bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparing Patterns for Transitioning the Care of Young Adults with Sickle Cell Disease Versus Hemophilia: The Toronto Experience
Authors: Simpson Ewurabena , Ward Richard , Kirby Melanie , Odame Isaac 
Published: October 2019

Abstract
Abstract 2072

Background:
The Hospital for Sick Children (HSC) in Toronto, Canada cares for more than 700 children with sickle cell disease (SCD), which is the largest Canadian population of children with SCD. Since 2009, the SCD Program at HSC has partnered with adult hematologists within the Red Blood Cell Disorders program at Toronto General Hospital (TGH) to develop a coordinated strategy for transitioning the care of young adults with SCD. Hemophilia is a chronic hematological condition which, like SCD, has a spectrum of disease severity that requires multidisciplinary follow up. At HSC, we care for nearly 200 patients with hemophilia A and B and have a long-established partnership with adult hematologists, which has led to an effective transfer of patients with hemophilia into adult care. In Ontario, adult health providers are remunerated according to a fee-for-service billing schedule. In contrast, pediatric subspecialists are mainly salaried under an alternate funding plan. Until 2010, adult hematologists who provided medical care for individuals with hemophilia received a significantly higher pay scale than those who cared for individuals with SCD. This was changed in July 2010 so that adult hematologists receive commensurate remuneration for services rendered for both hemophilia- and SCD-related medical care.

Objectives:
1. To compare the patterns for transitioning patients of varying disease severity within the pediatric and adult SCD and hemophilia programs in Toronto, Ontario.
2. To identify barriers and enablers that have influenced the transition of young adults with SCD within a universal health care system.

Methods:
Data for active, transitioned and inactive patients in the HSC and TGH clinical programs are maintained in a database at HSC. These patient numbers were gathered according to sickle cell genotype. Similar data were available for hemophilia patients who were transitioned from HSC to adult care. Chi-square analyses were used to compare the proportions of patients in the sickle cell and hemophilia programs that were transitioned between 2009 and 2011.

Results:

Conclusion:
The HSC-TGH- partnership has significantly reduced the number of youth with SCD who continue to be followed at HSC or are lost to follow up. However, a significant number of young adults within the HSC SCD program still need to be transitioned to adult care. For the sustainable expansion of this transitional care strategy, health policymakers must collaborate with tertiary and community level health care providers to develop a coordinated and distributed strategy for the effective delivery of comprehensive health care services for young adults with SCD.

Disclosures:
No relevant conflicts of interest to declare.
A High Throughput Screening System for the Identification of Novel Genes in the Regulation of Mammalian Erythropoiesis
Authors: Ji Peng , Wong Piu , Lodish Harvey F. 
Published: October 2019

Abstract
Abstract 2093
Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursors. This induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. A recent genome wide transcriptional profile study demonstrated that over 600 genes are up-regulated during erythropoiesis. Surprisingly, the roles that most of the identified genes play in erythroid cells are still unknown. Understanding the functions of these unknown genes in the erythroid cells is necessary to elucidate the pathogenesis of red cell disorders such as congenital dyserythropoietic anemias, fanconi anemia, aplastic anemia, megaloblastic anemia, as well as leukemia and myelodysplastic syndromes with leukemic or dysplastic erythroid features. The goal of our study is to identify novel genes involved in different stages of erythropoiesis. To achieve this goal, we developed a high-throughput flow cytometry based assay that simultaneously detects erythroid cell differentiation and enucleation. In this assay, mouse fetal liver erythroblasts were purified and infected with lentiviruses containing a mammalian shRNA knockdown library obtained from the Broad Institute. The infected cells were cultured in a 96-well plate. Over the following two days the unaffected cells fully differentiate with approximately 60% of the cells enucleated. However, those cells in which shRNAs have knocked down genes critical for erythropoiesis are expected to show alterations in differentiation and/or enucleation. The system was validated using lentiviruses expressing shRNAs against Gata1 and mDia2, known proteins that are involved in the early and late stages of erythropoiesis, which showed inhibitions of differentiation and enucleation, respectively. We have pre-screened the most up-regulated 100 genes that play unknown functions in the erythroid cells. Future studies will be focused on the identified novel genes on their functions in erythroid lineage commitment, Epo mediated signaling pathways, hemoglobin enrichment, chromatin condensation, and enucleation.

Disclosures:
No relevant conflicts of interest to declare.
HPFH From Klf1 Haploinsufficiency Combined to Transcriptional Heterozygous β-Thalassemia
Authors: Moi Paolo , Marini Giuseppina M. , Asunis Isadora , Porcu Loredana , Cabriolu Annalisa , Satta Stefania , Perseu Luciana , Maccioni Liliana , Cao Antonio , Galanello Renzo 
Published: October 2019

Abstract
Abstract 2151
KLF1 mutations are emerging as a frequent cause of hereditary persistence of hemoglobin F (HPFH). Although it has been initially proposed that KLF1 haplo-insufficiency could determine KLF1 related HPFH, following studies have shown that isolated mono allelic Klf1 defects do not or only occasionally raise HbF and that biallelic KLF1 mutations are needed for HPFH to fully develop. Here we present a different mechanism of HPFH in a Sardinian family with a rare genetic combination in which HPFH with HbF levels up to 20% is caused by Klf1 haploinsufficiency combined to a β-thalassemia transcriptional defect from a −87C to G mutation in the proximal CACCC box, the canonical Klf1 binding site. Other known determinants of HPFH in the Myb, γ-globin or Bcl11 gene or variation in the expression of Bcl11 could not account for the increased HbF expression in the compound heterozygotes compared to the simple KLF1 or βThal-87 heterozygotes siblings. Hence, in this family delayed hemoglobin switching and HPFH are not dependent upon the absolute reduction in the free Klf1 concentration, as suggested by current models, but from the reduction in the amount of CACCC bound Klf1. This observation suggests that CACCC bound Klf1 is the primary anchor site for the LCR and that Klf1 mediated bridging between LCR and β-globin promoter is the most important determinant for hemoglobin switching in humans.

Disclosures:
No relevant conflicts of interest to declare.
Only Dendritic Cells, but Not Lymphocytes, Acquired Tolerogenicity by PUVA-Treatment by Upregulation of Indoleamine 2,3-Dioxygenase (IDO)
Authors: Maeba Hideaki , Kuroda Rie , Niida Yo , Mase Shintaro , Araki Raita , Fujiki Toshihiro , Yachie Akihiro , Koizumi Shoichi , Nishimura Ryosei 
Published: October 2019

Abstract
Abstract 2176
Extracorporeal photopheresis (ECP) is a leukapheresis-based immunomodulatory therapy by exposing isolated white blood cells to photoactive 8-MOP (psoralen) and UVA radiation (PUVA) for refractory acute and chronic graft-versus-host disease (GVHD). Although the effects of PUVA treatment on immune system remain poorly characterized, we have reported that bone marrow-derived dendritic cells (BM-DCs), which are generated from bone marrow cells plus GM-CSF, acquired tolerogenicity by PUVA-treatment in mice. In mixed lymphocyte reaction (MLR) assays, strong tolerogenicity was observed when adding PUVA-DCs generated from the same strain of stimulator cells, or responder cells, or even from third-party (Figure 1) strain into MLR mixture. That is, the PUVA-treated DCs have tolerogenic function in a MHC-independent manner (52nd ASH), in part, due to the reduced expression levels of CD80 and CD86, both of which are costimulatory molecules for T cell activation. To clarify the mechanisms of how PUVA-treated DCs induce tolerogenicity in further detail, we performed MLR with the addition of neutralizing antibodies against IL-10 or TGF-β1 or both, which have immunosuppressive effects. Neutralization of immunosuppressive cytokines had no effects on MLR. We then hypothesized that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect. To this end, we performed MLR using transwell to prevent cell-to cell contact. MLR was not suppressed when transwell was used, suggesting that PUVA-DCs dominantly regulates the alloreaction in a cell contact-dependent manner. Next we compared the expression of the indoleamine 2,3-dioxygenase (IDO), which induces T-cell anergy by tryptophan depletion and by the production of metabolic byproducts collectively known as kynurenines, by real-time PCR between PUVA-DCs and BM-DCs. An increased IDO gene transcription level was observed in PUVA-DCs about 5 times more than in BM-DCs (p<0.01) as shown in Figure 2. This suggested that the PUVA-treated DCs induced tolerogenic function by depleting tryptophan in the immediate environment. The question we have to consider next is whether tolerogenic capacity is acquired only in DCs or other lymphocytes subpopulations, because recent rodent studies have shown that the transfer splenocytes treated with PUVA prevent GVHD. To test this, PUVA-treated CD4, CD8, and B220 cells from the same strain of stimulator cells were added into the MLR mixture. MLR was suppressed only when PUVA-DCs were added. In conclusion, PUVA-DCs, but not other lymphocytes subpopulations, acquired regulatory function in a MHC-independent manner by upregulation of IDO. Infusion of PUVA-treated DCs could have a great potential to treat lethal acute GVHD in clinical settings.

Disclosures:
No relevant conflicts of interest to declare.
CD4+ T-Cell Functions Are Modulated by the Extracellular ATP Concentration
Authors: Trabanelli Sara , Ocadlikova Darina , Gulinelli Sara , Idzko Marco , Curti Antonio , Virgilio Francesco Di , Ferrari Davide , Lemoli Roberto M. 
Published: October 2019

Abstract
Abstract 2190
Adenosine 5'-triphosphate (ATP) plays a pivotal role in several cellular processes, through specific cell membrane purinergic P2 receptors (P2Rs). During inflammation and tumor cell growth, cell necrosis causes the release of intracellular ATP into the extracellular space, thus increasing from low (1–10 nM) to high (5–10 mM) the concentration of extracellular ATP. For this reason, variations in the extracellular ATP concentration might activate/inhibit the immune system.
Here we investigated the role of ATP on CD4+ T-cell functions. We first demonstrated the expression of P2Rs for extracellular nucleotides in human activated CD4+ T cells and regulatory T cells (Tregs) We then show that physiological concentrations of extracellular ATP (i.e. 1–50 nM) do not affect both activated CD4+ T cells and Tregs. Conversely, supraphysiological concentrations of ATP show a bimodal effect on activated CD4+ T cells. Whereas 250 nM of ATP stimulates proliferation, cytokine release, expression of adhesion molecules and adhesion, high ATP concentration (i.e. 1 mM) induces apoptosis and inhibits activated CD4+ T-cell functions. On the contrary, at the same high concentration, ATP enhances the proliferation, adhesion, migration and immunosuppressive ability of Tregs. Similar results are obtained when activated CD4+ T cells and Tregs are exposed to ATP released by necrotized leukemic blasts.
The present results provide evidence that different concentrations of extracellular ATP modulate T cells according to their activation status. Therefore, high concentrations of ATP, compatible with fast-growing tumors or hyper-inflamed tissues, may have a key role in killing activated CD4+ T cells and in expanding Tregs.

Disclosures:
No relevant conflicts of interest to declare.
Bone Marrow Specimens From Patients with Immune Thrombocytopenia (ITP) Demonstrate Increased Megakaryocyte-Bound IgG and Increased T-Helper Cells
Authors: Toltl Lisa J. , Ross Catherine , Kelton John G. , Arnold Donald M. 
Published: October 2019

Abstract
Abstract 2233

BACKGROUND:
Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease characterized by low platelet counts and platelet autoantibodies. The mechanisms of ITP remain unclear although recent evidence suggests that megakaryocytes may be the target of immune destruction. To test this hypothesis we evaluated IgG-binding to megakaryocytes and B- and T-cells in bone marrow specimens of ITP patients and non-thrombocytopenic controls.

METHODS:
Histological slides of bone marrow biopsies from ITP patients and control patients were prepared from stored paraffin-embedded tissue blocks for blinded pathological assessment. Adult ITP patients (N=19) had a platelet count less than 100 x109/L (range 2 – 76 x109/L) as measured within 4 weeks before the date of bone marrow biopsy procurement without splenomegaly, myelodysplastic syndrome, lymphoproliferative disease, HIV, hepatitis B or C, drug-induced thrombocytopenia or prior treatment with thrombopoietin receptor agonists or other hematopoietic growth factors. Control patients (N=15) were adults with limited stage lymphoma or monoclonal gammopathy of undetermined significance with a normal platelet count (150 – 400 x109/L) who had a bone marrow biopsy for staging purposes which was reported as normal. Patients with prior use of anti-neoplastic agents or hematopoietic growth factors were excluded. Coded bone marrow biopsy sections were labeled with rabbit polyclonal anti-IgG, anti-CD4, anti-CD8, anti-CD20, and CD61 in serial sections, followed by streptavidin-biotin labeling techniques. The intensity of staining was assessed semi-quantitatively by an experienced hematopathologist for megakaryocyte, B-cell and T-cell numbers (increased, decreased, within normal limits) and megakaryocyte-bound IgG (negative, below 50% of cells positive or more than 50% of cells positive) blinded to diagnosis and platelet count.

RESULTS:
ITP bone marrows demonstrated greater IgG binding to megakaryocytes compared with controls [12/19 (63.16%) vs. 4/15 (26.67%), p=0.02], increased CD4+ cells [15/19 (78.95%) vs. 5/15 (33.33%), p=0.003] and marginally increased CD8+ cells [11/19 (57.89%) vs. 4/15 (26.67%), p=0.05]. B-cell numbers were not different between groups.

CONCLUSIONS:
Using bone marrow specimens from carefully selected ITP patients and controls, our study shows that IgG-bound megakaryocytes and T cells, especially T-helper cells, are increased in bone marrow of ITP patients compared with controls. These data provide evidence of megakaryocyte injury in ITP. Additional labeling with caspase-3 and TUNEL stains is planned to identify markers of apoptosis.

Disclosures:
Kelton: Amgen: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding. Arnold:Amgen: Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding; Talecris: Honoraria; Hoffmann-LaRoche: Research Funding.
Silent Pulmonary Embolism in Patients with Lower Limb Deep Vein Thrombosis
Authors: Tsoran-Rosenthal Inna , Sakharov Gleb , Brenner Benjamin , Rivron-Guillot Karine , VisonÁ Adriana , JimÉnez David , Monreal Manuel 
Published: October 2019

Abstract
Abstract 2306

Background:
One in every three patients with deep vein thrombosis (DVT) may have silent pulmonary embolism (PE), but its clinical relevance has not been thoroughly studied. Methods: We used the RIETE Registry data to compare the clinical characteristics, diagnostic tests, and 3-month outcome in 842 patients with proximal DVT in the lower limbs and silent PE at baseline, 1533 with DVT without PE, and 585 patients with DVT and symptomatic PE. Results: On admission, a minority of DVT patients (with or without silent PE) presented with hypoxemia (9.0% vs. 6.4%, respectively), or typical PE signs on the chest X-ray (25% vs. 22%) or electrocardiogram (23% vs. 17%). Patients with symptomatic PE more frequently presented with hypoxemia (30%) or had PE signs on the chest X-ray (41%) or electrocardiogram (37%). After the initial 15 days of follow up the incidence of PE was higher among patients with DVT and silent PE compared to those with symptomatic PE (0.95% vs. 0.17%).During the first 90 days of anticoagulant therapy, patients with DVT without PE had a lower incidence of recurrent PE than bleeding (1.0% and 2.9%, respectively). Of note, an excessive risk for bleeding was observed during the first 2 weeks of therapy among patients without PE. Incidence of recurrent PE and major bleeding was similar in DVT patients with silent PE (1.8% and 1.9%) and in those with symptomatic PE (2.6% and 2.7%). Conclusions: Most DVT patients with silent PE have no hypoxemia, chest X-ray signs or electrocardiographic evidence suggestive of embolism. Frequency of recurrent PE was higher among patients with silent PE at the initial 15 days of follow up. In contrast to other subgroups, in DVT patients without PE at baseline, the incidence of major bleeding far exceeded that of PE development during follow up.

Disclosures:
No relevant conflicts of interest to declare.
An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury
Authors: Adams Brian D. , Guo Shangqin , Bai Haitao , Xiao Changchun , Reddy E. Premkumar , Lu Jun 
Published: October 2019

Abstract
Abstract 2333
.
MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge.
Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro.
Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field.

Disclosures:
No relevant conflicts of interest to declare.
Addition of Rituximab to Chemotherapy Alters the Prognostic Impact of Tumor-Associated Macrophages in Diffuse Large B-Cell Lymphoma
Authors: Riihijärvi Sari , Pöyhönen Maria , Taskinen Minna , Karjalainen-Lindsberg Marja-Liisa , Leppä Sirpa 
Published: October 2019

Abstract
Abstract 2650

Background:
Tumor associated macrophages (TAM) have at least two potential roles in promoting tumor growth: suppression of immune responses and potentiation of angiogenesis. In numerous cancer types, including lymphomas, high M2 type TAM content has been associated with worse prognosis. Rarely, high TAM content correlates with better survival. We have recently shown that CD68 positive TAMs in DLBCL contribute to unfavorable survival after high dose chemotherapy. Here we have extended our analyses on M2 type macrophages and questioned how combination of rituximab with chemotherapy influences TAM-associated clinical outcome.

Patients and Methods:
Expression of CD163 and CCL18, which are primarily expressed in M2 type macrophages, were identified immunohistochemically from samples of 101 de novo DLBCL patients treated with rituximab in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-like regimen (immunochemotherapy). With a median follow-up of 65 months, (range 16–114 months), 5-year progression free survival (PFS) was 70% and overall survival (OS) 73%. 29 DLBCL patients previously treated with up front high dose chemotherapy served as a control group.

Results:
Correlation between CD163 and CCL18 positive TAMs was found (rs=0.427, p<0.001). In the Kaplan-Meier analyses the cutoff level of 67% was found to best discriminate between subgroups with different outcomes. Consistent with previous data, chemotherapy-treated patients with high CD163 or CCL18 positive TAM counts displayed a significantly inferior OS and PFS than the low group (Table). In contrast, after rituximab containing regimen, the patients with high CD163 and CCL18 positive TAM content tended to have favorable survival. Among the patients with low counts in both CD163 and CCL18 positive TAMs, PFS and OS were found to be significantly worse in comparison to others.

Conclusions:
In contrast to data on chemotherapy treated DLBCL or other lymphoma types, M2 type TAM content is associated with favorable prognosis in DLBCL patients after immunochemotherapy.

Disclosures:
No relevant conflicts of interest to declare.