scholarly journals Novel Biosensor–Based Analytic Device for the Detection of Anti–Double-Stranded DNA Antibodies

2007 ◽  
Vol 53 (2) ◽  
pp. 334-341 ◽  
Author(s):  
Alexander Buhl ◽  
Jochen H Metzger ◽  
Niels H H Heegaard ◽  
Philipp von Landenberg ◽  
Martin Fleck ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Dependent Manner ◽  
Surface Immobilization ◽  
Surface Binding ◽  
Concentration Dependent Manner ◽  
Spr Biosensor ◽  
Double Stranded Dna ◽  
Healthy Donors ◽  
Biosensor System ◽  
Biosensor Chip

Abstract Background: Patients with systemic lupus erythematosus (SLE) develop a wide variety of serologic manifestations, including double-stranded DNA autoantibodies (anti-dsDNA). The determination of the potentially pathogenic autoantibodies is diagnostically relevant. Methods: We developed a novel surface plasmon resonance (SPR) biosensor chip for studies of dsDNA and anti-dsDNA binding. A synthetic oligonucleotide was coupled to biotinylated human transferrin, hybridized with the complementary antistrand, and ligated with a human recombinant dsDNA fragment 233 bp in length. After surface immobilization of this antigenic construct, diluted sera from SLE patients and healthy donors were analyzed with the resulting SPR biosensor system. Results: This SPR biosensor allowed specific detection of anti-dsDNA. In pilot experiments, sera from SLE patients were distinguished from control sera. We also confirmed the specificity of this biosensor by supplementing anti-dsDNA–positive sera with salmon sperm DNA, which blocked the surface binding of anti-dsDNA in a concentration-dependent manner. Conclusions: An SPR biosensor monitors interactions in real time under homogeneous conditions, providing information about binding kinetics and affinities. Its applicability critically depends on the design of the solid-state surface of the sensor chips. Covalently immobilizing dsDNA as the antigen to the surface in a flow-through cell assured maximal stability for multiple serum injections and regeneration cycles. This technique, which adds a new analytic quality to existing methods, may be beneficial in the diagnosis and clinical monitoring of SLE.

scholarly journals Biosensor Analysis of β2-Glycoprotein I–Reactive Autoantibodies: Evidence for Isotype-Specific Binding and Differentiation of Pathogenic from Infection-Induced Antibodies

2007 ◽  
Vol 53 (6) ◽  
pp. 1137-1143 ◽  
Author(s):  
Jochen Metzger ◽  
Philipp von Landenberg ◽  
Marcus Kehrel ◽  
Alexander Buhl ◽  
Karl J Lackner ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Specific Binding ◽  
Parvovirus B19 ◽  
Laboratory Diagnosis ◽  
Antiphospholipid Antibody ◽  
Serum Samples ◽  
Additional Information ◽  
Glycoprotein I ◽  
Spr Biosensor ◽  
Biosensor System

Abstract Background: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-β2-glycoprotein I (β2GPI) autoantibodies (anti-β2GPI) and pathogen-specific β2GPI cross-reactive antibodies that occur transiently during infections. Methods: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-β2GPI in serum samples, affinity-purified human β2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to β2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with β2GPI-specific ELISA. Results: Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50–500, resonance units (RU) for anti-β2GPI ELISA-positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-β2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized β2GPI. In contrast to APS patient samples, no significant anti-β2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection. Conclusions: The SPR biosensor system enables specific detection of APS-associated β2GPI-reactive APL and differentiation from β2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-β2GPI responses in APS patient sera gives additional information regarding the influence of anti-β2GPI IgG and IgM isotype distribution.

scholarly journals DNA-hydrolysing activity of IgG antibodies from the sera of patients with schizophrenia

Open Biology ◽  
2015 ◽  
Vol 5 (9) ◽  
pp. 150064 ◽  
Author(s):  
Evgeny A. Ermakov ◽  
Ludmila P. Smirnova ◽  
Taisiya A. Parkhomenko ◽  
Pavel S. Dmitrenok ◽  
Nina M. Krotenko ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Mass Spectra ◽  
Intrinsic Property ◽  
Dnase Activity ◽  
Light Chains ◽  
Double Stranded Dna ◽  
Healthy Donors ◽  
Lower Molecular Mass ◽  
Autoimmune Processes ◽  
Dna Antibodies

It is believed that damage to the membranes of brain cells of schizophrenia (SCZ) patients induces the formation of autoantigens and autoantibodies. Nevertheless, the importance of immunological changes leading to the loss of tolerance to self-antigens in the genesis of SCZ has not been established. The MALDI mass spectra of the IgG light chains of 20 healthy donors were relatively homogeneous and characterized by one peak with only one maximum. In contrast to the healthy donors, the MALDI mass spectra of IgG light chains corresponding to 20 SCZ patients demonstrated, similarly to 20 autoimmune systemic lupus erythematosus (SLE) patients, two maxima of a comparable intensity. In addition, the MALDI spectra of the IgG light chains of five SLE and four SCZ patients contained a small additional brightly pronounced peak with remarkably lower molecular mass compared with the main one. DNase autoantibodies (abzymes) can be found in the blood of patients with several autoimmune diseases, while the blood of healthy donors or patients with diseases without a significant disturbance of the immune status does not contain DNase abzymes. Here, we present the first analysis of anti-DNA antibodies and DNase abzymes in the sera of SCZ patients. Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of SCZ patients. The sera of approximately 30% of SCZ patients displayed a higher content of antibodies (compared with 37% of SLE) interacting with single- and double-stranded DNA compared with healthy donors. Antibodies with DNase activity were revealed in 80% of the patients. These data indicate that some SCZ patients may show signs of typical autoimmune processes to a certain extent.

scholarly journals RNA-Containing Immune Complexes Formed by Anti-Melanoma Differentiation Associated Gene 5 Autoantibody Are Potent Inducers of IFN-α

2021 ◽  
Vol 12 ◽  
Author(s):  
Kaiwen Wang ◽  
Jiangfeng Zhao ◽  
Wanlong Wu ◽  
Wenwen Xu ◽  
Shuhui Sun ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn ◽  
Cytoplasmic Rna ◽  
Healthy Donors ◽  
Rna Immunoprecipitation ◽  
High Level

ObjectiveAnti-melanoma differentiation-associated gene 5 (MDA5) autoantibody is a distinctive serology hallmark of dermatomyositis (DM). As an autoantigen, MDA5 is a cytoplasmic RNA recognition receptor. The aim of this study was to address the question of whether the RNA-containing immune complex (IC) formed by MDA5 and anti-MDA5 could activate type I interferon (IFN) response.MethodPatients with anti-MDA5+ DM (n = 217), anti-MDA5− DM (n = 68), anti-synthase syndrome (ASyS, n = 57), systemic lupus erythematosus (SLE, n = 245), rheumatoid arthritis (RA, n = 89), and systemic sclerosis (SSc, n = 30) and healthy donors (HD, n = 94) were enrolled in our studies. Anti-MDA5 antibody was detected by line blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and Western blotting. Cytokine profiling was determined by multiplex flow cytometry, and IFN-α was further measured by ELISA. Type I IFN-inducible genes were detected by quantitative PCR (qPCR). RNA–IC binding was analyzed by RNA immunoprecipitation. Plasmacytoid dendritic cells (pDCs) derived from healthy donors were cultivated and stimulated with MDA5 ICs with or without RNase and Toll-like receptor 7 (TLR-7) agonist. The interaction between MDA5 ICs and TLR7 was evaluated by immunoprecipitation and confocal microscopy.ResultsAccording to our in-house ELISA, the presence of anti-MDA5 antibody in 76.1% of DM patients, along with 14.3% of SLE patients who had a lower titer yet positive anti-MDA5 antibody, was related to the high level of peripheral IFN-α. ICs formed by MDA5 and anti-MDA5 were potent inducers of IFN-α via TLR-7 in an RNA-dependent manner in vitro.ConclusionOur data provided evidence of the mechanistic relevance between the anti-MDA5 antibody and type I IFN pathway.

The CD40/CD40 Ligand (CD40L) Pathway Plays a Role in Primary Hemostasis and Platelet Function.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1562-1562
Author(s):  
Florian Langer ◽  
Susan B. Ingersoll ◽  
Ali Amirkhosravi ◽  
Todd V. Meyer ◽  
Farooq A. Siddiqui ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Lupus Erythematosus ◽  
Dense Granule ◽  
Soluble Form ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Platelet Surface ◽  
Knock Out ◽  
Primary Hemostasis

Abstract Previous studies have suggested a role for platelet CD40L in thrombosis and atherosclerosis. However, there are contradictory reports on the biologic activity of its soluble variant (sCD40L) and the involved receptor signaling pathways (CD40 vs αIIbβ3). Furthermore, CD40L mAb-associated thromboembolic complications in recent human and animal studies have raised additional questions about the pro-thrombotic properties of this molecule. This study was conducted to further investigate the function of the CD40/CD40L dyad in primary hemostasis and platelet function. CD40−/− and CD40L−/− mice and mice deficient for both genes (“double knock-out”) showed prolonged tail vein bleeding and platelet function analyzer (PFA-100) closure times as compared to their wild-type littermates, indicating an inherited defect in platelet function. Recombinant human sCD40L (rsCD40L), chemical cross-linking of which yielded a single reaction product compatible with a trimeric structure of the protein in solution, bound to CD40 on resting platelets and induced CD62P (P-selectin) expression in a concentration-dependent manner (0–5 μg/ml) from 1±1 to 23±5% positivity (means±SD, n=4–8; P<0.01). This response was completely abolished by CD40 mAb M3 and not affected by blocking the β3 integrin (CD61) with abciximab. In contrast, CD40 mAb G28-5 significantly enhanced rsCD40L-induced CD62P expression to 51±5%. This agonistic effect was strongest when G28-5 was added to the platelets after rsCD40L. Pre-incubation of rsCD40L with CD40L mAb M90 also had a potentiating effect, showing an inverted V-shaped dose response curve with maximum levels of CD62P+ platelets (60–95%) at a molar M90/rsCD40L ratio of 1/3. G28-5 and M90 alone had no effect, but their combination in the presence of rsCD40L proved synergistic. Experiments with corresponding F(ab’) fragments and the FcγRII-inhibitory mAb IV.3 demonstrated that G28-5- and M90-mediated additional platelet activation resulted from signaling through FcγRII cross-linked to CD40 and rsCD40L bound to CD40 on the platelet surface, respectively. The CD40L mAb TRAP-1 showed similar characteristics to M90, but its synergistic effect with rsCD40L was less pronounced. Platelet activation by rsCD40L in combination with G28-5 and/or M90 also induced morphological shape changes, fibrinogen binding, dense granule release, microparticle generation, and monocyte-platelet-conjugate formation. Interestingly, consistent with their platelet function-modulating effects, M3 but not G28-5 prolonged PFA-100 closure times of normal human blood. This work provides genetic evidence for a role of CD40 and CD40L in primary hemostasis. Our mechanistic studies further suggest that CD40-mediated platelet activation by CD40L, in its membrane and/or soluble form, may be at least partially involved in this process. The results also offer a potential explanation for the unexpected high incidence of CD40L mAb-associated thrombotic events in patients with systemic lupus erythematosus, an inflammatory autoimmune disease characterized by elevated levels of circulating sCD40L. The underlying pathomechanism, in which sCD40L as a platelet-derived, homotrimeric protein causes multimeric clustering of bivalent antibodies on the platelet surface with subsequent FcγRII-mediated platelet activation, resembles other known immune thrombophilias such as the heparin-induced thrombocytopenia (HIT) syndrome.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

Influence of Brain Gangliosides on the Formation and Properties of Supported Lipid Bilayers for Binding Kinetics Studies

2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg
Keyword(s):  
Lipid Bilayers ◽  
Binding Kinetics ◽  
Supported Lipid Bilayers ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Supported Lipid Bilayer ◽  
Head Groups ◽  
Lipid Diffusion ◽  
Kinetics Of ◽  
Brain Gangliosides

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.

Enhancement of hydroxyl radical generation by phenols and their reaction intermediates during ozonation

1998 ◽  
Vol 38 (6) ◽  
pp. 147-154 ◽  
Author(s):  
Hideo Utsumi ◽  
Sang-Kuk Han ◽  
Kazuhiro Ichikawa
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Spin Trapping ◽  
Active Species ◽  
Generation Rate ◽  
Peak Height Ratio ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Phenol Derivatives ◽  
Semiquinone Radicals

Generation of hydroxyl radicals, one of the major active species in ozonation of water was directly observed with a spin-trapping/electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) as a spin-trapping reagent. Hydroxyl radical were trapped with DMPO as a stable radical, DMPO-OH. Eighty μM of ozone produced 1.08 X 10-6M of DMPO-OH, indicating that 1.4% of •OH is trapped with DMPO. Generation rate of DMPO-OH was determined by ESR/stopped-flow measurement. Phenol derivatives increased the amount and generation rate of DMPO-OH, indicating that phenol derivatives enhance •OH generation during ozonation of water. Ozonation of 2,3-, 2,5-, 2,6-dichlorophenol gave an ESR spectra of triplet lines whose peak height ratio were 1:2:1. ESR parameters of the triplet lines agreed with those of the corresponding dichloro-psemiquinone radical. Ozonation of 2,4,5- and 2,4,6-trichlorophenol gave the same spectra as those of 2,5- and 2,6-dichlorophenol, respectively, indicating that a chlorine group in p-position is substituted with a hydroxy group during ozonation. Amounts of the radical increased in an ozone-concentration dependent manner and were inhibited by addition of hydroxyl radical scavengers. These results suggest that p-semiquinone radicals are generated from the chlorophenols by hydroxyl radicals during ozonation. The p-semiquinone radicals were at least partly responsible for enhancements of DMPO-OH generation.

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