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Background IgE antibodies specific for the major birch pollen allergen frequently cross-react with Bet v 1 homologous food proteins, for example Cor a 1 in hazelnut and Mal d 1 in apple. Specific immunotherapy with birch pollen ( BP- SIT) induces Ig G4 antibodies that inhibit Ig E binding to Bet v 1. However, information on cross-reactivity of BP- SIT-induced Bet v 1-specific Ig G4 antibodies with food allergens is limited. In this study, we investigated the kinetics of production, cross-reactivity, and Ig E-blocking activity of Bet v 1-specific Ig G4 antibodies emerging during conventional BP- SIT and whether Ig G4-epitopes overlapped with IgE epitopes. Methods IgE and Ig G4 levels specific for Bet v 1, Mal d 1, and Cor a 1 were determined in 42 birch pollen-allergic patients before and during BP- SIT. Inhibition of Ig E binding was studied by Ig E-facilitated antigen-binding assays and basophil activation tests. Furthermore, inhibition of Ig E-mediated activation of food allergen-reactive Bet v 1-specific T-cell lines was assessed. Competitive immunoscreening of phage-displayed peptides was applied to select mimotopes recognized by Ig E and Ig G4 antibodies, respectively. The resulting mimotopes were mapped on the surface of the 3 D structure of the allergens using a computer-based algorithm. Results BP- SIT significantly increased Bet v 1- and food allergen-reactive Ig G4 antibodies. In parallel, allergen-specific Ig E levels decreased significantly. Sera containing food allergen-reactive Ig G4 antibodies inhibited Ig E binding, basophil activation, and Ig E-mediated food allergen-induced T-cell proliferation. Predicted Ig E and Ig G4 epitopes on all allergens showed high overlap. Conclusion Our results indicate that BP- SIT may induce Bet v 1-specific Ig G4 antibodies that cross-react with related food allergens and inhibit Ig E binding by epitope competition. [ABSTRACT FROM AUTHOR]

Background Alternaria alternata is one of the most important allergenic fungi worldwide. Mannitol dehydrogenase (MtDH) has previously been shown to be a major allergen of Cladosporium herbarum and cross-reactivity has been demonstrated for several fungal allergens. Objective The present study's objective was to clone the MtDH from an A. alternata cDNA library, express and purify the recombinant non-fusion protein and test its IgE-binding properties. Methods A cDNA library prepared from A. alternata hyphae and spores was screened for mannitol dehydrogenase by DNA hybridization with the radioactively labelled C. herbarum homologue as a probe. The resulting clone was sequenced and heterologously expressed in Escherichia coli as a recombinant non-fusion protein, which was purified to homogeneity and analysed for its IgE-binding capacity. Results The coding sequence of the full-length cDNA clone comprises 798 bp encoding a protein with a molecular mass of 28.6 kDa and a predicted pI of 5.88. Protein sequence analysis revealed an identity of 75% and a homology of 86% between the MtDHs of A. alternata and C. herbarum. The functional mannitol dehydrogenase was expressed in the E. coli strain BL21(DE3) transformed with the vector pMW172 and purified to homogeneity. The enzyme catalyses the NADPH-dependent conversion ofd-fructose tod-mannitol. In IgE-ELISA and immunoblots, MtDH is recognized by 41% of A. alternata-allergic patients. In vivo immunoreactivity of the recombinant MtDH was verified by skin prick testing. Finally, inhibition-ELISA experiments confirmed cross-reactivity between the MtDHs of A. alternata and C. herbarum. Conclusion Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures. [ABSTRACT FROM AUTHOR]

The T cell reactivity to the major allergen of bee venom, phospholipase A2, has been thoroughly characterized. In contrast, only little is known about the human cellular response to major allergens from wasp venom.To characterize the human T cell response to antigen 5 fromVespula vulgaris, Ves v 5.Recombinant Ves v 5 was used to establish allergen-specific T cell lines (TCL) and T cell clones (TCC) from the peripheral blood of vespid-allergic and non-allergic individuals. Ves v 5-specific TCL were mapped for T cell epitopes using overlapping synthetic peptides representing the complete amino acid sequence of Ves v 5. Ves v 5-specific TCC were analysed for antigen-induced secretion of IL-4, IFN-γ and IL-10.Seventeen distinct T cell epitopes were recognized by allergic individuals among which Ves v 5181–192 was identified as a dominant T cell epitope. Partially different epitopes were observed in TCL from non-allergic subjects and the dominant epitope Ves v 5181–192 was not prevalent in these cultures. Ves v 5-specific TCC isolated from allergic individuals did not show the typical T helper type 2 (Th2)-like cytokine profile in response to specific stimulation, i.e. high amounts of IL-4 and low IFN-γ. TCC from non-allergic individuals showed a Th1-like cytokine pattern.Our findings provide evidence that the allergic T cell response to Ves v 5 is not Th2-dominated and that different immunogenic sites on this major wasp venom allergen are recognized by allergic and non-allergic individuals. [ABSTRACT FROM AUTHOR]

Background and Objective. The mechanisms operative in specific immunotherapy (SIT) of Type I allergy are not completely understood. In the present study we evaluated immunological changes during SIT in pollinosis. Method. Eight patients suffering from pollinosis (monosensitized to grass pollen) were treated with conventional SIT. All subjects had IgE specific for Ph1 p 1, a major allergen of timothy grass. In vitro changes in the immunological reactivity to grass pollen extract and to recombinant Ph1 p 1 were evaluated. Subjects were examined at three occasions: before, after 3 months and after 1 year of SIT. Results. Serological analysis revealed a marked increase of grass pollen- and Ph1 p 1-specific IgG, titres of specific IgE did not change significantly. Lymphoproliferative responses to grass pollen extract and rPh1 p 1 were reduced already after 3 months of treatment. Accordingly, the cloning efficiency for Ph1 p 1-specific T-cell clones (TCC) dropped markedly in all patients. The majority of allergen-specific TCC raised before SIT revealed a TH2-like pattern of cytokine production. TCC established after SIT revealed TH1 characteristics. This shift was due to a decrease in IL-4 rather than an increase of IFN-production by T cells. Investigations of the epitopes recognized by T cells before and after SIT did not reveal the outgrowth of new ('protecting') specificities. We could not observe induction of allergen-specific CD8+ lymphocytes (supressor cells). Conclusion Our data indicate that &mdash on the level of TH lymphocytes — SIT induces tolerance to the allergen and a modulation of the cytokine pattern produced in response to allergen stimulation. [ABSTRACT FROM AUTHOR]

Background Symptoms elicited by IgE mediated food allergy range from mild local to severe systemic reactions. Allergens in spices are particularly dangerous due to their hidden presence in many dishes. Objectives and Methods According to clinical observations, mugwort and birch pollen allergy, and hypersensitivity to spices are frequently associated, but the crossreacting compounds were not defined so far. We tested sera of 15 patients who experienced adverse reactions to spiced food and characterized their IgE-binding patterns on anise, fennel, coriander and cumin extracts through immunoblot and inhibition experiments. Results The use of anti-Bet v 1 (MoAb) and anti-profilin (rabbit) antibodies revealed the presence of crossreacting allergens in the tested spice extracts. Inhibition experiments showed that IgE-binding to allergens in Apiaceae spices could be blocked by preincubation of sera with rBet v I or rBet v 2 (birch profilin). Moreover, we detected crossreacting allergenic molecules in the molecular weight range of 60 kDa. IgE-binding to spice allergens occurred only with sera of 10/15 (66%) patients with allergy to pollen (birch, niugwort) and/ or celeriac. In five out of 15 (33%) patients with a history of adverse reaction to spices, but without pollen and celeriac allergy, no IgE-binding to any spice protein could be demonstrated. It is possible that these clinical reactions could be elicited by other types of hypersensitivity (Type II. Ill, IV), however, as spices contain highly reactive substances, the symptoms may most likely be classified as food-intolerant. Conclusions Bet v 1- and profilin-related allergens may, besides higher molecular weight allergenic molecules, be responsible for Type I allergy to anise, fennel, coriander or cumin, members of the Apiaceae. [ABSTRACT FROM AUTHOR]

Background Animal hair/dander proteins frequently cause Type I hypersensitivities. Species-specific and broadly cross-reacting allergens have been characterized in the past. Methods Sera from eight individuals suffering from symptoms due to exposure to deer and deer-derived products were investigated by immunoblotting. Extracts from deer, dog, cat, horse, rabbit and cow, respectively, were tested for IgE-binding. To reveal cross- reactivities patients' sera were preadsorbed with these extracts prior to testing with deer extract. Results Deer allergens with the molecular mass of 22 and 25 kD (major allergens), as well as 60 kD were identified. The 22 and 25 kD allergens are cross-reactive with the corresponding cow allergens. Conclusions Deer allergy is a rare sensitization mainly affecting persons exposed to deer, who displayed an atopic disposition. From our results it can be assumed that this hypersensitivity is partly associated with allergy to cow dander. [ABSTRACT FROM AUTHOR]

Background: Allergy to celery is often associated with sensitization to birch and;or mugwort pollen. Objective and Methods: In a multi-centre study. sera from 23 patients suffering from type I allergy to celery and 1 5 patients with positive celery RAST hut no clinical sensitization were compared. To examine whether cross-reactivity between celery and mugwort pollen includes cross-sensitization to birch pollen allergens, we determined cross-reacting structure in birch pollen. mugwort pollen and celery by means of immunoblotting. Inhibition studies were performed by preincubation of sent with extracts of birch pollen. mugwort pollen, and celery. Results: We identified three groups of proteins homologues of Bet v 1 and birch profilin (Bet v2) as well as a group of proteins with a molecular range of 46 to 60KD displaying IgE-cross-reactivity. which were shared by birch pollen and celery. Two of these groups of allergens (profilin and the 46 to 60 U) proteins) were also present in mugwort pollen. In this paper we demonstrate that most cross-reacting allergens present in mugwort pollen and celery can also be detected in birch pollen extract. Conclusion therefore we propose. Iron a serological point of view, to extend the mugwort-celery syndrome to the birch-mugwort-celery syndrome. [ABSTRACT FROM AUTHOR]

Background Allergic conjunctivitis is a common symptom amongst Type I (IgE-mediated) allergic diseases; and most frequently seen as rhinoconjunctivitis. However, the site of production and the significance of allergen specific IgE needs further elucidation. Objective We investigated whether the presence of IgE in tears of grass pollen allergic patients correlated with disease and clinical symptoms, whether the IgE binding pattern to the different grass pollen antigens was different in sera and tears, and whether IgA antibodies to grass pollen allergens were present in tears. Finally, we looked whether specific IgE was produced locally or was exudated from serum. Methods Sera from 44 grass pollen allergic patients suffering from either allergic rhinitis (n =11) or allergic rhinoconjunctivitis (n = 33) and from healthy controls (n =7) were used for the experiments. Binding of specific IgE and IgA antibodies to the different groups of grass pollen allergens (Phleum pratense) was evaluated by means of immunoblotting. Results Specific IgE was detected in sera as well as in tears of allergic patients, whereby tear-derived allergen-specific IgE exerted similar specificities to the corresponding IgE from serum. The correlation between symptoms of ocular allergy and the presence of allergen-specific IgE in tears was highly significant (P = 0.0001). In contrast, only a poor correlation was found between specific and/or total IgE in sera and the manifestation of ocular allergy (P = 0.73). Conclusion Allergen-specific IgE antibodies in tears seem to be produced locally rather than exsudated from serum. IgE in tears seems to be responsible for allergic conjunctivitis. IgA in tears cannot exert a protective function since the IgA antibodies recognize different antigens in a grass pollen (Phleum pratense) extract than IgE antibodies. The highly significant correlation between allergic conjunctivitis and the presence of specific tear IgE emphasizes the diagnostic value of immunoblots with tear IgE, especially in cases in which serum provides inconclusive results. [ABSTRACT FROM AUTHOR]

Background Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer from 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 intradermal 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. [ABSTRACT FROM AUTHOR]

The article discusses the functions of recombinant allergens in the diagnosis and treatment of atopic allergy. It highlights the allergen-specific monitoring of immunoglobulin antibodies against allergens in childhood including the characterization of cross-reactions in accordance with the molecular structures of allergens. It also discusses several approaches to the production of new molecules for the treatment of birch-pollen allergy.

Mugwort and birch pollen allergy are frequently associated with IgE-mediated hypersensitivity to celery and spices. We analyzed 22 sera from patients with the mugwort-birch-celery-spice syndrome for IgE binding to the spices pepper and paprika by immunoblotting, Immunoblot results revealed two major allergens of 28 and 60 kDa in pepper and a 23-kDa allergen together with allergens of higher molecular weight in paprika. In immunoblot-inhibition studies, crude mugwort, birch pollen, and celery extracts significantly reduced the IgE binding to pepper and paprika allergens. However, no inhibition was achieved with rBet v 1 and rBet v 2, suggesting that no homologs of these birch proteins act as allergens in pepper or paprika extracts, N-terminal sequence analysis of the 14- and 28-kDa pepper and 23-kDa paprika allergens revealed no homology to known allergens. The 28-kDa pepper allergen showed homology to a wheat germin protein, and the 23-kDa paprika allergen was identified as a homolog of a osmotin-like or pathogenesis-related protein in tomato. Therefore, we conclude that the IgE cross-reactivity in the mugwort-birch-celery-spice syndrome to the spices pepper and paprika is not caused by homologs of Bet v 1 and profilin, N-terminal amino acid sequence analysis of the main allergens in pepper and paprika indicate a relation to frequently occurring plant proteins. [ABSTRACT FROM AUTHOR]

Several in vitro and in vivo studies indicate that application of high doses of dominant T cell epitopes can induce a state of antigen-specific non-responsiveness (anergy). In the present study, we developed a murine model of an allergic immune response to Bet v 1, the major birch pollen allergen. Mice were sensitized by injection of rBet v 1 and the allergic state was proven by the presence of allergen-specific IgE and positive immediate-type skin tests to Bet v 1. In epitope mapping experiments, an immunodominant T cell epitope of Bet v 1 in BALB/c mice was identified by the use of overlapping peptides. This peptide (BV139) was subsequently employed for treatment. Two tolerization protocols were used; in one approach, the peptide was administered to naive mice before immunization (group BV139-S), in the second, already sensitized mice were treated (S-BV139). The results demonstrated that administering high doses of the dominant T cell epitope of Bet v 1 profoundly diminished T cell proliferation to the peptide in the BV139-S group, and to the peptide as well as to the whole protein in the S-BV139 group. Skin test reactivity to Bet v 1 was reduced in the BV139-S group. However, no differences in terms of specific antibody production between treated and untreated mice could be observed. This study provides evidence that administration of dominant T cell epitopes can down-regulate the allergen-specific T cell response. Proceeding on the assumption that the T lymphocyte response to allergens is crucial for the induction and maintenance of the allergic disease, a modulation of the immune response to allergens by treatment with T cell epitope peptides could represent a promising concept for immunotherapy in the future. [ABSTRACT FROM AUTHOR]

We analyzed the cDNA sequence data of complementarity determining regions (CDR3) of epitope mapped αβ TCR of T cell clones (TCC). The TCC were specific for the major timothy grass (Phleum pratense) pollen allergen Phl p 1 and were derived from the peripheral blood of seven unrelated grass pollen-allergic individuals. Each TCR recognized one of two immunodominant T cell epitopes, PP73 or PP103, of Phl p 1. Although a diversity of recombined V and J segments was observed, amino acid motifs as long as five residues were conserved among CDR3 loops of TCR from TCC of different atopic individuals specific for the same peptide. The conserved sequences could comprise as much as 60% of the CDR3. All amino acid residues of the motifs of the CDR3β and most of the CDR3α of all TCR used in this study were encoded by randomly added nucleotides. This indicates that they were specifically selected for by the peptide bound to the MHC class II molecule. For one selected patient, a larger number of TCC, specific for PP103, was analyzed. The TCR repertoire was limited to three different TCR. The same MHC class II molecule, DRB1*1301, was identified to present PP103 to each of the three TCR. [ABSTRACT FROM PUBLISHER]