bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clinical Features of De Novo CD25-Positive Diffuse Large B-Cell Lymphoma
Authors: Fujiwara Shin-ichiro , Muroi Kazuo , Hirata Yuji , Sato Kazuya , Matsuyama Tomohiro , Ohmine Ken , Suzuki Takahiro , Ozaki Katsutoshi , Mori Masaki , Nagai Tadashi , Ozawa Keiya 
Published: October 2019

Abstract
Abstract 2666

Background:
CD25 (the alpha chain of the IL-2 receptor) has been shown to be expressed in a small subset of normal B cells and hairy cell leukemia cells; however, its expression in diffuse large B-cell lymphoma (DLBCL) has not been examined. The aim of this study was to clarify the clinical features of CD25+ DLBCL. Patients and Methods: Fifty-five patients with newly diagnosed CD25+ DLBCL who were admitted to our hospital between 1993 and 2011 were retrospectively evaluated. Lymph node or related tissue biopsy specimens from the patients were analyzed using FCM combined with single- and two-color staining. CD25 expression was defined as positivity of >20% of clonal B cells with CD19 or 20 expression >80% in a gated region. Results: There were 30 males and 25 females, with a median age of 65 years (range, 27–88 years). They showed aggressive clinical features as follows: 37 patients older than 60 (67%), 45 with elevated LDH (82%), 42 with soluble IL-2 receptor higher than 2,000 U/ml (79.4%), 33 with advanced-stage disease (stage III or IV, 60%), 28 with more than one extra nodal site (51%), 31 at high/high-intermediate risk according to the international prognostic index (IPI) score (56.4%). Chromosomal abnormalities were identified in 27 (79.4%) of 34 patients and frequently at chromosomes 3 (n = 13), 6, 7, 8, 14 (n = 9, respectively), and 1 (n = 7). CD25 expression showed a mean of 60.2% (range, 21.2–97.4%), and was particularly higher in patients aged more than 65 years and with CNS involvement (66.8 vs. 52.8%, p= 0.03 and 88.2 vs. 61.8%, P <0.01, respectively). In two-color FCM analysis, the percentages (mean ± SD) of CD19+CD25+ and CD20+CD25+ cells were 63.7 ± 25.5% (n=13) and 55 ± 28.1% (n=14), respectively. Compared to the patients with de novo CD25+ follicular lymphoma (CD25>20%, CD19/20>80%, n = 7), CD25 expression was significantly higher in CD25+ DLBCL (59.1%) than in CD25+ follicular lymphoma (34.5%). The complete remission (CR), 4-year progression free survival (PFS) and overall survival rates in the patients treated with R-CHOP (n = 35) were 81, 47, and 64%, respectively. When CD25 expression levels were compared between patients in CR and alive and those in non-CR and deceased, a trend was shown (58.1 vs. 74.4%, P = 0.055, and 57 vs. 71.8%, P = 0.058, respectively). Conclusion: CD25+ DLBCL may constitute a distinct subgroup with aggressive clinical features. Because R-CHOP is less effective for these patients, an alternative approach such as R-CHOP + anti-CD25 immunotherapy is needed.

Disclosures:
No relevant conflicts of interest to declare.
Using the Zebrafish As a Tool for Modeling Systemic Mastocytosis,
Authors: Balci Tugce B , Coombs Andrew J , Grondin Chloe , Da'as Sahar I , Chute Ian , Leger Daniel , Ferrando Adolfo A. , Lewis Stephen , Berman Jason N 
Published: October 2019

Abstract
Abstract 3208
The zebrafish system provides many advantages in investigating intricate molecular pathways regulating vertebrate blood cell development and disease in vivo. We have been exploiting these assets to elucidate normal mast cell (MC) function and previously described the structural and functional characteristics of zebrafish MC equivalents (Dobson et al, Blood 2008, Da'as et al, Dev Comp Imm 2011). We have used this knowledge to develop transgenic zebrafish models of systemic mastocytosis (SM). SM is a pre-leukemic myeloproliferative disease that results from perturbed MC development and proliferation. Our recent studies have suggested that zebrafish MCs are uniquely dependent on Notch pathway signaling in contrast to that observed for other myeloid cell populations. Whole mount in situ hybridization (WISH) studies on the zebrafish Notch signaling mutant, mindbomb and notch1b “morphant” embryos both displayed decreased to absent carboxypeptidase A5 (cpa5) positive mast cells by WISH. Furthermore, wild type embryos treated with Compound E (CpdE), a γ-secretase inhibitor that inhibits Notch signaling, showed a similar phenotype. Given the role for Notch signalling in normal MC development, we wanted to see if driving the Notch pathway would result in a phenotype reminiscent of SM. Through a heat-shock inducible Gal4-UAS based system; we now demonstrate that over-expression of Notch signalling results in increased cpa5 positive mast cells in embryos as observed by WISH at 30, 36 and 48 hours post fertilization (hpf). Importantly, we were able to inhibit this increase and even reduce MC numbers below baseline levels in a dose-dependent manner using CpdE. Concurrently, we have established a transgenic zebrafish model of SM that ubiquitously expresses the human c-KIT D816V mutation under the zebrafish β-actin promoter. Beginning at 9 months of age, adult fish develop a number of skin and visceral lesions, many of which have been found to contain an abundance of MCs as identified by toluidine blue staining and tryptase immunohistochemistry. Transgenic embryos lack a developmental phenotype but demonstrate evidence of decreased phospho-histone H3 (pH3) signaling, suggesting additional mutations are required to progress to SM. In support of this G2/M arrest phenotype, microarray studies conducted on transgenic embryos revealed upregulation of p53 and cyclin G1. These studies have provided new insights into the role of Notch signaling in MC development and the opportunity to use the zebrafish as an in vivo model to identify and evaluate novel therapeutic strategies in MC diseases.

Disclosures:
No relevant conflicts of interest to declare.
Anti-Leukemic Effect of IMiDs® in AML Involves the Direct Cytotoxicity Effects in Addition to a Biased NK Activation,
Authors: Le Roy Aude , Vey Norbert , Fauriat Cyril , Orlanducci Florence , Bateman Scott , Olive Daniel 
Published: October 2019

Abstract
Abstract 3224
Lenalidomide is an immunomodulatory drug (IMiD®) that has shown efficacy in the treatment of patients with multiple myeloma (MM) and myelodysplastic syndrome with 5q deletion. This drug has several potential antitumor mechanisms of action, shown in MM (Görgün G et al. Blood. 2010). In addition to its direct anti-MM effect, lenalidomide also stimulates T-cell proliferation, IL-2 and interferon-γ (IFNγ) production (Corral LG et al.J Immunol. 1999), and enhances cytotoxic T lymphocyte (CTL) and natural killer (NK) effector cell activity against MM cells (Davies et al. Blood. 2001).
We examined the in vitro antileukemic effects of two IMiDs® (lenalidomide and another derivative of thalidomide, pomalidomide) in AML.
We have shown that both lenalidomide and pomalidomide treatment had an effect on two major mechanisms involved in the antileukemic action: on one hand, on the function of effector cells including NK cells, on the other hand, a direct toxic effect on leukemic blasts.
First, we have shown that these drugs induced rapid phenotypic modifications of NK cells, independently of other cells or cytokines. Our data favor a transition of these cells towards more immature NK cells (CD56bright phenotype) as indicated by the upregulation of CD56 and the downregulation of KIR and CD57 that are hallmarks of educated and differentiated NK cells. These drugs also modulated activating receptors (NKp30 and NKp46), chemokine receptors (CXCR4) and cytokine receptors (IL7R) by downregulating their expression. Lenalidomide and pomalidomide do not affect other NK cells surface molecules such as activating receptors (NKG2D, DNAM1), adhesion molecules (CD44) and homing receptors (CD62L, CXCR3 and CCR7).
Intriguingly, despite downregulation of NKp46 and moderate downregulation of NKp30, NK cells cytotoxicity against leukemia targets was conserved.
We also showed that IMiDs® had a direct effect on contact-dependent NK cells functions, independently of cytokines stimulation, inducing a specific undescribed profile CD107+/IFNγ−/TNFα++. This new subset has never been previously identified to our knowledge.
Finally, we observed a direct toxicity of IMiDs® on AML blasts that was independent on immune effectors including NK cells. This may be partly due to oxidative stress.
Altogether, these data indicate new interesting functions of IMiDs® that are able to modify the differentiation and functional status of the NK cells and act in addition directly on AML blasts. Further studies are needed to develop pre-clinical models as well as the identification of the basis of NK differentiation of this new NK subset.

Disclosures:
Bateman: Celgene: Employment.
Downstream Targets of PKCβII in Dendritic Cell Differentiation,
Authors: Spangler Haley , Farren Matthew R , Carlson Louise M , Lee Kelvin P 
Published: October 2019

Abstract
Abstract 3245
An important component of antitumor immunity is the antigen presenting dendritic cell (DC), which initiates innate and adaptive immune responses. It is therefore beneficial for cancer to inhibit the function of these DCs. Tumors often depress DC activity by inhibiting their differentiation, which results in an accumulation of immature DCs which are immunosuppressive and aid in tumor progression and immunologic evasion. Tumor-derived factors prevent hematopoietic progenitor cells (HPCs) from fully completing differentiation to a mature DC, but the intracellular mechanisms by which tumors do this remain largely unknown.
Previous studies have shown that the serine-threonine kinase, protein kinase C βII (PKCβII), is required for DC differentiation from HPCs. It has also been shown that the interferon regulatory factor 8 (IRF8) is necessary for DC differentiation from HPCs. Loss of either PKCβII or IRF8 results in an accumulation of immunosuppressive immature DCs, incapable of inducing immune responses, and capable of promoting tumor maintenance and progression. The signaling pathways both of these molecules have independently been shown to play critical roles in DC differentiation, and to be downregulated in tumor presence to promote survival. However, whether a relationship exists between the two molecules in this process has yet to be established.
In human and murine models, we have determined that inhibition of PKβCII with a specific molecular inhibitor, CGP 53353, prevented differentiation of lineage negative progenitor cells to DCs. The cells treated with CGP 53353 failed to upregulate DC markers analyzed by flow cytometry, and failed to induce allogeneic T-cell proliferation compared to progenitor cells treated with DC-inducing cytokines. We also found through q-PCR that the PKC agonist phorbol 12-myristate 13-acetate (PMA) upregulated IRF8 expression, and in turn PKCβII inhibition lead to a loss in IRF8 expression. Therefore we have demonstrated a role for PKCβII in DC differentiation in mice (which was previously uncharacterized), as well as identified a potential relationship between IRF8 and PKCβII signaling during differentiation. Identifying such a relationship would provide a signaling model for DC differentiation, allowing a better understanding of how DC differentiation is initiated and impaired. It may also provide signaling components to target with antitumor therapeutics in order to induce DC differentiation in a tumor environment.

Disclosures:
No relevant conflicts of interest to declare.
Prasugrel Aggravates the Inflammatory Response in a PG-PS-Induce Arthritis Model,
Authors: Garcia Analia E , Rico Mario C , Liverani Elisabetta , Kunapuli Satya P. , De La Cadena Raul A. 
Published: October 2019

Abstract
Abstract 3268
Prasugrel is a very potent and effective P2Y12 receptor antagonist, used primarily to prevent thrombotic events in patients managed for percutaneous coronary intervention (PCI). It is not recommended as prophylaxis antithrombotic therapy because prasugrel increases patient's risk of bleeding. Even though indication of prasugrel treatment has been limited to the PCI procedure, there are not reports of the effects of prasugrel in patients with associated inflammatory diseases that undergo for PCI. Thus, we evaluate the effects of prasugrel, for 21 days, in a peptidoglycan polysaccharide (PG-PS)-induced arthritis rat model. Lewis rats were randomly separated into four groups from which only half of the animals were induced with PG-PS. There was a significant increase in the inflammatory changes observed in the PG-PS-induced animals treated with prasugrel compared to the PG-PS-induced control including a) increase joint diameter associated with visible signs of inflammation (during days 4 to 6 [p<0.05] and days 13 to 21 [p<0.001]); b) microscopic studies of hind joints showed marked increase in histomorphometric measurements including leukocyte infiltration, synoviocyte hyperplasia, fibrosis and neovascularization (significant increase in vessel number and size); and c) significant increase in platelet count (1,047 ± 89.03 × 103 vs. 710.8 ± 52.65 × 103, p<0.05). Platelet factor 4 (PF4) plasma levels were increased in PG-PS induced control as well as in PG-PS treated with prasugrel animals, however no significant differences were observed between these two groups. Plasma levels of cytokines such as IL-1 beta, IL-6, MIP1 alpha, MCP1, and RANTES were increased in PG-PS-induced animals (both, control and prasugrel treated animals). Only MIP1 alpha levels were significantly increased with additional treatment of prasugrel (p<0.05). In contrast, IL-10 levels were lower in the PG-PS-induced treated with prasugrel (p<0.05). In summary, treatment with P2Y12 receptor antagonist drugs, such as prasugrel, increases the inflammatory changes in PG-PS-induced arthritis model. Further investigations are in need to elucidate the potential unwanted effect of the P2Y12 receptor antagonists in patients afflicted with inflammatory illnesses.

Disclosures:
No relevant conflicts of interest to declare.
KLF2 and KLF4 Are Essential Mediators of the Anti-Thrombotic Effects of Statins in the Presence of Antiphospholipid/Anti-ß2GPI Antibodies,
Authors: Allen Kristi L , Jain Mukesh K , McCrae Keith R. 
Published: October 2019

Abstract
Abstract 3272
Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLA). The majority of APLA are directed against phospholipid binding proteins, particularly β2GPI. Anti-ß2GPI antibodies activate endothelial cells and monocytes in a β2GPI-dependent manner through a pathway that involves NF-κB and leads to increased expression of adhesion molecules, tissue factor and proinflammatory cytokines. Krüppel-like factors (KLFs) regulate endothelial cell and monocyte responses to inflammatory stimuli; increased expression of these transcription factors inhibits proinflammatory and procoagulant gene expression, and maintains vascular homeostasis. We recently reported that anti-ß2GPI antibodies decrease the expression of KLF2 and KLF4 in endothelial cells (Allen et al, Blood 2011), promoting endothelial cell activation. Subsequent studies demonstrate that these antibodies decrease expression of KLF2 in monocytes as well. Statins have been proposed as a potential alternative to anticoagulation for APS patients, and stimulate the expression of KLFs. We hypothesized that the ability of statins to block endothelial cell activation in response to anti-β2GPI antibodies was mediated by KLFs. Treatment of endothelial cells and monocytes with 100 nM fluvastatin, lovastatin, or simvastatin upregulated KLF2 and KLF4 mRNA, even in the presence of anti-ß2GPI antibodies. In parallel, statin treatment inhibited the anti-β2GPI antibody-mediated induction of E-selectin, VCAM-1, and TF mRNA in endothelial cells, and ICAM-1 and TF mRNA in human monocytes. To assess the dependence of these effects on KLF expression, endothelial cells were pretreated with KLF2 or KLF4 siRNA prior to treatment with statins. siRNA-mediated inhibition of KLF expression completely blocked the ability of statins to prevent anti-β2GPI antibody-induced endothelial cell activation, as measured by adhesion molecule and TF mRNA levels and expression of E-selectin on the endothelial cell surface. Taken together, these data demonstrate that KLFs are critical modulators of the effects of statins on endothelial cells, and that increased expression of KLFs may represent a mechanism by which these drugs inhibit the activation of endothelial cells and monocytes by APLA/anti-β2GPI antibodies.

Disclosures:
No relevant conflicts of interest to declare.
Patients with Hermansky-Pudlak Syndrome Show Various Phenotypes Caused by Novel Mutations,
Authors: Sandrock Kirstin , Kurnik Karin , Ehl Stephan , Bidlingmaier Christoph , Nakamura Lea , Rombach Nina , Schäfer Sophie , Zieger Barbara 
Published: October 2019

Abstract
Abstract 3286

Background:
Hermansky-Pudlak syndrome (HPS) is a rare autosomal recessive disorder causing oculocutaneous albinism, bleeding disorder and ceroid lipofuscinosis. Platelets from HPS patients are characterized by impaired secretion of dense (δ)-bodies (CD63). Neutropenia and susceptibility to recurrent infections were exclusively observed in HPS2 patients so far. There are eight known human HPS genes (HPS1-HPS8), each leading to a particular clinical HPS subtype (HPS1-HPS8).

Patients/Results:
The patients show a typical HPS phenotype concerning oculocutaneous albinism and bleeding symptoms. In vivo-, in vitro bleeding time and platelet aggregometry analyses revealed impaired platelet function. We identified HPS3 in two Turkish brothers and HPS2 in a girl from the United Emirates. Both brothers with HPS3 demonstrated absence of platelet δ-granule secretion measured by flow cytometry analysis. A novel 1 bp-deletion in the HPS3 gene was identified in both brothers. In addition, one brother with HPS3 demonstrated psychomotoric retardation. MRI scan revealed cranial gliosis. Interestingly, array-CGH analysis revealed a 0.7 Mb deletion on chromosome 17 which had not been identified in the other brother and which seems to have caused the cranial gliosis.
The girl with HPS2 suffered from life-threatening bleeding after tonsillectomy leading to severe asphyxia, resuscitation and finally, to mental retardation. Flow cytometry analysis demonstrated impaired platelet δ-granule secretion with a typical pattern for HPS2. CD63 expression was already increased on resting platelets, but there was only little increase after thrombin stimulation. Interestingly, only a NK-CD107 partial degranulation defect was diagnosed. So far, clinical symptoms of immunodeficiency are not obvious. Molecular genetic analyses revealed a novel 2 bp-deletion in the last exon of HPS2 leading to a frameshift and a prolonged altered protein. The location of the deletion at the very C-terminal end may prevent a complete loss of the HPS2 protein leading to a less pronounced severity of immunodeficiency as in other HPS2 patients.

Conclusion:
Patients with oculocutaneous albinism should be investigated for increased clinical bleeding symptoms. In case of increased bleeding symptoms, analyses of primary hemostasis should be initiated to confirm HPS. Using flow cytometry analyses HPS2 can be distinguished from the other subtypes of HPS. Molecular genetic investigations should be performed to differentiate the various subtypes of HPS which is important for therapy and prognosis. The HPS3 patient`s mental retardation seemed to be caused by an additional deletion. The identification of the molecular genetic defect helps to understand the patients` various clinical phenotypes.

Disclosures:
Zieger: Novo Nordisk: Research Funding.
Parathyroid Hormone Stimulates Human Mesenchymal Stem Cells to Enhance the Expansion of Hematopoietic Stem Cells,
Authors: Yao Hisayuki , Miura Yasuo , Yoshioka Satoshi , Hayashi Yoshihiro , Hirai Hideyo , Maekawa Taira 
Published: October 2019

Abstract
Abstract 3407
Expanding hematopoietic stems cells (HSCs) has a great interest for successful engraftment of donor cells in hematopoietic stem cell transplantation (HSCT). In addition, mesenchymal stem cells (MSCs) show unique features with multi-differentiation, immune-modulation and tissue regeneration capabilities. Recent studies demonstrated the MSC-derived mature osteoblasts play important roles for the maintenance of HSC niche in the bone marrow (BM) microenvironment. We found that culture-expanded human MSCs support the expansion of HSCs and stimulation of MSCs with parathyroid hormone (PTH) further enhances the MSC-mediated expansion of HSCs.
Culture-expanded MSCs (2×104 cells/well) were seeded on a 24-well culture plate. Autologous purified CD34+ HSCs (0.6×103 cells/well) was applied on the MSC-grown plate and co-cultured in StemSpan Serum Free Expansion Medium (StemCell Technologies) supplemented with 100ng/mL SCF, 100ng/mL Flt-3 ligand, 50ng/mL TPO and 20ng/mL IL-3. After 10 days of co-culture of HSCs with MSCs, the number of CD34+ cells was increased to 9.0±1.6×103 (Fig. culture condition #2), whereas that cultured in the absence of MSCs was 0.98–8, −1 and □ {7 of the irradiation. The number of leukocytes was decreased to the bottom level around 6–8 days after the irradiation in both PTH-treated and non-treated (control) mice. However, the number of leukocyte showed a steep increase in PTH-treated mice and it reached maximal level on day 14 at 16±5.2×103/mL compared with that of 6.4±1.4×103/mL in the control mice. In vitro experiments confirmed that PTH has no direct effects on the expansion of HSCs. It is suggested that PTH has supportive effects on hematopoietic recover in vivo, probably in part, through acting on MSCs in BM microenvironment.
In conclusion, PTH stimulated MSCs to enhance HSC expansion mainly through contribution of surface membrane molecules. A two-step manipulation of MSCs with culture expansion and subsequent PTH stimulation showed enhancement of MSC-mediated expansion of HSCs. This work revealed the novel mechanisms underlying HSC expansion mediated by MSCs. In addition, this novel concept of MSC manipulation could provide HSCT procedure with new technology for effective engraftment of donor hematopoietic cells.

Disclosures:
No relevant conflicts of interest to declare.