bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Parallel Signaling Pathways Of Phosphatidylserine (PS) Exposure Downstream Of Platelet FcγRIIa
Authors: Andre Pierrette , McKenzie Steven E. , Bergmeier Wolfgang 
Published: October 2019

Abstract
Platelet FcγRIIa is an important component of heparin-induced thrombocytopenia and other immune-mediated thrombocytopenia and thrombosis syndromes. Platelet FcγRIIa, an ITAM receptor, signals not only to cause aggregation and secretion, but also to PS exposure in the platelet procoagulant response when platelets are co-stimulated via the GCPR PARs. The molecular mechanisms downstream of FcγRIIa that lead to PS exposure are incompletely understood. Recently, we (WB; Ahmad et al., JTH 2011; 9:2077) demonstrated that downstream of PAR and GPVI/FcRγ, another ITAM receptor, there are CalDAG-GEF I (CDGI)-dependent and CDGI-independent, ADP/P2Y12-dependent parallel signaling pathways to PS exposure. In this study, we investigated the molecular signaling requirements for PS exposure downstream of FcγRIIa + PAR dual stimulation.
We studied the exposure of PS in FcγRIIa transgenic (tg) mouse platelets following dual stimulation through FcγRIIa via anti-mouse CD9 and through PAR4 via PAR4 activating peptide (PAR4AP; AYPGKF). Washed platelets from FcγRIIa-tg mice and FcγRIIa-tg/CDG1-/- mice were stimulated with varying concentrations of anti-mouse CD9 and PAR4-AP (200uM) under static conditions and immediately measured for PS exposure by labeled Annexin-V in flow cytometry. We observed that at 0.5ug/ml of anti-mouse CD9 that the PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 70% of the FcγRIIa-tg platelets. However, when the platelets are pre-incubated with P2Y12 inhibitor MesAMP at 100uM, the PS exposure of the FcγRIIa-tg platelets is decreased by approximately 50% (n=3). For the FcγRIIa-tg/CDG1-/- platelets in the presence of MesAMP, PS exposure is completely abolished (n=3). This indicates that CDG1 contributes part of the signal that leads to PS exposure, while ADP/P2Y12 contributes the other CDGI-independent part of PS exposure downstream of FcγRIIa and GPCR dual stimulation. At a lower concentration of anti-mouse CD9 (0.25ug/ml; near threshold), the level of PS exposure of the FcγRIIa-tg/CDG1-/- platelets is approximately 80% of the FcgRIIa-tg platelets. In addition, at 0.25 ug/ml of anti-CD9, FcγRIIa-tg/CDG1-/- platelets pre-incubated with the P2Y12 inhibitor revealed no stimulated PS exposure. This observation indicates that ADP/P2Y12 plays a more significant role in PS exposure as the concentration of FcγRIIa stimulant nears threshold.
Eradication of procoagulant PS exposure may require targeting of both the CDGI-dependent and CDGI-independent pathways for optimum therapeutic benefit in HIT and other immune-mediated thrombocytopenia and thrombosis disorders. CDGI inhibitors useful in human platelets will allow translation of these findings.

Disclosures:
No relevant conflicts of interest to declare.
Utility Of The Immature Platelet Fraction In Predicting Recovery With Or Without Treatment In Pediatric Immune Thrombocytopenia
Authors: Ghemlas Ibrahim A. , Klaassen Robert J. , Barrowman Nick , Leung Elaine W. 
Published: October 2019

Abstract

Background
The Immature Platelet Fraction (IPF) is a novel parameter available on the Sysmex XE-Series Hematology analyzers. It can be expressed as both IPF percent (IPF %) and absolute IPF count (AIPF#). It has been shown that IPF% is higher in Immune Thrombocytopenia (ITP) than in the normal population, and correlates with bone marrow platelet production or thrombopoiesis.

Objectives
To evaluates the utility of the IPF parameters to predict treatment response or recovery in pediatric ITP patients.

Method
This is a retrospective, single institution study performed at a 165 bed tertiary care pediatric hospital (Children's Hospital of Eastern Ontario, Ottawa, Canada). We reviewed the medical charts and electronic databases of all patients with ITP who had measured IPF parameters between June 2011 and June 2013. The standard definitions and terminology of the International Working Group were used.
Patient age, phase of ITP at the time of the study (acute, persistent, and chronic), initial platelet count, IPF%, AIPF#, and type of therapy given (including observation alone) were recorded. For patients who responded to treatment or had spontaneous recovery without treatment, we analyzed the platelet count, IPF% and AIPF# on subsequent CBCs, then looked at the course of IPF% during platelet count recovery.
Comparisons between groups were performed using Kruskal-Wallis tests or Mann-Whitney tests, as appropriate. Association between measured blood parameters was measured using the Spearman correlation coefficient. Within-patient Spearman correlations were also used to study the association between IPF% and platelet counts during recovery. The combined significance of these associations was determined using Stouffer’s method.

Results

Conclusion
IPF measurements are easily available parameters that are useful in the management of ITP in pediatric patients. This study suggests the use of AIPF# as a predictive factor for recovery in pediatric ITP patients with and without treatment, and IPF% as a predictive factor for early recovery.

Disclosures:
No relevant conflicts of interest to declare.
Angelica Polysaccharide and TPO Have a Protective Effect On Hcmv-Induced Apoptosis In Megakaryocytes
Authors: Yang Mo , Chen Jian Liang , Ye Jie yu , Li Su yi , Liang En yu , Chong Beng 
Published: October 2019

Abstract
Human cytomegalovirus (hCMV) infection is often associated with thrombocytopenia. Megakaryocytes may be one of the major sites of hCMV infection, then inducing this cell apoptosis. Angelica Sinensis (Danggui) is an important ingredient of many commonly used herbal Medicine for promoting blood production. Our previous study has showed that the hematopoietic effect of Angelica Sinensis is related to its constituent, angelica polysaccharide (APS) (Yang M et al, J Ethnopharma, 2009). This present study investigated the anti-apoptotic effect of APS and TPO on hCMV-induced apoptosis in megakaryocytes. Human bone marrow mononuclear cells (MNC) or megakaryocytic cell line CHRF-288-11 and hCMV AD169 strain were co-cultured in this study. hCMV significantly inhibited the formation of CFU-MK as shown in three different concentrations of viral infection groups (103, 104 and 105 pfu/ml), compared with blank control and mock control (n=10, P<0.05). hCMV also significantly inhibited the growth of CHRF cells in these three different concentrations after incubation for 3 days, which compared with control group (n=10, P<0.01). hCMV DNA and mRNA were also positively detected in CHRF cells and the cells of CFU-MK with IS-PCR and RT-PCR respectively, while it was negative in blank and mock control groups. We further studied the effect of APS and TPO on CFU-MK formation. Results showed that APS (50 ug/ml) like TPO (50 ng/ml) enhanced hCMV-reduced CFU-MK (P=0.05, n=6). CHRF cells were also analyzed by Annexin V/PI with flow cytometry at day 3 after infection with hCMV AD169. The percentage of apoptotic cells in group of 103 pfu/ml was 19.0 ± 2.0%; The group of 104 pfu/ml was 23.0 ± 1.5%; The group of 105 pfu/ml was 28.0 ± 3.0%. The control group was 2.0 ± 0.5%. The apoptotic cells were confirmed by morphologic observation. In addition, apoptotic signals from megakaryocytic surface, cytoplasma and mitochondria were detected in hCMV infected cells by flow cytometry with Caspase-3 and JC-1 assay. Compared to mock infection control at day 5, Annexin-V positive cells population increased by 58%; active caspase-3 signal increased by 120% in viable cell population; and cell population with damaged mitochondial membrane showed a 5-times increase. Moreover, the anti-apoptotic effect of APS and TPO on CHRF cells was also demonstrated by using Annexin-V assay. Our studies showed that hCMV induces the apoptosis in megakaryocytes via mitochondrial and caspase-3 signaling, and angelica polysaccharide (APS) like TPO has a protective effect on hCMV-induced apoptosis in these cells.

Disclosures:
No relevant conflicts of interest to declare.
Blocking Protein Secretion and Degradation Is a Novel Treatment Strategy For Malignant Cells With High Protein Load
Authors: Lowe Eric , Maglathlin Rebecca , Lerner Alana , Taunton Jack , Kirk Christopher J. , Arastu-Kapur Shirin 
Published: October 2019

Multiple myeloma (MM) is the result of deregulated proliferation in malignant plasma cells that secrete an overabundance of a monoclonal immunoglobulin (Ig). The high level of Ig production in MM cells places a unique burden on the ubiquitin proteasome system (UPS) to maintain protein homeostasis and may explain why MM cells are among the most sensitive cell type to proteasome inhibition. We hypothesized that blocking protein secretion would further increase the protein load in MM cells and induce an anti-tumor response. In H929 and U266 cells, exposure to the proteasome inhibitors carfilzomib (CFZ) or bortezomib for 1 hour resulted in ubiquitination of several components of the Sec61 translocon, a hetero-trimeric channel present in the ER membrane central to protein secretion. Recently, cotransins were identified as a class of selective Sec61 inhibitors that block the translocation of specific nascent peptides into the ER. We determined the sensitivity to dual inhibition of protein secretion and degradation in 3 MM lines (NCI-H929, RPMI-8226, U266) and two plasma cell leukemia lines (JJN3, ARH-77) using the combination of CFZ and the cotransin CT08. We observed that 8226, H929, and JJN3 cells were sensitive to single agent CT08, with cell viability IC50 values ranging from 120 to 170 nM, while U266 and ARH-77 showed little sensitivity. CFZ IC50 values ranged from 1.5 to 6 nM across all 5 cell lines. CT08-sensitive cells treated with both agents, across a matrix of concentrations, showed a significant decrease in cell viability. For most concentrations, combination index (CI) values fell below 0.9, indicating synergistic activation of cell death, and reached levels of 0.19 (H929), 0.277 (JJN3), and 0.536 (8226). In resistant cells, CT08 potentiated (CI not evaluable) an anti-myeloma response when co-treated with CFZ, suggesting a significant interaction effect. CT08 also potentiated the cytotoxic effect of CFZ in the small cell lung cancer line DMS114 despite these cells being 10-fold less sensitive to CFZ than MM cell lines. In addition, CT08/CFZ combination treatment of U266 cells in co-culture with the bone marrow stromal cell line HS-5 overcame the protective effect of the stromal cells, as measured by Annexin V and propridium iodide staining. Taken together, these data suggest that dual Sec61 and proteasome inhibition represents a novel treatment strategy for malignant cells with high protein secretion like multiple myeloma.

Disclosures:
Lowe: Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Lerner:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership. Kirk:Onyx Pharmaceuticals: Employment, Equity Ownership. Arastu-Kapur:Onyx Pharmaceuticals, Inc.: Employment, Equity Ownership.
Host and Donor B Cell-Derived IL-10 Ameliorates Murine Graft-Versus-Host Disease
Authors: Weber Michael , Stein Pamela , Fillatreau Simon , Roers Axel , Schild Hansjörg , Radsak Markus P 
Published: October 2019

Allogeneic hematopoetic stem cell transplantation (HSCT) is the treatment of choice for a variety of hematologic malignancies. Graft-versus-Host disease (GvHD) is a key contributor to treatment related morbidity and mortality and consequently limits the efficacy of allogeneic HSCT. Interleukin 10 (IL-10) is a well-known cytokine with immunoregulatory and anti-inflammatory properties, also important in context of GvHD. B cells have been described as potent IL-10 producers in various situations. Here we show how host as well as donor derived B cells contribute to GvHD amelioration through IL-10 production.
We address the role of IL-10 in GvHD in an acute murine MHC mismatch model: Mice on a C57BL/6 background received bone marrow and CD90+ T cells from mice on a BALB/c background or vice versa. Transplantation experiments with IL-10 deficient donor or host cells clearly show the importance of donor derived IL-10 in general. To further dissect the cells contributing to IL-10 production in this situation we employed an IL-10 knock-in reporter mouse in which expression of eGFP is under control of the Il-10 locus. Lethal irradiation as used in the conditioning regiment before transplantation revealed B cells as major contributors of host derived IL-10. In addition, transfer of cells from reporter mice into preconditioned recipients showed also donor B cells as contributors to IL-10 production. A phenotypical characterization of the eGFP+ B cells exhibited a CD1d+TIM-1+CD5int phenotype, in line with immunoregulatory B cells. To finally confirm the relevance of B cells derived IL-10 in GvHD, we employed B6.B-IL-10-/- mice that have a B cell specific IL-10 knock-out as donors or recipients. Here we found a reduced survival associated with the incapability of the B cells to produce IL-10 in both cases.
Taken together, our results provide new insights in the mechanisms and the variety of cells contributing to the course of GvHD. An improved understanding of these aspects might help to pave the way for new treatment options to overcome current limitations of allogenic HSCT.

Disclosures:
No relevant conflicts of interest to declare.
Potential Role Of RUNX1 In The Pathogenesis Of Juvenile Myelomonocytic Leukemia (JMML)
Authors: Huang Hui , Bauer Daniel E. , Loh Mignon L. , Bhagat Govind , Cantor Alan B. , Kung Andrew L. 
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of young children. The only current curative treatment is bone marrow transplantation. Yet even with this aggressive therapy, ∼50% of children still die from their disease. Somatic mutations leading to constitutive activation of the tyrosine phosphatase Shp2 (also called PTPN11) or of RAS signaling occur in ∼90% cases of JMML. However, the transcription factors that act downstream of these aberrant signaling events have not been identified. We recently showed that RUNX1 is a direct interacting partner of Shp2 in megakaryocytic cells (Huang et al. 2012. Genes Dev 26: 1587-1601). Moreover, we showed that RUNX1 is normally negatively regulated by src-family kinase (SFK) mediated tyrosine phosphorylation in megakaryocytes and T-lymphocytes, and that Shp2 contributes to RUNX1 tyrosine dephosphorylation. We now show that overexpression of a mutant RUNX1 (RUNX1Y260F, Y375F, Y378F, Y379F, Y386F, “RUNX1-5F”), which is expected to mimic constitutive dephosphorylation by Shp2 in murine Lin- Sca-1+ c-kit+ (LSK) bone marrow cells is resistant to SFK-mediated tyrosine phosphorylation and leads to a dramatic expansion of CFU-M/CFU-GM and Gr1+Mac1+ cells in vitro and in vivo. In contrast, these effects are not seen when wild type RUNX1 or RUNX1Y260D, Y375D, Y378D, Y379D, Y386D (“RUNX1-5D”; mimicking constitutive RUNX1 tyrosine phosphorylation) are overexpressed. The RUNX1-5F expressing cells also have increased replating activity in serial colony forming assays, increased proliferation (BrdU incorporation), decreased apoptosis, and reduced cytokine dependence. This partially phenocopies conditional knock-in mice that express JMML associated activating Shp2 mutations. Flow sorted Gr1+Mac1+ cells from the RUNX1-5F transduced cultures expressed higher levels of the direct RUNX1 target gene PU.1, which plays a role in myelomonocytic growth, and Cyclin D1. To test whether RUNX1 is required for the myelomonocytic hyperproliferation in JMML, CD34+ peripheral blood cells from a patient with JMML and known activating Shp2 mutation (Shp2E76G) were lentivirally transduced with doxycycline-inducible RUNX1-5D or RUNX1-5F expression constructs and cultured under myeloid growth conditions. Upon doxycycline induction, the RUNX1-5D overexpressing cells (resistant to Shp2) exhibited at 32% reduction in BrdU incorporation. In contrast, the control RUNX1-5F expressing cells had no significant reduction in proliferation. These results are consistent with RUNX1 acting as an essential downstream target of activated Shp2 in JMML. As ERK mediated phosphorylation (downstream of RAS/MEK) is also known to increase RUNX1 activity, we propose that RUNX1 may be a common downstream transcriptional target of both activated Shp2 and RAS signaling in the pathogenesis of JMML.

Disclosures:
No relevant conflicts of interest to declare.
Donor-Recipient Matching For Killer Immunoglobulin-Like Receptor Genotypes Confers Protection Against Graft Versus Host Disease Without Affecting Disease Relapse After Allogeneic Hematopoietic Cell Transplantation
Authors: Faridi Rehan M , Kemp Taylor J , Lewis Victor A. , Berka Noureddine , Storek Jan , Khan Faisal 
Published: October 2019

Background
Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for malignant and non-malignant hematological disorders. Unfortunately, complications of HCT, primarily graft versus host disease (GVHD) and relapse of the underlying disease are substantial. In Alberta, all HCTs are performed with antithymocyte globulin conditioning and >95% of these use HLA matched donors. However, in spite of that approximately 35% Albertan HCT recipients die or suffer long-term from GVHD while another 20% die due to disease relapse. Unfortunately, strategies/treatments aiming to control GVHD in most cases lead to increase in the rate of disease relapse and infections. In the recent years, the natural killer (NK) cell genetic system has garnered substantial research interest as an immunogenetic system that significantly influences HCT outcomes. The complexity of NK cell function is modulated by a series of activating and inhibitory cell surface receptors known as the Killer Immunoglobulin-like Receptors (KIR). Here we set out to determine the influence of KIR matching between HCT donor and recipient pairs on allogenic HCT complications.

Study design
Hypothesizing that donor-recipient KIR gene/profile mismatch would affect the allo-HCT outcomes, we genotyped 92 (discovery cohort) and 111 (validation cohort) allo-HCT pairs by Luminex-based rSSO method. The KIR genotypes were classified into AA and Bx genotypes on the basis of KIR gene constitution of the individual. Donor (D) and recipients (R) were ‘matched' for KIR genotypes when both carried either AA (two copies of group A haplotypes) or B/x (at least one copy of group B haplotype) genotypes. Effect of KIR matching on significant GVHD (described as grade 2-4 acute GVHD or chronic GVHD needing systemic therapy) as well as disease relapse was analyzed using binomial regression and Kaplan-Meier statistics.

Results
As observed in both the discovery and validation cohorts, a significant protection against GVHD was conferred upon when both the donor and recipient were matched for the KIR genotypes (HR=2.224; p=0.01) without any effect on disease relapse (HR=1.098; p=0.934) (figure1).

Conclusions
In spite of D-R matching for major immunogenetic determinants like HLA, complications of allogeneic HCT are significant. Since GVHD represents a flip side to graft-vs-leukemia (GVL) reaction, strategies to decrease GVHD in most cases have resulted in increased relapse. Matching donor and recipients for KIR genotypes can offer a significant protection against GVHD without increasing the risk of disease relapse.

Disclosures:
No relevant conflicts of interest to declare.
Nitrite Levels Are Associated To Fetal Hemoglobin Concentration In Patients With Sickle Cell Anemia: Implications In Inflammatory Process
Authors: Pitanga Thassila Nogueira , Cerqueira Bruno A. V. , Boas Wendell Vilas , Santana Sanzio S. , Reis João O. , Goncalves Marilda Souza 
Published: October 2019

Introduction
Sickle cell anemia (SCA) is a genetical hemolytic disorder defined as chronic inflammatory disease. Nitrite (NO-2) in SCA (HbSS) patients may be associated with the hemolytic process while fetal hemoglobin (Hb F) presents protective effect in these patients. Serum NO-2 interferes with the role of Hb F in reducing the hemolytic process contributing to vaso-occlusive crises.

Aims
The aim of this study was to evaluate the correlation of the serum levels of nitrite with fetal hemoglobin, low density lipoprotein cholesterol (LDL-C) and triglycerides levels in HbSS patients in steady-state of the Centro de Referência em Doença Falciforme de Itabuna (CERDOFI), Bahia, Brazil.

Methods
Forty-two patients diagnosed with SCA were included at baseline. Diagnosis was confirmed by high-performance liquid chromatography (HPLC). Serum levels of nitrite were performed using Griess reaction, and fetal hemoglobin, LDL cholesterol and triglycerides levels were measured by biochemical methods. The experimental protocol was approved by the Researcher Board Committee on Human Research, Gonçalo Moniz Research Center and informed consents were signed by patients.

Results
Our results showed a significant increase in NO-2 (p<0.0001) in adult patients with SCA compared to the control group (healthy volunteers). Data demonstrated a negative correlation between nitrite and fetal hemoglobin (p=0.004, r=-0.627). No correlation was found between fetal hemoglobin or nitrite with LDL-Cl and triglycerides.

Conclusion
The results reinforce that the serum nitrite can predict the clinical disease course.

Disclosures:
No relevant conflicts of interest to declare.
The Effects Of Oral Iron Treatment On Reactive Oxygen Radicals, Protective Enzymes and Lipid Peroxidation In Women With Iron Deficiency Anemia
Authors: Kadikoylu Gurhan , Ozkan Arzu , Yenisey Cigdem , Yavasoglu Irfan , Bolaman Zahit 
Published: October 2019

Iron deficiency anemia (IDA) is the commonest of deficiency disease in the world. In IDA, hemoglobin synthesis is impaired, nonetheless the production of iron (Fe2) containing proteins such as cytochrom, myoglobin, catalase (CAT), and peroxidase is affected. While oxidative stress can play an important role on the development of IDA, lipid peroxidation may be induced. The aim of the study is to investigate the effects of oral iron treatment on reactive oxygen radicals (ROR), protective enzymes from ROR, and lipid peroxidation in women with IDA. This prospective Phase-IV study was approved by ethical committees of university and the Ministry of Health. Sixty-seven women (mean age 34±11 years) with IDA were enrolled to the study. Inclusion criteria were hemoglobin level < 11 g/dl, serum ferritin level < 16 ng/ml, and iron saturation <15%. Twenty-seven helathy women (mean age 27±7 years) were accepted as control group. Exclusion criteria of control group were anemia, acute and chronic infection, acute and chronic bleeding, pregnancy, malignancy, chronic inflammatory, and renal diseases. In both group, whole blood counts, hydrogen peroxide (H202) as ROR, glutathione (GSH), superoxide dismutase, glutathione peroxidase, glutathione reductase, CAT for protective enzymes from ROR, and malondialdehyde (MDA) for lipid peroxidation were evaluated. Ferrous fumarate 200 mg/day were given to the patients for 4 weeks. At the end of 4 weeks, all parameters were repeated. All results were compared with SPSS 15.0 using independent and paired t-tests. P values <0.05 were accepted as significant. In women with IDA, the levels of GSH (p<0.001), H202 (p<0.001), and MDA (p<0.05) were significantly higher than controls, but other parameters were not different in both groups (p>0.05). At the end of treatment, GSH and H202 levels significantly decreased (p<0.001 for both), but other parameters did not changed (p>0.05). In women with IDA, while H202 increased, lipid peroxidation was induced. Compensation of oxidative stress were ensured by GSH. Iron treatment reduced oxidative stress, but it did not affect lipid peroxidation and protective enzymes from ROR.

Disclosures:
No relevant conflicts of interest to declare.