bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antiphospholipid Antibodies Stimulate Release Of Endothelial Cell Microparticles Enriched In IL-1β
Authors: WU Meifang , Kundu Suman , McCrae Keith R. 
Published: October 2019

Abstract

Introduction
Microparticles are submicron vesicles released by activated or apoptotic cells. Previous studies from our laboratory and others have demonstrated elevated levels of endothelial cell (EC)-derived microparticles in the plasma of patients with antiphospholipid antibodies (APLA), which are associated with thrombosis and recurrent fetal loss. The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. APLA/anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner and stimulate the release of EC microparticles. Here, we demonstrate that these microparticles are enriched in active interleukin-1β (IL-1β) and may directly stimulate EC activation.

Methods
EC were treated with β2GPI and anti-β2GPI antibodies and microparticles were isolated by ultra-centrifugation. The treated EC, released microparticles and microparticle-free supernatant were analyzed for interleukin-1β (IL-1β) expression by western blot. In addition, we assessed the ability of microparticles derived from EC exposed to control IgG or anti-β2GPI antibodies, or microparticle-free supernatant that had been adsorbed with immobilized β2GPI to remove residual anti-β2GPI antibodies, to activate EC. Activation was defined by increased expression of cell surface E-selectin and phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4).

Results
Incubation of EC with β2GPI and anti-β2GPI antibodies stimulated the expression of pro-IL-1β. Interestingly, while EC contained exclusively pro-IL-1β, microparticles derived from these treated cells contained only the active, cleaved form of IL-1β. Moreover, extracellular IL-1β was found exclusively in microparticles, and was absent from microparticle-free conditioned medium Finally, IL-1β-containing microparticles derived from anti-β2GPI antibody-treated EC, but not microparticles released from EC exposed to only control IgG activated additional EC. Conditioned medium from anti-β2GPI antibody-treated EC that had been pre-adsorbed with immobilized β2GPI to remove residual anti-β2GPI antibodies also failed to stimulate EC activation.

Conclusions
EC exposed to β2GPI and anti-β2GPI antibodies stimulate the expression pro-IL-1β, which undergoes cleavage to an active form and is packaged in microparticles. These microparticles may directly induce activation of EC or synergize with anti-β2GPI antibodies to activate EC. We hypothesize that delivery of IL-1β and perhaps other inflammatory cytokines by microparticles derived from anti-β2GPI-exposed EC or monocytes may induce a systemic vascular inflammatory state in which anti-β2GPI antibodies may more readily stimulate thrombotic events.

Disclosures:
No relevant conflicts of interest to declare.
Platelet-Derived ERp57 Mediates Platelet Incorporation Into a Growing Thrombus By Regulation Of The αIIbβ3 Integrin
Authors: Wang Lu , Wu Yi , Zhou Junsong , Ahmad Syed S. , Mutus Bulent , Garbi Natalio , Hammerling Gunter , Essex David W 
Published: October 2019

Abstract
ERp57 is a member of protein disulfide isomerase (PDI) family. Although previous studies using antibodies have indicated ERp57 mediates platelet aggregation, the specific role of platelet-derived ERp57 in hemostasis and thrombosis is unknown. In this study we addressed this issue using a megakaryocyte/platelet specific ERp57-knockout mouse model (PF4-Cre/ERp57fl/fl). Although PF4-Cre/ERp57fl/fl mice had normal platelet counts and platelet glycoproteins expression, they had significantly prolonged tail-bleeding times, and thrombus occlusion times with FeCl3-induced carotid artery-injury. Using a mesenteric artery thrombosis model we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Upon stimulation with convulxin and thrombin, platelets lacking ERp57 had defective activation of the αIIbβ3 integrin and platelet aggregation. Addition of exogenous wild type ERp57 corrected the defect in aggregation implicating surface ERp57 in platelet aggregation. Moreover, using recombinant ERp57 mutants we found that the second active site of ERp57 targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn2+-treated platelets lacking β3 was substantially decreased, suggesting a direct interaction of ERp57 with αIIbβ3. Surface ERp57 activity in human platelets increased in response to stimulation with thrombin, concomitant with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus.

Disclosures:
No relevant conflicts of interest to declare.
An Analysis Of Ph-Like ALL In Japanese Patients
Authors: Kiyokawa Nobutaka , Iijima Kazutoshi , Yoshihara Hiroki , Ohki Kentaro , Kato Motohiro , Fukushima Takashi , Kikuchi Akira , Fujimoto Junichiro , Hayashi Yasuhide , Koh Katsuyoshi , Manabe Atsushi , Ohara Akira 
Published: October 2019

Abstract

Introduction
“Ph-like acute lymphoblastic leukemia (ALL)” or “BCR-ABL1-like ALL” is a recently identified subtype of high-risk B-cell precursor (BCP)-ALL with poor outcome and high frequency of IKZF1 deletion. They exhibit a gene expression profile similar to BCR-ABL1-positive (Ph1-)ALL but do not have BCR-ABL1 and other common chimeric genes. Recent study from others has identified rearrangements involving tyrosine kinase genes, such as ABL1, PDGFRB, JAK2, and CRLF2, by employing transcriptome sequencing using next generation sequencer. In the present study, we intended to explore the biologic characteristics and clinical outcomes of Ph-like ALL cases in Japanese patients.

Method
We investigated gene expression of 235 BCP-ALL cases enrolled on Tokyo Children Cancer Study Group (TCCSG) L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using both clustering analysis and Gene Set Enrichment Analysis (GSEA), a computational method that ascertains whether a given gene set is significantly enriched in a list of genes ranked by their correlation with a phenotype of interest. Four-year event-free survival (EFS) rate of the entire cases was 83.2%.

Results
Real-time PCR analysis revealed that 155 cases did not have common chimeric genes (“chimera-”), including BCR-ABL1, ETV6-RUNX1, E2A-PBX1, MLL-AF4, MLL-AF9, and MLL-ENL. Among “chimera-” cases, 11 (4.7% of the entire BCP-ALL cases) patients were identified as ALL with gene expression profile similar to that of Ph1-ALL by clustering analysis. Their 4-year EFS rate and the frequency of IKZF1 deletion were 66.7% and 80.0%, respectively.
On the other hand, GSEA detected 26 ALL cases with Ph-like signature (11.1%), including 9 cases those identified to have Ph-like phenotype by clustering analysis, and they exhibited 4-year EFS rate of 58.3% and IKZF1 deletion frequency of 38.1%. Interestingly, GSEA also revealed that 7 cases simultaneously possess ETV6-RUNX1-like signature. When we excluded 7 cases having ETV6-RUNX1-like signature and 5 cases with higher NOM p-value than 0.05, 14 cases were remained and they represented a highly unfavorable outcome (event-free survival 38.1%), while the frequency of IKZF1 deletion was 41.7%.
We performed transcriptome sequencing of ALL cases with Ph-like phenotype using next generation sequencer and identified rare and novel chimeric fusions involving tyrosine kinases, such as ABL1, as well as CRLF2-related chimeric fusions.

Conclusions
We identified the presence of ALL subset having Ph-like phenotype in Japanese patients. They represent unfavorable outcome. However, the diagnosis of “Ph-like ALL” varies according to the method to identify Ph-like phenotype. Therefore, the definition of “Ph-like ALL” needs more consideration.

Disclosures:
No relevant conflicts of interest to declare.
Serine 559 Of Glycoprotein Ibα Is Phosphorylated By Protein Kinase A Which Plays Key Roles In Regulating Von Willebrand Factor Binding, Surface Expression, and Signaling Of Glycoprotein Ib-IX
Authors: Zhang Jie , Zhang Weilin , Liu Guanglei , Zhao Lili , Yan Rong , Dai Kesheng 
Published: October 2019

Abstract
Conformational changes in von Willebrand factor (VWF) exposed at the injured vessel wall initiate glycoprotein (GP) Ib-IX-dependent platelet adhesion, and simultaneously trigger signaling cascades leading to integrin αIIbβIII activation, platelet spreading, and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the cytoplasmic domain of GPIbα and play key roles in regulating the VWF binding function of GPIb-IX. Several phosphoserine containing motifs in GPIb-IX complex, such as Ser609 of GPIbα and Ser166 of GPIbβ, have been confirmed to bind to 14-3-3ζ and involved in the regulation of VWF binding function. We have recently identified a novel 14-3-3ζ binding site located in the filamin A binding domain of GPIbα at Ser559. Phosphorylation of GPIbα at Ser559 not only is important for 14-3-3ζ binding, but also plays a key role in the regulation of VWF binding function of GPIb-IX. However, the protein kinase responsible for Ser559 phosphorylation remains unknown. Here we show that a peptide corresponding to the Arg557-Pro561 region of GPIbα and purified GPIbα could be phosphorylated at Ser559 by the catalytic subunit of protein kinase A (PKA). Ser559 phosphorylation was enhanced by PKA activators and reduced by PKA inhibitors in platelets and Chinese hamster ovary (CHO) cells expressing GPIb-IX. Furthermore, PKA inhibitor enhanced VWF binding to the CHO cells expressing GPIb-IX mutant replacing Ser559 of GPIbα (S559A) with alanine, while reduced in the CHO cells of Ser166 of GPIbβ (S166A) with alanine. GPIb-IX association with filamin A and membrane expression were enhanced in S559A cells, whereas reduced in S166A cells. The peptide disrupting the interaction of 14-3-3ζ with Ser559 of GPIbα inhibited platelet spreading on VWF matrix and GPIb-IX-VWF interaction-induced association of Src with GPIb-IX. These data indicate that Ser559 of GPIbα is phosphorylated by PKA which plays key roles in regulating VWF binding, surface expression, and signaling of GPIb-IX.

Disclosures:
No relevant conflicts of interest to declare.
The Presence Of 1 / 8 HLA Mismatch Do Not Hamper Survival After Allogeneic Stem Cell Transplantation Using Immunoprophylaxis With Sirolimus-Tacrolimus
Authors: Parody Rocío , López-Corral Lucía , Godino Oriana Lopez , Cadenas Irene García , Lourdes Vazquez , Martino Rodrigo , Martinez Carmen , Solano Carlos , Martinez Ariadna Perez , Barba Pere , Valcarcel David , Falantes Jose F. , Márquez-Malaver Francisco J , Caballero Dolores , Perez-Simón Jose Antonio 
Published: October 2019

Several studies from the IBMTR have shown that overall survival (OS) is reduced by 10% for each mismatched at obligatory locus (A,B,C or DRB1) after allogeneic hematopoietic stem cell transplant (Allo-HSCT) (Lee et al. Blood 07). In the setting of 7/8 mismatched allo HSCT, different T-cell depletion strategies are frequently used in an attempt to decrease GVHD assuming a higher risk of infections and relapse. Data from the IBMTR are mostly based on series of patients who received Cyclosporine /Tacrolimus (TKR) plus methotrexate as immunoprophylaxis. As far as we know, the effect of sirolimus (SRL)/TKR combination on overall outcomes of transplant, regardless HLA mismatched, has not yet been evaluated.
With this background, we retrospectively analysed a series of 151 adult patients who received SRL/TKR based reduced intensity (RIC)-alloHSCT in 6 Spanish centres, from January 2007 to September 2012 (median follow up in alive patients 19 months, range 4-47), comparing overall outcomes according HLA compatibility in obligatory locus A,B,C, DRB1: 8/8 (n:129) vs 7/8 (n:22). No in vivo or ex vivo T depletion was used.
Patients characteristics are not significantly different between both groups. Median age was 53 years (17-70) and the two most common diseases (56% of cases) were AML and NHL. The most frequent stem cell source was peripheral blood (94%) and 76% transplants were from volunteer unrelated donors. Forty nine per cet of patients had active disease at the time of transplant.
Comparing 8/8 vs 7/8 alloHSCT, non statistically significant differences were found in 3 years-OS (56,4% vs 69%, p=0.7), 1 year-non-relapse mortality [12 % (7-20) vs 14% (5-42), p=0.8], and 3 year-relapse rate: 28% (19-43) vs 20% (9-50), p=0.8, respectively. Cumulative incidence of acute GVHD was significantly higher in 7/8 alloHSCT [68% (49-94) vs 41% (32-51), p=0.004] but no differences were observed with respect to neither III-IV acute GVHD (5 vs 9%, p:0.6) nor global (35 vs 51%, p=0.6) and extensive (20 vs 30%, p=0.9) chronic GHVD in 8/8 vs 7/8, respectively.
The present study shows favorable outcomes of SRL/TKR combination in the setting of RIC alloHSCT, with an overall survival in the range of 55-70%%, and non significant differences in overall outcomes regardless the presence of any mismatched at obligatory locus. Comparative studies with a larger sesries is desirable to confirm these results.

Disclosures:
No relevant conflicts of interest to declare.
A Myeloablative Conditioning Regimen With Fludarabine Demonstrates Good Results In UCBT With Hematologic Malignancies, Especially Acute Lymphoblastic Leukemia
Authors: Tong Juan , Zimin Sun , Huilan Liu , Liangquan Geng , Changcheng Zheng , Tang Baolin , Kaidi Song , Wen Yao , Xin Liu , Ding Kaiyang 
Published: October 2019

Objectives
We retrospectively analyzed the safety and efficacy of a myeloablative conditioning regimen without anti-thymocyte globulin (ATG) or total body irradiation (TBI) but with fludarabine (FLU) in unrelated cord blood transplantation (UCBT) for 30 patients with hematologic malignancies.

Methods
The myeloablative conditioning regimen consisted of FLU, busulfan (BU) and cyclophosphamide (CY). All of the patients received Cyclosporine (CSA) and mycophenolate mofetil (MMF) as graft versus host disease (GVHD) prophylaxis.

Results
With this conditioning regimen, we achieved high engraftment rates (96.7%) and rapid hematopoietic reconstitution. Acute GVHD occurred in 12 cases of the 29 engraftment patients (41.4%), and 6 cases (20.7%) were of grade III-IV. Chronic GVHD only occurred in 1 of 28 evaluable patients (3.6%). Twenty-three patients (76.7%) became infected, and 3 cases (10.0%) died of severe infections. Cytomegalovirus (CMV) reactivation occurred in 70.0% of the patients, but no CMV diseases were observed, nor did any patients die of CMV infection. The cumulative incidence of relapse (6.7%) was significantly reduced, and none of the acute lymphoblastic leukemia (ALL) patients relapsed. The 3-year overall survival (OS) and event-free survival (EFS) rates were 73.3% and 70.0%, respectively, representing satisfactory survival. The 3-year OS and EFS of the ALL patients was 75.0%.

Discussion
This conditioning regimen resulted in a high engraftment rate, rapid myeloid reconstruction and a low incidence of infection. Although there were many patients with high-risk disease and disease progression, the regimen resulted in low relapse rates and good survival. None of the ALL patients relapsed after UCBT, indicating that this conditioning regimen could be applied to more patients with ALL.

Disclosures:
No relevant conflicts of interest to declare.
High Plasma Level Of Regenerating Islet-Derived 3-Alpha (Reg3a) Protein Predicts Grade 3-4 Lower Gastrointestinal Acute Graft-Versus-Host Disease and Poor Prognosis
Authors: Sun Aining , Cai Chengsen , Chen Guanghua , Wang Jun , Depei Wu 
Published: October 2019

Objective
To explore the feasibility of applying plasma Reg3α protein level for the diagnosis and prognosis of the lower gastrointestinal acute graft-versus-host disease(LGI-aGVHD).

Method
One hundred and three patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) in our hospital from December 2011 to December 2012 were included in this prospective study. Peripheral blood samples were collected from allo-HSCT recipients at -9d, 0d, +14d, +28d, at the time when they developed aGVHD, and at the one and four weeks after aGVHD therapy. The plasma concentrations of Reg3α protein were measured by ELISA assay.

Results
Of the 103 patients, 17 subjects never developed aGVHD symptoms (no-aGVHD), 27 subjects presented with non-aGVHD associated diarrhea, 10 subjects presented with isolated skin aGVHD, 17 subjects developed grades ±-II LGI-aGVHD, 32 subjects developed grades III-‡W LGI-aGVHD. The plasma concentrations of Reg3α in patients with LGI-aGVHD vs non-aGVHD diarrhea were 111.5(54.7-180.2) vs 23.9 (14.5-89.5) ng/ml respectively, with the difference being statistically significant (P<0.001). The plasma concentrations of Reg3α in 17 patients who had no response to therapy at 4 weeks for grades III-‡W LGI-aGVHD and 7 patients who experienced a complete or partial response were 137.2(51.7-205.4) and 679.4(122.3-896.8) ng/ml respectively, with the difference being statistically significant (P=0.028). All of the patients who had no response to therapy died of aGVHD-associated multiple organ failure. The area under the ROC curve was 0.902 when plasma concentration of Reg3α was set at 87.73 ng/ml. The sensitivity was 81.48% and the specificity was 82.86% when the critical value was used in diagnosis of grades III-‡W LGI-aGVHD. The probability of grades III-‡W LGI-aGVHD had statistical difference above and below 87.73 ng/ml after allo-HSCT( P<0.001).

Conclusion
The high level of plasma Reg3α protein after transplantation predicts grades III-‡W LGI-aGVHD. In grades III-‡W LGI-aGVHD patients receivingimmunosuppressive treatment at four weeks, the high level of plasma Reg3α protein correlates withs a poor prognosis. The plasma concentrations of Reg3α can be used as a specific biomarker of LGI-aGVHD.

Disclosures:
No relevant conflicts of interest to declare.
TPO exerts a protective effect on iron-overload induces apoptosis in cardiomyocytes via mitochondrial pathways
Authors: Yang Mo , Chan Shing , Ye Jie yu , ChiFung Chan Godfrey 
Published: October 2019

Thalassaemia companied with iron-overload is common in Hong Kong. Iron overload induced cardiomyopathy is the commonest cause of morbidity and mortality in b-thalassaemia patients. One of the causes of cardiac failure is chronic iron overload of blood transfusion. Some studies showed that iron overload can cause toxic effect in heart cells. Iron-overload induced cardiomyopathy damages are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes. Our previous study showed TPO has cardiac protective effect (Li et al, Circulation, 2007). In this study, we demonstrated firstly that iron induced oxidative stress can cause apoptosis in cardiomyocytes. By using H9C2 cells, we showed that iron increased reactive oxygen species (ROS) production (n=3) and reduced cell viability in a dose-dependent manner (0-0.6 mM) (n=6). Apoptotic cells were found to be significantly increased under iron treatment (0.3 mM, 72 hrs) in the AnnexinV/PI assay (n=6). The expression of active caspase-3 significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/mL). The ROS production was significantly decreased with the addition of TPO at 50 ng/mL (n=3). Dot-plot analysis of AnnexinV/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6), indicating a trend in attenuation of the drop of mitochondrial membrane potential. Our findings suggest that iron-overload lead to generation of ROS which further induces apoptosis in cardiomyocytes via mitochondrial pathways and TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and mitochondrial pathway in cardiomyocytes.

Disclosures:
No relevant conflicts of interest to declare.
A New Mutation Of The ALAS2 Gene In a Chinese Family With X-linked
Authors: Cui Rui , Zhu Guoqing , Xu Zefeng , Zhang Yue , Huang Gang , Xiao Zhijian 
Published: October 2019

X-linked sideroblastic anemia (XLSA; OMIM 301300) is one of the most common inherited sideroblastic anemia, which results from mutations in the erythroid-specific isozyme of 5-aminolevulinate synthase. An amino acid (AA) substitution for arginine at the 452 AA position of the ALAS2 protein is the most frequent mutation, which has been found in approximately one-quarter of patients with XLSA. Here we first report a 3-generation pedigree including 10 individuals with a K156E substitution in ALAS2 gene in China. A 34 year-old man, complained with a microcytic anemia (hemoglobin (Hb) 84 g/dl, MCV 63.8 fl and MCH 16.5pg), increased ferritin serum (3123ng/ml) and transferrin sat (93%), and dyserythropoiesis with 50% of ring sideroblasts (RS) in BM. A diagnosis of XLSA was made. On treatment with 300mg/d pyridoxine and iron chelation therapy with deferoxamine allowed a correction of anemia and reduction of the S-ferritin (1096 ng/ml) in the proband. The peripheral blood samples had been extracted from proband and his family members. All exons containing exon-intron boundaries of ALAS2, SLC25A38 and GLRX5, the promoter region and intron 8 of ALAS2 were amplified and directly sequenced. He is a heterozygous for ALAS2 K156E substituion. The pedigree of the proband's family is shown in Figure 1, while hematologic and iron status data for the family members are reported in Table 1. All 4 heterozygous females from this family showed marginally increased red-cell distribution width (RDW) without phenotypic expression.Figure 1The pedigree of the probandFigure 1. The pedigree of the probandTable1Clinical and hematological features of the pedigreeSubjectSex/age,yHb g/dLMCV fLRDW%Ferritin μg/LALAS2I-1F/6811585.221.4136.7+II-1F/381288912.532.89-II-2M/348463.832.83123+II-3F/3213691.914.816.8-III-1M/2015288.212.1123.6-III-2F/1812384.813.321.84-III-3M/71449212.951.9-III-4F/101338513.716.8+III-5F/812681.418.831.2-
In summary, this is a novel K156E substitution in ALAS2 gene discovered in a 3-generation pedigree in China. Furthermore, early diagnosis of XLSA allows preventing the complications of the iron overload by chelators or by iterative phlebotomies.

Disclosures:
No relevant conflicts of interest to declare.