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  • 1
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells.Objective: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood inononuclear colls (PBMC) from pollen allergic patients and healthy control individuals.Methods: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profiling (Bet v II) and recombinant timothy grass pollen allergens. Phi p I, Phi p II, and Phi p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. Reults PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected.Conclusion: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Ribosome-inactivating proteins (RIPs) are expressed in many plants. Because of their anti-infectious and anti-proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur.Objective Stimulated by our study, a group of RIP researchers reported that some of the employees had suspected allergy to RIPs.Methods and Results We tested their sera in ELISA on natural RIPs. Specific IgE in four subjects were found against dianthin30, gelonin, momordin, PAP-S, saporin, ricin and volkensin. In contrast, asparin and lychnin did not show any IgE binding. When separating extracts of plants containing the toxins in SDS-PAGE, RIPs appeared to be the predominant constituents. Interestingly, among the other plant proteins, they were exclusively recognized by IgE in immunoblot. RIPs derived from close botanical families share high sequence homologies. Nevertheless, in IgE inhibition experiments with human sera, cross-reactivity between RIPs also derived from non-related plants could be demonstrated.Conclusion We conclude that sensitization and IgE induction to RIPs may occur upon exposure. This has to be considered when applying them in therapy against malignancies or viral infections.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background and objective Biocompatible and biodegradable microparticles have gained interest as antigen delivery systems during the recent years. We investigated whether biodegradable poly(d,l-lactic-co-glycolic) acid (PLGA) nanospheres could be used as allergen vehicles for few-shot therapy of type I allergy.Methods The major birch pollen allergen Bet v 1 was encapsulated in PLGA nanospheres (PLGA-Bet v 1). We examined the antigenicity and the immune response to PLGA-Bet v 1 in a BALB/c mouse model.Results The antigenicity of Bet v 1 was largely unaffected by PLGA entrapment. When BALB/c mice were immunized subcutaneously with PLGA-Bet v 1, they formed allergen-specific IgG antibodies, but did not develop hypersensitivity to Bet v 1, as shown by type I skin tests. To evaluate their therapeutic potential, PLGA-Bet v 1 with or without Al(OH)3 or non-entrapped Bet v 1 with Al(OH)3 were used for single-shot treatment of sensitized mice. Both groups treated with PLGA-Bet v 1 developed high levels of Bet v 1-specific IgG2a antibodies (P〈0.01), whereas IgG1 levels decreased significantly (P〈0.01). Moreover, T cells from mice treated with PLGA-Bet v 1 showed IFN-γ and IL-10 production. The synthesis of these cytokines was enhanced in the groups where Al(OH)3 had been added to the vaccine formulation.Conclusion Allergen-loaded PLGA nanoparticles modulate an ongoing Th2 response in the BALB/c mouse model, as demonstrated by down-regulation of IgG1 and production of IFN-γ and IL-10. Our data strongly suggest that PLGA nanospheres can advantageously be used for formulations of allergen extracts or allergen derivatives for the few-shot treatment of type I allergy.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Patients suffering from allergic rhinoconjunctivitis and dyspnoea during summer may exhibit these symptoms after contact with flowers or dietary products of the elderberry tree Sambucus nigra.Objective Patients with a history of summer hayfever were tested in a routine setting for sensitization to elderberry. Nine patients having allergic symptoms due to elderberry and specific sensitization were investigated in detail. We studied the responsible allergens in extracts from elderberry pollen, flowers and berries, and investigated cross-reactivity with allergens from birch, grass and mugwort.Methods Sera from patients were tested for IgE reactivity to elderberry proteins by one-dimensional (1D) and 2D electrophoresis/immunoblotting. Inhibition studies with defined allergens and elderberry-specific antibodies were used to evaluate cross-reactivity. The main elderberry allergen was purified by gel filtration and reversed-phase HPLC, and subjected to mass spectrometry. The in-gel-digested allergen was analysed by the MS/MS sequence analysis and peptide mapping. The N-terminal sequence of the predominant allergen was analysed.Results 0.6% of 3668 randomly tested patients showed positive skin prick test and/or RAST to elderberry. IgE in patients' sera detected a predominant allergen of 33.2 kDa in extracts from elderberry pollen, flowers and berries, with an isoelectric point at pH 7.0. Pre-incubation of sera with extracts from birch, mugwort or grass pollen rendered insignificant or no inhibition of IgE binding to blotted elderberry proteins. Specific mouse antisera reacted exclusively with proteins from elderberry. N-terminal sequence analysis, as well as MS/MS spectrometry of the purified elderberry allergen, indicated homology with ribosomal inactivating proteins (RIPs).Conclusion We present evidence that the elderberry plant S. nigra harbours allergenic potency. Independent methodologies argue for a significant homology of the predominant 33.2 kDa elderberry allergen with homology to RIPs. We conclude that this protein is a candidate for a major elderberry allergen with designation Sam n 1.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background and objective In previous studies we have generated mimotopes of Bet v 1, the major birch pollen allergen, by biopannings of phage-display random peptide libraries. In the present study, we analysed the humoral and cellular immune response to Bet v 1-mimotopes.Methods The mimotope CFPYCYPSESA, designated Bet mim 1, was used for intraperitoneal immunizations of BALB/c mice in phage-displayed form. For examination of the humoral immune response, enzyme-linked immunosorbent assay (ELISA) experiments were applied. Stimulation capacities were investigated in cultured mouse splenocytes and in humoral Bet v 1-specific T cell clones.Results We demonstrated that the Bet mim 1-induced murine antibody response against Bet v 1 was predominated by the IgG1 isotype. In these mice only the phage-displayed mimotopes, but neither the allergen nor the synthetic Bet mim 1-mimotopes were able to stimulate proliferation of cultured splenocytes. Using Bet v 1-specific T cell clones of allergic patients, phage-displayed and synthetic mimotopes were unable to stimulate T cell proliferation. Moreover, tolerance induction to Bet v 1 in mice by intranasal administration of Bet mim 1-phages or Bet mim 1-peptide failed.Conclusion Taking these results together, our data indicate that Bet mim 1 mimics a Bet v 1-epitope on the B cell but not on the T cell level. We suggest that the phage itself is responsible for the recruitment of T cells providing bystander help in the formation of a mimotope-specific humoral response.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1365-2222
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer froin allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis. leading to death.Objective The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy.Methods A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradennal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting.Results Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA. Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I.Conclusion Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in 〉 95% of birch pollen allergic patients.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Human natural killer (NK) cells express low-affinity Fc immunoglobulin G (IgG) receptor (FcγRIIIA/CD16). The binding of monomeric IgG (mIgG) and F(ab′)2 fragments of 3G8 anti-CD16 monoclonal antibody (mAb) to FcγRIIIA was investigated by flow cytometry. Over 90% of NK cells bound endogenous IgG, and during incubation at 37 °C, the FcγRIIIA occupancy decreased slowly. Approximately 90% of NK cells bind mIgG or F(ab′)2 fragments of 3G8 anti-CD16 mAb. The calculated half-time (T1/2) of in vitro mIgG dissociation from FcγRIIIA was 130 min. By cross-linking the mIgG ligand with F(ab′)2 fragments of anti-human IgG antibody, the T1/2 decreases to 85 min. In kinetics study, it has been shown that 125I-mIgG bound to FcγRIIIA is slowly released in the culture supernatant, maybe eluted at acid pH, or partially internalized and degraded. The binding of IgG to FcγRIIIA was increased by 53.8% on cells cultured in the presence of RU36156, a matrix metalloproteinase (MMP) inhibitor. Furthermore, an increase in phosphorylation of Lyn tyrosine kinase, after cross-linking of mIgG-FcγRIIIA complex, was observed on NK cells treated with RU36156. When the FcγRIIIA was occupied by mIgG, the capacity of NK cells to kill K562 target cells was decreased by RU36156, because the MMP inhibitor protects CD16 from proteolysis. Our data demonstrate that binding of mIgG to human NK cells is followed by ligand dissociation and/or internalization, enzymatic degradation and exocytosis. The RU36156 MMP inhibitor protects FcγRIIIA from cleavage, augments NK-cell activation and may interfere in their killing capacity.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1398-9995
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Background:  Germins and the related germin-like proteins (GLPs) are glycoproteins expressed in many plants in response to biotic and abiotic stress. To test the potential impact of germins and GLPs, recombinant germin from Triticum aestivum (tGermin) and GLPs from Arabidopsis thaliana (tGLP), both produced in transformed tobacco plants, were used.Methods:  Sera from 82 patients with type I allergy to birch, grass or mugwort pollen and/or wheat were tested in immunoblot for IgE binding to tGermin and tGLP, and the IgE reactivity after chemical and enzymatic deglycosylation was analysed. The biological activity of tGermin and tGLP was determined in a histamine release assay and in skin prick testing (SPT).Results:  In an immunoblotting assay, 24 out of 82 tested sera (29.26%) from allergic patients showed IgE-binding to tGermin, and 18 of these sera (21.95%) displayed also IgE-binding to tGLP. The deglycosylation experiments indicated that glycan moieties contribute significantly to the IgE-binding of tGermin and tGLP. Both tGermins and tGLP induced specifically histamine release in an invitro assay as well as in SPT.Conclusion:  Our in vitro and in vivo findings demonstrate that germin and GLPs are capable to bind IgE most likely via carbohydrate determinants, and represent allergenic molecules.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, U.K. and Cambridge, USA : Blackwell Science Ltd
    Scandinavian journal of immunology 43 (1996), S. 0 
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: In vitro cultured cells derived from human bone marrow stimulated with macrophage-colony stimulating factor (M-CSF) or granulocyte-macrophage-colony stimulating factor (GM-CSF) were investigated for their immunophenotypic surface characteristics by flow cytometry. Approximately 80–90% of the cells showed morphological and histochemical features of macrophages and bore CD11a, CD11b, CD11c, CD14, CD29, CD32, CD33, CD44, CD45, CD54, CD64, CD71, HLA-DR antigen. The expression of CD4, CD25 and CD45RO were detected only in low density. Bone marrow-derived macrophages were negative for CD8, CD45RA, CD16 and CD23. Functionally, macrophages were able to phagocytose opsonized Escherichia coli, but no oxidative bursts were induced either by fmlp or by opsonized bacteria. Cell surface phenotyping revealed a CD pattern very similar to monocyte-derived phagocytes, and was partially distinct from resident stromal macrophages.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1365-3083
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The expression of Fc receptors for IgG (FcγR) and IgA (FcαR) and of various other antigens on the human monocytic cell line U937 and peripheral blood monocytes, under stimulation with human recombinant tumour necrosis factor-α (TNF-α) and other cytokines, was investigated by flow cytomelry. TNF-α, as well as interferon-γ (IFN-γ) or interleukin-6 (IL-6) had a significant up-regulating effect on U937 expression of FcγRI/CD64. Furthermore, the action of TNF-α was augmented by IL-6, and more evidently by IFN-γ. IFN-α alone had only a marginal effect, but was able to increase the TNF-α-driven FcγRI expression. In contrast to U937 cells, TNF-α did not enhance significantly FcγRI expression on human monocytes. Interestingly, on both U937 cells and monocytes. FcαR was augmented markedly by TNF-α. Furthermore, TNF-α induced the expression of HLA-DR and HLA-DP antigens on monocytes and U937 cells. The expression of FcγRII/CD32, FcγRIII/CD16. CD14, complement receptor type 1 (CR1/CD35). CR4 (CD11c/CD18), and MHC class-I antigens, was not influenced significantly by TNF-α. The results of this study show that TNF-α may act on human mononuclear phagocytes, alone or in combination with other cytokines, by modulating the expression of various cell-surface antigens.
    Type of Medium: Electronic Resource
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