Home About us Contact
|
Home About us Contact | ||
|
|
||
Overlapping Peptides (overlapping + peptide)
Selected AbstractsCloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitorJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2004K. Verbeke Summary., This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor-1 (PAI-1) neutralizing single-chain variable fragment 56A7C10 (scFv-56A7C10). ScFv-56A7C10-wt exhibits a similar affinity (KA = 1.01 ± 0.3 × 109 m,1) and PAI-1 inhibitory capacity (90 ± 6% PAI-1 inhibition at a 16-fold molar excess and IC50 = 44 ± 14 ng mL,1) as MA-56A7C10 (KA = 1.43 ± 0.4 × 109 m,1, 90 ± 2% PAI-1 inhibition at a 16-fold molar excess and IC50 = 122 ± 26 ng mL,1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv-56A7C10-mutants (n = 26) were analyzed for their PAI-1 binding and PAI-1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI-1 inhibitory capacities (IC50 , 418 ng mL,1), confirmed by reduced affinities (14-, 17-, 7-, 9- and 16-fold reduced, respectively, vs. scFv-56A7C10-wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI-1 inhibitory properties (IC50 , 160 ng mL,1) and decreased affinities (i.e. 4-, 9-, 3-, 3- and 2-fold reduced affinity, respectively, vs. scFv-56A7C10-wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three-dimensional model of scFv-56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI-1 neutralizing compounds. [source] Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infectionHEPATOLOGY, Issue 6 2003Kazushi Sugimoto Hepatitis C virus (HCV) has a high propensity for persistence. To better define the immunologic determinants of HCV clearance and persistence, we examined the circulating HCV-specific T-cell frequency, repertoire, and cytokine phenotype ex vivo in 24 HCV seropositive subjects (12 chronic, 12 recovered), using 361 overlapping peptides in 36 antigenic pools that span the entire HCV core, NS3-NS5. Consistent with T-cell-mediated control of HCV, the overall HCV-specific type-1 T-cell response was significantly greater in average frequency (0.24% vs. 0.04% circulating lymphocytes, P = .001) and scope (14/36 vs. 4/36 pools, P = .002) among the recovered than the chronic subjects, and the T-cell response correlated inversely with HCV titer among the chronic subjects (R = ,0.51, P = .049). Although highly antigenic regions were identified throughout the HCV genome, there was no apparent difference in the overall HCV-specific T-cell repertoire or type-1/type-2 cytokine profile relative to outcome. Notably, HCV persistence was associated with a reversible CD4-mediated suppression of HCV-specific CD8 T cells and with higher frequency of CD4+CD25+ regulatory T cells (7.3% chronic vs. 2.5% recovered, P = .002) that could directly suppress HCV-specific type-1 CD8 T cells ex vivo. In conclusion, we found that HCV persistence is associated with a global quantitative and functional suppression of HCV-specific T cells but not differential antigenic hierarchy or cytokine phenotype relative to HCV clearance. The high frequency of CD4+CD25+ regulatory T cells and their suppression of HCV-specific CD8 T cells ex vivo suggests a novel role for regulatory T cells in HCV persistence. [source] Role of pathogenic T cells and autoantibodies in relapse and progression of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis in LEW.1AV1 ratsIMMUNOLOGY, Issue 1pt2 2009Yoh Matsumoto Summary Accumulating evidence suggests that T cells and autoantibodies reactive with myelin oligodendrocyte glycoprotein (MOG) play a critical role in the pathogenesis of multiple sclerosis (MS). In the present study, we have tried to elucidate the pathomechanisms of development and progression of the disease by analysing T cells and autoantibodies in MOG-induced rat experimental autoimmune encephalomyelitis (EAE), which exhibits various clinical subtypes mimicking MS. Analysis using overlapping peptides revealed that encephalitogenic epitopes resided in peptide 7 (P7, residue 91,108) and P8 (residue 103,125) of MOG. Immunization with MOGP7 and MOGP8 induced relapsing,remitting or secondary progressive EAE. T cells taken from MOG-immunized and MOGP7-immunized rats responded to MOG and MOGP7 and sera from MOG-immunized rats reacted to MOG and MOGP1. Significant epitope spreading was not observed at either T-cell or antibody levels. Interestingly, sera from MOGP7-immunized rats with clinical signs did not react to MOG and MOG peptides throughout the observation period, suggesting that disease development and relapse in MOGP7-induced EAE occur without autoantibodies. However, MOGP7 immunization with adoptive transfer of anti-MOG antibodies aggravated the clinical course of EAE only slightly. Analysis of antibodies against conformational epitope (cme) suggests that anti-MOGcme may play a role in the pathogenicity of anti-MOG antibodies. Collectively, these findings demonstrated that relapse of a certain type of MOG-induced EAE occurs without autoantibodies but that autoantibodies may play a role in disease progression. Relapses and the progression of MS-mimicking EAE are differently immunoregulated so immunotherapy should be designed appropriately on the basis of precise information. [source] Characterization of the immune response against hepatitis C infection in recovered, and chronically infected chimpanzeesJOURNAL OF VIRAL HEPATITIS, Issue 6 2002M. T. Shata summary.,The immune response to hepatitis C virus (HCV) is believed to be critical in determining the outcome of the disease. In this study we have analysed epitope recognition, cytokine profile, and anti-HCV antibody responses in chronically HCV-infected and recovered chimpanzees. Quantitative measurement of anti-HCV antibody in HCV-infected chimpanzees revealed that the response in HCV- recovered chimpanzees peaked within 4,20 weeks. In contrast, the anti-HCV antibody responses in chronically HCV infected chimpanzees did not peak until 100,200 weeks after infection, and decreased gradually thereafter. T cell proliferation assays measuring responses to pooled HCV proteins revealed significant increases in the 3H-uptake during the early stages of infection in recovered chimpanzees in comparison to the chronically infected ones. Class I-restricted epitopes of the core, and NS3 proteins of HCV were analysed using 9,10 mer overlapping peptides covering the core and NS3 proteins, and IFN-, ELISPOT technique. Our data indicated early and broad class-I restricted core, and NS3 protein epitope recognitions in HCV-recovered chimpanzees but not in chimpanzees that had been chronically infected. Additionally, dominant epitopes recognized early in infection (8 weeks) were no longer recognized later in infection (followed up to 64 weeks). Cytokines profiling revealed a 50-fold increase in TNF-, secretion in the supernatant of core-specific CD8 memory cells of the chronically infected chimpanzees in comparison to the recovered ones. In summary, multiple parameters correlate with HCV recovery in chimpanzees. [source] Immunological characterization of a Blo t 12 isoallergen: identification of immunoglobulin E epitopesCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009J. Zakzuk Summary Background Differences in the IgE response to isoallergens could have clinical implications; therefore, its analysis will contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases. Several isoforms have been described from mites but there is no information about the clinical impact of Blomia tropicalis isoallergens. Objective To evaluate the differences in the IgE response against two Blo t 12 isoallergens. Methods The IgE-binding properties of Blo t 12 isoallergens were analysed by ELISA, a skin prick test and ELISA cross-inhibition. Epitope mapping was performed using synthetic overlapping peptides. Fold recognition methods were used to model the chitin-binding domain of the two isoallergens. Results The frequency and strength of the IgE response were greater for Blo t 12.0101 than for Blo t 12.0102. Three IgE-binding areas were identified in Blo t 12.0101; one of them included two residues that are different in Blo t 12.0102. Modelling of the chitin-binding domains of these allergens predicted that they have structural differences that could influence antibody recognition of one of these epitopes. Conclusion In silico structural analysis and immunological characterization of Blo t 12 reveals that allergen polymorphism influences IgE reactivity. Blo t 12.0101 is the most IgE-reactive isoform in Cartagena. The identified IgE epitopes could be mutated to obtain hypoallergenic molecules of potential use for immunotherapy. [source] Analysis of sequential immunoglobulin E-binding epitope of Japanese cedar pollen allergen (Cry j 2) in humans, monkeys and miceCLINICAL & EXPERIMENTAL ALLERGY, Issue 2 2003Y. Tamura Summary Background Japanese cedar (Cryptomeria japonica; CJ) pollinosis has been reported to occur naturally in Japanese monkeys (Macaca fuscata) as well as in humans. Most human patients and monkeys with pollinosis have specific IgE for Cry j 2, a major allergen of CJ pollen. Objective The main purpose of this study was to identify IgE B cell epitopes of Cry j 2 using a synthetic peptide in humans, monkeys and mice. Methods We synthesized 38 overlapping peptides that span the entire length of Cry j 2. We examined the B cell epitopes of Cry j 2 that are recognized by IgE in the sera of human patients and monkeys with pollinosis and immunized mice using synthetic peptides of Cry j 2. We also examined the reaction of Cry j 2-specific mouse monoclonal IgG antibodies to the peptides. Furthermore, we conducted a histamine release assay with leucocytes from a pollinosis patient using human serum albumin (HSA) conjugated with the peptides as a B cell epitope. Results We found that 16 of the 20 pollinosis patients who had specific IgE to Cry j 2 also exhibited IgE reaction with some Cry j 2 peptides. Of these 16 patients, 10 exhibited IgE reaction with Cry j 2 peptide no. 13 (121GQCKWVNGREICNDRDRPTA140). Five of the seven monkeys with CJ pollinosis exhibited a reaction with peptide no. 13. Furthermore, IgE in mice immunized with Cry j 2 and two mouse monoclonal IgG antibodies reacted with peptide no. 13. Peptide no. 13-conjugated HSA showed the release of histamine from basophils. Furthermore, to determine the minimum epitope in peptide no. 13, we conducted an enzyme-linked immunosorbent assay inhibition test. The core of the epitope in humans, monkeys and mice was 124KWVNGREI131. Conclusion We found that 124KWVNGREI131 is an important B cell epitope recognized by IgE in humans, monkeys and mice. [source] Novel B and T cell epitopes of chicken ovomucoid (Gal d 1) induce T cell secretion of IL-6, IL-13, and IFN-,CLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2001E. Holen Background Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. Objectives The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. Methods Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. Results Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61,74, had the ability to bind IgE. Another peptide, OM 101,114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41,56, OM 71,84, OM 131,144 and OM 171,186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-,, IL-6, IL-4, IL-13, IL-10 and TNF-, in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-,, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71,84 and 51,64) when TCLs from two non-allergic donors were used. Conclusions One novel B cell epitope (OM 61,74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1,14 to 71,84, the overlapping peptide-pairs OM 121,134 and OM 131,144 and peptides OM 161,174 and 171,186. Peptides OM 1,14 and 171,186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11,24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-, and particularly IL-6. [source] Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot studyCLINICAL MICROBIOLOGY AND INFECTION, Issue 6 2006D. Goletti Abstract A previous case-control study reported that an in-vitro interferon (IFN)-, response to early secreted antigenic target (ESAT)-6 selected peptides was associated with active tuberculosis (A-TB). The objective of the present pilot study was to evaluate the diagnostic accuracy of this assay for TB disease in a clinical setting. An IFN-, ELISPOT assay was performed on samples from patients with suspected A-TB using two peptides selected from ESAT-6 protein and three peptides selected from culture filtrate 10 (CFP-10) proteins. The results were compared with those obtained by two commercially available assays approved for diagnosis of TB infection (T SPOT-TB and QuantiFERON-TB Gold) which use ESAT-6/CFP-10 (RD1) overlapping peptides. Sensitivity to the RD1 selected peptides was 70% (positive for 16 of 23 patients with microbiologically diagnosed A-TB) and specificity was 91% (positive for three of 32 controls). In contrast, the sensitivity and specificity were 91% and 59%, respectively, for T SPOT-TB, and were 83% and 59%, respectively, for QuantiFERON-TB Gold. The RD1 selected peptides assay had the highest diagnostic odds ratio for A-TB. Thus, the results suggest that an assay based on RD1 selected peptides has a higher diagnostic accuracy for A-TB in a clinical setting compared with commercially available assays based on RD1 overlapping peptides. [source] | ||||||||||