bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Determinants of Lymph Node Microenvironment Induced Host Immune Tolerance In CLL: Role for CAV1, PTPN6, and PKCβ In the Process
Authors: Gilling Christine , Mittal Amit , Nganga Vincent , Palmer Vicky , Weisenburger Dennis D. , Bierman Philip , Bociek R. Gregory , Swanson Patrick , Joshi Shantaram
Published: October 2019

Abstract 1367

Previously, we have shown that gene expression profiles (GEP) of CLL cells from lymph nodes (LN), bone marrow (BM), and peripheral blood (PB) are significantly different from each other. Among the major pathways associated with differential gene expression, a “tolerogenic signature” involved in host immune tolerance is significant in regulating CLL progression. The genes associated with the tolerogenic signature are significantly differentially expressed in patient LN-CLL compared to BM-CLL and PB-CLL, suggesting that LN-CLL cells induce this immune tolerance. From 83 differentially expressed genes identified by GEP that are associated with immune dysregulation, we selected eleven genes (CAV1, PTPN6, PKCb, ZWINT, IL2Ra, CBLC, CDC42, ZNF175, ZNF264, IL10, and HLA-G) for validation studies to determine whether these genes are also dysregulated in the Emu-TCL1 mouse model of CLL. The results demonstrate a trend of upregulation of these genes as determined by qRT-PCR in the LN-tumor microenvironment. To further evaluate the kinetics of selected gene expression during tumor progression, we determined the expression levels of Cav1, Ptpn6, and Pkcb at 12, 24, and 36 weeks of CLL development in the Em-TCL1 mouse model. We found that the expression of all three genes increased as a function of age, indicating a correlation of gene expression with disease progression. In addition, as CLL progressed in these mice there was a marked decrease in CD4+ and CD8+ T cells. The murine data were further validated using CLL cells from the same patients with indolent versus aggressive disease indicating a similar trend in expression as CLL progressed (n=4). Furthermore, patient data analyzed by Kaplan Meier analyses of the expression levels of the selected genes indicated a significant association between down-regulation of PTPN6 (p=0.031) and up-regulation of ZWINT (p<0.001) with clinical outcome as determined by a shorter time to treatment (p<0.05). Functional analysis by knockdown of CAV1 and PKCb in primary patient CLL cells determined by MTT assay showed a decrease in proliferation following knockdown of these genes (p<0.005). Protein-interaction modeling revealed regulation of CAV1 and PTPN6 by one another. Additionally, the PTPN6 protein regulates B cell receptor (BCR) signaling and subsequently the BCR regulates PKCb. Therefore, these data from both mice and humans with CLL, argue that an aggressive disease phenotype is paralleled by expression of genes associated with immune suppression. In particular, evidence presented here suggests, dysregulation of CAV1, PTPN6, ZWINT, and PKCb expression promotes CLL progression.

Disclosures:

No relevant conflicts of interest to declare.
The Receptor Tyrosine Kinase Inhibitors Vatalanib and Pazopanib Possess Strong Inhibitory Effect on Tumor Growth In a CLL-Like Xenograft Mouse Model
Authors: Paesler Julian , Gehrke Iris , Razavi Regina , Poll-Wolbeck Simon Jonas , Hallek Michael , Kreuzer Karl-Anton
Published: October 2019

Abstract 1393

As chronic lymphocytic leukemia (CLL) is still mainly treated using unspecific chemotherapy, new targeted therapies are highly desirable. Vatalanib and Pazopanib are potent orally available receptor tyrosine kinase inhibitors with high selectivity towards the vascular endothelial growth factor (VEGF)-receptor. VEGF-signaling is critically involved in the microenvironment-mediated prolonged survival of CLL cells. We investigated the efficacy and selectivity of both compounds in CLL cells and tested their effect on CLL like tumor xenografts in nude mice. Primary CLL cells (n=20) and healthy B-cells (n=5) were treated with Vatalanib and Pazopanib ranging from 1 to 200 μM. Both substances were tested for their apoptosis-inducing efficiency in a CLL cell coculture with the bone marrow-derived stromal cell line HS5 (n=4), which provides a strong survival support for CLL cells and mimick the in vivo situation. Apoptosis induction was monitored flow cytometrically by Annexin V-FITC/PI staining and by immunoblotting of caspases and PARP. Further, both compounds were administered daily via oral gavage at 100 mg/kg bodyweight to nude mice engrafted with CLL-like JVM3 cells for 21 days (n=10/group). Both substances effectively induced apoptosis in primary CLL cells with lethal concentration (LC) 50 of 48.4 μM and 32.7 μM for Vatalanib and Pazopanib, respectively. Healthy B-cells were largely unaffected. Furthermore, both substances showed similar apoptosis-inducing capacity when CLL cells were maintained on a survival-supporting HS5 feederlayer. In a CLL-like xenograft mouse model tumor growth was significantly inhibited by 76% and 77% after a treatment period of 21 days for Vatalanib and Pazopanib, respectively. In two mice complete tumor eradication could be observed after treatment with Vatalanib. No adverse side effects were observed during the treatment period. We conclude that the receptor tyrosine kinase inhibitors Vatalanib and Pazopanib have strong and selective anti-neoplastic activity towards CLL cells. As they are orally available and in general well tolerated they deserve further evaluation as a potential new therapeutic option for treatment of CLL.

Disclosures:

No relevant conflicts of interest to declare.
A Novel Factor XII Mutation, FXII Tokyo (R84P), Causing Factor XII Deficiency In a Patient with Hereditary Spastic Paraplegia
Authors: Matsuki Eri , Miyakawa Yoshitaka , Okamoto Shinichiro
Published: October 2019

Abstract 1404

Hereditary factor XII (FXII) deficiency is a clinically asymptomatic, autosomal recessive disorder. We have experienced a rare case of FXII deficiency in a patient with hereditary spastic paraplegia (HSP). This is the first report in the literature of the coexistence of these two disorders together with the analysis of their genetic cause.

The patient, who had no major bleeding episodes, presented with a prolonged APTT of 104.9 seconds. Further evaluation revealed decreased FXII activity of less than 10% and FXII antigen level of 17% with negative results on mixing study. Based upon these findings, she was diagnosed as having hereditary FXII deficiency. Her family history was positive for HSP on her mother's side, the genetic diagnosis of which had been confirmed in her younger daughter. Pedigree analysis showed that her two daughter's coagulation profiles were all within normal limit.

Sequencing of the 14 exons and intron/exon boundaries of the FXII gene revealed a novel missense mutation at exon 4 that substitutes arginine 84 to proline (R84P), which we named FXII Tokyo. The mutation status was also confirmed by restriction fragment length polymorphism using restriction enzyme BtgZI. To elucidate the molecular mechanism of FXII deficiency, wild type and R84P mutant FXII cDNA were transiently expressed in CHO cells. We performed Western blot analysis, and found that secretion but not synthesis of R84P mutant protein was markedly reduced compared to wild type.

Altogether, these results indicated that R84P mutation might impair the intracellular transport or secretion of FXII protein of the cells and could be a useful tool for the analysis of structure-function relationship and intracellular protein transport of FXII protein in future.

Disclosures:

No relevant conflicts of interest to declare.
Inhibitor Development In Patients with Mild and Moderate Hemophilia A: Results From a Single Centre.
Authors: Repesse Yohann , Gautier Philippe , Borel-Derlon Annie
Published: October 2019

Abstract 1407

The development of factor VIII (FVIII) inhibitors is usually considered uncommon among patients with mild and moderate hemophilia A (HA) and less frequent than in patients with severe HA. We report here the prevalence of FVIII inhibitors and their caracteristics in 167 patients with mild and moderate HA followed in Caen Hemophilia Treatment Centre (Table). FVIII molecular defects were identified by direct sequencing in 167 patients including 30 and 137 with mild and moderate HA, respectively. Following FVIII concentrates infusions, FVIII inhibitors occured in 7.8% of patients (13/167). Fifteen percent (2/13) were low-responding inhibitors. The risk of inhibitor development appeared to be associated with high-risk FVIII genotypes clustered in the A2 and C2 domains, especially p.Arg2150His (50%) and p.Arg593Cys mutations. Interestingly, we described inhibitor development associated with novel missense-mutations (p.Tyr1786Ser, p.Asp115Tyr and -219C>T substitutions in FVIII gene promoter). In addition, high regimen infusion of FVIII concentrates appeared as risk factor for FVIII inhibitors development. Indeed, 60% (8/13) developped FVIII inhibitors following massive infusion of FVIII concentrates associated with FVIII:C levels above 1.2 UI/dL. Inhibitors in mild HA usually cross-react with endogenous factor VIII reducing the circulating basal FVIII:C level and are associated with more bleeding events. Similarly, we observed the evolution of bleeding patterns in our cohort to severe phenotypes. Bleedings were treated with FVIII concentrates and bypassing therapies (activated FVII and activated-prothrombin complex). About 25% (3/13) of these inhibitors disappeared spontaneously. Induction of Immune Tolerance (ITI) protocoles with high doses of FVIII were initiated for 7 high-responding patients with a success rate of 85 % (6/7). However, inhibitors persisted long-term and remained troublesome in 1 of these patients despite of ITI protocole. For two patients, immunosuppressive treatment with corticosteroids was started. Inhibitors disappeared and the levels of FVIII:C became detectable within 6 months. Development of FVIII inhibitors, their disappearance and the efficacy of ITI regimen seem to be different from our experience between patients with mild or moderate HA and severe HA.

Disclosures:

No relevant conflicts of interest to declare.
Use of a Population Pharmacokinetic Model of ADVATE In Pediatric and Adult Patients with Hemophilia A Permits Limited Blood Sampling for Individual Dose Tailoring.
Authors: Oh MyungShin , Björkman Sven , Schroth Phillip , Fritsch Sandor , Collins Peter W , Fischer Kathelijn , Blanchette Victor S. , Casey Kathleen M. , Spotts Gerald , Ewenstein Bruce M.
Published: October 2019

Abstract 1416

Introduction:

A population pharmacokinetic (PK) model of a recombinant FVIII (rFVIII) was established on ADVATE® (Antihemophilic Factor (Recombinant), Plasma/Albumin-Free Method) studies in pediatric and adult patients with hemophilia A. The objective of this analysis was to evaluate the effect of reduced PK sampling time points on the estimated PK parameters in the population PK model.

Patients and Methods:

Plasma FVIII activity PK data were collected for 3 ADVATE® clinical trials in previously treated patients: 184 full PK data sets (11 time points) for 100 adults/adolescents, aged 10 to 65 years, and from 52 reduced sample PK data sets (5 time points) for 52 children, aged 1 to 6 years. A population PK analysis was conducted on a two-compartment structure model and the covariate effect of age and weight was explored.

Four reduced sampling scenarios from the full 10 post-infusion sampling time points, were investigated: 1) Reduced to 4 (1 hr, 9 hr, 24 hr, and 48 hr), 2) Reduced to 3 (6 hr, 24 hr, and 48 hr), 3) Reduced to 2 (6 hr and 24 hr), and 4) Reduced to 1 sampling time points (24 hr post-infusion). After applying the reduced sampling on a random 10% of sampling set at a time in the population PK model, the differences in model estimates and individual PK estimates between full and reduced sampling, were evaluated.

Results:

The two-compartment population PK model adequately described the data. Clearance (CL) was significantly correlated with age and body weight and central volume of distribution was also related with body weight.

Absolute deviations (%) from the estimates using full PK sampling in the Individual PK estimates (CL, Vss, and Half-life) using each of the reduced sampling time points were showed in the below table.

Conclusions:

It appears that PK parameters estimated using population PK model are robust to reduced sampling time points. Accurate measurement of PK on reduced samples gives patients and clinicians the opportunity to design treatment regimens that are better tailored to individuals.

Disclosures:

Oh: Baxter: Employment. Björkman:Baxter: Consultancy; Octapharma: Consultancy. Schroth:Baxter: Employment. Fritsch:Baxter: Employment. Collins:NovoNordisk: Consultancy, Honoraria, The EACH2 registry was funded by Novonordisk; Baxter Healthcare: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Fischer:Baxter: Consultancy; NovoNordisk: Consultancy. Blanchette:Bayer: Consultancy; Baxter: Research Support. Casey:Baxter: Employment. Spotts:Baxter: Employment. Ewenstein:Baxter Bioscience: Employment.
Preclinical Safety of Baxter's Recombinant Factor VIIa
Authors: Dietrich Barbara , Kubik Susan , Auer Wilfried , Reipert Birgit , Horling Frank , Ehrlich Hartmut J. , Scheiflinger Friedrich , Schwarz Hans Peter , Muchitsch Eva-Maria
Published: October 2019

Abstract 1417

Baxter has developed a recombinant FVIIa (rFVIIa) product for the treatment of patients with hemophilia A and hemophilia B with inhibitors. Baxter's new rFVIIa is produced by a genetically engineered Chinese hamster ovary (CHO) cell line in a cell culture medium free from any animal or human proteins. The objective of this preclinical study-program was to evaluate the safety of Baxter's rFVIIa in different species.

To assess the anaphylactoid and thrombogenic potential of rFVIIa and its effects on blood pressure, cardiac and respiratory function, and the coagulation system the preclinical program included studies in established models of safety pharmacology in telemetered cynomolgus monkeys, rabbits, normal and spontaneously hypertensive rats, and guinea pigs. Furthermore, studies on single and repeated dose toxicity (hemophilia A mouse, rat, cynomolgus monkey), local tolerance (rabbit), and comparative immunogenicity (mouse) were conducted. Commercially available licensed rFVIIa served as active reference item.

Treatment with rFVIIa did not result in any anaphylactoid reaction, and had no adverse effect on the cardiovascular or respiratory system or on hematology parameters. Thrombogenicity studies showed no greater thrombogenic potential of Baxter's rFVIIa than the commercially available rFVIIa product. Single dose toxicity studies in mice, and repeated administration of Baxter's rFVIIa in rats and cynomolgus monkeys, administered at doses of 2.7 mg/kg, confirmed the safety of the new product. As expected, due to the pharmacological action of rFVIIa, high doses of either recombinant FVIIa product resulted in exaggerated pharmacological effects leading to thrombus formation in some organs, which are well- known effects for this class of compound. Furthermore, Baxter's rFVIIa was well tolerated at the injection site after intravenous injection. Results obtained in comparative immunogenicity studies demonstrate that Baxter's new rFVIIa and the commercially available rFVIIa product have a similar immunogenicity profile.

In summary, Baxter's rFVIIa has a safety profile that supports the evidence necessary for proceeding with human trials.

Disclosures:

Dietrich: Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Auer:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.
Inhibitor Eradication In Acquired Factor VIII Inhibitor: A Case Series
Authors: Badari Ambuga , Bernstein Zale P , Logue Gerald L. , Patel Anush , Miller James , Tran Tan , Keshavamurthy Chandana
Published: October 2019

Abstract 1423

Acquired factor VIII (FVIII) inhibitor is an uncommon condition, with an incidence of ≂f1.5 cases/million/year, as reported by Collins et al. (Blood 2007. 109: p. 1870–1877) in a two year observational study in the UK. However, this is a serious condition, characterized by auto-antibodies against circulating factor VIII and has high mortality from bleeding. Controlling the acute bleeding event with FVIII inhibitor bypassing agents like recombinant activated factor VII or activated prothrombin complex concentrates is of utmost importance. Equally important is the eradication of inhibitor, in order to restore normal hemostasis. There are several approaches for inhibitor eradication, including, but not limited to, the use of Corticosteroids, Cyclophosphamide, Azathioprine, Vincristine, IVIG, Cyclosporine, Mycophenolate and, recently, Rituximab, either singly, or, in different combinations. However, there is no single standard approach. We report 4 cases of acquired FVIII inhibitor from our center that were initially treated with factor VIII bypassing agents. Inhibitor eradication was achieved by using a combination of Rituximab, Prednisone and Cyclophosphamide.

Complete and sustained remission was achieved in all 4 patients (100%).All 4 patients tolerated the therapy well. None of the patients had significant adverse effects like neutropenia, hemorrhagic cystitis or other complications, either immediately or during follow up. Thus, the combined use of Cyclophosphamide, Rituximab and Prednisone seems to be an effective and safe regimen for inhibitor eradication in patients with acquired FVIII inhibitor.

Disclosures:

Off Label Use: Rituximab.For th purpose of auto-antibody supression.
Relevance of Intact Von Willebrand Factor (VWF) Triplet Structure for VWF Function.
Authors: Schwartz Bruce A. , Fuchs Birte , Kannicht Christoph , Solecka Barbera , Kröning Mario
Published: October 2019

Abstract 1425

Introduction:

The characteristic multimer pattern of plasmatic von Willebrand factor (VWF) results from asymmetric cleavage by the processing metalloprotease ADAMTS13 between Y1605/M1606 within the VWF A2 domain. In normal plasma, characteristic species of various multimeric sizes with flanking satellite bands (triplets) encircling the major band on VWF multimer gels are present. The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Even though the distribution of VWF satellite bands is significantly altered in some types of von Willebrand disease (VWD) and several commercial VWF concentrates, the impact of triplet structure on VWF function has not been investigated so far.

Methods:

Four commercially available VWF concentrates were analyzed with respect to ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by the fluorescence resonance energy transfer (FRET) assay. VWF zymogram gels were used to test for ADAMTS13 activity. Samples composed of different VWF triplet distribution but comparable VWF multimers were obtained by fractionation of plasmatic VWF using heparin affinity chromatography. VWF affinity to collagen was measured by surface plasmon resonance (SPR).

Results:

VWF concentrates markedly differed in their content of ADAMTS13 antigen and activity. A higher ADAMTS13 content correlated with an increased portion of the proteolyzed faster migrating VWF triplet band. The degree of VWF proteolysis, i.e. lack of an additional N-terminal fragment, correlated with a decreased collagen binding level measured by SPR.

Conclusion:

Proteolytic cleavage of N-terminal domains of VWF resulting in a higher content of faster migrating satellite bands affects the function of VWF. The impact of VWF N-terminal domains on collagen binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated.

Disclosures:

Schwartz: Octapharma: Employment. Fuchs:Octapharma: Employment. Kannicht:Octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment.
The Effect of Anti-GPIIIa49-66 Antibody on Megakaryocyte Differentiation.
Authors: Wang Jianhui , Pan Ruimin , Nardi Michael A. , Li Zongdong
Published: October 2019

Abstract 1434

We previously reported that patients with early-onset HIV-1 ITP develop a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cells for platelets and may contain similar signal pathways, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5 fold in in vitro culture of mouse bone marrow Lin-/- cells driven by thrombopoietin (TPO). We also observed a 3 fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody we generated. However, we could not detect ROS release in DCFH-loaded MEG-01 cells treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of MK cell line L8057 induced by TPO. In fact, we found a dose dependent increasing of the percentage of αIIb integrin positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 treated by various concentration of H2O2 (from 5 to 20μM). Thus, our data suggests that ROS is not involved in the inhibition of MK differentiation induced by anti-GPIIIa49-66, in contrast to the effect that this antibody has on mature platelets. We therefore conclude that the anti-GPIIIa49-66 antibody dysregulates ROS independent β3 integrin signaling to inhibit MK differentiation.

Disclosures:

No relevant conflicts of interest to declare.
IgG Antibodies Directed towards a Single Antigenic Surface In the Spacer Domain of ADAMTS13 Predominate In Plasma of Patients with Acquired TTP.
Authors: Pos Wouter , Fijnheer Rob , Vidarsson Gestur , Voorberg Jan
Published: October 2019

Abstract 1438

Background:

The majority of patients diagnosed with thrombotic thrombocytopenic purpura (TTP) have primarily autoantibodies directed towards the spacer domain of ADAMTS13. Previously, we have shown that residues Arg660, Tyr661 and Tyr665 are involved in binding of anti-ADAMTS13 antibodies. Inspection of the three-dimensional structure of the spacer domain reveals that Arg658 and Phe592 are in close proximity to Arg660, Tyr661 and Tyr665.

Objective:

In this study we explored whether Arg658 and Phe592 contribute to a previously identified antigenic surface in the spacer domain. We also addressed Ig class distribution as well as the contribution of antibodies directed towards the carboxy-terminal TSP2-8 and CUB1-2 domains in a large cohort of patients.

Methods:

The epitope specificity of anti-spacer domain directed antibodies was examined using two recombinant anti-spacer domain directed antibodies I-9 and II-1 and plasma of 48 patients with acute acquired TTP by means of immunoprecipitation of ADAMTS13 variants containing single or multiple alanine substitutions. Reactivity of patient-derived antibodies directed towards the TSP2-8 and CUB1-2 domains was also addressed by immunoprecipitation. Quantitative Ig class analysis was performed using human monoclonal antibody II-1 as an internal reference.

Results:

Plasma from all 48 patients included in this study contained anti-ADAMTS13 antibodies of subclass IgG; plasma of 5 patients contained anti-ADAMTS13 IgM and in plasma of 9 patients had anti-ADAMTS13 antibodies of subclass IgA1. No anti-ADAMTS13 antibodies of subclass IgA2 were observed. Plasma of some patients contained anti-TSP2-8 (∼17%) and anti-CUB1-2 (∼35%) domain directed antibodies. Analysis of anti-spacer domain antibodies revealed that residues Arg568 and Phe592 contribute to an antigenic surface in the spacer domain. The majority of patients (90%) lost the reactivity towards the spacer domain of ADAMTS13 through the introduction of multiple alanine substitutions at positions Arg568, Phe592, Arg660, Tyr661 and Tyr665.

Conclusions:

IgG antibodies directed towards a single antigenic surface in the spacer domain of ADAMTS13 predominate in plasma of patients with acquired TTP.

Disclosures:

No relevant conflicts of interest to declare.
Mycophenolate Mofetil Therapy Is a Steroid Sparing In Childhood and Young Adult Chronic ITP and Evans Syndrome.
Authors: Naji Zaher , Rajpurkar Madhvi , Savasan Sureyya , Chu Roland , Chitlur Meera , Callaghan Michael
Published: October 2019

Abstract 1441

Chronic ITP and Evans syndrome are diseases characterized by autoantibody formation with resultant destruction of platelets or both platelets and red blood cells on neutrophils, respectively. Affected patients are at risk of life threatening bleeding complications and/or life threatening anemia. Conventional therapies are often ineffective or transiently effective and have significant toxicity. One of the most commonly used therapeutic strategies employs chronic corticosteroid administration with the attendant weight gain, hypertension, hyperglycemia, bone loss, infection risk, mood changes and protean other undesirable side effects. Mycophenolate Mofetil (MMF) is an immunosuppressive agent with a favorable side effect profile. It is converted to the active metabolite, mycophenolic acid, and interferes with purine metabolism in T-lymphocytes, effectively killing many of these cells and down-regulating autoimmune phenomena. With approval of our local IRB /HIC we retrospectively reviewed the charts of 11 chronic ITP/Evans syndrome patients who had received MMF, all such patients treated at a large urban Children's Hospital. Clinical variables included age, sex, duration of disease, steroid use, IVIG use, Anti-D use, platelet counts, hemoglobin concentrations and reticulocyte percentages. These data were analyzed using paired t-tests, one-sample t-test and descriptive statistics. The 11 patients ranged in age from 9–22 years old, with a mean age of 15 years. The mean time from diagnosis of disease was 41.8 months with a range of 6–95 months. There were 5 female subjects, 6 Evans syndrome patients and 5 with chronic ITP alone. The median platelet count over the 6 months prior to MMF was 70×109/L, (18-223), with a median of 90×109/L with (27-145) during the first 6 months of MMF therapy (p=0.4). In the Evans syndrome group, the mean hemoglobin prior to MMF was 10.9 g/dL, (9.1-14.6), with a mean of 12.1 g/dL, (6.9-16.7) on MMF (p=0.54). Similarly, the mean reticulocyte percentage was 2.7%, (0.5-14) prior to MMF, with a mean of 2.3%, (0.3-8.9) during MMF therapy. The mean total dose of steroids used in the 6 months prior to MMF was 84.2 mg/kg (prednisone equivalent), (11.2-170), compared to 62.2 mg/kg, (0-193.8) on MMF. 9 of 11 patients had reductions in steroid requirement by an average of 49.3% (p=0.0013). During the first 6 months of MMF both IVIG and anti-D usage decreased from total doses for the entire group of 17 and 3 doses to 8 and 1 dose, respectively. None of the patients experienced severe bleeding episodes or side effects more serious than a transient rash while on MMF. These data suggest that MMF may have a role as a steroid sparing therapy in the treatment of chronic ITP and Evans syndrome.

Disclosures:

Off Label Use: mycophenolate mofetil for ITP Evans syndrome.
Evaluation of Platelet Dysfunction In Children: Improved Detection and Efficiency
Authors: Nugent Diane J. , Roberts Ryan , Nakagawa Peggy
Published: October 2019

Abstract 1446

Currently, there is no single assay that will detect platelet function abnormalities in all individuals. We prospectively studied 369 patients with a strong history of bruising and mucosal membrane bleeding for possible platelet dysfunction following documentation of normal Von Willebrand antigen and activity levels. In an effort to evaluate platelet function under more diverse conditions we chose to simultaneously evaluate 1) aggregation using platelet rich plasma and light transmission aggregometry (LTA), 2) adhesion under high shear using the Platelet Function Analyzer (PFA-100, ADP-collagen, and Epinephrine-collagen cartridges) and 3) platelet initiated clot formation using heparinized whole blood and the two standard mapping agonists, arachadonic acid (AA) and ADP (Hemascope Thromboelastograph Analyzer).

Of the 369 platelet evaluations performed, 87 patients (24%) were found to be normal in all three assays with all agonists. On repeated assays with increased attention to medication and food history, an additional 152 patients were felt to have a transient or acquired dysfunction which improved or normalized on further testing. Leaving 130 patients with persistent bleeding and documented abnormalities on one or more of the assays used. There were no patients with only Collagen (CN) or arachadonic acid (AA) aggregation alone on LTA. Using LTA, there was one patient with combined CN and ADP aggregation defect only, another with AA and EPI absent aggregation only, and one with only AA and Thrombin receptor agonist peptide (TRAP) aggregation abnormalities. A summary of the remaining 127 patients are displayed in the table below:Abnormal AssayLTA EPILTA ADPLTA ADP + EPILTA ADP, EPI CNLTA ADP, EPI, AALTA ADP, EPI CN, AAPFA and PLT Mapping ONLYNumber201240841825M/F3M/17F6M/6F16M/24F1M/7F1M/3F8M/10F12M/13FPFA1M/2F4M7M/4F01M01M/3FPlt Mapping2M/2F1M/2F3F1F009M/7FBoth PFA and Mapping1M0001F2M/9F2M/3F

Summary: By using a combination of three assays, we were able to identify 25 additional patients with significant platelet dysfunction detected with abnormal platelet mapping or PFA-100 despite normal light transmission aggregometry. Patients with the most abnormalities on aggregation, also demonstrated abnormal adhesion and platelet initiated clot formation. However, the use of PFA-100 and/or platelet mapping alone would miss the majority of patients with aggregation defects. In the future, the unique combination of platelet function defects as measured by these assays, and future technologies, will not only improve detection, but also facilitate phenotype to genotype associations and expedite mutational analysis.

Disclosures:

Off Label Use: Rituximab to treat ITP.
HDAC11 Inhibits T-Cell Response to Alloantigens and Reduces Gvhd In Mice.
Authors: Wang Dapeng , Cheng Fengdong , Yu Yu , Semple Kenrick , Peng Lirong , Telles Elphine , Luetteke Noreen , Seto Edward , Anasetti Claudio , Sotomayor Eduardo M. , Yu Xue-Zhong
Published: October 2019

Abstract 1466

Background:

Histone acetyltransferases and histone deacetylases (HDAC) regulate gene expression through acetylation-deacetylation of histones. HDACs are the target of a family of compounds known as HDAC inhibitors, which have been shown to suppress pro-inflammatory cytokines and reduce acute graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect after allogeneic bone marrow transplantation (BMT) in mice. However, the role of individual HDAC members in the development of GVHD is not clear. Recently, HDAC11, the newest member of the HDAC family has emerged as an important transcriptional regulator of inflammatory responses in antigen-presenting cells (APCs)1. Here, we evaluated the role of HDAC11 on APCs and T cells in the allogeneic BMT setting in mice with genetic disruption of HDAC11.

Method:

Proliferation of wild-type (WT) and HDAC11 knock-out (KO) T cells in response to allogeneic antigens was compared by [H3] thymidine incorporation assay. Using the same method, we also tested the antigen presentation ability of WT and HDAC11 KO APCs. For in vivo studies, we used a clinical relevant mouse model of BMT: C57BL/6 (B6) ® BALB/c. To evaluate the role of HDAC11 in the function of T cells and APCs, WT and KO mice on B6 background were used as donors and recipients, respectively. Recipient survival was monitored daily and GVHD symptom was evaluated at least twice a week. HDAC11 KO mice were supplied by Merck and Co., Inc.

Results:

In vitro, HDAC11 KO T cells proliferated stronger than WT T cells under the stimulation of allogeneic APCs. Recipients of HDAC11 KO T cells lost significantly more body weight (p < 0.05), and died significantly sooner than those of WT T cells (p < 0.01). The pathologic score of KO recipients was higher than that of WT recipients in each of GVHD target organs including lung, liver, small intestine and colon. Mechanistically, we found that there were significantly more total and IFNγ-producing donor T cells in the recipients of KO cells than those of WT cells (p < 0.05). Collectively, HDAC11 KO T cells have higher activity in response to alloantigens in vitro and induced more severe GVHD in vivo compared to WT T cells. In contrast, KO and WT APCs had a similar ability to stimulate allogeneic T cells in vitro, and no significant difference in GVHD development was observed in WT or KO recipients after allogeneic BMT.

Conclusion:

HDAC11 negatively regulates T-cell function, but has no significant effect on APC function. This finding provides a rationale to promote T-cell immunity or tolerance by inhibiting or enhancing HDAC11, respectively.

1 Villagra et al. Nature Immunology, 10:92-100, 2009.

Disclosures:

No relevant conflicts of interest to declare.
Glia Maturation Factor-Gamma Negatively Modulates TLR4 Signaling In Macrophages Induced by Lipopolysaccharide (LPS).
Authors: Aerbajinai Wulin , Chin Kyung , Lee Hyun Woo , Zhu Jianqiong , Rodgers Griffin P.
Published: October 2019

Abstract 1482

Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response attracts much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response in macrophages. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We show here that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for ∼2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease.

Disclosures:

No relevant conflicts of interest to declare.
PSTPIP2 Limits Osteoclast Precursor Differentiation and Inflammation-Associated Bone Loss.
Authors: Chitu Violeta , Nacu Viorel , Charles Julia F , Ketchum Halley , Haskell Cristopher , Nandi Sayan , Stanley E. Richard
Published: October 2019

Abstract 1489

Chronic inflammatory conditions are frequently associated with bone loss. However, the common pathways linking these processes are still poorly defined. Here we show that PSTPIP2, an F-BAR domain containing protein, negatively controls inflammation and osteoclast formation. Mutations in Pstpip2 lead to autoinflammatory disease and secondary generalized osteopenia. Dentine disc implants are more rapidly and extensively resorbed in PSTPIP2-deficient mice than in wt controls due to increased osteoclast formation on the implant. Bone marrow and spleens of cmo mice contain increased numbers of multipotent c-Kit+ c-fmslo Mac-1lo myeloid precursors that give rise to more osteoclasts in vitro than cells from wt mice. All these phenotypes are corrected by administration of the CCR1 antagonist BX471. Our results identify PSTPIP2 as an essential limiting factor of myelopoiesis and osteoclast differentiation and MIP-1α as a mediator of inflammation-induced bone loss in PSTPIP-2 deficient mice.

Disclosures:

No relevant conflicts of interest to declare.
Neutrophil Proteinase (PR3) Regulates Neutrophil Transendothelial Cell Migration.
Authors: Kuckleburg Christopher , Tilkens Sarah , Santoso Sentot , Newman Peter J.
Published: October 2019

Abstract 1492

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions where receptor-ligand interactions between these cells promotes leukocyte diapedesis. Neutrophils contain several different proteases which are thought to play a role in aiding in transendothelial cell migration, either by degrading extracellular matrix components or junctional proteins, or by inducing endothelial cell activation. Proteinase 3 (PR3) is a serine protease stored in azurophil granules that is released by activated neutrophils and can rebind to the neutrophil expressed cell surface protein NB1 (CD177). The neutrophil marker NB1 is expressed on a subset of neutrophils (∼50%) and has recently been demonstrated to be a heterophilic binding partner for PECAM-1, a protein highly expressed at endothelial cell junctions. Disrupting NB1-PECAM interactions has been reported to significantly inhibit neutrophil transmigration. Because of the critical role of NB1 in neutrophil transmigration we believe that the interactions between NB1 and PECAM-1 have the potential to localize PR3 to endothelial cell junctions where it may aid in leukocyte transmigration.

For this study we sought to test the hypothesis that NB1-PR3 interactions contribute to neutrophil transmigration. Human umbilical vein endothelial cells (HUVEC) were cultured on transwell membranes, treated with IL-1β, TNFα or fMLP and then incubated with NB1+ or NB1- PMN. Using flow cytometry we observed that transmigration alone resulted in a significant increase in PR3 expression on NB1+ but not NB1- neutrophils. Using a pan-serine protease inhibitor (AEBSF) total neutrophil transmigration was significantly inhibited. However, using a highly specific PR3 inhibitor (Elafin) we observed a selective inhibition in NB1+ but not NB1- neutrophil transmigration on IL-1β stimulated HUVEC. Similarly, antibodies against the NB1 recognition site on PECAM (Ig domain 6) inhibited neutrophil transmigration of NB1+ but not NB1- cells. Interestingly, in the presence of different stimuli (TNFα, fMLP), neutrophil transmigration was significantly less dependent on NB1-PECAM interactions and inhibition of PR3 activity did not inhibit transmigration. This is despite the fact that PR3 expression was highly up-regulated on NB1+ neutrophils incubated with either of these stimuli or following neutrophil transmigration.

In conclusion, the serine protease PR3 appears to play a significant role in the transmigration of NB1+ but not NB1- neutrophils. Likewise, the contribution of NB1 and PR3 in neutrophil transmigration is regulated in a stimulus-dependent mechanism which involves NB1 interactions with PECAM. These data therefore suggest that NB1 and PR3 may regulate recruitment of a neutrophil subset in response to specific inflammatory signals and this regulation may play a role in modulating the immune response.

Disclosures:

No relevant conflicts of interest to declare.
Cytokine Profile of Patients with Invasive Apsergillosis- Preliminary Results.
Authors: Ceesay M. Mansour , Kordasti Shahram , Berry Lisa , Smith Melvyn , Wade Jim , Mufti Ghulam J. , Pagliuca Antonio
Published: October 2019

Abstract 1500

Invasive Aspergillosis (IA) is an important cause of morbidity and mortality in haemato-oncology patients undergoing haematopoietic stem cell transplantation (HSCT) or chemotherapy resulting in prolonged cytopenia. The host cytokine response to fungal infection in this immunocompromised state is a crucial component of susceptibility and response to IA. The aim of this study was to assess if a unique cytokine profile exist for patients who developed IA compared to those who didn't.

All adult haemato-oncology patients enrolled on the Aspergillosis study, a prospective cohort study of adult haemato-oncology patients undergoing HSCT or chemotherapy, have baseline and fortnightly follow up serum samples profiled for 30 inflammatory cytokines using multiplex bead immunoassays by Luminex 100™ instrument. The cytokines measured were: EGF, Eotaxin, FGF, G-CSF, GM-CSF, HGF, IFN-α, IFN-γ, IL-1RA, IL-1β, IL-2, IL-2R, IL-4, IL5, IL6, IL7, IL-8, IL-10, IL-12p40/p70, IL-13, IL-15, IL-17, IP-10 (CXCL10), MCP-1, MIG, MIP-1α, MIP-1β, Rantes, TNF-α and VEGF.

The first hundred patients enrolled with sufficient follow up data were included in this initial analysis. The median (range) age was 52.5 (19-73) years and M/F ratio was 58/42. The main diagnosis were AML/MDS (40), Myeloma (23), NHL (17) and aplastic anaemia (9) treated by allogeneic HSCT (43), autologous HSCT (32), chemotherapy (20), and immunosuppressive therapy (5). The diagnosis of invasive fungal infection was based on the revised 2008 EORTC/MSG criteria. The incidence of proven and probable infection was 17% and possible infection accounted for 12%. Eight patients were excluded from analysis because of lack of proper baseline sample prior to starting chemotherapy/transplant or had infective episodes that proved to be due to IA. Using a longitudinal logistic regression model (R2= 0.4355, p<0.001) and using each patient as a cluster from baseline to follow-up periods and adjusting for age, sex, underlying diagnosis and treatment received, the following were found to be statistically significantly different between those with proven/probable IA compared to those patients with no evidence of IA: lower IL-10 (P= 0.042), lower IL-12 (P=0.017), higher IL-6 (P=0.001), lower IL-15 (P= 0.026), and higher CXCL-10 (P=0.046). Age correlated significantly with IA (P= 0.014) but not gender (P=0.693).

This preliminary data suggests that there is a significant difference in the cytokine profile between patients with proven/probable IA compared to those with no evidence of fungal infection. Further analysis will be performed on the rest of the cohort to confirm this.

Disclosures:

No relevant conflicts of interest to declare.
The Evolution Between 2001 and 2010 In the Uk Costs of Resolving a Mild to Moderate Bleeding Episode with Pd-aPCC Versus rFVIIa In Haemophilia A Patients with inhibitors.
Authors: Sewpaul Paresh , Ashley Donna , Riley Paul
Published: October 2019

Abstract 1518

Introduction:

Haemophilia A is a rare inherited bleeding disorder characterised by spontaneous or trauma-related bleeding, most commonly into the joints, leading to pain, swelling and limited movement. Some 30–50% of patients with haemophilia A develop inhibitors to their standard treatment, comprised of FVIII concentrates. There are two bypassing agents used for the management of bleeding episodes in patients with haemophilia A with inhibitors in the UK: recombinant factor VII (rFVIIa, NovoSeven®) and plasma-derived activated prothrombin complex concentrate (pd-aPCC, Feiba®).

Objective:

A systematic review of the cost-effectiveness of pd-aPCC versus rFVIIa was published in 2003. Since the analysis, costs of bypassing agents in the UK have changed significantly (pd-aPCC +30% from ≤0.57/U to ≤0.74/U; rFVIIa -11% from ≤0.51/mcg to ≤0.46/mcg). The present study examines the evolution in costs of resolving a mild to moderate bleeding episode with treatment initiated outside hospital.

Methods:

A decision analytic model estimated the expected costs and outcomes of resolving a mild-moderate bleeding episode in patients with haemophilia A with inhibitors. The model compared three treatment regimens in patients with a mean weight 70kg. Each regimen comprised first-, second- and third-line treatment: (1) pd-aPCC-pd-aPCC-rFVIIa; (2) pd-aPCC-rFVIIa-rFVIIa; and (3) rFVIIa-rFVIIa-rFVIIa. Efficacy and dosing of pd-aPCC and rFVIIa were estimated from the published literature, with rFVIIa assumed to resolve 92% of mild-moderate bleeds when used as first and second line treatment and pd-aPCC resolving 79% and 88% of mild-moderate bleeds when used first and second line respectively. Patients whose bleeding episodes were not resolved with first-line treatment were admitted to hospital for second- and third-line treatment. Costs included in the economic model were bypassing agent costs and hospitalisation costs.

Results:

The different treatment regimens result in different total costs for the resolution of a single mild-moderate bleeding episode with up to three lines of treatment: (1) GBP 13,542 (EUR 15,180); (2) GBP 12,985 (EUR 14,556); and (3) GBP 8,569 (EUR 9,605). Since 2001, the costs of regimens 1 and 2 have increased by 26% and 18%, but regimen 3 has decreased by 11% in the UK.

Conclusion:

Divergence in the price of pd-aPCC and rFVIIa since 2001 has increased the cost-effectiveness of rFVIIa. The results reinforce the fact that effective first-line treatment with rFVIIa results in lower overall treatment costs due to a reduced need for further treatment and associated hospitalisation costs. Adopting rFVIIa as first-line treatment is cost-saving and more effective compared to reserving rFVIIa as a second- or third-line treatment option.

Disclosures:

Sewpaul: Novo Nordisk : Employment. Ashley:Novo Nordisk : Employment. Riley:Novo Nordisk Ltd: Employment.