilla , Lazo-Langner Alejandro , Kovacs Michael J. 
Published: October 2019

Abstract
Abstract 3132
Poster Board III-69
Women with a past history of venous thrombosis are at higher risk for venous thromboembolism (VTE) during and after pregnancy. The highest risk period is the first four weeks post partum. For a woman whose previous event was secondary to a major transient risk factor the antepartum risk of recurrent VTE is low whereas for women whose previous event was idiopathic the antepartum risk is higher. In our institution, for women whose previous event was secondary to a major transient risk factor, standard treatment is to follow closely with no thromboprophylaxis antepartum and then receive either prophylactic low molecular weight heparin (LMWH) or warfarin for six weeks postpartum. For women whose previous event was idiopathic or who were on warfarin at the time of becoming pregnant they receive prophylactic LMWH antepartum with a scheduled delivery and are put back on warfarin if they have long term anticoagulation needs or LMWH for six weeks if they don't have longterm anticoagulation needs. We report the outcome for our patients from 1997 to 2008. All patients were followed for the duration of pregnancy and for 6 weeks postpartum for pregnancy outcome, recurrent VTE, and major bleeding in the thrombosis clinic of London Health Sciences Centre. There were a total of 90 women; 30 women (mean age 30.6 years) with a history of previous secondary thrombosis with 37 pregnancies and 60 women (mean age 29.5 years) with past idiopathic thrombosis with 99 pregnancies. For the secondary group there was 1 episode (2.70%; 95% CI 0.48-13.82) of antepartum recurrent VTE whereas for the idiopathic group there were 3 episodes (3.03%; 95% CI 1.04-8.53). There was no statistical difference between groups (p=1.0). There were no episodes of postpartum VTE recurrence or major hemorrhage for either group. For the secondary group there was 1 fetal loss at 23 weeks (2.7%; 95% CI 0.48-13.82) whereas for the idiopathic group there were 6 fetal losses at 8, 10, 10, 11, 22, 37 weeks gestation (6.06%; 95% CI 2.81-12.60). There was no statistically significant difference between groups (p=0.77). This retrospective review suggests that for pregnant women with a past history of VTE, a strategy of no antepartum prophylaxis for previous secondary thrombosis and antepartum prophylactic LMWH for previous idiopathic thrombosis as well as prophylactic LMWH or warfarin postpartum is efficacious and safe.

Disclosures
Off Label Use: low molecular weight heparin for prevention of VTE in pregnancy.