bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation Of Natural Killer Cells By Soypeptide Lunasin and Cytokine: Implication In Cancer Immunotherapy For Lymphoma
Authors: Tung Chun-Yu , Kyazike Sharifah , Lewis David , Han Ling , Kolb Alexander , Pelloso David , Sinn Anthony , Pollok Karen E. , Srivastava Shivani , Robertson Michael J. , Chang Hua-Chen 
Published: October 2019

Abstract

Introduction
Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that chemotherapy-treated lymphoma patients have acquired deficiency of Signal Transducer and Activator of Transcription 4 (STAT4), which results in defective IFNg production during clinical immunotherapy. With the goal of further improvement in cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that exhibits immunostimulatory effects on natural killer (NK) cells.

Experimental Design
PBMCs of healthy donors and chemotherapy-treated lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine.

Results
Adding lunasin to IL-12- or IL-2-cultuted NK cells demonstrated synergistic effects in the induction of IFNG and genes involved in cytotoxicity. The expression level of CD16 and granzyme B was increased in CD56-bright population of NK cells following stimulation with lunasin and cytokine. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNg production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines had higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. Moreover, lunasin augmented antibody-dependent cellular cytotoxicity (ADCC) of NK cells against anti-CD20 coated human B-lymphoma cell line. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci.

Conclusion
We have discovered a novel use of lunasin that exerts synergistic effects together with IL-12 or IL-2. This synergism leads to stronger NK-mediated anti-tumor activity, suggesting the potential clinical use of lunasin to augment the therapeutic responses in cytokine-based immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.
Molecular Dynamics As a Computational Tool To Analyze Lymphocyte Alloreactivity In Hematopoietic Transplant Recipients
Authors: Guerrero Estefania Garcia , Moreno Irene Diaz , De la rosa Miguel Angel , Pérez Simón Jose Antonio , Quintana Antonio Diaz 
Published: October 2019

Abstract

Introduction
The generation of the immune response requires the recognition of peptides presented by the major histocompatibility complex (MHC) through the T cell receptor (TCR). In the hematopoietic transplantation context, T cells (LT) from the donor recognize foreign MHC or own MHC bound to foreign peptides (pMHC), generating an alloimmune response. Currently, the molecular mechanisms of LT alloimmune activation are unknown. In order to analyze the molecular interactions between peptides, MHC and TCR, we have implemented Molecular Dynamics techniques. We have compared immunologically reactive complexes (HLA-A2/TAX/TCR-A6; HLA-A2/HUD/TCR-A6) to non/weakly reactive complexes (HLA-A2/V7R/TCR-A6; HLA-A2/P6A/TCR-A6; HLA-A2/Y8A/TCR-A6).

Methods
Starting structures of two reactive complexes were downloaded from the PDB database and used to model mutations known to lead to different degrees of immune reactivity. Dynamics simulations were performed and analyzed using the program AMBER version 9. The simulation time was approximately 10 ns. Further analysis was carried out using the script ARO (Díaz-Moreno et al. 2009) in the VMD Tk console.

Results
A total of 17 MD trajectories have been reckoned, to simulate the behavior of isolated components of the different MHC-TCR complexes. Analysis of the fluctuations shows that pMHC binding barely restrains TCR motions, affecting mostly to CDR3 loops. Opposite, pMHC displayed substantial changes in its dynamics upon comparing its free versus ternary form (pMHC-TCR). Furthermore, taking as reference the positions of the MHC´s helices in free binary structures (MHC-peptide) and comparing them to the positions in ternary structures (pMHC-TCR), we can observe that in reactive complexes MHC exhibits a higher variability than in non-reactive complexes, suggesting that the MHC must tighten and acquire a position further away from its position in free binary form.
We also analyzed the position of the peptide in the groove of MHC and we found that at position 5 (aa aromatic) the peptide is diverted although its position in the groove of MHC seems to be unrelated to the reactivity of the interaction TCR-pMHC.

Conclusions
The MHC shows strong changes in its molecular dynamics upon binding TCR, decreasing its mobility. The structure of MHC is slightly perturbed in reactive complexes, but not in non-reactive ones.

Financial support
Spanish MINECO (BFU2009-07190/BMC, BFU2012-31670/BMC) the Andalusian Government (BIO198) and Instituto de Salud Carlos III (PFIS - FI12/00189 and FIS PI11/02366)

Disclosures:
No relevant conflicts of interest to declare.
In Vitro Study Of The Mechanisms Involved In The Bone Marrow Mesenchymal Stromal Cell Modulatory Effect On B Cell Function
Authors: Amati Eliana , Bassi Giulio , Di Trapani Mariano , Liotta Francesco , Annunziato Francesco , Perbellini Omar , Ricciardi Mario , Pizzolo Giovanni , Scupoli Maria Teresa , Krampera Mauro 
Published: October 2019

Abstract
Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators of T cell activation and proliferation, mainly through the production of partially defined soluble factors, including the IFN-g-induced tryptophan-degrading enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is still controversial, due to both biological peculiarities of B cells, which do not produce IFN-γ, a key MSC-triggering cytokine, and to different and poorly comparable experimental approaches.
Human purified B cells, either resting or activated for 4 days with a stimulation cocktail (CD40 ligand + enhancer polyhistidine mAb MAB050 + rhIL-2 + mouse F(ab’)2 anti-human IgM/IgA/IgG + CpG oligodeoxynucleotide 2006) were co-cultured with MSC, either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-γ + TNF-α for 48 hours), or with MSC supernatants. CD27-positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and intracellular activation status (through signaling network analysis by Phosphoflow) were assessed. Our results showed that MSC are normally supportive cells, not intrinsically capable of suppressing B cell proliferation, and require inflammatory priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair significantly activated B cell growth in a cell contact-independent manner, as supernatant is sufficient to provide maximal inhibition of B cell proliferation. B cell inhibition by MSC is not related to either induction of B cell apoptosis or early signaling events necessary for B cell activation. In addition, IDO pathway triggered in IFN-γ-primed MSC seems to have a role also in B cell inhibition by interfering with the tryptophan metabolism.
Overall, B cell behavior following the interaction with MSC depends on the functional state of both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation.

Disclosures:
No relevant conflicts of interest to declare.
Influenza Virus-Containing Immune Complexes Activate Platelets Through FcγRIIa Resulting In Bioactive Lipid Synthesis and Microparticle Release
Authors: Boilard Eric , Pare Guillaume , Nathalie Cloutier , Dubuc Isabelle , Rousseau Matthieu , Flamand Louis 
Published: October 2019

Abstract
After red blood cells, platelets represent the most abundant cell lineage in blood, where they sentinel the vasculature and play crucial functions in haemostasis and the prevention of bleedings. Platelets also express a vast array of immune mediators and receptors, suggesting that they can also be regarded as tiny immune cells capable of the recognition of pathogens. Indeed, platelets express functional Toll-like receptors and are thought to actively participate to innate immunity. During severe cases of H1N1 influenza A virus infection, circulating platelets display markers of activation. The specific platelet activation triggers during Influenza infection remain however unknown. In this study, we incubated human platelets with H1N1 influenza A virus (IAV) and monitored platelet activation. We found that IAV is a highly potent inducer of de novo lipid mediators production and a trigger for the release of microparticles from platelets. This activation process takes place independently of the Toll-like receptor 4 and requires the presence of serum, pointing to the contribution of soluble factor(s) present in blood. We observed that the virus scaffolds with immunoglobulin G to form large immune-complexes (∼1micron in size) that activate platelets through the engagement of FcgRIIA. Accordingly, the serum of naïve mice housed in a pathogen-free facility is ineffective at initiating human platelet activation when incubated in the presence of IAV. Intrigued by the fact that all the tested human sera were capable of forming immune complexes with IAV, we hypothesized that significant antibody cross-reactivity between different influenza viruses was sufficient to promote the formation of virus-containing immune complexes. Using in vivo approaches, we found that the antibodies from H3N2 influenza virus-immunized mice generate immune complexes when put in presence of H1N1, activating human platelets and transgenic mouse platelets that express FcgRIIA. Taken together, our observations demonstrate that beyond their activities in haemostasis and innate immunity, platelets can also play a role during the active stage of adaptive immune responses against pathogens through FcγRIIa activation.

Disclosures:
No relevant conflicts of interest to declare.
Impaired Hemostasis Leads To Alveolar Hemorrhages and Increased Mortality In Influenza A Infection
Authors: Antoniak Silvio , Tatsumi Kohei , Milner Justin , Beck Melinda , Mackman Nigel 
Published: October 2019

Abstract

Objective
Viral infections lead to activation of coagulation which enhances inflammatory responses. For instance, influenza A is associated with activation of coagulation and the increased risk for thrombotic events, such as myocardial infarction. Protease-activated receptor 4 (PAR-4) is the main functional receptor on mouse platelets. Here, we investigated the role of tissue factor (TF) and platelets in influenza A infection in mice.

Methods
We used male LowTF mice, which express 1% of normal TF levels, PAR-4 deficient mice and respective control mice. All mice were infected intranasal with influenza A H1N1/PR8. Changes in body weights and survival were analyzed up to 14 days after infection. In addition, bronchoalveolar lavage (BAL) was collected and analyzed 7 days after virus challenge.

Results
LowTF mice exhibited increased loss of body weights compared to littermate control mice between day 4 and 6 after infection (P<0.05). At day 7, BAL of LowTF had increased levels of free hemoglobin compared to infected control mice (2.60±0.39 g/dL vs. 0.05±0.02 g/dL, P<0.05). In addition, LowTF lungs exhibited increased protein levels in the BAL compared to controls (1.52±0.18 mg/mL vs. 0.70±0.26 mg/mL, P<0.05). Lung histology revealed that LowTF mice had extensive hemorrhages. The impaired hemostasis resulted in increased mortality in LowTF mice compared to control mice (90% vs 30%, P<0.004, N=9-10, Figure). Similarly, mice with defective thrombin signaling in platelets (PAR-4-/-) exhibited visible lung hemorrhages with increased hemoglobin levels in the BAL (P<0.05) at day 7 and increased mortality compared to controls (57% vs 10%, P<0.03, N=9-14, Figure).

Conclusions
TF mediated activation of coagulation and PAR-4 dependent platelet activation are essential for pulmonary hemostasis during influenza A infection.

Disclosures:
No relevant conflicts of interest to declare.
Inhibition Of Anti-b2GPI-Dependent Thrombus Enhancement In The Laser-Induced Thrombosis Model In Mice
Authors: Kolyada Alexey , Porter Andrew , Beglova Natalia 
Published: October 2019

Abstract
B2GPI is the major antigen for pathogenic antibodies in antiphospholipid syndrome (APS). The presence of anti-b2GPI antibodies correlates with increased risk for thrombosis and pregnancy loss in patients with APS. In animal models of thrombosis, anti-b2GPI antibodies cause the increase in thrombus size after injury to the vessel wall. We engineered a molecule, A1-A1, that prevents the binding of b2GPI to anionic phospholipids and ApoER2, and studied how it inhibits prothrombotic properties of anti-b2GPI antibodies in vivo using the laser-induced arterial thrombosis model. Lupus-prone (NZWxBXSB)F1 male mice express chronic autoimmune anti-b2GPI antibodies. In (NZWxBXSB)F1 mice, infusion of A1-A1 at a dose of 4 ug/g of body weight caused a decrease in thrombus size of more than 85% as compared to thrombus size in the absence of A1-A1. Similarly, A1-A1 inhibits prothrombotic properties of b2GPI/anti-b2GPI antibody complexes in wild type mice following acute infusion with anti-b2GPI positive APS IgG. The thrombus reduction was due to the binding of A1-A1 to b2GPI, because infusion of LA6, a structural homologue of A1 that does not bind b2GPI, did not affect thrombus size. We did not observe thrombus inhibition by A1-A1 in the absence of anti-b2GPI antibodies, indicating that A1-A1 does not affect normal thrombus formation in the absence of anti-b2GPI antibodies.

Disclosures:
No relevant conflicts of interest to declare.
Heparin-Induced Thrombocytopenia: A Rapid, Reliable and Practical, Functional Flow-Cytometric Assay
Authors: Deutsch Varda , Cipok Michal , Kay Sigi , Levy Yvette , On Shoshana Bar , Kirgner Ilya , Naparstek Elizabeth , Tomer Aaron 
Published: October 2019

Abstract

Background
Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin treatment, associated with morbidity and mortality. HIT is characterized by thrombocytopenia and thrombotic complications secondary to the formation of antibodies (Abs) against heparin-platelet-factor 4 (PF4) complexes. The pathologic mechanism involves the binding of the heparin-immune-complex to the platelet-Fc-receptor, resulting in platelet activation, aggregation, and rapid elimination. The diagnosis of HIT requires laboratory confirmation. Common laboratory testing is based on immune detection of antibodies directed against the PF4/heparin complex (ID-H/PF4-PaGIA or ELISA). However, these assays suffer from methodological limitations, especially low specificity, as compared to the platelet functional assays. The “gold standard” functional test for detecting of platelet-activating antibodies is the radioactive [14C] serotonin-release assay (14C-SRA) (Sheridan D, et al, Blood. 1986;67:27-30, Kelton JG, et al.,Blood.1988;72:925-30). However, the assay includes the use of a radiolabeled biological probe and requires considerable expertise to obtain reliable results. Consequently, its use is limited to research laboratories.

Aim
To overcome the methodological limitations associated with current assays, we modified a functional flow-cytometry assay (FCA), which exhibits high sensitivity and specificity (Tomer, A. Br J Haematol, 1997;98: 648-656 , Tomer, A., et al, Am J Hematol, 1999;61: 53-61). This assay, similar in concept to the 14C-SRA, determines the capacity of the patient's serum to activate platelets in the presence of heparin, using a fluorescent probe.

Methods
Consecutive samples from 254 patients clinically suspected for HIT were tested. The FCA assay was compared with the standard ID-H/PF4-PaGIA antigenic assay (DiaMed, Switzerland) with two dilutions to assess specificity (Nellen, V., et al.,Haematologica, 2012;97: 89-97).

Results
Of the total 254 samples tested, 48 (19%) were positive by PaGIA, compared to 13 (5.1%) positive by the functional FCA (Table 1). The number of PaGIA positive samples was reduced to 24 (9.4%) by 1:16 dilution, and to 14 (5.5%) by 1:32 dilution. All FCA positive samples were positive at all PaGIA dilutions (relative sensitivity 93%). Thirty PaGIA negative samples were all negative by the FCA (relative specificity 100%).

Conclusion
The results suggest that the functional FCA is a practical, sensitive, and highly specific test for the reliable diagnosis of HIT.

Disclosures:
No relevant conflicts of interest to declare.
Four-Factor Prothrombin Complex Concentrate (4-PCC) Effectively Reverses Edoxaban Induced Bleeding In a Rabbit Model Of Acute Injury
Authors: Herzog Eva , Kaspereit Franz , Krege Wilfried , Pragst Ingo , Morishima Yoshiyuki , Schulte Stefan , Dickneite Gerhard 
Published: October 2019

Abstract
This study was designed to determine whether the four-factor Prothrombin Complex Concentrate (4-PCC) Beriplex® P/N (Kcentra™) can effectively reverse bleeding following edoxaban administration in a rabbit kidney injury model.
The oral direct and selective factor Xa inhibitor edoxaban (Daiichi Sankyo) is currently available in Japan for the prophylaxis of venous thromboembolism (VTE) in patients undergoing major orthopedic surgery and is undergoing investigation in phase III trials for the prevention of stroke in patients with atrial fibrillation and the treatment and secondary prevetion of VTE. The primary complication of any available anticoagulant therapy is the risk of bleeding. Rapid reversal of anticoagulation may be necessary in patients requiring emergency treatment due to uncontrolled bleeding. PCCs are frequently used to reverse the effect of vitamin K antagonists such as warfarin and have also been suggested to be potentially effective in reversing the effects of the new oral anticoagulants.
The present study was therefore designed to further investigate the ability of 4-PCC to reverse the anticoagulant action of edoxaban using an in vivo rabbit model of acute bleeding.
Animals were treated with a high intravenous bolus dose of edoxaban (1200 µg/kg) leading to increased and prolonged bleeding following standardized kidney injury compared to vehicle administration. Parallel monitoring of biomarkers of haemostasis showed that edoxaban treatment resulted in the prolongation of prothrombin time (PT), activated partial thromboplastin time, and whole blood clotting time (WBCT), and affected the thrombin generation parameters thrombin peak, endogenous thrombin potential (ETP), lagtime and time to peak. Subsequent administration of the 4-PCC (25-75 IU/kg) resulted in a dose-dependent reversal of edoxaban induced bleeding as indicated by reduced time to haemostasis and total blood loss. Both parameters achieved statistical significance compared to placebo at the 4-PCC dose of 50 IU/kg under fully blinded study conditions. The biomarkers correlating best with 4-PCC mediated edoxaban anticoagulation reversal included PT, WBCT and ETP.
In summary, 4-PCC treatment effectively decreased edoxaban induced hemorrhage in an animal model of acute bleeding at clinically relevant dose levels.

Disclosures:
Herzog: CSL Behring GmbH: Employment. Kaspereit:CSL Behring GmbH: Employment. Krege:CSL Behring GmbH: Employment. Pragst:CSL Behring GmbH: Employment. Morishima:Daiichi Sankyo: Employment. Schulte:CSL Behring GmbH: Employment. Dickneite:CSL Behring GmbH: Employment.
The Transfer Of Dabigatran and Rivaroxaban Across a Dually Perfused Isolated Human Placental Cotyledon – Implications For Therapy In Pregnancy
Authors: Bapat Priya , Kedar Reuven , Melamed Nir , Matlow Jeremy , Lubetsky Angelika , Aleksa Katarina , Berger Howard , Koren Gideon 
Published: October 2019

Abstract

Background
Anticoagulant therapy is often required in cases of high-risk pregnancy for the prophylaxis of venous thromboembolism following surgery, atrial fibrillation, and congestive heart failure, and for the prevention of pregnancy loss in thrombophilic women. During pregnancy, the concentrations of many blood-clotting factors rise, thereby increasing the need for anticoagulants that are safe to use throughout gestation. Dabigatran (Pradaxa®) and rivaroxaban (Xarelto®) are newer generation oral anticoagulants that are increasingly being prescribed to women of reproductive age for the treatment of thromboembolic disorders. Dabigatran acts by directly inhibiting thrombin, and rivaroxaban acts as a direct factor Xa inhibitor. However, the information regarding fetal safety and placental transfer of these drugs is currently lacking. If there is limited transfer of either drug across the placenta, then it may not increase the risk of bleeding in the fetus. The objective of this study was to determine the transplacental kinetics of dabigatran and rivaroxaban.

Methods
Placentae were obtained with informed consent after elective caesarean section of healthy term pregnancies in Toronto, Ontario. The transplacental transfer of dabigatran and rivaroxaban were separately assessed using ex vivo dual perfusion of an isolated human placental cotyledon. Dabigatran, at a concentration of 35 ng/ml, was added to the maternal circulation at the start of the experimental phase. Maternal and fetal samples were taken throughout the pre-experimental (1 h) and experimental (3 h) phases for measurement of dabigatran and markers of placental viability. Separate placenta perfusions with rivaroxaban were conducted at an initial maternal concentration of 250 ng/ml. The perfused drug was measured in maternal and fetal samples using liquid chromatography-mass spectrometry (LC/MS).

Results
There was slow transfer of dabigatran from the maternal to fetal circulation. The fetal-to-maternal (F:M) concentration ratio was 0.33 ± 0.13 after 3 hours (n=3). In contrast, the transfer of rivaroxaban from maternal to fetal circulation was much more rapid, as characterized by a F:M ratio of 0.72 ± 0.12 at 3 hour (n=4), suggesting rapid equilibrium between maternal and fetal circulations. Placental viability markers for all perfusions were within normal ranges.

Conclusions
This is the first direct evidence of the transfer of dabigatran and rivaroxaban across the human placenta from the mother to fetus. It suggests less fetal exposure to dabigatran.

Disclosures:
No relevant conflicts of interest to declare.