bluuebunny/crossref_metadata_2025_split huggingface dataset:
Integrated Genotyping and mRNA Expression Profiling of Killer Immunoglobulin-Like Receptors.
Authors: Cremer Anja , Heider Ute , Tomiuk Stefan , Arendt Andreas , Schmitz Jurgen , Bosio Andreas , Huppert Volker , Biervert Christian 
Published: April 2020

Abstract
Natural killer (NK) cells belong to a subgroup of lymphocytes (CD3-CD56+) which play an important role in the cellular immune response against virus-infected cells and tumors. The activity of NK cells is regulated by a balance of triggering and inhibitory receptors, including Killer Ig-like Receptor (KIR) molecules which interact with specific HLA class I molecules, predominantly HLA-C, on target cells. The 17 known KIR genes are divided into two classes: activating KIRs and inhibitory KIRs. There is strong evidence that inhibitory KIR mismatch between donor and recipient improves the outcome of haploidentical hematopoietic stem cell transplantation (HSTC) in leukemia patients (Ruggeri et al. 2002). In addition, the KIR-HLA constellation is assumed to have an influence on the severity of graft versus host disease (GvHD). Whether these activities of NK cells are clinically important and to what extent these processes are mediated only by KIR-HLA class I interactions remains to be determined. In human populations, KIR gene haplotypes vary in the number and type of KIR genes they contain. Further diversification is observed by expanded allelic polymorphism at the individual genes. In general, KIR haplotypes contain 7–12 genes plus 2 pseudogenes. Extra KIR heterogeneity is provided at the expression level: different subsets of NK cells within an individual express different KIRs. Recently, it was shown that KIR genotyping alone does not seem to be sufficient for donor KIR assessment because of the lack of gene expression in approximately one-fourth of the individuals for one of the inhibitory KIRs that recognize the three major groups of MHC class I ligands (Leung et al. 2005). KIR phenotyping by flow cytometry using monoclonal antibodies is insufficient due to the lack of specific monoclonal antibodies. For trustworthy analysis, one has to combine KIR genotyping with mRNA expression profiling and flow cytometry. Therefore, we developed a new set of sequence-specific primers (SSP). This primer set can be applied to perform either KIR genotyping or mRNA expression profiling despite the high degree of identity of the genes (80–90%, sometimes more than 95%). The primers of each KIR gene (15 genes and 2 pseudogenes) cover all allelic variants annotated by the IPD KIR Sequence Data Base (status quo July 05). Using this primer set, we genotyped 25 individuals, and compared the results with other sets of KIR primers published elsewhere. Additionally, we show the mRNA expression profile employing the same set of new primers. We confirmed these results on the protein level by flow cytometry.
CD8+ T Cell Responses to the Polycomb Protein EZH2 in Patients with Haemopoietic Malignancies.
Authors: Kalk Emma , Piper Karen , Pratt Guy , Young Lawrence , Steele Jane , Moss Paul 
Published: April 2020

Abstract
Polycomb Group (PcG) genes encode transcriptional repressors that have been implicated in stem cell survival and cell fate determination (Lessard J et al. Blood. 1998). They are the human homologues of the Drosophila Polycomb and Posterior sex comb genes where they maintain segment-specific expression of the homeotic (Hox) genes. Hox gene products are transcription factors involved in cell fate determination and have been implicated in normal haematopoiesis and leukaemic transformation. In mammals, the PcG genes are highly conserved and essential for normal embryonal development and cell cycle control, as well as the regulation of haematopoiesis. Two distinct PcG gene subsets have been identified which demonstrate consistent and often mutually exclusive expression in haemopoietic cells, the patterns correlating with phenotypically distinct populations in the lymph nodes and thymus. BMI-1 and EZH2 are representative examples of these two subsets of PcG genes. BMI-1 expression is essential for stem cell self-renewal whereas EZH2 is required for cell proliferation. Disrupted expression of both of these genes has been demonstrated in a range of haemopoietic and non-haemopoietic malignancies and appears to deliver a proliferative advantage to the cell.
Humoral and T cell immunity to BMI-1 and EZH2 has been observed in patients with hepatocellular and prostate carcinoma, suggesting that these proteins may act as tumour antigens. However, CD8+ T cell responses to peptides derived from the proteins have also been demonstrated in healthy controls indicating that T cell tolerance might not be complete.
Using the cytokine secretion assay and HLA-tetramer staining, HLA A*0201-restricted CD8+ T cell responses to an EZH2-derived peptide have been identified in 7 out of 19 (37%) healthy controls. The frequency of IFNg-secreting cells ranged from 0.001–1.6% of the total CD8+ T cell pool. In the patient cohort, 4 out of 11 donors responded to the peptide (frequency 36%) with a frequency representing between 0.002 and 0.03% of CD8+ T cells. The frequency of the response is therefore comparable between the two groups. The potential role of regulatory CD4+CD25+ T cells in controlling auto-reactive immune responses to PcG proteins is being assessed. Depletion of this population can result in an increased expansion of the tumour-reactive T cell response in vitro.
Thus, although ectopic expression of polycomb proteins is often seen in haemopoietic malignancy there is currently little evidence for a tumour-selective immune response in such patients.
Cas-L/Hef1 Is Required for Marginal Zone B Cell Maintenance and Lymphocyte Trafficking.
Authors: Seo Sachiko , Asai Takashi , Saito Toshiki , Suzuki Takahiro , Ichikawa Motoshi , Ogawa Seishi , Kurokawa Mineo , Chiba Shigeru , Hirai Hisamaru 
Published: April 2020

Abstract
Cas-L (Crk-associated substrate lymphocyte type) which is also known as Hef1 (human enhancer of filamentation 1) was first identified as a protein tyrosine-phosphorylated upon stimulation of b1 integrin. Cas-L possesses a single Src homology (SH) 3 domain and multiple YXXP motifs (substrate domain) as a member of Cas protein family, and is well expressed in peripheral lymphocytes. Previous studies suggest that Cas-L might be involved in Bcr-Abl positive leukemia and adult T cell leukemia. However, the biological function of Cas-L in lymphocytes is little known. We generated Cas-L-deficient mice using a gene targeting strategy. The mice showed a deficit of marginal zone (MZ) B cells and a decrease of cell number in secondary lymphoid organs. To elucidate the mechanism of the MZ B cell defect, the reciprocal bone marrow transfer assays were performed. The results revealed that the defect of MZ B cells in Cas-L-deficient mice is cell autonomous. Next, we analyzed B cell receptor signaling by measurement of intracellular Ca2+ concentration and lymphocyte proliferation. However, we could not find any significant differences between wild type and Cas-L-deficient mice. Cas-L-deficient lymphocytes showed reduced chemotactic response to CXCL12 and CXCL13. The adhesion assay also showed the decreased adhesiveness to VCAM-1 and ICAM-1, which are important for retention of MZ B cells in spleen. Moreover, we found that the lymphocyte trafficking to spleen and lymph nodes was altered in Cas-L-deficient mice. Thus, Cas-L affects homeostasis of MZ B cells and peripheral lymphoid organs, which is considered to be relevant to impaired lymphocyte migration and adhesion.
Standardization of Functional Immune Cell-Based Assays: An Integral Aspect to Vaccine and Biologic Development and Validation.
Authors: Wilkinson Julie , Smith Cecilia , D’Costa Sybil , Rabellino Enrique 
Published: April 2020

Abstract
The utility of the ex-vivo evaluation of immune cell functionality in the context of a) Determining an efficacious vaccine strategy for infectious diseases/cancer, b) Determining a tolerance profile in autoimmunity and transplantation, and c) understanding the basic mechanisms of immune cell responses in disease pathogenesis is well recognized.
However, the benefit of these assays as surrogate markers of immune cell activity in vivo has not been fully realized due to the variable nature of these in vitro assays which is particularly pronounced in T cell functional assays. This variability arises from a variety of factors ranging from choice of assay, source of the cells, the sample processing methodology (isolation, freezing, thawing, and culturing), sample staining protocol for the chosen assay and ultimately data analysis, and data reduction. With a view to reducing variability and standardizing targeted steps of T cell functional assays, an automated methodology for simultaneous staining and analysis of multiple intracellular cytokines and cytotoxicity markers via flow cytometry was developed and validated. A 5-color flow cytometry assay (2–3 surface markers; 2 intracellular markers) was developed to characterize the restricted polyclonal (SEB/CD28) and antigen specific (CEF peptide pool) cytokine and cytotoxic profile response in human PBMCs. A modification to available sample preparation instruments was performed that enabled the automated pipetting, incubation, and staining of intracellular and surface molecules of stimulated human peripheral blood mononuclear cell populations (PBMC) for flow cytometric analysis.
Statistically significant reductions in both inter and intra assay variability was observed in the automated methodology as compared to the manual assay with improvements in CVs for positive cell numbers and mean fluorescence intensity. For example, the inter assay CVs for IFNg cytokine producing CD4+ T cell populations improved from approximately 15 to 5, while the mean fluorescence intensity improved nearly 5 fold with automation. Importantly, the automated methodology furnished comparable responses in percent positive cytokine/cytotoxicity profiles as compared to the manual method while reducing the “handson” sample preparation and analysis time from 2 hours to 20 minutes. With the standardization of functional assays, other sources of variability in assays result can now be addressed specifically e.g. specimen handling, freezing, thawing, culturing, or biological. Standardized multiparametric functional profiling of the cells thus reveals the complex nature of the immune response and lends credence to their use as surrogate markers of efficacy and functionality.
Platelet Apoptosis Events Can Be Triggered by Shear Stresses and Chemical Agonists.
Authors: Leytin Valery , Mykhaylov Sergiy , Allen David J. , Freedman John J. 
Published: April 2020

Abstract
Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10-44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIbα downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from α-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.
SEPT11, a Novel Platelet Septin Partners with the Platelet Septin SEPT5.
Authors: Röseler Sabrina , Bläser Susanne , Bartsch Ingrid , Rempp Hansjörg , Bauer Heike , Lieber Mareike , Busse Anja , Zieger Barbara 
Published: April 2020

Abstract
The septin SEPT11 is a novel member of the highly conserved septin family. Septins are GTP-binding proteins which form heteropolymeric complexes. In non-dividing cells (such as platelets and neurons) septins are implicated in exocytosis. The septins SEPT4, SEPT5 and SEPT8 are expressed in platelets. Platelets from a SEPT5 knockout mouse showed an altered serotonin secretion and platelet aggregation suggesting that SEPT5 is involved in secretion in platelets. Transmission electron microscopy of platelets revealed that SEPT4, SEPT5 and SEPT8 are localized surrounding alpha-granules suggesting that the three septins may be components of the septin complex in platelets and contribute in such a way to platelet biology. Activation of platelets by agonists resulted in the translocation of SEPT4 and SEPT8 to the platelet surface indicating a possible functional role of these proteins in granular secretion. Previously, we had identified the interaction of the septins SEPT5 and SEPT8. The aim of this study was to identify other interaction partners of the human septin SEPT5. Using the yeast two-hybrid system we now demonstrate that SEPT11 partners with SEPT5. Western analysis revealed that SEPT11 is also expressed in platelets. The molecular interaction of SEPT11 with SEPT5 was verified by precipitation of the SEPT5/SEPT11 complex from lysates of human platelets. In addition, using Western analysis and immunofluorescence analysis, the co-expression of SEPT5 and SEPT11 is shown in human endothelial cells (HUVECs). The Western analyses of various mouse tissues showed that the expression pattern of SEPT11 matches with the expression pattern of SEPT5 suggesting that SEPT11 and SEPT5 are components of the corresponding cell specific septin complex. Since SEPT5 and SEPT11 are members of the same septin complex and since SEPT5 has been reported to play an important role in exocytosis, the SEPT5/SEPT11-complex may be involved in exocytosis in platelets and HUVECs.
Properties of Soluble His-Tagged rGPIbα1–483 Wild-Type and Increase-of-Function Mutants.
Authors: Biswas Tapan K. , Miller Jonathan L. 
Published: April 2020

Abstract
The extracellular domain of human platelet GPIbα contains the binding site for von Willebrand factor (vWF) within the amino-terminal 45 kDa region comprising approximately 300 amino acids. In the present studies, we have used CHO cells to synthesize a longer fragment of His1-Phe483, representing nearly the complete extracellular domain of GPIbα, with an additional 8 histidine residues added at the carboxyl end to provide recognition by his-tag reagents. Both wild-type and the increase-of-function mutants Gly233→Val233 and/or Met239→Val239 were employed. For studies of antibody binding, modulator-induced vWF binding, or antibody inhibition of this vWF binding, capture of the recombinant GPIbα could be accomplished by nickel chelate binding of the his-tag. Both botrocetin and ristocetin induced saturable binding of vWF by ELISA, as demonstrated by HRP-conjugated anti-vWF polyclonal antibody. In the case of the different increase-of-function mutants, significant vWF binding was seen even in the absence of added modulator. Inhibition of botrocetin-induced vWF binding was greater for wild-type than for the Met239→Val239 GPIbα mutant with some mabs (e.g., AS-2), whereas the degree of inhibition was essentially equivalent with other mabs (e.g., C-34). When a rabbit polyclonal anti-his antibody was used to immobilize the recombinant proteins instead of the nickel chelate system, binding of vWF and of conformation-dependent mabs was impaired. However, when this same antibody was biotinylated and then captured on a streptavidin plate, excellent capture and subsequent binding characteristics of the recombinant GPIbα were restored. With both this latter system and with the nickel chelate system, a highly sensitive functional vWF activity assay could be established, using botrocetin-induced binding of vWF from human plasma. Linearity of binding was observed between 0.1–0.6% normal plasma, with saturation occurring around 2%. Whereas higher concentrations of plasma actually appeared to displace bound GPIbα to the nickel chelate, the streptavidin-biotin-anti-his attachment method appeared more resistant to such effects. The ELISA-based assay systems developed in this study accordingly provide an and effective approach to study functional interactions of both wild-type and mutant forms of GPIbα with its ligand vWF and also with antibodies directed against different epitopes within the entire extracellular domain of this receptor molecule. The high sensitivity, reproducibility, and rapidity of the assay system also offer the possibility of a useful test for functional assay of plasma vWF.
Expression and Functional Analysis of Disintegrin from Agkistrodon Actus Venom.
Authors: Bai Xia , Fu Jianxin , Su Jian , Dai Lan , Zhang Wei , Ding Kaiyang , Wang Zhaoyue , Ruan Changgeng 
Published: April 2020

Abstract
Disintegrin is a kind of small native peptides derived from the snake venom, containing RGD sequences. Its interaction with integrins, such as aIIbb3, anb3 and a5b1, can inhibit platelet aggregation. hinder the process of angiogenesis and tumor metastasis. In order to further investigate the function of disintegrin in platelet aggregation, cell adhesion and angiogenesis, the sequence encoding the disintegrin domain of agacutin was fused with enhanced green fluorencent protein(eGFP) and inserted in plasmid pQE-30. The recombinant protein was expressed in E.Coli M15 after induction by IPTG, amounting to 38% of the total bacterial protein. The cells expressing integrin such as breast cancer cell line MDA-MB-231 and endothelial cell line EA.hy926 can specifically bind to the recombinant GFP-disintegrin. Flow cytometry showed that the recombinant protein could bind to platelet specfically and could compete with anti-b3 integrin monoclonal antibody CD61. Moreover, the recombinant protein could inhibit platelet aggregation induced by collagen and ADP in a dose-dependent manner, but had no ability to impede the platelet aggregation induced by Ristocetin. Meanwhile, it could inhibit cell (MDA-MB-231 and EA.hy926) adhesion to fibronectin with inhibition rate of 69% and 48%, respectively. Finally, it could induce apoptosis of EA.hy926 endothelial cells and inhibit the angiogenesis on chicken chorioallantoic membrane mode. As a result, it is demonstrated that GFP-disintegrin has the combined quality of its disparate components and can serve as a novel tool for study of tumor and angiogenesis. In addition, the recombinant protein can efficiently inhibit platelet aggregation and angiogenesis, which will be useful for designing the potential drug for anti-thrombosis and anti-tumor metastasis.
Inhibition of Angiogenesis by a Novel Neutralizing Antibody Against Human Vascular Endothelial Growth Factor Receptor 3 (VEGFR-3).
Authors: Chen Hao , Ding Xiu-Yun , Gao Yuan , Ren Fei-Yao , Li Hui , Gao Jian-En , Sun Qi-Hong 
Published: April 2020

Abstract
Vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligands, vascular endothelial growth factor-C (VEGF-C) and D (VEGF-D), are the major molecules involved in the development of the embryonic vascular system and pathological lymphangiogenesis. Throughout embryogenesis, VEGFR-3 is expressed in most endothelial cells, whilst being restricted to lymphatic vessels later in development. This receptor plays a significant role in the normal development of blood and lymphatic vessels. In the present studies, we generated a novel panel of 17 monoclonal antibodies (mAbs) against the human VEGFR-3 and characterized their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. In CAM angiogenesis experiments, the angiogenesis induced by recombinant GST-VEGF-D was decreased in the presence of antibody BDD073. These data indicate that this novel neutralizing antibody against human VEGFR-3 not only might be a potential compounds in blocking the VEGF-D-induced angiogenesis and lymphangiogenesis, but also be a tool for the investigations of biology of VEGFR-3 and analysis of lymphatic vessels in malignant tumors and their metastases.