bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome Variation and Donor-Recipient Compatibility in Graft-Versus-Host Disease
Authors: McCarroll Steven A 
Published: October 2019

Abstract
Abstract SCI-12
Graft-versus-host disease (GVHD) after hematopoetic stem cell transplantion (HSCT) arises from differences between the genomes of donor and recipient. The fact that GVHD is common when HSCT involves HLA-identical siblings but almost never occurs when HSCT involves identical twins has long indicated that the human genome harbors histocompatibility loci outside the HLA locus. An important, unresolved question has involved what specific genomic loci might have potent effects on transplantation outcomes. Although GVHD is a disease of genome compatibility, it was until recently impossible to know all the genomic differences between any specific donor and recipient, or among the individuals in a population. 1. An ongoing revolution in genome analysis methods is making it possible to characterize genome variation almost completely. I will first describe what the complete sequencing of large numbers of human genomes in the 1000 Genomes Project is revealing about how antigen repertoire varies among the individuals in a population, or between any particular candidate donor and recipient. These results have surprising implications for research strategies to identify the genetic basis of histocompatibility barriers in human transplantation. 2. In this and earlier work, we have found a surprising number of genome polymorphisms that remove entire protein-coding genes from people's genomes. Many of these gene deletion alleles are sufficiently common that individuals inherit them from both parents and therefore lack completely an entire protein that is expressed in other individuals. The immune system of such an individual may tend to recognize epitopes encoded by that gene as alloantigens. Colleagues from the Dana Farber Cancer Institute, the Helsinki University Central Hospital and Finnish Red Cross, the University of Michigan, the Fred Hutchinson Cancer Research Center and the Broad Institute recently came together to evaluate the relationship of six of these gene deletions to GVHD in clinical cohorts. We found that donor-recipient mismatch for one of these gene deletions – deletion of the UGT2B17 gene – associated in multiple clinical cohorts with the occurrence of acute GVHD after HSCT. This same gene has been found to give rise to histocompatibility antigens in individual HSCT patients, offering a molecular and cellular mechanism for its association with GVHD. 3. Finally, I will describe a strategy for identifying the genetic bases of histocompatibility barriers among individuals, based on our emerging understanding of how the human genome varies in populations.

Disclosures:
No relevant conflicts of interest to declare.
New Strategies to Define Regulators of Fetal Hemoglobin
Authors: Orkin Stuart H. 
Published: October 2019

Abstract
Abstract SCI-17
The beneficial effect of hemoglobin F (HbF, α2γ2) expression on the phenotypes of sickle cell disease and the β-thalassemias has been recognized for many years. Therefore, retardation of the fetal-to-adult (HbF to HbA, α2b2) switch or reactivation of g-globin expression in erythroid cells of adults has been a long sought goal. Despite molecular analysis of the human β-globin gene cluster and intense study of transgenic mice harboring human globin genes, the nuclear factors mediating hemoglobin switching and maintaining γ-globin gene silencing have remained unknown. The situation has changed recently with the success of new genetic tools, including genome-wide association study (GWAS), in the identification of candidate gene regions in which genetic variation correlates with HbF levels. Three genomic regions have been fingered: the β-globin cluster itself, the BCL11A gene on chromosome 2, and the HB1SL-Myb region on chromosome 6. Taken together, these regions account for ∼40% of the variation in HbF levels. Functional studies have shown that BCL11A encodes a dose-sensitive, zinc-finger repressor that is required for maintenance of silencing of γ-globin in adult erythroid cells. BCL11A also controls the developmental switches from embryonic to adult β-like globins in the mouse and fetal to adult globin in human transgenes harbored in the mouse. BCL11A binds downstream of the γ-globin gene and in the locus control region, but not at the γ-promoters. Mechanistic studies reveal that BCL11A cooperates with another factor, SOX6, that binds within the γ-promoters. The identification of genetically validated regulators has therapeutic implications for directed reactivation of HbF in adults. For stage-specific repressors, such as BCL11A, inhibition of their expression, or interference with their function or interactions with other regulators, could constitute new therapeutic strategies. While transcription factors have been generally considered “undruggable”, advances in high-throughput screening of complex libraries of small molecules and in the use of RNA interference in vivo may provide new platforms for discovery and refinement of HbF inducers. The presentation will illustrate some of these potential approaches.

Disclosures:
No relevant conflicts of interest to declare.
Safety and Efficacy of 90Y Ibritumomab Tiuxetan (Zevalin®) for Untreated FollicularNon-Hodgkin's Lymphoma (FL) Patients, An Italian Cooperative Study
Authors: Pica G. , Nati S. , Vitolo Umberto , Galimberti Sara , Zinzani Pier Luigi , Cascavilla Nicola , Petrini Mario , Carella A. M. 
Published: October 2019

Abstract
Abstract 100
90Y ibritumomab tiuxetan (Zevalin®) combines the targeting advantage of monoclonal antibody with the radiosensitivity of FL. Previous studies showed that Zevalin resulted safe and highly effective in relapsed/refractory indolent NHL, irrespective to prior treatment with rituximab.
Based on these results, we designed a multicenter trial to evaluate the safety and the efficacy of “upfront” single-agent Zevalin in FL. The primary endpoint was the incidence of responses in terms of overall remission rate (ORR) and complete remissions (CR). The secondary endpoints were the treatment safety by monitoring hematology and biochemistry parameters as well as adverse events.
Fifty patients, with a median age of 59 years (range, 35–81), were treated. Forty-eight percent had bone marrow involvement (<25%) and 14% an elevated LDH. Thirty-four percent of patients had high risk FLIPI. Forty-six patients were also assessed by qualitative and quantitative PCR for Bcl2/IgH or IgH clonal rearrangement, for total 30 cases PCR-positive (65.2%).

Results:
The ORR was 93% (45/48) with a CR rate of 82% (41/48). Twenty-six patients, who were PCR-positive at diagnosis, were assessed at week 14. Twenty out of 26 (77%) became PCR-negative. After a median follow up of 24 months, the 2-year EFS for all patients was 85%; moreover, 15 patients (55%), who were PCR-positive at diagnosis, maintain PCR negativity.
As expected, the main toxicity was moderate myelosuppression, with 30% and 26% of patients developing Grade 3/4 neutropenia and thrombocytopenia, respectively. Very few patients required platelets transfusion (4%) or growth factor use (6%). None of the patients experienced grade 3/4 non hematologic toxicity.
In conclusion, Zevalin is highly effective and safe treatment for newly diagnosed FL patients. In the next future, the role of radioimmunotherapy - i.e. including optimal sequencing with chemotherapy - should be established in randomized studies.

Disclosures:
No relevant conflicts of interest to declare.
Antioxidant Vitamins C and E Supplementation Does Not Improve the Hemolytic Profile of Sickle Cell Anemia Patients: A Randomized, Double-Blind, Placebo-Controlled Trial
Authors: Arruda Martha Mariana A S , Mecabo Grazielle , Rodrigues Celso Arrais , Matsuda Sandra S , Rabelo Iara Baldim , Figueiredo Maria Stella 
Published: October 2019

Abstract
Abstract 1063

Background:
Erythrocytes of sickle cell anemia (SCA) patients continuously produce larger amounts of pro-oxidants than normal cells, and oxidative stress seems to play a significant role in the pathophysiology of this disease. In erythrocytes, oxidative stress primarily affects the membrane and results in hemolysis. The use of antioxidants in vitro reduces the generation of pro-oxidants, which could prevent adhesion and phagocytosis of oxidized erythrocytes.

Aims:
To evaluate the impact of antioxidant vitamins C (vitC) and E (vitE) supplementation in the hemolytic profile in SCA patients.

Patients and methods:
Homozygous SCA or S-β-thalassemia patients, over 18 years, were randomly assigned to receive VitC 1,400 mg + VitE 800 mg per day or placebo, administered orally for 180 days. Pregnant women and patients with iron overload out of chelation therapy were excluded. Patients were evaluated clinically and blood samples were collected at days 0 and 180 for complete blood count, automated reticulocyte count, indirect bilirubin, lactate dehydrogenase (LDH), haptoglobin, uric acid, and serum levels of VitC and VitE.

Results:
Overall, 83 patients were enrolled (44 vitamins, 39 placebo). The median (range) age was 27 (18–68) years, and 53 (64%) were female. There were no significant differences between the two groups as regards clinical complications of SCA or baseline laboratorial tests and serum vitC and vitE. Sixty percent of the patients were VitC deficient (30% with severe deficiency), 70% were VitE deficient (33% with severe deficiency) and 44% were deficient in both vitamins. Vitamin supplementation increased VitC from 27.2 to 62.6 μMol/L (p<0.0001) and VitE from 13.9 to 20.2 μMol/L (p<0.0001). No changes in vitC or vitE were observed in patients receiving placebo. No significant changes in hemoglobin levels, hematocrit, mean corpuscular volume were observed in either group. However, patients receiving vitamin supplementation presented a significant increase in the median reticulocyte count (from 152 to 195 ×106/μL, p=0.01), LDH (from 396 to 425 U/L, p=0.018), indirect bilirubin (from 1.45 to 1.73 mg/dL, p<0.0001), and uric acid (from 4.75 to 5.15 mg/dL, p=0.02), and a decrease in the haptoglobin levels (from 3.95 to 3.45 mg/dL, p=0.06).

Conclusion:
Supplementation with vitamins C and E did not improve anemia and, surprisingly, increased markers of hemolysis in patients with SCA and S-β-thalassemia. The exact mechanisms to explain our findings and their clinical significance remain to be determined.

Disclosures:
No relevant conflicts of interest to declare.
Platelet Microparticles Increase Pro-Angiogenic Properties of Prostate Cancer Cell Lines
Authors: Dashevsky Olga , Shai Ela , Varon David 
Published: October 2019

Abstract
Abstract 1147
Platelets are key players in hemostasis, but are also involved in fundamental processes of vascular biology as angiogenesis, tissue regeneration and tumor metastasis. Platelet Microparticles (PMP) are vesicular membrane fragments shed from activated platelets, containing the platelet cytoplasmic content with a typical platelet membrane.
Prostate cancer is the most frequently diagnosed noncutaneous cancer in men in western countries. A major factor of the life-threatening course of this disease is the high rate of metastasis. Circulating tumor cells encounter platelets and may activate them, resulting in a production of microparticles.
We have previously reported a pro angiogenic effect of PMP, both in vitro and in vivo. We also demonstrated that prostate cancer cells that were pre-incubated with PMP acquired invasive properties. This was shown by rapid and transient increase of MMP-2 mRNA level and increased secretion of metalloproteinase-2 (MMP-2) as demonstrated by gelatin zymography.
In the present study, in vivo experiments of injection of prostate cancer cells pre-incubated with PMP to the mouse tail vain showed more rapid localization of cancer cells to the lungs, compared to untreated cancer cells. Microarray assay of prostate cancer cells pre-incubated with PMP has revealed significant differences in transcriptional regulation of genes related to cell growth, survival, tumorigenicity, invasiveness, and angiogenesis that suggested a more aggressive feature of the cells. We then investigated effect of PMP on release of the pro-angiogenic cytokine IL-8 from prostate cancer cells. RT-PCR demonstrated that protein release was accompanied by increase of IL-8mRNA and changes in regulation of related transcription factors. We have shown the involvement of the signaling molecules PI3, p38 and ERK in synthesis and expression of IL-8 from prostate cancer cells and revealed the contribution of the platelet-derived lipid mediator sphingosine 1-phosphate (S1P) and angiopoietin 1 (Ang-1) to the process.
Better understanding of the role of PMP in cancer development and identification of the mechanisms responsible for prostate cancer progression and aggression by the tumor microenvironment may lead to the development of novel effective therapies for metastatic disease.

Disclosures:
No relevant conflicts of interest to declare.
Strong Correlation of High Endogenous Plasma Levels of Tissue Factor Pathway Inhibitor (TFPI) with Poor Blood Clotting and Corrective Action of BAX 513 Under FVIII-Inhibited Conditions
Authors: Dockal Michael , Gorczyca Monika , Knappe Sabine , Palige Michael , Ehrlich Hartmut J. , Scheiflinger Friedrich , Jilma Bernd 
Published: October 2019

Abstract
Abstract 1201
TFPI is the major physiological inhibitor of the extrinsic pathway of blood coagulation. Thus, blocking TFPI activity is a possible approach to treat bleeding disorders such as hemophilia A and B. BAX 513, a fucoidan derived from the brown seaweed L. japonica, and other non-anticoagulant sulfated polysaccharides (NASPs) improve coagulation in hemophilic blood and plasma (Liu et al. Thromb Haemost 2006;95:68). This procoagulant activity of NASPs can at least partially be attributed to the inhibition of TFPI. For this study, we predicted that endogenous TFPI levels are associated with decreased hemostatic capacity under hemophilic conditions. We correlated clotting parameters measured by global hemostatic assays with the total and full-length (FL) TFPI levels in human plasma samples in the absence or presence of BAX 513.
Tissue factor-triggered coagulation of normal and FVIII-inhibited plasma or blood from 30 healthy subjects was monitored by calibrated automated thrombograph (CAT) assay and rotation thromboelastometry (ROTEM). The experiments were performed with or without factor VIII blockade with a goat anti-human Factor VIII antibody to simulate acquired hemophilia. The total and FL-TFPI plasma levels were quantified by ELISA. We observed longer clotting and clot formation times by ROTEM and decreased thrombin generation in the presence of high TFPI levels in normal plasma (p<0.05). These correlations became substantially stronger when FVIII was inhibited (reaching up to r2= 0.64; p<0.001). In the presence of BAX 513 (0.1–1.2 μg/mL), most plasma samples showed a pro-coagulant response with an at least 50% increase in thrombin peak and a decrease in time to peak. The BAX 513-mediated increase in thrombin generation correlated strongly with the individual FL-TFPI plasma level. The effect was even more apparent when FVIII was inhibited, where the extrinsic pathway is the only driver of thrombin formation.
These data indicate that high endogenous TFPI levels critically compromise hemostasis in acquired factor VIII deficiency and that TFPI is a threshold factor for the intrinsic amplification of thrombin formation. As low TFPI levels are associated with increased blood clotting, TFPI inhibition is a rationale treatment approach in patients with hemophilia. Importantly, our findings validate the inhibition of TFPI activity as a mode of action for BAX 513 and related pro-coagulant compounds.

Disclosures:
Dockal: Baxter Innovations GmbH: Employment. Gorczyca:Medical University of Vienna: Employment. Knappe:Baxter Innovations GmbH: Employment. Palige:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.
Kinase Domain Mutations in JAK2V617F Confer Resistance to the Novel JAK2 Inhibitor Ruxolitinib
Authors: Deshpande Anagha , Reddy Mamatha M. , Schade Georg , Ray Arghya , Chowdary Tirumala K. , Griffin James D. , Sattler Martin 
Published: October 2019

Abstract
Abstract 125
The JAK2V617F kinase mutation is frequently associated with myeloproliferative neoplasms (MPNs) and thought to be instrumental for the overproduction of myeloid lineage cells. Ruxolitinib (INCB018424) and several additional small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would cause resistance to ruxolitinib. Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would lower sensitivity towards ruxolitinib, using a cell-based assay. We found 5 different non-synonymous point mutations that conferred drug resistance, including Y931C, G935R, R938L, I960V and E985K. Cells containing mutations had a 9 to 33-fold higher EC50 for ruxolitinib. Results were analyzed by evaluating the three-dimensional structure of ruxolitinib bound to the ATP binding pocket of the monomer JAK2 kinase domain, using a docking algorithm. The inhibitor is held in the pocket by polar contacts and extensive hydrophobic interactions with several residues that line the binding pocket. A880, L855, V863 and M929 interact with the inhibitor on one side, and V911 and L983 provide hydrophobic interactions on the other side. The pyrazol ring of ruxolitinib appears to have a π-π interaction with the Y931 ring. Most mutations that were identified in our screen are either residues that interact with ruxolitinib or are in proximity to the binding pocket and hence are likely to indirectly alter inhibitor binding. The structural analysis indicates that there are additional residues in the kinase domain that may be critical for optimal binding of ruxolitinib and point mutations at these sites may also alter the affinity of the drug. Our analysis further suggests that ruxolitinib and other JAK2 inhibitors may bind in a similar way to the kinase domain. Consequently, these identified mutations conferred cross-resistance to all JAK2 kinase inhibitors tested in cell growth assays, including AZD1480 (5.6 to >7-fold higher EC50), TG101348 (2.2 to 2.8-fold increase in EC50), lestaurtinib (CEP-701) (2.6 to 3.3-fold increase in EC50) and CYT-387 (5.1 to 7.4-fold increase in EC50). Surprisingly, introduction of the ‘gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (4-fold increase in EC50). In addition, the E985K and Y931C mutations reduced the sensitivity to ruxolitinib and offered a growth advantage to cells during treatment, compared to native JAK2V617F expressing cells. These results suggest that the development of point mutations in JAK2 could result in clinical resistance to a wide variety of JAK2 inhibitors currently in clinical trials in patients with myeloproliferative neoplasms.

Disclosures:
No relevant conflicts of interest to declare.
A T Cell Receptor Signaling Mutant Reveals Two Distinct Populations of IL-17 Producing CD4+ T Cells
Authors: Kim Jiyeon S , Smith-Garvin Jennifer E , Jordan Martha S , Koretzky Gary A 
Published: October 2019

Abstract
Abstract 186
Interleukin-17 (IL-17) producing CD4+ T cells (Th17 cells) are essential for immune responses in mucosal and epithelial sites which are the first line of host defense. Th17 cells play a critical role in the pathogenesis of many inflammatory and autoimmune diseases, and the role of IL-17 and Th17 cells in cancer has recently become the focus of extensive investigation. Most studies to date have focused on elucidating the cell extrinsic requirements for differentiation of Th17 cells from naïve CD4+ T cells in peripheral effector sites. Here we report an unconventional population of Th17 cells, “natural Th17 cells” (nTh17), that acquire effector function during development in the thymus, thereby distinguishing them from conventional Th17 cells which require antigen encounter and differentiation in the periphery. We show that these nTh17 cells are present and indeed develop in the thymus using fetal thymic organ culture. nTh17 cells express surface markers consistent with an innate and/or activated phenotype and their development is dependent on selection by MHC class II in the thymus. Yet unlike conventional CD4+ T cells, MHC class II expression on thymic cortical epithelium is not sufficient for their development, rather expression on medullary epithelium is necessary. In addition, T cell receptor (TCR) repertoire analysis of nTh17 cells revealed unique characteristics in TCR gene usage compared to conventional Th17 cells. A mouse model with a mutation in the TCR signaling protein SLP76 (SLP76 Y145F mice) further highlights the difference between the two distinct Th17 populations. SLP76 Y145F mice have increased numbers of nTh17 cells in the thymus compared to wild-type mice. However, peripheral naïve CD4+ T cells from these mice showed severely defective IL-17 production when cultured in vitro under conditions promoting Th17 cell differentiation. This defect was reflected in vivo as CD4+ T cells in the small intestinal lamina propria of SLP76 Y145F mice fail to produce IL-17. Using mixed radiation bone marrow chimeras, we found that the aberrant Th17 phenotype in the thymus and periphery of SLP76 Y145F mice is cell-intrinsic. Finally, adoptive transfer of purified nTh17 cells into RAG-deficient host mice revealed preferential homing of nTh17 cells to thymus and lung compared to other comparison/competitive populations that were co-transferred. Taken together, our data suggest a distinct population of Th17 cells that have characteristics of innate lymphocytes that function at the interface between innate and adaptive immunity. Understanding the biology of nTh17 cells will provide insight into the recently identified Th17 cells in human thymi and umbilical cord blood.

Disclosures:
No relevant conflicts of interest to declare.