House dust mites as potential carriers for IgE sensitization to bacterial antigens.

BACKGROUND: IgE reactivity to antigens from Gram-positive and Gram-negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis. METHODS: Plasma samples from patients with AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified HDM bodies, and the IgE-reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments. RESULTS: IgE reactivity to bacterial antigens was significantly more frequent in patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune-blotted HDM extract, and the presence of IgE-reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments. CONCLUSION: House dust mites (HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens.

Intranasal administration of allergen increases specific IgE whereas intranasal omalizumab does not increase serum IgE levels-A pilot study.

BACKGROUND: Administration of the therapeutic anti-IgE antibody omalizumab to patients induces strong increases in IgE antibody levels. OBJECTIVE: To investigate the effect of intranasal administration of major birch pollen allergen Bet v 1, omalizumab or placebo on the levels of total and allergen-specific IgE in patients with birch pollen allergy. METHODS: Based on the fact that intranasal allergen application induces rises of systemic allergen-specific IgE, we performed a double-blind placebo-controlled pilot trial in which birch pollen allergic subjects were challenged intranasally with omalizumab, placebo or birch pollen allergen Bet v 1. Total and allergen-specific IgE, IgG and basophil sensitivity were measured before and 8 weeks after challenge. For control purposes, total, allergen-specific IgE levels and omalizumab-IgE complexes as well as specific IgG levels were studied in subjects treated subcutaneously with either omalizumab or placebo. Effects of omalizumab on IgE production by IL-4/anti-CD40-treated PBMCs from allergic patients were studied in vitro. RESULTS: Intranasal challenge with Bet v 1 induced increases in Bet v 1-specific IgE levels by a median of 59.2%, and this change differed significantly from the other treatment groups (P = .016). No relevant change in allergen-specific and total IgE levels was observed in subjects challenged with omalizumab. Addition of omalizumab did not enhance IL-4/anti-CD40-induced IgE production in vitro. Significant rises in total IgE (mean IgE before: 131.83 kU/L to mean IgE after: 505.23 kU/L) and the presence of IgE-omalizumab complexes were observed after subcutaneous administration of omalizumab. CONCLUSION: Intranasal administration of allergen induced rises of allergen-specific IgE levels, whereas intranasal administration of omalizumab did not enhance systemic total or allergen-specific IgE levels.

Molecular allergen profiling in horses by microarray reveals Fag e 2 from buckwheat as a frequent sensitizer.

BACKGROUND: Companion animals are also affected by IgE-mediated allergies, but the eliciting molecules are largely unknown. We aimed at refining an allergen microarray to explore sensitization in horses and compare it to the human IgE reactivity profiles. METHODS: Custom-designed allergen microarray was produced on the basis of the ImmunoCAP ISAC technology containing 131 allergens. Sera from 51 horses derived from Europe or Japan were tested for specific IgE reactivity. The included horse patients were diagnosed for eczema due to insect bite hypersensitivity, chronic coughing, recurrent airway obstruction and urticaria or were clinically asymptomatic. RESULTS: Horses showed individual IgE-binding patterns irrespective of their health status, indicating sensitization. In contrast to European and Japanese human sensitization patterns, frequently recognized allergens were Aln g 1 from alder and Cyn d 1 from Bermuda grass, likely due to specific respiratory exposure around paddocks and near the ground. The most prevalent allergen for 72.5% of the tested horses (37/51) was the 2S-albumin Fag e 2 from buckwheat, which recently gained importance not only in human but also in horse diet. CONCLUSION: In line with the One Health concept, covering human health, animal health and environmental health, allergen microarrays provide novel information on the allergen sensitization patterns of the companion animals around us, which may form a basis for allergen-specific preventive and therapeutic concepts.

Molecular characterization of wheat allergens specifically recognized by patients suffering from wheat-induced respiratory allergy.

BACKGROUND: Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour-Baker's asthma). OBJECTIVE: The objective of this study was the molecular and immunological characterization of new recombinant wheat allergens and to evaluate their usefulness for the diagnosis of allergy to wheat. METHODS: A T. aestivum cDNA library was constructed and screened with serum IgE from patients suffering from wheat allergy to identify cDNAs coding for new wheat allergens. The allergen-encoding cDNAs were expressed in Escherichia coli and purified to homogeneity. IgE reactivity of recombinant proteins was analysed in RAST-based, non-denaturing dot blot experiments and by ELISA with sera from wheat allergic patients and their allergenic activity was assessed in basophil degranulation experiments. RESULTS: We report the molecular characterization, recombinant expression and purification of five wheat allergens, a thioredoxin h isoform, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin. Homologous proteins were identified by sequence comparisons in various plants. 1-Cys-peroxiredoxin appeared to be the most relevant of the newly identified wheat allergens according to prevalence of IgE recognition and results from basophil degranulation experiments. It showed IgE cross-reactivity with seed proteins from barley, rye, rice, maize, soy, oat and spelt. 1-Cys-peroxiredoxin, glutathione transferase and dehydrin were mainly recognized by patients with baker's asthma but not wheat-induced food allergy. CONCLUSION AND CLINICAL RELEVANCE: The characterized recombinant wheat allergens may be useful for the development of serological tests which allow the discrimination of different clinical manifestations of wheat allergy.

The role of T-cell reactivity towards the autoantigen α-NAC in atopic dermatitis.

BACKGROUND: A subgroup of patients with atopic dermatitis (AD) produces IgE autoantibodies to human proteins which may be present in inflamed skin and perpetuate cutaneous inflammation. OBJECTIVES: In order to investigate mechanisms of 'autoallergy' for AD we studied T-cell responses to the autoallergen Hom s 2, the human transcriptional coactivator α-nascent polypeptide-associated complex (α-NAC). METHODS: Specific proliferation of blood lymphocytes from 30 patients and 12 healthy control individuals was investigated by flow cytometry. The proliferation of skin- and blood-derived T cells was assessed in limiting-dilution assays. T-cell clones (TCC) were generated from peripheral blood and from biopsies of lesional skin of patients with AD and the phenotype and cytokine patterns were determined. RESULTS: α-NAC-specific T-cell responses were detected in patients and control individuals. α-NAC induced a significantly higher proliferation of CCR4+ (compared with CCR4-) and CLA+ (compared with CLA-) T cells from the circulation. Limiting-dilution assays revealed a high proliferation of blood and skin-infiltrating lymphocytes in the presence of α-NAC compared with control cultures. α-NAC-specific TCC generated from lesional skin of AD predominantly produced interferon-γ and some TCC also produced interleukin-17. The cytokine pattern of α-NAC TCC may contribute to keratinocyte apoptosis and eczema formation in AD. CONCLUSIONS: α-NAC-specific TCC can be generated from blood and lesional skin of patients with AD. These TCC produce not only Th2 but also Th1 cytokines which may explain the Th1 phenotype of inflammation in AD.

Immunocytochemical localisation of actin and profilin in the generative cell of angiosperm pollen: TEM studies on high-pressure frozen and freeze-substituted Ledebouria socialis Roth (Hyacinthaceae).

Actin was demonstrated for the first time at the EM level in the generative cell of mature angiosperm pollen by using immuno-gold labelling of high-pressure frozen and freeze-substituted Ledebouria socialis Roth anthers. In addition, profilin, an actin-monomer binding protein, is shown to coexist in the generative cell. We attribute the detection of actin and profilin to the applied cryomethods which yield a much better preservation of ultrastructure and antigenicity of delicate cytoskeletal constituents than conventional fixation techniques. Actin labelling was observed within the cytoplasm of the generative cell and became especially clear in close vicinity to microtubular bundles. Filamentous structures congruent with the actin labelling patterns do occur, but are not a frequent feature. Profilin was localised throughout the cytoplasm.

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation.

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50-100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.

Identification of pronp1, a tobacco profilin gene activated in tip-growing cells.

In plant cells, several cellular processes depend on rapid reorganization of a dynamic network of actin cytoskeletal elements in response to internal and environmental stimuli. Profilins, ubiqitous eukaryotic actin monomer-binding proteins with highly conserved three-dimensional structures, regulate the actin cytoskeleton and are considered to link the microfilament system with signal transduction pathways. Plant profilins have been grouped into two distinct classes, gametophytic (pollen-specific) and sporophytic. Here we report the isolation of a profilin gene that seems to be activated during tip growth of specialized cells of gametophytic as well as sporophytic origin. Identification of a genomic DNA clone containing a tobacco profilin gene, pronp1, and analysis of the pronp1 promoter-uidA fusion gene in transgenic Nicotiana tabacum plants revealed a prominent expression of pronp1 in mature pollen and elongating pollen tubes and significant activity in root hairs of developing seedlings. This expression pattern was distinct from that of any other profilin gene isolated so far. Pronp1 thus represents a unique profilin gene that is activated at the transcriptional level in two kinds of tip-growing cells, pollen tubes and root hairs, both of which require rapid organization of the actin cytoskeleton. The isolation of such a gene has fundamental importance for our understanding of modulation of the actin cytoskeleton at the molecular level.

High-level expression in Escherichia coli and purification of recombinant plant profilins: comparison of IgE-binding capacity and allergenic activity.

Because of their structural similarity and ubiquitous distribution as actin binding proteins, plant profilins represent important cross-reactive allergens for almost 20% of patients suffering from Type I allergy to pollen and other plant products. The cDNAs coding for three birch profilin variants (Tyr44, Glu47, and Asn47), timothy grass profilin, and three tobacco profilin isoforms (ntprof1-3) were expressed at high levels in Escherichia coli as non-fusion proteins. The recombinant plant profilins were purified to homogeneity by poly (L-proline) affinity chromatography and showed comparable capacity to bind IgE-antibodies from profilin allergic patients. All recombinant plant profilins elicited dose-dependent histamine release from basophils of a profilin allergic patient and induced immediate type skin reactions. It is concluded that profilins from different plant species share IgE-epitopes and allergenic properties. Plant profilins therefore constitute a family of functional pan-allergens which may substitute each other for diagnosis and specific immunotherapy.