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Статті в журналах з теми "Beta2 glycoprotein 1 antibody"
1
Maggiorini, M., A. Knoblauch, J. Schneider, and EW Russi. "Diffuse microvascular pulmonary thrombosis associated with primary antiphospholipid antibody syndrome." European Respiratory Journal 10, no. 3 (March 1, 1997): 727–30. http://dx.doi.org/10.1183/09031936.97.10030727.
Повний текст джерелаАнотація:
Thromboembolism is a well-known complication of the hypercoagulable state associated with antiphospholipid (aPL) antibodies. Acute respiratory failure (ARF) with diffuse pulmonary infiltrates has been reported in only a few patients with aPL antibodies. We describe a 49 year old patient with spiking fever, livedo reticularis, mild haemoptysis and ARF. Chest radiography revealed diffuse bilateral pulmonary infiltrates, and high resolution computed tomography (CT) revealed patchy distribution of areas of ground-glass attenuations. Pulmonary emboli were excluded with angiography. Lung biopsy revealed diffuse microvascular thrombosis, without capillaritis. High serum levels of anticardiolipin (aCL) antibodies were found. The patient's condition improved dramatically after intravenous infection of 1 g methylprednisolone on three consecutive days, followed by 50 mg prednisone orally. The rapid improvement following the administration of glucocorticosteroids suggests that anticardiolipin associated microvascular thrombosis, without inflammatory lesions, may depend on an interference with beta2-glycoprotein I (beta2=GPI) by anticardiolipin.
2
de Laat, Bas, Sander B. Meijer, Carel M. Eckmann, M. van Schagen, Koen Mertens, and Jan A. van Mourik. "Antiphospholipid Antibodies with LAC Activity That Bind beta2-Glycoprotein I Cause Increased Resistance Against Activated Protein C." Blood 110, no. 11 (November 16, 2007): 3622. http://dx.doi.org/10.1182/blood.v110.11.3622.3622.
Повний текст джерелаАнотація:
Abstract Background: The antiphospholipid syndrome is characterized by the occurrence of vascular thrombosis combined with the presence of antiphospholipid antibodies (aPL) in plasma of patients. Recently it was published that aPL with lupus anticoagulant activity (LAC), caused by anti-beta2-glycoprotein I (beta2GPI) antibodies, highly correlate with a history of thrombosis. aPL-related resistance against activated protein C (APC) is one of the proposed mechanism responsible for thrombosis. We investigated a possible correlation between a beta2GPI-dependent LAC and increased APC-resistance in a population of 22 plasma samples with LAC activity. Methods: Twenty-two LAC-positive plasma samples were tested for beta2GPI-dependence (titration of cardiolipin into an APTT-based assay), increased APC-resistance, anti-beta2GPI IgG/IgM antibodies, anti-prothrombin IgG/IgM antibodies and anti-protein C IgG/IgM antibodies. In addition, a monoclonal anti-beta2GPI antibody and patient-purified IgG (both with LAC activity) were diluted in plasma with/without protein C and tested for occurrence of a beta2GPI-dependent LAC (normalization of clotting time by the addition of cardiolipin). To study aPL-induced APC-resistance in more detail, surface plasmon resonance analysis was used to investigate binding between APC and beta2GPI in the presence/absence of a mouse-derived monoclonal anti-beta2GPI antibody. Results: Eleven plasma samples that displayed a beta2GPI-dependent LAC also showed increased APC resistance. In contrast, only 1 of the 11 plasma samples with a beta2GPI-independent LAC displayed increased APC-resistance. None of the other serological parameters (antibodies against beta2-glycoprotein I, prothrombin or protein C) displayed the same association with increased APC resistance as a beta2-glycoprotein I dependent LAC. Furthermore, we found a linear correlation between the potency of a beta2GPI-dependent LAC and the level of APC-resistance. When a monoclonal anti-beta2GPI antibody and a patient-purified IgG were tested for a beta2GPI-dependent LAC, both antibodies did not display a beta2GPI-dependent LAC when diluted in protein C deficient plasma. In literature it has been proposed that direct binding of beta2GPI to APC results in a decreased activity of APC. By using surface plasmon resonance analysis, we found that beta2GPI displayed a higher affinity for coated APC in the presence of the monoclonal anti-beta2GPI antibody (4 nM) compared to beta2GPI alone (400 nM). Conclusion: The results of this study indicate that by adding cardiolipin into an APTT-based clotting assay, one can detect beta2GPI-dependent LAC based on increased resistance against APC. Increased resistance against activated protein C might result from direct binding of beta2GPI to activated protein C. In conclusion, our observations indicate a direct correlation between a major clinical symptom of APS (thrombosis), a diagnostic assay (beta2GPI-dependent LAC) and a potential mechanism responsible for thrombosis in the antiphospholipid syndrome (increased APC-resistance).
3
Ram S, Bharath, Monisha Harimadhavan, Shilpa Prabhu, Karthick R G, Devi Prasad Shetty, and Sharat Damodar. "Acquired and Inherited Thrombophilia Testing in Patients with Chronic Thromboembolic Pulmonary Hypertension: Value of Testing in an Academic Health Center." Blood 138, Supplement 1 (November 5, 2021): 4256. http://dx.doi.org/10.1182/blood-2021-148655.
Повний текст джерелаАнотація:
Abstract Introduction Chronic thromboembolic pulmonary hypertension (CTEPH) is classed as group 4 in the present classification of pulmonary hypertension. The pathophysiology of CTEPH is complex, mainly is a consequence of prior acute pulmonary embolism with failure of thrombi to resolve and the recent recognition of added small vessel changes which impacts long-term outcomes even after surgical management. The role of thrombophilia testing in this condition has been debated. Hence, we here analyzed the utility of thrombophilia testing in CTEPH from a center in a developing country. Methods This is a single institution (Narayana Health City, Bangalore); retrospective study including patients ≥ 18 years of age who underwent thrombophilia workup in a diagnosis of CTEPH from January 2019 till July 2021. Tests done to evaluate thrombophilia included factor V Leiden; prothrombin F20210A mutation; MTHFR gene mutation; Protein C, S, and antithrombin deficiency; lupus anticoagulant, anti-beta2 glycoprotein I (IgM and IgG) and anticardiolipin antibody (IgM and IgG); hyperhomocysteinemia and anti-nuclear antibody testing (ANA-IF). The study was approved by the ethics committee of the institute and was carried out in accordance with the principles of the declaration of Helsinki. Results and discussion The study included 56 patients with a median age of 37 years (range 23-50), and 36 (64%) were males. Patients with recurrent venous thrombosis included 37 (66%), with the majority having thrombosis at 2 sites (53%; 22 patients with associated deep vein thrombosis). A family history of thrombosis was present in 4 patients. The majority of patients received vitamin K antagonists (76%), with the rest receiving direct oral anticoagulants (DOAC). Among the tests sent for acquired thrombophilia, ANA-IF and antiphospholipid antibody (APLA) were most frequently evaluated (94%). ANA-IF and APLA tests were positive in 5.6% and 30.1%, respectively. Among the APLA tests, Anti-beta2 glycoprotein I (IgM or IgG) was the most commonly detected antibody (13/46), followed by anticardiolipin antibody (IgG or IgM) (9/43) and lupus anticoagulant (7/40). Double and triple positive APLA were present in 3 and 4 patients, respectively. Homocysteine levels were high in 93.7% though only 16 patients were tested in this cohort. Among the tests for inherited thrombophilia, genetic tests (factor V Leiden, prothrombin F20210A mutation, and MTHFR gene mutation) were tested in only ~50%. Twenty-three percent were positive for heterozygous MTHFR followed by MTHFR compound heterozygous (10%) and heterozygous factor V Leiden heterozygous (10%). Antithrombin III, protein C, and S were tested in ~30% of patients. Antithrombin III was low in only 1 patient, with protein C and S assays being normal in all the patients. The cost analysis was calculated, showed a median of $364 (₹ 27,055) was spent per person on thrombophilia workup. The median cost incurred per patient for inherited thrombophilia workup was $232 (₹ 17,300) and for acquired thrombophilia was $132 (₹ 9814), respectively. Conclusion This single-institution study on thrombophilia workup in CTEPH patients reveals that APLA was the most commonly performed test with high positivity rates of 30.1%. Among the inherited thrombophilia, the positivity rate of MTHFR mutation was highest (33.3%), with other tests having a low positivity rate (0-10%). Hence, we would recommend APLA testing in all patients with CTEPH considering its high positivity and clinical utility. Testing for other thrombophilias should be pursued judiciously especially in economically restrictive settings. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
4
Kolyada, Alexey, Alfredo De Biasio, and Natalia Beglova. "Probing Anticoagulant Fondaparinux for Interference with Prothrombotic B2GPI/Anti-B2GPI Antibody Complexes." Blood 118, no. 21 (November 18, 2011): 1209. http://dx.doi.org/10.1182/blood.v118.21.1209.1209.
Повний текст джерелаАнотація:
Abstract Abstract 1209 Background: The presence of autoimmune antibodies directed to beta2-glycoprotein-I (B2GPI) often leads to thrombosis in antiphospholipid syndrome (APS). Heparin, low molecular weight heparin (LMWH) and fondaparinux are commonly used for prophylaxis and treatment of thromboses in APS. These drugs bind and activate antithrombin III to inactivate blood clotting proteases. Fondaparinux is a synthetic pentasaccharide matching a specific sequence within heparin interacting with antithrombin. Aim: We investigated if fondaparinux can bind B2GPI and ameliorate prothrombotic properties of B2GPI/anti-B2GPI antibody complexes. Results: We found that fondaparinux interacts with B2GPI and that the binding is dominated by electrostatic interactions. We measured the binding affinity by monitoring changes in the intrinsic fluorescence of domain V of B2GPI (B2GPI-DV) upon titration with fondaparinux. In the presence of 100 mM NaCl, the binding affinity was about 1.5 uM and stoichiometry of the binding is 1:1. Using solution NMR spectroscopy, we determined that the binding interface of the complex is centered on Lys251 of B2GPI-DV. This observation was confirmed by site-directed mutagenesis. The Lys251/Asp mutant fails to bind B2GPI-DV. Interestingly, the binding site for fondaparinux on B2GPI does not overlap with the major binding site for heparin. Cellular activation by the binding of B2GPI/anti-B2GPI antibody complexes with cell-surface receptors (among them ApoER2, a lipoprotein receptor from the LDLR family) and interference with the protective function of annexin V on anionic phospholipids expressed on the surfaces of activated cells are two potential prothrombotic mechanisms of B2GPI/antibody complexes. We found that fondaparinux does not prevent the association of the ligand-binding modules from ApoER2 with B2GPI-DV. Therefore, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes with lipoprotein receptors. Neither fondaparinux, nor heparin and LMWH were effective in inhibiting the binding of B2GPI/anti-B2GPI antibody complexes to cardiolipin-coated plates suggesting that these drugs do not prevent the destructive effect of B2GPI/antibody complexes on antithrombotic function of annexin V. Conclusions: At therapeutic concentrations, fondaparinux forms only small number of complexes with B2GPI, given that the binding affinity of the complex is in a micromolar range. When bound to B2GPI, fondaparinux does not interfere with the binding of B2GPI/anti-B2GPI antibody complexes to lipoprotein receptors and anionic phospholipids. Disclosures: No relevant conflicts of interest to declare.
5
Beglova, Natalia, and Chang-Jin Lee. "Mapping Interactions Between B2GPI and the Lipoprotein Receptors by Solution NMR Spectroscopy." Blood 112, no. 11 (November 16, 2008): 2018. http://dx.doi.org/10.1182/blood.v112.11.2018.2018.
Повний текст джерелаАнотація:
Abstract Antiphospholipid syndrome (APS) is an autoimmune disease characterized by venous or arterial thrombosis and recurrent pregnancy loss. Beta2 glycoprotein I (B2GPI) is the major target for these antibodies. Current evidence suggests that B2GPI acquires pathological properties leading to APS after association with antibodies. It was demonstrated that APS related antibodies induce activation of endothelial cells, monocytes and platelets, although the identity of a cell-surface receptors that transmit the antibody-induced signaling inside cells remain unclear. Biochemical studies have shown that the receptors of the low-density lipoprotein receptor (LDLR) family are all capable to bind B2GPI/antibody complexes. These receptors are abundantly expressed by many cell types. The ligand-binding activity of the lipoprotein receptors is confined primarily to structurally homologous LA modules, which are arranged in sequence to form a ligand-binding domain. We investigated at the atomic level the features of the ligand-binding LA modules required for interaction with B2GPI. We sought to determine whether the same residues that form the ligand-binding pocket on the LA modules in the crystal structure of a RAP/LA3-4 complex also recognize B2GPI. We demonstrated that the same residues that form the ligand-binding pocket on LA4 in the crystal structure of the RAP/LA3-4 complex comprise the contact site for B2GPI. The domain 5 from B2GPI (B2GPI-D5) was expressed and labeled for NMR studies. The residues of B2GPI-D5 that are perturbed when B2GPI-D5 was titrated with LA4 are located at the C-terminus of domain 5. Using NMR relaxation measurements, we determined that B2GPI-D5 binds LA4 with 1:1 stoichiometry. The estimated Kd of the B2GPI-D5/LA4 complex is about 30 uM. NMR experimental data was used to build a docking model of the complex.
6
Xu, Jinfeng, Daijuan Chen, Yuan Tian, Xiaodong Wang, and Bing Peng. "Antiphospholipid Antibodies Increase the Risk of Fetal Growth Restriction: A Systematic Meta-Analysis." International Journal of Clinical Practice 2022 (January 31, 2022): 1–10. http://dx.doi.org/10.1155/2022/4308470.
Повний текст джерелаАнотація:
Objective. Antiphospholipid syndrome (APS) is a chronic autoimmune disease with a high prevalence in females. Published data have identified pregnant women with APS may suffer from recurrent miscarriage, fetal death. However, the association between antiphospholipid antibody (aPL) and fetal growth restriction (FGR) remains controversial. This study aims to systematically review the literature on population-based studies investigating an association between aPL and FGR. Methods. The literature was searched on 1 November, 2021, using Ovid MEDLINE, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL), following the MOOSE checklist. Study inclusion criteria focused on peer-reviewed published articles that reported an association between aPL and FGR. Quality assessment was performed based on the Newcastle-Ottawa scale. The between-study heterogeneity was assessed by the Q test. Publication bias was assessed by funnel plots. Results. Twenty-two studies (with 11745 pregnant women) were included in the final analysis. Pooled odds ratio for association of aPL, anticardiolipin antibodies (ACA), anti-beta2 glycoprotein 1 antibodies (β2GP1), and FGR was 1.26 (95%CI 1.12, 1.40), 2.25 (95%CI 1.55, 2.94), and 1.31 (95% CI 1.12, 1.49), respectively. Lupus anticoagulant (LA) did not increase the chance of FGR (OR 0.82, 95%CI 0.54, 1.10). Conclusions. Our meta-analysis showed that aPL increased the risk of FGR. The risk of FGR varies with the aPL types. ACA and β2GP1 are strongly associated with FGR. There are currently insufficient data to support a significant relationship between LA and FGR.
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Krueger, Irena, Lothar Gremer, Lena Mangels, Meike Klier, Kerstin Jurk, Dieter Willbold, Hans H. Bock, and Margitta Elvers. "Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases." Arteriosclerosis, Thrombosis, and Vascular Biology 40, no. 10 (October 2020): 2391–403. http://dx.doi.org/10.1161/atvbaha.120.314902.
Повний текст джерелаАнотація:
Objective: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. Conclusions: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.
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Demir, S., J. Li, L. Magder, and M. A. Petri. "SAT0203 SINGLE LAC POSITIVITY VERSUS DOUBLE AND TRIPLE POSITIVITY FOR THROMBOSIS IN SLE." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1044.1–1044. http://dx.doi.org/10.1136/annrheumdis-2020-eular.2762.
Повний текст джерелаАнотація:
Background:The antiphospholipid syndrome (APS) is defined by the development of venous and/or arterial thromboses, and pregnancy morbidity, in the presence of antiphospholipid antibodies (aPL); lupus anticoagulant, moderate-to-high titer anticardiolipin (aCL) and anti-β2-glycoprotein (aB2GPI). It has been suggested that the incidence of thromboembolic events were significantly higher in the triple positive subjects, and the rate of pregnancy loss was also significantly much higher in double positive subjects (1). On the other hand several studies showed that LAC is more highly associated with thrombosis risk (2).Objectives:We aimed to investigate the risk of thrombosis in Systemic Lupus Erythematosus (SLE) patients with single LAC positivity versus double and triple positivity in Hopkins Lupus Cohort.Methods:The Hopkins Lupus Cohort is a prospective longitudinal cohort of SLE patients ongoing since 1987. This analysis was based on cohort experience from 2003 through October 2019. Anticardiolipin and anti-Beta2 glycoprotein were defined as positive when the antibody titer exceeded 20 units. The lupus anticoagulant was determined by dilute Russell’s viper venom time (dRVVT) and confirmatory mixing studies, if prolonged. It is defined as positive if a patient had a dRVVT of 45 or more second and a positive confirm ratio of more than 1.4. For each aPL, we defined the patient as positive at a given month of follow up if they ever had a positivity in previous measures. The relationships between thrombosis and aPL were adjusted for number of prior aPL assessment.Results:There were 805 patients with a complete profile of 7 antiphospholipid antibodies, with a total of 73417 person months (6118 person years) of follow up. For any thrombosis when compared to patients with LAC positivity only, double positivity with any isotypes [1.15(0.50, 2.66) p=0.7484] and triple positivity with any isotypes [1.68(0.74, 3.80), p=0.2145] showed a higher point estimates but statistically not significant (Table 1).Table 1.Single, double and triple positive patterns and the risk of any thrombosis.PatternNumber of eventsPerson-yearsRate per 1000 person-yearsadjusted RR (95% CI)p-valueLAC positivity only1063315.81.00 (Ref)Never any aPL33258112,80.73(0.36, 1.47)0.3819any aCL positivity only67937,60.43(0.16, 1.18)0.1028any aB2GPI positivity only751713,50.78(0.30,2.05)0.6195any aCL and aB2GPI positivity only540412,40.71(0.24,2.04)0.5211LAC and ACL positivity740617,31.15(0.50,2.66)0.7484LAC and aB2GPI positivity1249024,5Triple positivity01470,01.68(0.74,3.80)0.2145Conclusion:We found that triple or double positive aPL profiles are not superior to single LAC positivity in their association with any thrombosis in SLE patients.References:[1]Pengo V, Ruffatti A, Del Ross T, Tonello M, Cuffaro S, Hoxha A, Banzato A,Bison E, Denas G, Bracco A, Padayattil Jose S. Confirmation of initial antiphospholipid antibody positivity depends on the antiphospholipid antibody profile. J Thromb Haemost. 2013 Aug;11(8):1527-31.[2]Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk factors for thrombosis than anticardiolipin antibodies in the antiphospholipid syndrome: a systematic review of the literature. Blood. 2003; 101(5):1827-32.Disclosure of Interests:Selcan Demir: None declared, Jessica Li: None declared, Laurence Magder: None declared, Michelle A Petri Grant/research support from: GSK, Eli Lilly and Company, Consultant of: Eli Lilly and Company
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Porter, Andrew, and Natalia Beglova. "Pharmacokinetics, Serum Stability and Immunogenicity of A Polypeptide Inhibitor of B2GPI/Antibody Complexes Tested in Mice." Blood 120, no. 21 (November 16, 2012): 2205. http://dx.doi.org/10.1182/blood.v120.21.2205.2205.
Повний текст джерелаАнотація:
Abstract Abstract 2205 Background: Antiphospholipid syndrome (APS) is an autoimmune disease with clinical features of thrombosis and pregnancy loss. Beta2-glycoprotein I (B2GPI) is the major antigen for APS-related antibodies. We engineered a polypeptide consisting of two ligand-binding A1 modules from the ApoE receptor 2. Previously, we demonstrated that this polypeptide, A1-A1, preferentially binds B2GPI/antibody complexes compared to B2GPI alone and efficiently inhibits the binding of B2GPI/antibody complexes to negatively charged phospholipids. Therefore, A1-A1 effectively interferes with two pathological mechanisms of B2GPI/antibody complexes: the binding to anionic phospholipids and ApoER2. In order to use A1-A1 to study pathological mechanisms of B2GPI/antibody complexes in vivo, we tested its pharmacokinetic, serum stability and immunogenicity in mice. To visualize A1-A1, we labeled it with a fluorescent probe Atto-488 attached to the N-terminus. Results: We monitored clearance of A1-A1 from the circulation after intraperitoneal and intravenous administration. After intraperitoneal administration, the concentration of A1-A1 in the blood reached its maximum at 30 min after injection and cleared from the blood in 6–8 hours. When A1-A1 was injected intravenously, 14% of A1-A1 remained in the blood 1 hour after administration and decreased to 4% in 3 hours. We assessed the binding of A1-A1 to serum proteins in both mouse and human serum by gel-filtration chromatography. Chromatograms of A1-A1 in both mouse and human serum collected just after mixing of A1-A1 with serum were almost identical to those collected after 2 hours of incubation at 37° C. About 90% of A1-A1 stays free from serum proteins. Previously, we demonstrated that A1-A1 has a favorable stability in human serum. More than 35% of A1-A1 remained in human serum after 15 days of incubation at 37° C. Here, we determined whether A1-A1 is cleaved by proteases in mouse serum. Degradation of A1-A1 was monitored by the reversed-phase HPLC by comparing the peak corresponding to intact A1-A1 and A1-A1 incubated with serum for 2 hours at 37° C. After incubation with mouse serum, A1-A1 eluted at the same time as intact A1-A1 and the intensity of the elution peak did not decrease, indicating that A1-A1 remains intact in mouse serum. To evaluate immunogenecity of A1-A1, we immunized mice with A1-A1, A1-A1 in the presence of adjuvant and A1-A1 conjugated to a carrier protein. A1-A1 did not induce detectable anti-A1-A1 IgG production even in the presence of adjuvant or carrier protein. Conclusions: A1-A1 has favorable properties for use in vivo. It stays in the circulation for more than one hour following intravenous injection. After intraperitoneal administration, A1-A1 is rapidly absorbed into blood and cleared in about 6 hours. A1-A1 is resistant to both human and mouse proteases, has low immunogenicity and its amount in the blood is not depleted by binding to serum proteins. Disclosures: No relevant conflicts of interest to declare.
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Lenting, Peter J., Janine J. Hulstein, Bas de Laat, Ronald H. Derksen, Rob Fijnheer, and Philip G. de Groot. "Beta2-Glycoprotein I Interferes with von Willebrand Factor-Dependent Platelet Adhesion." Blood 108, no. 11 (November 16, 2006): 415. http://dx.doi.org/10.1182/blood.v108.11.415.415.
Повний текст джерелаАнотація:
Abstract Patients with the antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity with the presence of antibodies against phospholipid-binding proteins, such as beta2-Glycoprotein (beta2-GPI) or prothrombin. In particular, antibodies against beta2-GPI strongly correlate with thrombotic complications. One model that explains this correlation involves an antibody-induced gain-of-function of beta2-GPI, which results in prothrombotic properties of this plasma protein. In an alternative model, beta2-GPI may display anti-thrombotic properties that disappear in an antibody-dependent manner. Of course, both possibilities may occur simultaneously. It should be noted, however, that little is known about the physiological function of beta2-GPI. Several in vitro-based studies have pointed to beta2-GPI as a potential inhibitor of fibrinolysis and/or the intrinsic pathway of coagulation. In the present study, we investigated the hypothesis that beta2-GPI affects platelet function. Addition or immune-depletion of beta2-GPI from platelet-rich plasma had little effect on ADP- or collagen-induced platelet aggregation, whereas it did modulate ristocetin-induced platelet aggregation. In a system employing washed platelets, beta2-GPI completely inhibited platelet aggregation induced by VWF/ristocetin or by a recombinant VWF type 2B mutant (VWF/R1306Q). This suggests that beta2-GPI is directed to the von Willebrand (VWF)-glycoprotein Ib axis. Indeed, beta2-GPI interfered with platelet adhesion to VWF-coated coverslips under conditions of flow as well, resulting in a 2-fold reduction of platelet coverage. In direct binding studies, wt-VWF displayed weak binding to immobilized beta2-GPI. However, conversion of latent VWF into a platelet-binding conformation (either via ristocetin or via a type 2B mutation) induced efficient, dose-dependent and saturable binding of VWF to beta2-GPI. By using a number of recombinant deletion-mutants and individual domains, we found that binding was mediated by the VWF A1 domain. Interestingly, anti-beta2-GPI antibodies isolated from patients with the antiphospholipid syndrome not only interfered with the interaction between beta2-GPI and VWF, but also neutralized the inhibitory effect of beta2-GPI towards VWF-dependent platelet aggregation. Finally, we analysed plasma of antiphospholid-syndrome patients for the presence of VWF that is in its platelet-binding conformation by using a specific nanobody (Hulstein et al (2005) Blood106:3035). Levels of active VWF were increased 1.7-fold in patients compared to healthy individuals or patients that lack beta2-GPI-directed antibodies (p<0.0001). This increase in active VWF is similar to that observed in patients with acquired TTP. In conclusion, we demonstrate that beta2-GPI interacts preferentially with the platelet-binding conformation of VWF, thereby inhibiting VWF-dependent platelet adhesion and aggregation. Anti-beta2-GPI antibodies obtained from antiphospholipid-syndrome patients reverse this anti-thrombotic effect of beta2-GPI. We propose that beta2-GPI functions as a natural first-line barrier that prevents small amounts of active VWF to interact with platelets.
Дисертації з теми "Beta2 glycoprotein 1 antibody"
1
Gatenby, Paul, Gytis Danta, Roger Tuck, Colin Andrews, and Carolyn Hawkins. "Cerebrovascular disease associated with antiphospholipid antibodies: more questions than answers." Master's thesis, BioMed Central, 2006. http://hdl.handle.net/10440/104.
Повний текст джерелаАнотація:
Neurological syndromes occur in a significant number of patients with antiphospholipid antibodies. The
optimal management for these patients however remains uncertain.
Our study is a descriptive analysis looking retrospectively at 45 patients who presented to the principal
tertiary referral centre in the Australian Capital Territory, with either cerebral arterial or venous
thrombosis for which there was no obvious cause for their presentation when initially reviewed. The
diagnosis was based on the clinical findings made by one of three neurologists attached to our centre.
Radiological findings and the presence of either IgM or IgG anticardiolipin antibodies, IgG anti-beta-2
glycoprotein 1 antibodies or a lupus anticoagulant were then documented.
In this group of patients three subgroups were identified:
1. Individuals that fulfilled the Sapporo Classification Criteria
2. Individuals with transiently positive antiphospholipid antibodies and
3. Individuals with persistently low positive antiphospholipid antibodies.
The most interesting of these three groups are those individuals with transiently positive antiphospholipid
antibodies. A potential cause for presentation was identified in only one patient of this group with
documented infective endocarditis and bacteraemia. Comparison with the other two groups suggested
that there was little in terms of clinical presentation, radiological findings or intercurrent risk factors for
thrombotic disease to distinguish between them. With disappearance of antiphospholipid antibodies, the
individuals within this group have not had further thrombotic events.
Our observations emphasise the problems that continue to exist in relation to the occurrence of
cerebrovascular disease in the context of antiphospholipid antibodies and the optimal management of
these stratified groups. Our findings also raise an as yet unanswered question as to the signficance of these
transiently positive antiphospholipid antibodies. In the absence of significant intercurrent risk factors our
findings would suggest that in the group we describe that they are likely to be of clinical significance.
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Swers, Jeffrey Seth. "Isolation and engineering of a high affinity antibody against P-selectin glycoprotein ligand-1 (PSGL-1)." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/32323.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2005.Vita.
Includes bibliographical references (leaves 87-91).
We aim to develop novel protein antagonists of P-selectin adhesion as anti- inflammatory therapeutics. Blocking P-selectin adhesion is particularly attractive because this adhesion mediates leukocyte rolling which occurs early in the inflammatory cascade before extensive tissue damage caused by the amplification of inflammation by proinflammatory cytokines. Currently, no subnanomolar antagonists of selectin adhesion are available. The low affinity of current antagonists results in the need for frequent administration and large doses in order to obtain inhibition. High affinity antagonists are desirable because they can be administered in smaller amounts thus reducing the risk of harmful side effects and reducing production costs. Our approach for developing high affinity antagonists is to combine error prone PCR and in vivo homologous recombination to mimic in yeast the broad spectrum of mutagenic strategies exploited by B cells such as somatic hypermutation, receptor revision (... CDR replacement), receptor editing (chain shuffling), and amino acid insertions and deletions. Together with yeast surface display and flow cytometric screening (FACS), this approach has been used to effect at least a five order of magnitude affinity improvement in a single chain antibody (scFv) directed against the N-terminal 19 amino acids of P-selectin glycoprotein ligand- 1 (PSGL- 1). Three rounds of engineering were performed after an initial pool of binders was isolated from a non-immune scFv library. Chain shuffling was found to be important for generating an improved mutant in the first round of engineering.
(Cont.) For the final round of engineering, four different libraries were generated: one with random mutations, one with preferential replacement of the ... CDR1, one with preferential replacement of the ... CDR1 and the ... CDR2, and one with preferential replacement of the light chain. All of these methods produced two order of magnitude affinity improvements except the light chain exchange library. However, the CDR exchange libraries gave equivalent affinity improvement despite the fact that they were 77 fold smaller than the random mutagenic library. In addition, an insertion in CDR2 of the VH was isolated in the best binder from both of the CDR exchange libraries and this mutation could not have been found through random mutagenesis. These results suggest that chain shuffling is best used when the affinity of the antibody to be matured is weak (> 1 [mu] M). In addition, receptor revision is an equally robust method as random mutagensis for the generation of ultra-high affinity binders. The best antibody from the library with preferential replacement of ... CDR1 and ... CDR2 was converted to an IgG and characterized. It was found to better inhibit P-selectin binding to PSGL-1 than the commercially available antibody KPLI in a static adhesion assay and an in vitro rolling assay. Our integrated approach, made possible by in vivo homologous recombination in yeast, decreases the likelihood of convergence upon a single high affinity solution and increases the probability of obtaining an antibody with desired secretory properties and therapeutic potential. This facile method for combining all the mutational strategies used in nature should prove as a valuable tool in the antibody engineering field.
by Jeffrey Seth Swers.
Ph.D.
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Jenette, Kankanamge Pushpa. "Studies on the Epitope of Rabies Virus Glycoprotein Recognized by a Monoclonal Antibody #1-30-44." 京都大学 (Kyoto University), 2003. http://hdl.handle.net/2433/149174.
Повний текст джерела
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Khati, Makobetsa. "Macrophage-HIV interactions : aptamers against the gp120 surface envelope glycoprotein of the macrophage tropic strains of HIV-1." Thesis, University of Oxford, 2002. http://ora.ox.ac.uk/objects/uuid:a32becf2-bf5d-4428-b598-e8057d977fbd.
Повний текст джерелаАнотація:
HIV-1 has evolved a number of strategies in response to current anti-retroviral drugs and the selection pressure of humoral and cellular immunity. In particular, R5 viral strains that are essential for AIDS pathogenesis are very resistant to neutralization by antibodies. Therefore, the aim of this thesis was to develop synthetic nucleic acid ligands, aptamers, against gp120 of an R5 strain of HIV-1, with a view of using aptamers as novel neutralization molecules and analytical tools to study HIV-1 entry into target cells. The central hypothesis of this thesis was that aptamers by virtue of their small size and slow dissociation rates, compared to antibodies, would easily access and bind occluded gp120 neutralization sites. Using the SELEX protocol and SPR technology, I isolated 2'-Fluoro-pyrimidine-RNA aptamers against HIV-lBa-L monomeric gp120. Most of these aptamers not only bound gp120 with high affinities but also neutralized R5 primary isolates in human PBMC by 1,000 to 100,000-fold, truly unprecedented when compared with natural ligands such as antibodies. Some aptamers, like B4, defined a conserved site of gp120 that could not mutate to escape neutralization following stringent selection, in vitro, for breakthrough virus. This was consistent with subsequent findings that B4 aptatope (binding site) overlaps a poorly immunogenic but highly conserved CD4-induced epitope as determined by competition with 17b and 48d mAbs that map to this neutralization epitope on the gp120. This study was thus the first of its kind to describe neutralization of HIV-1 primary isolates by a ligand against the CD4-induced epitope. Most intriguing, although B4 potently neutralized HIV-1Ba-L infection in PBMC, which is a mixed T cell and macrophage population, it modestly neutralized infection of the same virus in a purified culture of macrophages. These findings are intriguing in that they suggest that aptamers could be used to dissect unique sites on the virus that interact with target cell surface in ways that have not been revealed heretofore, and would help understand better HIV-1 entry pathways, especially in macrophages. Thus neutralizing aptamers such as these could be exploited to provide leads in developing alternative anti-HIV-1 drugs and a deeper understanding of the molecular interactions between the virus and its host cell.
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van, Diepen Michiel Theodoor. "Generation and characterization of HIV-1 subtype C candidate vaccines that will induce high titre antibody responses to HIV-1 envelope glycoprotein." Doctoral thesis, Faculty of Health Sciences, 2021. http://hdl.handle.net/11427/33091.
Повний текст джерелаАнотація:
Despite huge strides being made towards decreasing the number of individuals getting newly infected with HIV-1, and in reducing AIDS-related deaths, unfortunately current predictions are that the 2020 UNAIDS goals (90-90-90 targets, where 90% of people living which HIV-1 are diagnosed as such, from which 90% will will receive sustained antiretroviral therapy, resulting in viral suppression in 90% of these individuals by 2020) are out of reach. This of course means that the numbers of newly infected indivuals and AIDS-related deaths will be above the target derived from the 2020 UNAIDS goals. The development of an effective HIV vaccine could therefore be an important step towards realising these objectives. In work done for this thesis, a heterologous HIV-1 vaccine platform regimen was developed using antigen sequences from the predominant circulating HIV-1 subtype (subtype C) in South Africa. Specifically, this involved use of the envelope glycoprotein sequence of the CAP256 superinfecting virus (CAP256_SU) from the CAPRISA 002 cohort, and a mosaic Gag sequence which resulted in robust autologous Tier 2 neutralisation of CAP256_SU pseudovirions. The envelope glycoprotein sequence was modified so as to replace the native leader sequence with the tissue plasminogen activator leader, the furin cleavage site with a glycine rich flexible linker, and to introduce an I559P mutation. DNA and modified vaccinia virus Ankara (MVA) vaccines were generated where Env was truncated to gp150, thereby retaining the transmembrane domain and a partial cytoplasmic tail (Env). The Env sequence for the protein vaccine was further trimmed by removal of the transmembrane domain to give gp140, leading to a soluble, secreted protein (soluble Env). This allowed for the latter vaccine to be affinity purified using lectin (soluble Env (GNL)), and after generating stable cell lines, soluble Env yields were high enough to enable size exclusion chromatography which allowed isolation of the trimeric fraction of Env as determined by molecular weight (soluble trimeric Env). A Cterminal His-tagged version of soluble Env was generated as well. Surprisingly, the folding of Env-His was inferior to soluble Env, with a switch in profile from mainly trimeric Env to mainly monomeric Env. Nevertheless, soluble Env-His (GNL) and soluble trimeric Env-His were assessed for the presence of Env broadly neutralising antibody (bnAb) epitopes in an ELISA assay. The V3-glycan supersite (binding of bnAbs PGT128 and PGT135), the CD4-binding site (VRC01) and the V2-glycan site (PG9) were detected for both Env-His (GNL) and soluble trimeric proteins, whereas low signals for PG16, PGT145 and CAP256-VRC26.08, bnAbs which specifically recognise Env trimers in a native-like conformation, were only detectable for soluble trimeric Env-His. Soluble Env (GNL) was subsequently used as a protein vaccine in rabbits to test the immunomodulatory effects of the two adjuvants AlhydroGel (similar to alum) or the MF59-like squalene-based oil-in-water nano-emulsion AddaVax. Soluble Env (GNL) adjuvanted in AlhydroGel resulted in improved immune response in rabbits, with significantly higher serum binding antibodies to soluble Env (GNL) and scaffolded CAP256 V1V2-loop in comparison to AddaVax and unadjuvanted protein. Furthermore, significantly higher neutralisation titres to Tier 1A subtype C virus (MW965.26), in combination with an improved breadth to subtype C Tier 1A and 1B viruses, were observed in the AlhydroGel group. However, no neutralisation of Tier 2 viruses was detected. Nonetheless, AlhydroGel was selected as the best protein adjuvant for all further rabbit immunogenicity studies. Furthermore, in all subsequent experiments, soluble trimeric Env was used as a protein vaccine. DNA and recombinant MVA vaccines were generated using a membrane anchored gp150 (Env) with the aim that co-expression with mosaic Gag (GagM) would lead to the incorporation of Env into Gag virus-like particles (VLPs). Electron microscopy of cells expressing Env+GagM from DNA and recombinant MVA vaccines verified VLP formation from these constructs, and the presence of Env was observed in VLPs purified using a two-step OptiPrep gradient centrifugation protocol. The presence of Env bnAb epitopes in cellular membrane-bound Env was verified by qualitative immunofluorescent microscopy of live-cell stainings and a quantitative FACS assay. The same bnAb epitopes as for the Env protein vaccine were detectable, including bnAbs recognising only native-like Env trimers (PG16, PGT145 and CAP256-VRC26_08). However, expression levels of native-like Env trimers were lower, at approximately 20% when normalised to VRC01. These HIV-1 DNA, rMVA and soluble trimeric Env protein vaccines were tested in different heterologous vaccine platform immunogenicity studies in rabbits. These consisted of either priming with two recombinant MVA vaccines and boosting with three protein vaccines (MMPPP), or priming with DNA vaccines followed by two MVA vaccines, followed by two protein vaccines (DDMMPP). Furthermore, the inclusion of GagM into the DNA and MVA vaccines was compared to use of Env alone. Both vaccine regimens resulted in binding antibodies to soluble trimeric Env and a scaffolded CAP256 V1V2-loop; however, these were induced by MVA and protein vaccines, but not by DNA vaccines. Despite the lack of Env binding antibodies after DNA vaccination, better neutralisation was observed for the DDMMPP regimen compared to MMPPP, resulting in higher sera neutralisation titres towards vaccinematched, autologous Tier 2 CAP256_SU virus. Most encouragingly, when compared to Env alone, the inclusion of Gag (Env+GagM) into DNA and MVA vaccines improved the immunogenicity of the DDMMPP regimen even further. For Env+GagM DDMMPP, more animals developed Tier 2 neutralising antibodies, and improved titres, whereas Tier 2 neutralisation in general started to develop after fewer vaccinations, as for most rabbits this was observed after the second MVA inoculation. In an attempt to improve the spike density of Env on VLPs and the plasma membrane, two Env chimaeras were made replacing parts of gp41 with the corresponding elements of influenza A H5 haemagglutinin (HA2) (Env:HA2 chimaeras). Increased Env spike density was observed in a previous study using this strategy for the gp41 transmembrane domain and cytoplasmic tail (gp140HA2tr). A similar construct was generated here for CAP256_SU and a second chimaera was included replacing the whole of gp41 with HA2 (gp120HA2). Surprisingly, in experiments where VLPs were purified from OptiPrep gradients or the whole-cell bnAb FACS assay conducted with these Env:HA2 chimaeras, there was no evidence of increased spike density on VLPs or the plasma membrane as compared to Env. Furthermore, the folding of Env was severely impacted, especially regarding gp120HA2 where no binding of PG16, PGT145 and CAP256 VRC26.08 - bnAbs recognising native-like Env trimers - was observed. Although results for gp140HA2tr was improved over gp120HA2, in general the data for gp150 (Env) was superior in both the bnAb live-cell staining and FACS assay. Consequently, when both Env:HA2 chimaeras in combination with GagM were tested in the DDMMPP regimen, no improvement was observed with regard to autologous Tier 2 neutralisation. For rabbits receiving gp120HA2, no animals developed Tier 2 nAbs, whereas for gp140HA2tr, Tier 2 neutralisation in general developed later and to lower titres compared to Env+GagM. In conclusion, different HIV-1 DNA, recombinant MVA and protein vaccines were generated and characterised both in vitro and in vivo, leading to a vaccination regimen that induced both high titre Env binding and vaccine-matched Tier 2 neutralising antibodies in rabbits. Furthermore, a new Env sequence, the first from the South African CAPRISA cohort, has been added to the small list of Env sequences that can induce Tier 2 neutralisation.
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Yuan, Tingting, and 袁婷婷. "Identification of intermediate antibodies of broadly neutralizing HIV-1 human monoclonal antibody b12 and characterization of variable loops of HIV-1 envelop glycoprotein." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/196445.
Повний текст джерела
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Johnson, Jacklyn. "Properties of HIV-1 env and human seminal fluid that determine virus inhibition by antibodies and microbicides." Diss., University of Iowa, 2019. https://ir.uiowa.edu/etd/6966.
Повний текст джерелаАнотація:
Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection that leads to acquired immunodeficiency syndrome (AIDS). Approximately 36 million people worldwide are living with HIV-1, which is commonly acquired through sexual contact. Antiviral therapies control disease progression, but do not eliminate this virus from the host. Thus, global efforts are focused on developing vaccines that prevent HIV-1 transmission. Such vaccines are based on eliciting the production of protective antibodies that target the envelope glycoproteins (Envs) of this virus. Unfortunately, HIV-1 immunization trials have shown limited efficacy. A better understanding of the antibody-mediated inactivation process is needed to improve vaccine strategies. In this work we describe two novel factors that contribute to HIV-1 inactivation. First, we show that structural stability of the Env protein determines its sensitivity to vaccine-elicited antibodies. Different interactions within Env contribute to its stability. Perturbation of the Env-stabilizing interactions by physical and chemical treatments enhances sensitivity of HIV-1 to antibodies. Second, we found that the chemical composition of the transmission medium affects Env inhibition by antibodies and other inhibitory agents. Semen is the most common vehicle for HIV-1 transmission. This medium contains high concentrations of the sugar fructose. We found that semen fructose competitively blocks binding of antiviral agents that target sugar residues on Env. Together, this work advances our understanding of the mechanism that underlies HIV-1 inactivation by vaccine-elicited antibodies and provides novel strategies to enhance their potency.
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Howard, Christopher Bruce. "Identification of epitopes on the Dengue virus type 4 envelope glycoprotein involved in neutralisation by antibodies." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16401/1/Christopher_Howard_Thesis.pdf.
Повний текст джерелаАнотація:
Dengue virus (DENV) is the causative agent of dengue fever (DF), the most prevalent arthropod-borne viral disease in the world and therefore is considered an emerging global health threat. The four DENV serotypes (DENV-1, DENV-2, DENV-3 and DENV-4) that infect humans are distinguished from one another by unique antigenic determinants (epitopes) on the DENV envelope (E) protein. The E protein is the primary antigenic site of the DENV and is responsible for inducing neutralising antibody (Ab) and cell mediated immune response in DENV infected hosts. The DENV E protein also mediates attachment of virions to host cell receptors and entry of virions into host cells by membrane fusion.
The study of epitopes on DENV E protein is necessary for understanding viral function and for the design of unique polyvalent vaccines capable of inducing a neutralising antibody response against each DENV serotype. Reverse genetics using infectious cDNA clones has enabled the construction of functional intertypic DENV, where the E protein of one DENV serotype is put in the genetic background of a different DENV serotype. In addition, observations from our laboratory indicate that chimeric E proteins, consisting of E protein structural domains from different DENV serotypes can fold into functional proteins. This suggests that there is potential to engineer viruses with intertypic DENV E proteins as potential DENV vaccine candidates, which is the long term goal of studies within our research group. However, if a chimeric E protein was to be constructed containing epitopes involved in antibody mediated neutralisation of each DENV serotype, then knowledge of the location of these epitopes on the E protein of each DENV serotype would be essential.
Prior to this study, monoclonal antibodies (MAbs) had been used to identify epitopes involved in antibody mediated neutralisation on the E protein of all DENV serotypes, except DENV-4. The primary objective of this study was to identify epitopes on the DENV-4 E protein involved in neutralisation by antibodies. In order to achieve this objective, a panel of 14 MAbs was generated against DENV-4 in BALB/c mice and characterised using various serological and functional assays.
The identification of DENV-4 specific neutralising MAbs in the panel was essential for subsequent experiments aimed at determining antigenic domains, structural domains or specific epitopes (peptides or amino acids) involved in the neutralisation of DENV-4.
The majority of MAbs (11/14) generated against DENV-4 recognised the E protein. The remaining three MAbs reacted with the non-structural (NS) 1 protein. The majority of MAbs against the E protein were DENV or Flavivirus group reactive, but four MAbs were DENV-4 specific. All MAbs against the E protein recognised conformationally dependent epitopes and were able to capture DENV-4 in an enzyme linked immuno-adsorbent assay (ELISA).
Eighty percent (9/11) of the anti-E MAbs produced for this study neutralised infection of cells by DENV-4 in vitro. Three of the neutralising MAbs (F1G2, 18F5 and 13H8) were DENV-4 specific and also demonstrated the strongest neutralisation activity of the panel, reducing DENV-4 infectivity by 100-1000 fold. The amount of virus neutralised by the MAbs was not related to the avidity of the MAbs. The DENV-4 specific MAbs F1G2, 18F5 and 13H8 were used to identify epitopes involved in neutralisation of DENV-4.
The MAbs that effectively captured DENV-4 were used in competitive binding assays (CBAs) to determine spatial relationships between epitopes and therefore define antigenic domains on the DENV-4 E protein. The CBAs indicated that the epitopes recognised by the panel of MAbs segregated into two distinct domains (D4E1 and D4E2) and both contained epitopes involved in neutralisation. CBAs incorporating human serum from DENV-4 infected patients suggested that the MAbs recognised the same, or spatially related, epitopes in domain D4E2 as antibodies from humans who had experienced natural dengue infections, indicating the clinical relevance of such epitopes for the development of DENV vaccines. The reactivity of the capture MAbs with low pH treated DENV-4 was also evaluated in an attempt to identify epitopes that might be more accessible during low pH-mediated virus fusion. Only one of the MAbs (13H8) recognised an acid resistant epitope.
Initial attempts to identify epitopes on the DENV-4 E protein involved in neutralisation followed the traditional epitope mapping approach of selecting subpopulations of DENV-4 which escaped neutralisation by MAbs. These attempts were unsuccessful so a variety of strategies for mapping epitopes were used including DENV-4 variant analysis and site directed mutagenesis of the DENV-4 E protein, MAb screening of chimeric DENV-3/4 E proteins and MAb screening of a bacterial peptide display library.
DENV-4 variants including DENV-4 isolates from different geographical locations or chemically mutagenised DENV-4 were screened with neutralising MAbs to identify neutralisation escape mutant (n.e.m.) viruses. Site directed mutagenesis of the DENV-4 E protein confirmed whether amino acid changes identified in DENV-4 n.e.m.s were essential for the binding of neutralising MAbs to an epitope.
The MAb screening of DENV-4 variants identified n.e.m.s with amino acid changes at residues E95, E96, E156, E157, E203, E329 and E402 of the DENV-4 E protein. Site directed mutagenesis of the DENV-4 E protein identified two epitopes recognised by the DENV-4 specific neutralising MAbs F1G2 and 18F5 at specific amino acid residues within domains II and III of the DENV-4 E protein. No specific epitopes were identified for the MAb 13H8; however this MAb did recognise domain I and II of the DENV-4 E protein, when screened against DENV-3/4 chimeric DENV E proteins.
The first epitope, which was recognised by the MAb F1G2, contained residue E95 which was located in domain II of the DENV-4 E protein. The aspartate (Asp) to alanine (Ala) change at E95 prevented the binding of F1G2 to the DENV-4 E protein. The binding of F1G2 to the E95 residue was confirmed using the pFlitrX bacterial peptide display library, which demonstrated binding of F1G2 to a peptide homologous with residues E99-E104. No peptides recognised by 13H8 and 18F5 were identified by this method. The MAb F1G2 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric DENV E proteins. This implied that F1G2 may be recognising a discontinuous epitope consisting of domains II and III.
The second epitope, which was recognised by MAb 18F5, contained residue E329 which was located in domain III of the DENV-4 E protein. The alanine (Ala) to threonine (Thr) change at E329 prevented the binding of 18F5 to the DENV-4 E protein. MAb 18F5 also bound to the domain III region (E300-E495) of the DENV-4 E protein when screened against DENV-3/4 chimeric E proteins, thus confirming the E329 epitope.
The potential mechanisms by which the DENV-4 specific MAbs neutralise virus infection were evaluated by the virus overlay protein binding assay (VOPBA). The binding of MAb 18F5 to a domain III (E329) epitope of the DENV-4 E protein and the binding of MAb F1G2 to domain II (E95, E99-E104) and domain III epitopes (chimeric E protein) of the DENV-4 E protein, prevented the attachment of DENV-4 to a 40 kDa C6/36 cell protein. In contrast the binding of MAb 13H8 to domains I and II of the DENV-4 E protein did not prevent attachment of DENV-4 to the same protein.
This was preliminary evidence that the binding of domain III epitopes by the MAbs F1G2 and 18F5 may be important in preventing virus attachment. The binding of MAb 13H8 to domains I and II, and the ability of this MAb to recognise DENV-4 treated at low pH, suggested that MAb 13H8 may block epitopes exposed at low pH that are required for low pH mediated virus fusion to host cell membranes.
Overall, the different methods used in this study identified epitopes involved in the neutralisation of DENV-4. The distribution of epitopes involved in neutralisation throughout the DENV-4 E protein were similar to the distribution of epitopes involved in neutralisation on the DENV-1, 2 and 3 E proteins. This suggested that it might be possible to elicit neutralising antibodies against multiple DENV serotypes using chimeric E-proteins derived from two or more DENV serotypes and therefore, facilitate the design of novel tetravalent DENV vaccines.
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Matz, Julie. "Développement de fragments d' anticorps simple-domaine inhibiteurs ciblant les protéines structurales et enzymatiques du VIH 1." Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4027.
Повний текст джерелаАнотація:
Le VIH-1 est l'agent infectieux qui cause le SIDA. De nombreuses thérapies existent pour combattre le SIDA mais aucune ne permet son éradication et des résistances apparaissent. Le développement de nouvelles thérapies est donc nécessaire. Les anticorps simple-domaine (sdAb) de lamas présentent les propriétés idéales pour le développement de molécules neutralisantes. Des lamas ont donc été immunisés avec Vpr et les formes native, ou induite par un miniCD4, du trimère de gp140 (partie extracellulaire de l'enveloppe (Env)). Des banques de sdAbs ont ensuite été construites et des sélections par phage display et par double hybride ont été réalisées. Trois sdAbs se liant au site de liaison du co- récepteur de l'Env et un sdAb se liant au site de liaison du CD4 ont ainsi été sélectionnés. Ces sites sont conservés mais difficile d'accès pour des immunoglobulines conventionnelles. Ces sdAbs ont ensuite été caractérisés par ELISA, SPR et cytométrie de flux pour leur capacité de liaison à différentes Env, et en « single round assay » pour leur capacité de neutralisation d'un large spectre (LS) de pseudovirus. Des protéines multidomaines (plusieurs sdAbs reliés par un linker) ont ensuite été construites et testées pour leur neutralisation. Plusieurs de ces molécules, neutralisant un LS de virus, pourraient être utilisées dans des microbicides. La stabilité caractéristique des sdAbs, même en absence de formation de pont disulfure, par exemple dans un environnement réducteur tel que le cytoplasme, est primordiale dans le développement d'anticorps intracellulaires (intrabodies)HIV-1 is the infectious agent of AIDS. Numerous therapies exist to fight AIDS, but they are not able to eradicate it, and resistances appear. So, new therapy development is necessary. Single-domain antibodies (sdAb) of llamas have ideal properties to develop neutralizing molecules. So, llamas have been immunized with Vpr and with free or miniCD4 induced trimeric gp140 (extracellular part of the envelope (Env)). SdAb libraries have been built and selections were done by phage display and yeast two hybrid. Three sdAbs targeting the co-receptor binding site of the Env and one sdAb targeting the CD4 binding site have been selected. These sites are conserved but inaccessible by conventional immunoglobulins. These sdAbs have been characterized by ELISA, SPR and FACS for their ability to bind different Env and by single-round assay for their neutralization ability. Multimeric proteins (linked sdAbs) have been built and tested for their neutralization ability. Several of these molecules are able to neutralize a broad spectrum of pseudoviruses. They can be used in microbicides. The characteristic stability of these sdAbs, even without disulfide bound formation, ie into reducing environment, as the cytoplasm, is primordial for intracellular antibody (intrabody) development. One sdAb anti-Vpr has been selected using the Sos Recruitment System (SRS), an yeast two-hybrid system allowing detection of cytoplasmic protein-protein interactions. This sdAb is able to alter the localization of its antigen into eukaryotic cells. It is a proof of concept ot the use of SRS in the selection of intracellularly functional sdAbs
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Pereira, Ivânio Alves. "Aterosclerose na artrite reumatóide e sua associação com auto-imunidade humoral." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5145/tde-03052007-101757/.
Повний текст джерелаАнотація:
Objetivos: Muitas questões permanecem sobre as causas da aterosclerose acelerada nos pacientes com doenças inflamatórias sistêmicas como a artrite reumatóide (AR). Estudos na população geral sugeriram que além da inflamação existe uma participação patogênica da auto-imunidade na aterosclerose e discutem a possível associação dos anticorpos contra fosfolípides e proteínas de choque térmico (Hsp). O objetivo deste estudo foi investigar a presença de anticorpos contra fosfolípides, beta2-glicoproteína 1 (beta2-gp1), lipoproteína lipase (LPL) e Hsp em pacientes com AR e avaliar a associação entre estes anticorpos com a presença de aterosclerose subclínica de carótidas. Métodos: Anticorpos contra cardiolipina (aCL) IgG e IgM, beta2-gp1 IgG, IgM e IgA , Hsp 60 e Hsp 65 foram testados por ELISA em um grupo de 71 pacientes com AR comparado com 53 indívíduos controles não portadores de AR, de idade e sexo similar. Foram excluídos os pacientes com HAS, diabetes melitos e os fumantes em ambos os grupos. Níveis de lipoproteínas, parâmetros clínicos da AR, questionário de avaliação de saúde (HAQ), escore de atividade da doença (DAS) 28, velocidade de hemossedimentação (VHS) e proteína C reativa (PCR) foram avaliadas. A associação entre a presença dos anticorpos aCL, beta2-gp1, Hsp 60 e Hsp 65 com os parâmetros clínicos de atividade da doença, com a presença das placas de aterosclerose e com a medida da espessura íntimomedial (IMT) da carótida comum, usando ultra-som (US) modo B de alta resolução foram pesquisadas. Resultados: A idade média no grupo com AR foi 48,93 ± 12,31 vs. 45,37 ± 9,37 no grupo controle saudável (p = 0,20); 90,1% no grupo com AR eram do sexo feminino vs. 86,8% no grupo controle (p = 0,56); índice de massa corporal (IMC) foi 25,72 ± 4,57kg/m² no grupo com AR vs. 26,40 ± 4,52kg/m² no grupo controle (p = 0, 69); Os níveis de colesterol, LDL, triglicerídeos e a relação CT/HDL não foram diferentes quando comparamos os 2 grupos (p > 0,05). O nível de HDL foi maior no grupo com AR vs. grupo controle com 60,56 ± 14,40mg/dl e 54,52 ± 11,55 respectivamente (p = 0,05). A média da medida da IMT foi 0,721 ± 0,16 mm na AR e 0,667 ± 0,14mm no grupo controle, e a IMT dos pacientes com AR foi maior naqueles com idade acima dos 50 anos (P < 0,001). No grupo com AR, 14,1% dos pacientes tinham placas nas carótidas vs. 1,9% dos indivíduos saudáveis (p = 0,02) e no grupo com AR, as placas foram mais frequentes nos pacientes acima dos 50 anos (p = 0,004). No grupo AR, 5,6% tinham anticorpos aCL IgG vs. 3,8% no grupo controle (p > 0,05); 14,1% apresentavam aCL IgM vs. 7,5% (p > 0,05); 43,7% tinham anti-beta2-gp1 IgA vs. 40,8% no grupo controle (p > 0,05). A prevalência de anti-beta2-gp1 IgG e IgM e anti-LPL não foi diferente entre os pacientes com AR e o grupo controle ( p > 0,05). A presença dos anticorpos anti-Hsp 60 e 65 na AR e no grupo controle não foram diferentes (p > 0,05), mas os títulos de anticorpos contra Hsp 65 e beta2-gp1 IgM foram maiores no grupo com AR ( p = 0,007 e p = 0,03 respectivamente). Nós não encontramos associação entre a presença e os títulos dos anticorpos aCL IgG e IgM, beta2-gp1 IgG, IgM e IgA, LPL e Hsp 60 e 65 com a presença de placas nas carótidas ou com a medida da IMT (p > 0,05). Discussão: Este estudo confirma achados anteriores da maior prevalência de aterosclerose carotídea nos pacientes com AR e sua correlação com idade, colesterol e LDL. Embora tenha se encontrado uma tendência a maior presença de anticorpos nos pacientes com AR, não houve relação entre a presença da aterosclerose mais prevalente nos pacientes com AR, com a auto-imunidade dirigida contra cardiolipina, beta2-gp1 ou Hsp.Purpose: Many questions remain unanswered about the causes of accelerated atherosclerosis in patients with inflammatory systemic diseases such as rheumatoid arthritis (RA). Some studies have suggested the role of autoimmunity besides inflammation in the pathogenesis of atherosclerosis in general population and have also discussed the possible association with antibodies directed to phospholipids and heat shock proteins (Hsp). The aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoprotein1 (beta2-gp1), lipoprotein lipase (LPL) and Hsp in RA subjects and evaluate the association between these antibodies with the presence of subclinical carotid atherosclerosis. Methods: Tests to antibodies against cardiolipin (aCL) IgG and IgM, beta2-gp1 IgG, IgM and IgA ,Hsp 60 and Hsp 65 were done by ELISA test in a group of 71 RA subjects compared with 53 age and sex-matched non-RA subjects. Smoking, diabetic and hypertensive patients were excluded in both groups. The lipoprotein levels, clinical parameters of RA, Health Assessment Questionnaire (HAQ), Disease Activity Score (DAS) 28, Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were evaluated. The association between the presence of antibodies against cardiolipin, beta2-gp1 and Hsp 60 and 65 with the clinical parameters of disease activity in RA, and with the presence of plaques and mean intimo-medial thickness (IMT) of common carotid using high-resolution B-mode ultrasound were assessed. Results: Mean age in RA group was 48.93 ± 12.31 vs. 45.37 ± 9.37 in healthy control group (p = 0.20); 90.1% were women in RA group vs. 86.8% in healthy control (p = 0.56); body mass index (BMI) were 25.72 ± 4.57 in RA group vs. 26.40 ± 4.52 in healthy control (p = 0.69). The levels of cholesterol, LDL, triglycerides, CT/HDL didn t have difference between the two groups (p > 0.05). The HDL was higher in RA group vs. control group with 60.56 ± 14.40mg/dl and 54.52 ± 11.55 respectively (p = 0.05). The mean IMT was 0.721 ± 0.16mm in RA and 0.667 ± 0.14mm in control group, and the IMT was higher in patients older than 50 years among RA subjects (p < 0.001). In RA subjects, 14.1% had carotid plaques vs. 1.9% in healthy controls (p = 0.02). In RA group, the carotid plaques were more frequent in patients older than 50 years (p = 0.004). In RA group, 5.6% had antibodies against cardiolipin IgG vs. 3.8% in control group (p > 0.05); 14.1% in RA group had anti-cardiolipin IgM vs. 7.5% (p > 0.05); 43.7% had anti-beta2-gp1 IgA vs 40.8% in control group (p > 0.05). The presence of anti-beta2-gp1 IgG and IgM, and anti-LPL didn t have significant difference between the groups (p > 0.05). The prevalence of antibodies to Hsp 60 and Hsp 65 were similar in RA and in control group (p > 0.05), but the titers of antibodies against Hsp 65 and beta2-gp1 IgM were higher in RA group (p = 0.007 and p = 0.03 respectively). We didn t find relationship between antibodies against cardiolipin IgG and IgM, or beta2-gp1 IgG, IgM and IgA, LPL, Hsp 60 and 65 with mean IMT or plaque carotid (p > 0.05). Discussion: This study confirms the great prevalence of carotid atherosclerosis in RA subjects and its correlation with age, cholesterol and LDL. Although it was found a tendency to have more autoantibodies in RA subjects, there weren t any link between atherosclerosis in RA with autoimmunity against cardiolipin, beta2-gp 1, LPL or Hsp.
Частини книг з теми "Beta2 glycoprotein 1 antibody"
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Raby, Anne, Karen Moffat, and Mark Crowther. "Anticardiolipin Antibody and Anti-beta 2 Glycoprotein I Antibody Assays." In Haemostasis, 387–405. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-339-8_32.
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McGuckin, Michael A., and David J. Thornton. "Detection and Quantitation of Mucins Using Chemical, Lectin, and Antibody Methods." In Glycoprotein Methods and Protocols, 45–55. Totowa, NJ: Humana Press, 2000. http://dx.doi.org/10.1385/1-59259-048-9:045.
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Richardson, M. D., L. A. McTaggart, and G. S. Shankland. "A Rapid Double Antibody Biotin-Streptavidin Elisa for Aspergillus Fumigatus Glycoprotein Antigen." In Fungal Antigens, 386. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-0773-0_59.
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Hand, P. Horan, M. O. Weeks, J. Greiner, A. Thor, D. Colcher, C. Szpak, W. Johnston, and J. Schlom. "Potential Clinical Application of a Monoclonal Antibody to a Tumor Associated Glycoprotein (TAG-72)." In Monoclonal Antibodies and Breast Cancer, 108–18. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4613-2617-5_8.
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Stoeva, Stanka, Gerald Grübler, Wolfgang Rönspeck, Hartmut Echner, and Wolfgang Voelter. "gp41 Envelope glycoprotein partial sequence with high sensitivity against HIV-1 antibody positive sera." In Peptide Chemistry 1992, 668–70. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-1474-5_190.
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Gallagher, Thomas M., and Michael J. Buchmeier. "Monoclonal Antibody-Selected Variants of MHV-4 Contain Substitutions and Deletions in the E2 Spike Glycoprotein." In Advances in Experimental Medicine and Biology, 385–93. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4684-5823-7_53.
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Utiger, Anna, Max Rosskopf, Franco Guscetti, and Mathias Ackermann. "Preliminary Characterization of a Monoclonal Antibody Specific for a Viral 27 kD Glycoprotein Family Synthesized in Porcine Epidemic Diarrhoea Virus Infected Cells." In Coronaviruses, 197–202. Boston, MA: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4615-2996-5_31.
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Stoeva, Stanka, Gerald Grübler, Wolfgang Rönspeck, Hartmut Echner, and Wolfgang Voelter. "Optimized solid phase peptide synthesis of a 41 AA residue peptide sequence of gp 41 envelope glycoprotein with significant high sensitivity against HIV-1 antibody positive sera." In Peptides 1992, 856–57. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-1470-7_393.
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Ernste, Floranne C. "Rapid-Onset Weakness and Numbness in a Patient With Systemic Lupus Erythematosus." In Mayo Clinic Cases in Neuroimmunology, edited by Andrew McKeon, B. Mark Keegan, and W. Oliver Tobin, 120–21. Oxford University Press, 2021. http://dx.doi.org/10.1093/med/9780197583425.003.0038.
Повний текст джерелаАнотація:
A 33-year-old woman with systemic lupus erythematosus, diagnosed 2 years prior and treated with hydroxychloroquine, sought care for a 4-week history of pain and paresthesias in her low back and lower extremities. She described a bandlike sensation of numbness starting in her midback which descended to both legs. Her symptoms progressed to constipation and inability to urinate adequately. She reported difficulty with ambulation. Over the course of 1 week of hospitalization, urinary and fecal incontinence developed. On examination, she was alert and appropriately oriented. She had a malar rash and swelling of the metacarpophalangeal joints consistent with bilateral hand synovitis. Neurologic examination indicated hyperreflexia with brisk patellar and Achilles tendon reflexes bilaterally. She had trace motor weakness of the hip flexors, quadriceps, and hamstrings. She had loss of pinprick and temperature sensation in the lower extremities, extending beyond the saddle area to the T12 dermatome. Vibration perception and proprioception were preserved. She had a positive Babinski sign in the left foot. Her cerebellar examination showed slowing of rapid alternating movements in the left hand. Magnetic resonance imaging of the lumbosacral spine indicated subtle T2 signal change of the intramedullary conus and enhancement of the cauda equina nerve roots. Cerebrospinal fluid analysis showed an increased protein concentration. Two white blood cells/µL were found in the cerebrospinal fluid. The serum antinuclear antibody was strongly positive, and the anti–double-stranded DNA antibody level was greater than 1,000 IU/mL. The serum complement levels were low. Lupus anticoagulant, beta-2 glycoprotein antibodies, and antiphospholipid antibodies were increased, at greater than twice the upper limits of normal. Electromyography indicated multiple sacral radiculopathies. The patient was diagnosed with autoimmune myeloradiculitis as a neuropsychiatric manifestation of systemic lupus erythematosus (neuropsychiatric systemic lupus erythematosus). The patient received methylprednisolone followed by prednisone, with a gradual taper. Her hospital course was complicated by the development of deep venous thromboses in the bilateral lower extremities. She was started on heparin and transitioned to warfarin therapy. She started mycophenolate mofetil. Hydroxychloroquine was continued. At a 24-month follow-up visit, the patient remained in neurologic remission. Neuropsychiatric systemic lupus erythematosus events consist of a heterogeneous array of neurologic and psychiatric disorders including intractable headaches, cognitive dysfunction, psychosis, seizure disorders, transverse myelitis, aseptic meningitis, cranial neuropathies, and acute inflammatory demyelinating polyneuropathy.
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Jitprapaikulsan, Jiraporn, M. Tariq Bhatti, Eric R. Eggenberger, Marie D. Acierno, and John J. Chen. "A Woman With Subacute Painful Vision Loss." In Mayo Clinic Cases in Neuroimmunology, edited by Andrew McKeon, B. Mark Keegan, and W. Oliver Tobin, 3–6. Oxford University Press, 2021. http://dx.doi.org/10.1093/med/9780197583425.003.0001.
Повний текст джерелаАнотація:
A 51-year-old White woman sought care for vision loss 1 week after a nonspecific upper respiratory tract infection. She reported pain in both eyes exacerbated by eye movement, which lasted for several days, followed by bilateral vision loss to the level of counting fingers–only vision. Optic neuritis was diagnosed, and she was treated with 1 g intravenous methylprednisolone for 3 days. Her vision improved substantially, and the pain resolved during the corticosteroid treatment. However, 1 week later, she woke up with right eye pain and vision loss. She was again treated with 5 days of intravenous methylprednisolone, with visual improvement nearly back to baseline. Two weeks later, she had recurrence of painful vision loss in both eyes. A diagnosis of chronic relapsing inflammatory optic neuropathy was made. Tests for serum angiotensin-converting enzyme, antineutrophil cytoplasmic antibody, antinuclear antibody, Lyme disease, syphilis, tuberculosis, and aquaporin-4-immunoglobulin G antibodies were negative. Serum was definitively positive for myelin oligodendrocyte glycoprotein-immunoglobulin G antibodies at a titer of 1:1,000. Myelin oligodendrocyte glycoprotein-immunoglobulin G–associated recurrent optic neuritis was diagnosed. After her diagnosis of recurrent corticosteroid-dependent optic neuritis associated with myelin oligodendrocyte glycoprotein-immunoglobulin G positivity, the patient was treated with 5 days of intravenous methylprednisolone. The eye pain resolved, and her vision returned to normal. At follow-up evaluation, the patient’s visual acuity, color vision, and visual fields were normal in both eyes, but there was mild bilateral optic disc pallor. She has not had recurrent demyelinating episodes while on chronic immunotherapy. Optic neuritis is an inflammatory demyelination of the optic nerve manifesting as acute to subacute vision loss, classically associated with pain with eye movement. The long-term prevention and prognosis depend on the cause of the optic neuritis.
Тези доповідей конференцій з теми "Beta2 glycoprotein 1 antibody"
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Folts, J. D. "A MODEL OF ACUTE PLATELET THROMBUS FORMATION IN STENOSED CORONARY AND CAROTID ARTERIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643712.
Повний текст джерелаАнотація:
There is currently a great deal of interest in the diagnosis and treatment of unstable angina and silent ischemia.Many feel that these syndromes are due, in part, to periodic accumulation of platelet thrombi which subsequently embolize.In addition, anti-piatelet therapy is also considered necessary for patients after coronary artery bypass grafts (CABG'S), balloon angioplasty, and thrombolysis. Currently the two antiplatelet agents most commonly prescribed for the patient conditions mentioned above are aspirin (ASA), alone or in combination with dipyridamole (Dip). ASA reduces cardiac events in patients with unstable angina, and prolongs CABG graft patency. The addition of Dip to ASA therapy is very confusing since most studies done compared ASA + Dip to placebo. In several studies however,when an ASA group was compared to an ASA + Dip group there was no significant difference.We have developed and will describe ananimal model of coronary artery stenosis in the dog and the pig, or carotid arterystenosis in the monkey and the rabbit, with intimal damage, that simulates some ofthe conditions that exist in patients with coronary or carotid artery disease. The artery to be studied is dissected outand blood flow is continuously measured with an electromagnetic flowmeter probe. As acute platelet thrombus formation (APTF) developes in the stenosed lumen, the blood flow declines to low levels, producing ischemia until the thrombus emobolizesdistally resulting in abrupt restoration of blood flow. These cyclical flow reductions (CFR's), when they occur in the coronaries, produce ECG changes identical to those observed in patients with silent ischemia and unstable angina. They also produce significant transient regional dyskinesis of the ventricular wall, which resolves when blood flow is restored. Histologic examination of myocardial tissue in the bed distal to the stenosis shows focal areas of ischemic change presumably caused by the embolized platelet emboli.We have examined factors which exacerbate the size and frequency of these CFR"ssuch as; IV infusion of epinephrine (E) 0.4 μg/kg/min for 15 min, ventilating the animals with cigarette smoke, infusing nicotine IV, or placing chewing tobacco under the tongue.We have examined four groups of agentswhich prevent APTF in our model.1. Antiplatelet agents including ASA, indomethacin, ibuprofen and several other NSAI agentsas well as several experimental thromboxane synthetase inhibitors. These agents all block the production of TXA2and inhibit APTF in our model. Unfortunately the IV infusion of E reinstates APTtemporarily (by another biochemical pathway) until the E is metabolized. High (2-4 mg/kg) doses of Dip, alone or with sub threshold dose of ASA does nothing to I APTF.However,0.6mg/kg of chi orpromaz i ne abolishes APTF in all four species and protects agents renewal of APTF by E.2. Dietary Substances In our model, caffeine 10 mg/kg, or the extract from two garlic cloves, or enough ethanol to achieve a blood alcohol level of 0.07 mg% all significantly inhibit or abolish APTF in our model.3. Metabolic Inhibitors POCA, an oral hypoglycemic agent, which inhibits mitochondrial beta oxidation of fatty acids also inhibits APTF in our model possibly by reducing ATP production in the platelet.4. We have studied a monoclonal antibody(developed by Dr. Barry Coller) to the platelet I Ib�I I la glycoprotein receptor where fibrinogen binds platelets into aggregates and ultimately leads to APTF. This antibody 0.3 mg/kg/completely inhibits APTF, and also strongly inhibits in vitro platelet aggregation in response to either ADP or collagen given alone or each combined with E. This antibody is the most potent inhibitor of APTF that we have studied.
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Yamamoto, N., H. Kitagawa, K. Tanoue, and H. YAmazaki. "AN EPITOPE OF A MONOCLONAL ANTIBODY (TM83) AGAINST GLYCOPROTEIN lib/Ilia COMPLEX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644881.
Повний текст джерелаАнотація:
GPIIb/lIIa is forming a heterodimer complex on the human platelet membrane.Many monoclonal antibodiesagainst GPIIb/IIIa complex obtained elsewhere react with neither GPIIb nor GPIIIa separately when GPIIb/IIIa is blotted to filter after SDS-PAGE. Therefore the epitope of GPIIb/IIIa complex is not identified actually. Our attemption is to clarify the epitope recognized by a monoclonal antibody against GPIIb/IIIa designated TM83. TM83 (30 yg/ml) inhibited collagen-, ADP-, or thrombin-induced aggregation, but it did not inhibit ATP-secretion induced by 0.01 U/ml of thrombin. TM83 also inhibited fibrinogen-binding approximately to 50 % of total binding. The binding of 125 I- TM83 to platelets decreased to b0% of controlwhen platelets were incubated in the presence of 1 mM EDTA at 37°C for 30 min. However the incubation at25°C for 30 min did not change any binding capacity of 125 I-TM83 to platelets. Thus thebinding of TM83to platelets was dependent on both temperature and calcium concentration in surrouding medium, suggesting that TM83 bound to GPIIb/IIIa complex.If the small amounts of epitope of GPIIb/IIIa complex is not injured during SDS-PAGE and blotting, we may identify clearly the epitope of GPIIb/IIIa complex. For this aim, GPIIb/IIIa complex was extracted carefully in the presence of 1 mM calcium by the phase separationusing Triton X-11U, and was run on SDS-PAGE in the presence of 100 yM calcium. Western-blot of the membrane preparation showed that 125 I-TM83 was incorporated into both GPIIb and GPIIIa on Durapore filter. Further radio-crossed immunoelectrophoresis showed that I-TM83 was incorporated only into immunoprecipitin of GPIIb/IIIa complex in the presenceof 1 mM calcium. While after addition of 25 mM EDTA to the membrane preparation containing 1 mM calcium,125i-tm83 wasmainly incorporated into GPIIb/IIIa complex as well, however very faint radioactivity fromthe immunoprecipitin corresponding to GPIIIa was observed, but the radioactivity from GPIIb was not identified. While there is a discrepancy inour result, it must be further studied whether one monoclonal antibody can recognize or not two kinds of GPs, GPIIb and Ilia, which are formed in complex in physiological condition.
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Karpouzas, George, Sarah Ormseth, Elizabeth Hernandez, Joel Estis, John Todd, and Matthew Budoff. "THU0611 SERUM HIGHLY-SENSITIVE CARDIAC TROPONIN-I AND ANTI-BETA2-GLYCOPROTEIN-1 IGA ANTIBODIES AT BASELINE PREDICT CORONARY PLAQUE PROGRESSION IN RHEUMATOID ARTHRITIS." In Annual European Congress of Rheumatology, EULAR 2019, Madrid, 12–15 June 2019. BMJ Publishing Group Ltd and European League Against Rheumatism, 2019. http://dx.doi.org/10.1136/annrheumdis-2019-eular.6767.
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Farache Trajano, Luiza, Rebecca Moore, and Quentin Sattentau. "The Presence of Chemical Cross-Linking Stabilises HIV-1 Envelope Glycoprotein Trimer Antigens in a Model of Intramuscular Immunisation." In Building Bridges in Medical Science 2021. Cambridge Medicine Journal, 2021. http://dx.doi.org/10.7244/cmj.2021.03.001.4.
Повний текст джерелаАнотація:
Background: The HIV-1 envelope glycoprotein (Env) is the target of antigen design for antibody- based vaccination. In 2019, four trimeric Env vaccines entered an experimental trial: ConM, ConS, and their cross-linked counterparts. The trimers were formulated with MPLA adjuvant. Studies have demonstrated that adjuvants trigger neutrophil infiltration. Neutrophils activate and degranulate releasing proteases, namely elastase and cathepsinG. Aims: To assess the stability and immunogenicity of these vaccines in the presence of adjuvant- recruited neutrophils and their proteolytic enzymes. Methods: Trimers were incubated with commercially-sourced proteases. To analyse stability, samples were reduced, denatured and separated using gel electrophoresis. To assess antibody binding, a trimer-protease incubation was followed by an ELISA. To establish more physiologically relevant conditions, harvested neutrophils were exposed to various adjuvants. The supernatant, shown to contain elastase, was incubated alongside the vaccines. The reducing and denaturing gels, as well as the ELISA, was repeated. Results: Gel analysis revealed that un-crosslinked trimers underwent significant digestion whereas cross-linking conferred enhanced stability. In the presence of neutrophil-sourced protease-containing-supernatant, trimers displayed resistance to digestion. The differential stability profile of Env trimers when exposed to commercially sourced compared to supernatant- derived proteases may be due to the inhibitory effect of human serum on elastase. Antibody epitopes were maintained in vitro. Conclusion: The vaccine antigens are sensitive to enzymatic degradation. This is reduced by cross-linking and human serum.
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Imura, J., N. Suzuki, K. Higashi, M. Tubokura, and K. Shirasawa. "EFFECTS OF PLASMA MEMBRANE GLYCOPROTEIN OF PLATELETS ON THE AGGREGATION ACTIVITY OF THE STORED CELLS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644593.
Повний текст джерелаАнотація:
Platelet aggregation activity is gradually reduced depending on the duration of storage. Platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa) complex is intimately related to the aggregation activity. We intended, therefore, to analyse a sequential change in plasma membrane glycoprotein of stored platelets by means of flow cytometry (FCM). On this occasion, aggregation activity of the platelets was also studied. Concentrated human platelets (150×104 cell/μl) were stored in the bag containing citrate-phosphate-dextrose at 22 °C for 24 to 72 hours. The bags were either kept in a flat position without agitation or continuosly stirred by a tumbler agitator (6rpm), or a flat bed rotator (30rpm). At the beginning of each experiment, fresh platelets separated from healthy donor were used as control group. The remaining platelets were washed twice with the 0.38% sodium-citrate dissolved in the lOmM of PBS. After incubation of the suspended platelets in the Tyrode's buffer solution at 37 °C for 30 minutes, they were fixed with 1% paraformaldehyde at 4 C for 2 hours, and were then incubated with anti-human GP IIb/IIIa mouse monoclonal antibody (5μg/ml) at 37 °C for 1 hour. The platelets, thus treated with primary antibody, had undergone further incubation with fluorescein-conjugated goat anti-inouse IgG immunoglobulin at 37°C for 1 hour. After analysis of labelled platelets on the Coulter EPICS V, the positive rate was estimated by counting 5×104 cells. In each group, aggregation activity of platelets induced by ADP (100μM) was measured by an aggregometer.The positive rate was significantly decreased in the stored platelets compared with those in the control group. In addition, the positive rate was more decreased in the non-agitated group than in the agitated group. No difference, however, occurred in the rate from the agitated groups. Moreover, the aggregation activity in each group was well compatible with the positive rate from FCM.It is finally suggested that the decrease in aggregation activity of the stored platelet is due to the reduction in the functioning receptor sides of GPIIb/IIIa on the platelet surface.
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Yamamoto, K., H. Kawasaki, K. Suzuki, K. Tanoue, G. Kosaki, and H. Yamazaki. "AMINO ACID SEQUENCE OF THE THROMBIN-BINDING SITE ON PLATELET GLYCOPROTEIN Ib." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642924.
Повний текст джерелаАнотація:
Amino acid sequence of the thrombin-binding site on platelet glycoprotein (GP) Ib was determined and the effects of the synthesized analogous peptides on thrombin-induced aggregation were studied. Crude platelet membrane fraction solubilized with 5mM dithiothreitol and 0.2% Tween 20 was applied to a WGA-Sepharose. The bound fractions were eluted with 0.3M N-actylglucosamine, then applied to a TM60 ( a monoclonal antibody against GP Ib)-Affi-gel column. Only one GP was bound to the column and was eluted with 50mM glycine-HCl, pH3.0, containing 0.2% Tween 20. SDS-PAGE showed a single band of 130kDa, corresponding to GP Ib alpha chain (GP Ibα). When the purified GP Ibα was digested with trypsin, two fragments (94kDa . and 43kDa) were obtained. The 43kDa fragment was shown to bind to both affinity colomns of TM60- and thrombin-Affi-gel, while the 94kDa fragment did not bind to either Affi-gel. When the same experiment was performed using chymotrypsin, three fragments (94kDa, 45kDa and 39kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45kDa and 39kDa) were bound to both columns. However, on. thrombin-Affi-Gel column, 39kDa fragment was found in both unbound and bound fractions. It showed that the 45kDa fragment interacts with thrombin with a higher affinity than the 39kDa fragment. These results indicate that the thrombin-binding site is located on the "tail" portion of GPIba, especially on a chymotryptic cleavage site.Then 43kDa tryptic fragment was purified and its partial amino acid sequence was analyzed using gas phase amino acid sequence analyzer. Based on its amino acid sequence, several analogous oligopeptides were synthesized. Three peptides (#1-10, #11-23, #17-28) inhibited 0.05 U/ml thrombin-induced aggregations of washed human platelets in dose-dependent manners. IC50 were in the range of L50-550μM for each of these peptides.
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Ikeda, Y., M. Murata, Y. Araki, M. Yamamoto, K. Watanabe, M. Ichitani, K. Sakai, I. Itagaki, and Y. Mori. "BINDING OF FIBRINOGEN TO PLATELET GLYCOPROTEIN (GP) IIb/IIIa IS CRUCIAL FOR SHEAR-INDUCED PLATELET AGGREGATION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643527.
Повний текст джерелаАнотація:
It is well known that human platelets can aggregate in vitro under certain shear stress without adding aggregating inducers. However, the mechanism of this shear-induced platelet aggregation has not been clarified yet. In this paper, we have investigated the role of fibrinogen and GP IIb/IIIa in shear-induced platelet aggregation. Citrated human platelet-rich plasma (PRP) was subjected to controlled shear stress levels in a polycarbonate cone and plate viscometer at 37°C for 2 minutes. After shearing the particle count was measured by an electronic particle counter. Particles with sizes from 3 to 20 μ cum were considered as single platelets. In unsheared PRP most of the particles were single platelets, but platelet doublets and platelet fragments larger than 3 μ cum were also counted. After exposure to shear rate of 3,600 - 9,000 sec−1 , the particle counts were decreased in a shear rate dependent manner, while LDH leakage from platelets was not significantly increased and 3H-serotonin release was 2-7%. Scanning electronmicroscopy clearly showed the presence of large platelet aggregates when the particle counts were decreased. Platelets from two patients with thrombasthenia and one patient with afibrinogenemia, however, failed to aggregate at a shear rate of 9,000 sec−1. Shear-induced aggregation was inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM). When fibrinogen was added to PRP from a patient with afibrinogenemia, shear-induced aggregation became evident as seen in normal platelets. Apyrase and hirudin showed no effect on shear-induced aggregation. Indomethacin (100 μM) and TXA2 synthetase inhibitor, OKY-046 (100 μM) markedly inhibited aggregation, while TXA2 competitive inhibitor, ONO-3708 (100 μM) exhibited only partial inhibition.Our results indicate that binding of fibrinogen to GPIIb/lIIa is also crucial for shear-induced platelet aggregation and that the exposure of fibrinogen receptor on GPIIb/IIIa may partially depend upon TXA2 synthesis in platelets.
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Yamamoto, M., Y. Ando, K. Watanabe, H. Iri, Y. Araki, M. Murata, H. Murakami, K. Satoh, and Y. Ikeda. "IMPORTANCE OF LARGE VON WILLEBRAND FACTOR (vWF) MULTIMERS IN vWF INTERACTION WITH PLATELET GLYCOPROTEIN IIb/IIIa." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644096.
Повний текст джерелаАнотація:
Recently it has been reported that, in addition to binding to glycoprotein (GP) lb, vWF also interacts with GPIIb/IIIa, although the physiological relevance of this interaction is not completeley clear. In this paper, we have investigated the role of different size of vWF multimers in vWF-mediated platelet aggregation. Different size of vWF multimers were purified from human plasma through Sephacryl S-1000 column according to the method of Fowler et al. Fractions were analysed by SDS-agarose gel electrophoresis by the method of Ruggeri et al. When each fraction was examined for ristocetin cofactor activity (RCo), only larger multimers exhibited significant RCo. The maximum extent of ristocetin-induced platelet agglutination by larger multimers (10 μg/ml) was 80%, while that of intermediate and lower multimers at the same concentration was 20% and 0%, respectively. Each fraction was then added to washed platelet suspensions in the presence of 10 μM ADP and 0.3 mM CaCl2. Only larger multimers induced platelet aggregation, while intermediate and lower multimers failed to induce platelet aggregation. The maximum extent of aggregation in the presence of larger multimers (10 μg/ml) was 70% of that in the presence of fibrinogen instead. Similar experiments were peformed using platelet-rich plasma from a patient with afibrinogenemia in stead of washed normal platelets. ADP caused significant aggregation only when purified vWF larger multimers or fibrinogen was added. This vWF-mediated aggregation was completely inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM).Our results indicate that larger multimers of vWF play major roles in vWF interaction with GPIIb/IIIa.
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Berndt, M. "STRUCTURE AND FUNCTION OF THE GLYCOPROTEIN Ib-IX COMPLEX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643729.
Повний текст джерелаАнотація:
At high shear flow, the adhesion of platelets to the exposed vascular subendothelium requires von Willebrand factor (vWF) and is dependent upon a specific platelet membrane adhesion receptor, the human platelet membrane glycoprotein (GP) Ib-IX complex. Recent evidence suggests that vWFbinding to the GP Ib-IX complex plays an important role in other key aspects of hemostasis and thrombosis such as shear-induced platelet aggregation and the interaction of platelets with fibrin.Studies in our laboratory with a seriesof murine monoclonal antibodies directed against epitopes on GP lb, GP IX, or against complex-specificepitopes indicate that GP lb and GP IX exist in the intact platelet membrane as a native heterodimer complex(-25,000 copies/platelet). By analysis onSDS-polyacrylamide gels, GP lb has an apparent molecular weight of 170,000 and cnsists of two disulfide-linked subunits, GP Iba (Mr = 135,000) and GP Ibβ (Mr = 25,000),whilst GP IX has an equivalent molecularweight under both nonreducing and reducing conditions (Mr = 22,000).The ±-chain ofGP lb has a central macroglycopeptide core (Mr =90,000) which is highly glycosylated. At each end of themacroglycopeptide region is a domainsensitive to proteolytic cleavage. Cleavage at the end proximal to the platelet membrane, e.g. by calpain, Serratia marcescens metalloprotease and trypsin, generates two fragments :a Mr =130,000 highly glycosylated fragment termed glycocalicin anda membrane-associated region consisting ofa Mr -25,000 fragment that remains disulfide-linkedto GP Ibβ and associated with GP IX. In resting platelets, the membrane-associated region spans the lipid bilayer linking the GP Ib-IX complex to the platelet endoskeleton via actin-binding protein. This membrane-associated region also contains the domain(s) recognized by quinine/quinidine drug-dependent antibodies. Cleavage at the plasma end of the macroglycopeptide, e.g. by human leukocyte elastase, generates a poorly glycosylated Mr = 45,000 fragment of GP Ibα (peptide tail region) and a heavily glycosylated Mr = 100,000 fragment that remains disulfide-linked to GP Ibg and associated with GP IX. Platelets lacking the N-terminal peptide tail region of GP Iba fail to agglutinate with ristocetin and vWF and show a delayed response to a-thrombin.Polyclonal and monoclonal antibodies against this region also inhibit both these platelet responses suggesting that the peptide tail region contains the binding sites for both α-thrombinand vWF. Rotary shadowingelectron microscopy of purified GP Ib-IX complex shows the structure to be highlyasymmetric with each complex existing asa flexible rod with a globular domain at each end. The overall length of the complexwas =60 nm.The smaller globular domain (peptidetail region) has a diameter of =9nm; the larger globular domain (membrane-associated region), a diameter of =16 nmWe have recently examined whetherthe human platelet GP Ib-IX complex is the receptor for the ristocetin-dependent binding of vWF by reconstitution with the purified components using a solid-phasebead assay. Our approach was to indirectlybind and orientate the GP Ib-IX complex onthe beads via a monoclonal antibody directed against the membrane-associated region of the complex (FMC 25, epitope on GP IX).Immunobeads were chosen as the insoluble matrix because they are uniform in size (=10μm in diameter), impermeable,specifically designed for the coupling of IgG, and because, like platelets, the beads have a net negative charge atneutral pH.Specific binding of 125I-labelled human vWF tothe GP Ib-IX complex-coated immunobeads was strictly ristocetin-dependent with maximal binding occurring atristocetin concentrations >1 mg/ml. Ristocetin-dependent specificbinding of 125I-labelled vWF was saturable.Scatchardanalysis revealed a single classof binding sites for vWF with purified GP Ib-IX complex.Monoclonal antibodies against the Mr = 45,000 peptide tail region ofGP lb which stronglyinhibitthe ristocetin-dependent binding ofvWF toplatelets also strongly inhibited the ristocetin-dependent binding of vWFto the GP Ib-IX coated beads. Monoclonalantibody against either themacroglycopeptide or membrane-associated regions of the GPIb-IX complex did not inhibit the ristocetin-dependent binding of vWF to platelets or to the GP Ib-IX complex-coated beads. Similar functional correlations were obtained with anti-vWFmonoclonal antibodies. The reconstitutiondata therefore confirm the functional roleof the GP Ib-IX complex as a major plateletvWF receptor. The region ofthe vWF molecule involved in binding to the GP Ib-IX complex has been localized toa Mr =50,000 domain towards theN-terminal end of the vWF subunit. The reconstitution assay should prove useful in the further definition of active peptides of vWF that bind tothe human platelet GP Ib-IX complex.
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Metzelaar, M. J., H. K. Nieuwenhuis, and J. J. Sixma. "DETECTION OF ACTIVATED PLATELETS WITH MONOCLONAL ANTIBODIES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643829.
Повний текст джерелаАнотація:
Blood tests reflecting in-vivo activation of platelets are potentially useful in evaluating patients with thrombotic diseases. Recently monoclonal antibodies have been described that react preferentially with activated platelets. We prepared an IgG2b antibody, designated RUU-AP 2.28, that reacted with a 53.000 MW protein that is located in a special subclass of platelet granules in unstimulated platelets and that is exposed on the surface of activated platelets. Increased numbers of platelets that expressed the 2.28 antigen on their surface were observed in patients undergoing cardiopulmonary bypass and in patients with acute deep venous thrombosis. The percentage of RUU-AP 2.28 positive platelets in the circulation was 3,9 ± 2.7 (SD)% in the controls, (n = 20), 24.6 ± 13.5% in patients after cardiopulmonary surgery (n = 10) and 8.5% in patients with acute deep venous thrombosis (n = 2).In order to detect also earlier stages of platelet activation, such as secretion-independent phenomena, we produced new monoclonal antibodies by fusing spleen cells from Balb/c mice, immunized with thrombin stimulated, paraformaldehyde fixed platelets, with Ag 8653 myeloma cells. As a screening assay we used an ELISA with freshly fixed platelets or fixed thrombin-activated platelets. We detected six monoclonal antibodies (RUU-AP 1-6) specific for thrombin-activated platelets. The results of the ELISA were confirmed by flow cytofluorometry.None of the antibodies inhibited platelet aggregation induced by ADP, collagen or ristocetin. Ascites of IgGl antibody RUU-AP 3 reacted with normal thrombin-activated platelets but did not react with thrombin-activated platelets from a patient with Glanzmann’s disease. In addition antibody RUU-AP 3 reacted with normal platelets stimulated with 1 pM of ADP. These data suggest that antibody RUU-AP 3 detects a secretion-independent conformational change in the platelet membrane glycoprotein IIb-IIIa complex.