976 resultados para Anti-transglutaminase antibody
Resumo:
Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.
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Glycan-binding antibodies form a significant subpopulation of both natural and acquired antibodies and play an important role in various immune processes. They are for example involved in innate immune responses, cancer, autoimmune diseases, and neurological disorders. In the present study, a microsphere-based flow-cytometric immunoassay (suspension array) was applied for multiplexed detection of glycan-binding antibodies in human serum. Several approaches for immobilization of glycoconjugates onto commercially available fluorescent microspheres were compared, and as the result, the design based on coupling of end-biotinylated glycopolymers has been selected. This method requires only minute amounts of glycans, similar to a printed glycan microarray. The resulting glyco-microspheres were used for detection of IgM and IgG antibodies directed against ABO blood group antigens. The possibility of multiplexing this assay was demonstrated with mixtures of microspheres modified with six different ABO related glycans. Multiplexed detection of anti-glycan IgM and IgG correlated well with singleplex assays (Pearson's correlation coefficient r = 0.95-0.99 for sera of different blood groups). The suspension array in singleplex format for A/B trisaccharide, H(di) and Le(x) microspheres corresponded well to the standard ELISA (r > 0.94). Therefore, the described method is promising for rapid, sensitive, and reproducible detection of anti-glycan antibodies in a multiplexed format.
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BACKGROUND: beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [glycoprotein (GP)IIb-GPIIIa], and in angiogenesis via endothelial alpha(V)beta(3). Cross-reactive ligands with antiaggregatory and proangiogenic effects, both desirable in peripheral vasculopathies, have not yet been described. OBJECTIVES: In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments, with emphasis on beta(3)-integrins. METHODS: Recombinant Fab fragments were obtained by phage display technology. Specificity, affinity and IC(50) were determined by immunodot assays, enzyme-linked immunosorbent assay (ELISA), and Scatchard plot analysis, and by means of human umbilical vein endothelial cells (HUVECs). Functional analyses included ELISA for interaction with fibrinogen binding to GPIIb-GPIIIa, flow cytometry for measurement of activation parameters and competitive inhibition experiments, human platelet aggregometry, and proliferation, tube formation and the chorioallantoic membrane (CAM) assay for measurement of angiogenic effects. RESULTS: We observed specific and high-affinity binding to an intact GPIIb-GPIIIa receptor complex of two human Fab autoantibody fragments, with no platelet activation. Dose-dependent fibrinogen binding to GPIIb-GPIIIa and platelet aggregation were completely inhibited. One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody (mAb) 7E3, whereas the other Fab fragment bound to cultured HUVECs, suggesting cross-reactivity with alpha(V)beta(3), and also demonstrated proangiogenic effects in tube formation and CAM assays. CONCLUSIONS: These Fab fragments are the first entirely human anti-GPIIb-GPIIIa Fab fragments with full antiaggregatory properties; furthermore, they do not activate platelets. The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies.
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While cancer is one of the greatest challenges to public health care, prostate cancer was chosen as cancer model to develop a more accurate imaging assessment than those currently available. Indeed, an efficient imaging technique which considerably improves the sensitivity and specificity of the diagnostic and predicting the cancer behavior would be extremely valuable. The concept of optoacoustic imaging using home-made functionalized gold nanoparticles coupled to an antibody targeting PSMA (prostate specific membrane antigen) was evaluated on different cancer cell lines to demonstrate the specificity of the designed platform. Two commonly used microscopy techniques (indirect fluorescence and scanning electron microscopy) showed their straightforwardness and versatility for the nanoparticle binding investigations regardless the composition of the investigated nanoobjects. Moreover most of the research laboratories and centers are equipped with fluorescence microscopes, so indirect fluorescence using Quantum dots can be used for any active targeting nanocarriers (polymers, ceramics, metals, etc.). The second technique based on backscattered electron is not only limited to gold nanoparticles but also suits for any study of metallic nanoparticles as the electronic density difference between the nanoparticles and binding surface stays high enough. Optoacoustic imaging was finally performed on a 3D cellular model to assess and prove the concept of the developed platform.
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Intravenous immunoglobulin (IVIG) is the first line treatment for Guillain–Barré syndrome and multifocal motor neuropathy, which are caused by anti-ganglioside antibody-mediated complement-dependent cytotoxicity. IVIG has many potential mechanisms of action, and sialylation of the IgG Fc portion reportedly has an anti-inflammatory effect in antibody-dependent cell-mediated cytotoxicity models. We investigated the effects of different IVIG glycoforms on the inhibition of antibody-mediated complement-dependent cytotoxicity. Deglycosylated, degalactosylated, galactosylated and sialylated IgG were prepared from IVIG following treatment with glycosidases and glycosyltransferases. Sera from patients with Guillain–Barré syndrome, Miller Fisher syndrome and multifocal motor neuropathy associated with anti-ganglioside antibodies were used. Inhibition of complement deposition subsequent to IgG or IgM autoantibody binding to ganglioside, GM1 or GQ1b was assessed on microtiter plates. Sialylated and galactosylated IVIGs more effectively inhibited C3 deposition than original IVIG or enzyme-treated IVIGs (agalactosylated and deglycosylated IVIGs). Therefore, sialylated and galactosylated IVIGs may be more effective than conventional IVIG in the treatment of complement-dependent autoimmune diseases.
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Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26–2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26–2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26–2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26–2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26–2F. Furthermore, the capacities of cAb 26–2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26–2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.
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The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab–Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.
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Active immunization with the amyloid β (Aβ) peptide has been shown to decrease brain Aβ deposition in transgenic mouse models of Alzheimer's disease and certain peripherally administered anti-Aβ antibodies were shown to mimic this effect. In exploring factors that alter Aβ metabolism and clearance, we found that a monoclonal antibody (m266) directed against the central domain of Aβ was able to bind and completely sequester plasma Aβ. Peripheral administration of m266 to PDAPP transgenic mice, in which Aβ is generated specifically within the central nervous system (CNS), results in a rapid 1,000-fold increase in plasma Aβ, due, in part, to a change in Aβ equilibrium between the CNS and plasma. Although peripheral administration of m266 to PDAPP mice markedly reduces Aβ deposition, m266 did not bind to Aβ deposits in the brain. Thus, m266 appears to reduce brain Aβ burden by altering CNS and plasma Aβ clearance.
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The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.
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The Eph receptor tyrosine kinases and their membrane-bound ephrin ligands form a unique cell-cell contact-mediated system for controlling cell localization and organization. Their high expression in a wide variety of human tumors indicates a role in tumor progression, and relatively low Eph and ephrin levels in normal tissues make these proteins potential targets for anticancer therapies. The monoclonal antibody IIIA4, previously used to isolate EphA3, binds with subnanomolar affinity to a conformation-specific epitope within the ephrin-binding domain that is closely adjacent to the low-affinity ephrin-A5 heterotetramerization site. We show that similar to ephrin-A5, preclustered IIIA4 effectively triggers EphA3 activation, contraction of the cytoskeleton, and cell rounding. BIAcore analysis, immunoblot, and confocal microscopy of wild-type and mutant EphA3 with compromised ephrin-A5 or IIIA4-binding capacities indicate that IIIA4 binding triggers an EphA3 conformation which is permissive for the assembly of EphA3/ephrin-A5-type signaling clusters. Furthermore, unclustered IIIA4 and ephrin-A5 Fc applied in combination initiate greatly enhanced EphA3 signaling. Radiometal conjugates of ephrin-A5 and IIIA4 retain their affinity, and in mouse xenografts localize to, and are internalized rapidly into EphA3-positive, human tumors. These findings show the biological importance of EphA3/ ephrin-A5 interactions and that ephrin-A5 and IIIA4 have great potential as tumor targeting reagents.
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Infliximab is an antibody that neutralizes TNF-α and is used principally by systemic administration to treat many inflammatory disorders. We prepared the antibody mimetic Fab-PEG-Fab (FpFinfliximab) for direct intravitreal injection to assess whether such formulations have biological activity and potential utility for ocular use. FpFinfliximab was designed to address side effects caused by antibody degradation and the presence of the Fc region. Surface plasmon resonance analysis indicated that infliximab and FpFinfliximab maintained binding affinity for both human and murine recombinant TNF-α. No Fc mediated RPE cellular uptake was observed for FpFinfliximab. Both Infliximab and FpFinfliximab suppressed ocular inflammation by reducing the number of CD45+ infiltrate cells in the EAU mice model after a single intravitreal injection at the onset of peak disease. These results offer an opportunity to develop and formulate for ocular use, FpF molecules designed for single and potentially multiple targets using bi-specific FpFs.
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Purpose: To develop liposome formulations containing monoclonal antibody anti-HER2 (MabHer2), and Paclitaxel (PTX). Methods: Seven different liposomal systems containing PTX, or MabHer2 or a combination of PTX and MabHer2 were made using lipid film hydration technique and sonication. The effects of liposome preparation conditions and extraction methods on antibody structure were investigated by polyacrylamide gel electrophoresis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The characteristics of the liposomes were determined by a zetasizer, while drug-loading efficiency was evaluated by high-performance liquid chromatography. The cytotoxic effect of the liposome formulations was evaluated on MDA-MB-453 (HER2+) and MCF-7 (HER2-) breast cancer cell lines by MTT assay. Results: The antibody was not significantly affected by the stress conditions and the method of extraction. The particle size of liposomes was < 200 nm while the amount of incorporated PTX was 97.6 % for liposome without cationic agent and 98.2 % for those with cationic agent. Recovery of MabHer2 was 94.38 % after extraction. Combined PTX/MabHer2 liposome was more toxic on HER2 overexpressing positive MDA-MB-453 cell line than PTX-loaded liposomes and MabHer2. MabHer2 and combined PTX/MabHer2 liposomes showed no toxic effects on HER2 overexpressing negative MCF-7 cells relative to cationic PTX-loaded liposomes. Conclusions: This results obtained show that PTX can be encapsulated successfully into liposoma systems and that owing to Her2 specific antibody, these systems can be delivered directly to the target cell.
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Background: The treatment of B-cell acute lymphoblastic leukemia (B-ALL) has been enriched by novel agents targeting surface markers CD19 and CD22. Inotuzumab ozogamicin (INO) is a CD22-calicheamicin conjugated monoclonal antibody approved in the setting of relapse/refractory (R/R) B-ALL able to induce a high rate of deep responses, not durable over time. Aims: This study aims to identify predictive biomarkers to INO treatment in B- ALL by flow cytometric analysis of CD22 expression and gene expression profile. Materials and methods: Firstly, the impact on patient outcome in 30 R/R B-ALL patients of baseline CD22 expression in terms of CD22 blast percentage and CD22 fluorescent intensity (CD22-FI) was explored. Secondly, baseline gene expression profile of 18 R/R B-ALL patient samples was analyzed. For statistical analysis of differentially expressed genes (DEGs) patients were divided in non-responders (NR), defined as either INO-refractory or with duration of response (DoR) < 3 months, and responders (R). Gene expression results were analyzed with Ingenuity pathway analysis (IPA). Results: In our patient set higher CD22-FI, defined as higher quartiles (Q2-Q4), correlated with better patient outcome in terms of CR rate, OS and DoR, compared to lower CD22-FI (Q1). CD22 blast percentage was less able to discriminate patients’ outcome, although a trend for better outcome in patients with CD22 ≥ 90% could be appreciated. Concerning gene expression profile, 32 genes with corrected p value <0.05 and absolute FC ≥2 were differentially expressed in NR as compared to R. IPA upstream regulator and regulator effect analysis individuated the inhibition of tumor suppressor HIPK2 as causal upstream condition of the downregulation of 6 DEGs. Conclusions: CD22-FI integrates CD22-percentage on leukemic blasts for a more comprehensive target pre-treatment evaluation. Moreover, a unique pattern of gene expression signature based on HIPK2 downregulation was identified, providing important insights in mechanisms of resistance to INO.