bluuebunny/crossref_metadata_2025_split huggingface dataset:
Platelet-Derived Growth Factor (PDGF)-BB Mediates Survival of Leukemic Large Granular Lymphocyte Via An Autocrine Regulatory Pathway
Authors: Yang Jun , Liu Xin , Nyland Susan , Zhang Ranran , Thomas Kendall , Jarbadan Ruth Nancy , Loughran Thomas P. 
Published: October 2019

Abstract
Tumor cells may stimulate their own proliferation through an autocrine mechanism by simultaneously producing growth factors and growth factor receptors. Proto-oncogene Platelet derived growth factor (PDGF) is a multifunctional molecule with roles in cell growth, proliferation, chemotaxis, actin reorganization and apoptosis resistance. PDGF exerts its cellular effects by inducing homo- or hetero-dimeric complex of α- or β-tyrosine kinase receptors, mediating its receptor tyrosine kinase (PDGF-β RTK) auto-phosphorylation, and consequently initiating downstream survival signaling pathway activation cascades. The importance of PDGF ligand and receptor activities in antigenactivated lymphoid malignancies is illustrated by their expression that has only been observed in HTLV-1/2 –immortalized human T lymphocytes, but not in normal T or NK cells. Large granular lymphocyte leukemia (LGL) is similarly defined as an antigen-activated malignancy. Same data suggest that LGL leukemia may be associated with an HTLV–like retrovirus infection. Since the LGL leukemia antigen is postulated to originate from a retroviral immunogen with HTLV-like properties, we investigated the role of PDGF in T- and NK-LGL leukemia. We report that LGL leukemia cells from both T and NK subtypes simultaneously express PDGF-BB and its receptor-β at both mRNA and protein levels. PDGF-BB mediates PDGF-β RTK auto-phosphrylation and consequently initiates downstream survival pathway activation cascade. We observed that PI3 kinase, Src family kinase and their downstream PKB/Akt pathways are constitutively activated in PBMC from LGL leukemia patients. Concurrent inhibition of both PI3 kinase and Src kinase pathways results in an additive effectiveness in induction of apoptosis via inhibition of PKB/Akt and MEK/ERK phosphorylation. In conclusion, PDGF-BB and PDGF receptor-β are highly expressed in LGL leukemia cells. Constitutive expression of PDGF-BB isoform from LGL leukemia cells may possibly be the result of chronic retroviral infection or retroviral antigenic stimulation. Co-expression of PDGF-BB and its receptor-β plays a central role in mediating PDGF/RTK auto-phosphorylation, and its downstream survival pathway activation cascades via an autocrine regulatory mechanism in LGL leukemia.
Resistance to Annexin A5 Anticoagulant Activity and Specificity for Anti-B2GPI Domain I Antibodies in Obstetric Antiphospholipid Syndrome
Authors: Hunt Beverley J , Wu Xiao-Xuan , de Laat Bas , Arslan Alan A , Parmar Kiran , Stuart-Smith Sarah , Rand Jacob H 
Published: October 2019

Abstract
Background Obstetric antiphospholipid (aPL) syndrome (APS) is defined by the presence of persisting aPL with previous unexplained recurrent first trimester pregnancy loss, &/or intrauterine death, &/or placental dysfunction (severe pre-eclampsia, intrauterine growth retardation or placental abruption). The placental anticoagulant protein Annexin A5 (AnxA5) is expressed on the surfaces of placental syncytiotrophoblasts where it has been postulated to play a thrombomodulatory role. aPL antibodies have been demonstrated to reduce the quantity of AnxA5 on these cells and accelerate plasma coagulation. Resistance to AnxA5 anticoagulant activity has previously been demonstrated in APS patients with thrombotic histories. We have investigated whether plasma samples from patients with the obstetric APS also interfere with AnxA5 anticoagulant activity and also whether this correlated with antibody recognition of domain I on β2GPI.
Methods Plasmas were obtained from 30 healthy non-pregnant donors (median age 34, range 18–55) and 70 healthy non-pregnant women with previous obstetric APS (median age 39, range 24–58), and assayed for AnxA5 resistance and anti-β2GPI domain I antibodies, using previously published assays.
Results Women with obstetric APS had significant reduction of AnxA5 anticoagulant ratios compared to controls (median 216%, range 130–282% vs 247%, range 217–283%, p<0.0001) and also elevated levels of antidomain I IgG (median optical density: 0.056, range 0.021–0.498 vs 0.042, range 0.020–0.323, p=0.002). Patients in the lowest tertile of AnxA5 anticoagulant ratios had an odds ratio for obstetric APS of 121.1 (95% confidence interval of 6.5–2240.2) and patients in the highest tertile of antidomain I IgG had an odds ratio for obstetric APS of 3.4 (95% confidence interval of 1.2–9.7). There was a significant correlation between reduction of AnxA5 anticoagulant activity and elevation of antidomain I IgG (r=−0.49, p<0.0001).
Conclusions. Women with obstetric APS are more likely to have AnxA5 resistance and also anti-domain I IgG antibodies than the control groups. These results are consistent with the hypothesis that aPL antibody-mediated disruption of AnxA5, via recognition of domain I of β2GPI is a mechanism for pregnancy morbidity & mortality in obstetric APS.
Role and Regulation of CSF-1-Induced CSF-1 Receptor Interchain Disulfide Bonding in Receptor Activation in Macrophages
Authors: Stanley E. Richard , Xiong Ying , Song Da , Yu Wenfeng , Yeung Yee-Guide 
Published: October 2019

Abstract
Colony stimulating factor-1 (CSF-1) is the major regulator of tissue macrophage development and function. The effects of CSF-1 are mediated by the CSF-1 receptor (CSF-1R), a class III receptor tyrosine kinase belonging to the PDGF receptor family. To study CSF-1R structure/function in macrophages using both genetic and proteomic approaches, we developed a novel, CSF-1R-deficient, mouse bone marrow macrophage (BMM) line (MacCsf1r−/−) (Yu et al., J. Leuk. Biol.84: in press, 2008). MacCsf1r−/− macrophages are maintained in GM-CSF. Retroviral expression of the wild type CSF-1R fully rescued the CSF-1-induced survival, proliferation, differentiation and morphological characteristics, which resemble those of primary BMM. We have studied CSF-1-induced covalent modifications of the receptor in MacCsf1r−/− cells. Activation of the CSF-1R involves ligand-induced receptor dimerization and trans-phosphorylation of the cytoplasmic domains. Besides tyrosine phosphorylation, CSF-1 stimulation results in disulfide bonding of the CSF-1R dimers and cysteine mutagenesis experiments revealed that specific residues in the kinase domain are involved. Inhibition of the disulfide bond formation significantly compromised CSF-1-induced CSF-1R tyrosine phosphorylation, indicating its requirement for full receptor activation. Analysis of the regulation of CSF-1-induced CSF-1R interchain disulfide bonding revealed the participation of third party molecules with additional CSF-1R post-translational modifications. The importance of ligand-induced disulfide bonding for class III receptor tyrosine kinase activation may not be restricted to the CSF-1R as interchain disulfide bonding of PDGF receptor dimers is also ligand-induced.
Severe Malaria and Leptospirosis Are Associated with a Deficiency of the Von Willebrand Factor Cleaving Protease, ADAMTS13
Authors: Lowenberg Ester C , Cohen Sophie , van den Born Bert-Jan H , Meijers J.C.M. , Levi Marcel , Dondorp Arjen M 
Published: October 2019

Abstract
The thrombotic microangiopathy observed in severe malaria and leptospirosis shows resemblance to that of thrombotic thrombocytopenic purpura (TTP) and haemolytic uremic syndrome (HUS), respectively. The pathophysiology of these complications remains incompletely understood. TTP and (in some cases) HUS are associated with a deficiency of ADAMTS13, a metalloprotease that cleaves large prothrombotic VWF multimers. We hypothesized that ADAMTS13 may be deficient in cerebral malaria and leptospirosis, and that the severity of ADAMTS13 deficiency may be associated with the degree of endothelial activation. We investigated a series of patients with cerebral malaria (n=9) and leptospirosis (n=18). Patients with scrub typhus (n=12) representing a disease that is accompanied by endothelial activation, but without the clinical features of TTP-HUS, and healthy blood donors served as controls. Baseline characteristics did not significantly differ between study groups, except for BUN values which were higher in cerebral malaria than in leptospirosis (p-value 0.046). The Glasgow Coma Scale (GCS) score was evidently lower in cerebral malaria, with a median score of 4 (interquartile range 3–6) while subjects with leptospirosis, scrub typhus and normal controls had (near) maximum scores of 15. ADAMTS13 levels were significantly reduced in both cerebral malaria and leptospirosis, compared to scrub typhus and normal controls, with a significantly more severe deficiency in cerebral malaria than in leptospirosis (data in figure). VWF antigen and VWF propeptide levels were elevated in all three disease groups in comparison to healthy controls (p-values < 0.001), and most markedly increased in cerebral malaria. ADAMTS13 activity inversely correlated with both VWF antigen and VWF propeptide levels (p-values <0.001 and 0.005). No significant correlation was apparent between ADAMTS13 activity and hemoglobin (Hb) concentration or platelet count. Free Hb levels were not elevated in any of the patient groups, hence it seems highly unlikely that free Hb is involved in the observed reduction of ADAMTS13 activity in cerebral malaria and leptospirosis. The results of this study indicate that ADAMTS13 levels are decreased in patients with cerebral malaria and leptospirosis providing direct support to the hypothesis of a role of ADAMTS13 in the pathogenesis of the thrombotic microangiopathy associated with these diseases. Further research seems warranted to establish the role of ADAMTS13 deficiency in the development and the severity of these diseases, and to evaluate its prognostic significance. Potentially, ADAMTS13 function might serve as a risk marker of disease severity and it may be speculated that the administration of exogenous (recombinant) ADAMTS13 can be used as an adjunctive therapeutic strategy in the clinical management of patients with cerebral malaria and severe leptospirosis.
Figure Figure
Staphylococcal Superantigen-Like 5 Activates Platelets, and Supports Platelet Adhesion Under Flow Conditions, Which Is Mediated by GPIb- Alpha and Alpha-IIb-Beta-3
Authors: Weeterings Cees , de Haas Carla JC , Vughs Mignon M , de Groot Philip G , van Strijp Jos , Lisman Ton 
Published: October 2019

Abstract
Introduction - Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus Aureus and has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Since PSGL-1 shows close homology to platelet glycoprotein Ib-alpha (GPIb), and as platelets have been shown to play an important role in neutrophil chemotaxis, we investigated whether SSL5 also interacts with platelets.
Results - When purified recombinant SSL5 was added to washed platelets in an aggregometry set-up, full and irreversible aggregation was observed even in the absence of exogenous fibrinogen. Proteolysis of the extracellular part of GPIb-alpha or the addition of dRGDW abrogated platelet aggregation. SSL5 was shown to interact with glycocalicin, a soluble GPIb-alpha fragment, and binding of SSL5 to platelets resulted in GPIb-mediated signal transduction as evidenced by translocation of 14-3-3zeta to the actin cytoskeleton. When a mixture of isolated platelets and red cells was perfused over immobilized SSL5 at a shear rate of 300 s−1, large stable platelet aggregates were observed. Platelet adhesion was substantially reduced by proteolysis of GPIb-alpha or addition of dRGDW. In addition, real-time video analysis showed that initial tethering of the platelets was abrogated by proteolysis of GPIb-alpha. Furthermore, when whole citrated blood was perfused over immobilized SSL5, real-time video analysis revealed that platelets activated with the PAR-1 activating peptide SFLLRN made transient contacts with the immobilized SSL5, although no permanent contacts were observed. Finally, SSL5 was shown to interact with endothelial cell matrix (ECM) from cultured human umbilical vein endothelial cells. The interaction of SSL5 with ECM enhanced aggregation of platelets from whole blood to this ECM.
Discussion - Here, we show that SSL5 is capable of activation, adhesion and aggregation of platelets, in which both GPIb-alpha and alpha-IIb-beta-3 play a role. We hypothesize that platelet activation by SSL5 could be important in colonization of the vascular bed and evasion of the immune system.
GP-VI Disruption Confers Additional Protection against Arterial Thrombosis in PAR-4 Deficient Mice
Authors: Cornelissen Ivo , De Candia Erica , Coughlin Shaun R 
Published: October 2019

Abstract
Following vascular injury, platelets are recruited and activated by adhesive proteins exposed on the subendothelium (including collagen), released agonists and locally generated thrombin. In mice, protease activated receptor 4 (PAR-4) mediates the platelet signaling response to thrombin. Previous studies showed that platelets from PAR-4 null mice do not respond to thrombin and while platelet thrombi do form at the site of injury in PAR-4 deficient animals, they do not propagate and extend into the lumen like thrombi in wild-type mice. The activation pathway responsible for this juxtamural platelet accumulation remains unknown. The collagen exposed on the subendothelium activates and recruites platelets at the site of injury by interacting with membrane glycoprotein (GP) VI. Therefore, the collagen-GP-VI pathway is a putative candidate for the platelet thrombus formation near the vessel wall. Using hirudin as a thrombin inhibitor in mice with disrupted GP-VI expression, it has been previously shown that GP-VI signaling and thrombin activation pathways may cooperate during thrombus growth. To more precisely evaluate the interplay between the thrombin/PAR-4 and collagen/GP-VI signaling pathways, we crossed mice lacking PAR-4 and GP-VI, and subsequently compared bleeding time and arterial thrombus formation in PAR-4:GP-VI double or single deficient animals. All genetic combinations were born at the expected mendelian distribution. Double deficient females carried offspring to term and no bleeding was observed during parturition. Pups lacking both receptors demonstrated transient perinatal abdominal bleeding which resolved rapidly. However, we observed prolonged tail bleeding times in double deficient animals compared to their single deficient littermates. Furthermore in a FeCl3-induced carotid thrombosis model, mice lacking both receptors were completely protected compared to their littermates. This study suggests that there is an interaction between the thrombin and collagen activating pathways in the setting of hemostasis and thrombosis.
Figure Figure Figure Figure
M1 Macrophages Eliminate Lymphoma Cells Through the Vitamin D Dependent Antimicrobial Peptide Cathelicidin
Authors: Bruns Heiko , Fabri Mario , Maurberger Anna , Pasemann Shirin , Fahrenwaldt Cornelia , Wilke Andrea , Moi Stefanie , Daudt Heidi , Amann Kerstin , Büttner Maike , Gaipl Udo , Mackensen Andreas , Gerbitz Armin 
Published: October 2019

Abstract
Abstract 1098
Macrophages make up the bulk of the leukocyte infiltrate in most solid tumors. These so-called tumor associated macrophages (TAMs) are thought to be recruited by tumor-derived chemokines and converted to a tumor-permissive state. TAMs are called M2, or alternatively-activated macrophages, to distinguish them from M1, or classically-activated macrophages. Of interest, these TAMs are not able to fight the tumor; instead, they play a tumor-supporting role via their ability to promote angiogenesis and metastases. In Burkitt's lymphoma infiltration of the tumor masses by macrophages is a characteristic morphological hallmark, while the phenotype and the relevance of TAMs as part of the stroma are poorly understood. Using a murine model of spontaneous Burkitt's lymphoma (λ-hu-c-myc transgenic mice), we therefore characterized macrophages infiltrating lymphoma masses and show here that infiltrating macrophages display an anti-inflammatory M2-phenotype characterized by the expression of surface markers, such as CD206. In addition, they secrete TGFβ and IL-10, which are known not only to support B-cell growth, but suppress T-cell immunity at the tumor site. In vitro analysis of human macrophages generated from PBMC either by GM-CSF to generate the M1 phenotype, or by M-CSF to obtain the M2 phenotype, revealed that both types display a distinct cytotoxic potential. When incubated together with different Burkitt's lymphoma cells, M2 macrophages did not efficiently lyse lymphoma cells. In contrast, M1 macrophages revealed a strong cytotoxic activity towards lymphoma cells, in a TNF, TRAIL, FasL and NO independent manner, as shown by antibody blocking studies. Investigating this mechanism further, we found that M1 macrophages, in contrast to M2 macrophages, express high levels of the Vitamin D inducible Cathelicidin antimicrobial peptide. Strikingly, we demonstrate that cathelicidin has a cytotoxic effect on lymphoma cells, but not on non-malignant B-cells.
Of note, excess supplementation of Vitamin-D increased the cytotoxicity of M2 macrophages against lymphoma cells. We conclude that Vitamin D may therefore point into a therapeutic option to influence a tumor stromal component that greatly impacts on survival and immunogenicity of malignant B-cells.

Disclosures:
Mackensen: Micromet Inc.: Research Funding.
Paralog 4 HOX Genes HOXA4 and HOXB4 Expand Pro-B Cells in Vitro
Authors: Fournier Marilaine , Lebert-Ghali Charles-Etienne , Hassawi Mona , Bijl Janetta Jacoba 
Published: October 2019

Abstract
Abstract 1299
HOX genes are known for their involvement in self-renewal of normal and malignant hematopoietic stem cells (HSC). Enforced expression of HOXB4 leads to HSC expansion in vitro and in vivo without leukemia development. We have recently shown that overexpression of its paralog HOXA4 also resulted in an increase of HSCs and myeloid progenitors in vitro. HOXA4 HSC are fully functional and reconstitute mouse chimeras with normal ratios of mature cells in the periphery. Interestingly, although the mature B-cell compartment is not enhanced, the overexpression of HOXA4 resulted in a 10-fold expansion of B-cell progenitors in the bone marrow (BM). Moreover, the number of more primitive WW-IC was not affected by the overexpression of HOXA4, indicating that this gene specifically expands IL-7 responsive B-cell progenitors. Based on these observations in vivo, we sought to determine if paralog 4 HOX genes can expand B-cell progenitors in vitro. To test this B220+ cells were sorted from BM of healthy wild type mice and transduced with MSCV retroviral vectors for HOXA4-GFP, HOXB4-GFP or control-GFP. Cells were cultured in B-cell specific medium and their growth in response to IL-7 was followed for three weeks. We observed a 15- and 10-fold increase of growth for HOXA4 and HOXB4 pro-B cells compared to control, respectively, within 16 days. FACS analysis confirmed the pro-B cell phenotype of all three cultures. These cells are currently being tested for their repopulation capacity of the B-cell compartment in irradiated hosts. To further investigate potential implication of these genes in oncogenic transformation HOXA4 and HOXB4 were overexpressed in E2APBX1 B-cells derived from E2APBX1 transgenic mice. We showed that the overexpression of HOXA4 or HOXB4 induced a strong expansion of E2APBX1 pro-B cell in vitro (2381- and 1090-fold difference over the control after 23 days, respectively), leading to an immortalization of the culture. Despite this huge expansion these cells were incapable to initiate leukemia upon transfer into irradiated recipients. B-CFC assays showed that the growth of HOXA4 or HOXB4 E2APBX1 pro-B cell cultures was supported by a strong expansion of B-cell progenitors (6138- and 15307-fold difference over the control after 20 days, respectively). Taken together, these results indicate that IL-7 responsive pro-B-cell progenitors are sensitive to paralog 4 HOX genes, an effect which is dramatically enhanced in the context of E2APBX1. Lack of clear oncogenic potential puts HOXA4 and –B4 forward as promising candidates to expand B-cell progenitors in vitro. These cells could potentially be used for B-cell complementation therapy in BM transplantation recipients.

Disclosures:
No relevant conflicts of interest to declare.
Mechanism of the Elevated UPR in CN Patients but Not in CyN Patients Carrying Same ELANE Mutations
Authors: Nustede Rainer , Kuznetsova Inna , Welte Karl , Skokowa Julia 
Published: October 2019

Abstract
Abstract 14
Several studies found that in patients with severe congenital neutropenia (CN) harboring mutations in the ELANE gene mutated NE protein induced unfolded protein response (UPR) leading to elevated apoptosis and diminished differentiation of myeloid cells. However, it is unclear, why UPR was not detected in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations, which have been found in CN patients. Several UPR components have been identified in mammalian cells, which include three transducers (IRE1, PERK, and activating transcription factor 6 (ATF-6) as well as one master regulator (BiP/GRP78). BiP is known to be regulated by ATF6. The activation of ATF6 and its target genes (GADD34, CHOP and BiP) in CN patients has not been studied yet. We were able to detect significantly elevated levels of ATF6 and BiP in myeloid cells of CN patients with ELANE mutations, in comparison to CyN patients and to healthy individuals. Therefore, we investigated the mechanism of UPR and activation of ATF6 and ATF6 target genes in CN patients in comparison to CyN patients. We transduced the myeloid cell lines HL60 and NB4 with lentiviral constructs contained either wild type (WT) ELANE cDNA, or mutated (MUT) ELANE cDNA and measured mRNA and protein expression of ATF6 as well as mRNA expression of ATF6 target genes. We compared the effects of three ELANE mutations: C42R, V145-C152del (both mutations presented in CN patients, but not in CyN patients) and S97L (typical for CN and CyN patients) with WT ELANE. We found that in both cell lines only C42R ELANE MUT, but not V145-C152del ELANE MUT or S97L ELANE MUT induced expression of ATF6, GADD34, CHOP and BiP, as compared to control transduced cells. Furthermore, we hypothesize that degradation of mutated NE protein by Secretory Leukocyte Protease Inhibitor (SLPI) might be involved in UPR induction. However, we detected only very low levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN), as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. The lack of the NE inhibitor, SLPI in CN patients may further contribute to elevated UPR triggered by ELANE MUT and normal levels of SLPI in CyN patients might protect from ELANE MUT-induced UPR. Indeed, inhibition of SLPI using SLPI-specific shRNA led to a significantly elevated expression levels of ATF6, GADD34 and BIP, as compared to ctrl shRNA transduced cells. More importantly, co-transduction of NB4 cells with SLPI shRNA in combination with ELANE S97L MUT (which is common for both CN and CyN patients), but not with WT ELANE led to elevated levels of ATF6, GADD34 and BIP. In summary, different ELANE mutations have different effects on UPR as judged by ATF6 activation and the level of ELANE-triggered UPR is regulated by SLPI.

Disclosures:
No relevant conflicts of interest to declare.