Oral challenge with pasteurized egg white from Gallus domesticus.

BACKGROUND: Since raw egg may cause digestive toxic infections, we assessed whether pasteurized raw egg white is as reliable as fresh raw egg white in the diagnosis of egg allergy. METHODS: Thirty-two egg-allergic children were challenged with both pasteurized and fresh raw egg white. Open challenges were carried out with increasing doses of pasteurized raw egg white and fresh raw egg white administered every 60 min. RESULTS: Eleven children (34.4%) had positive challenges with pasteurized raw egg white. Twenty-one children (65.62%) who tolerated pasteurized raw egg white also had a negative challenge with fresh raw egg white. If the challenge with pasteurized raw egg white resulted positive, the study was stopped. The protein profile and IgE-binding capacity of both pasteurized and fresh egg white were almost identical as observed by SDS-PAGE and IgE immunoblotting. In the IgE immunoblotting-inhibition and ImmunoCAP-inhibition assays, both extracts behaved in a similar way. CONCLUSIONS: We did not find any relevant allergenic differences between fresh and pasteurized egg white. This study supports the use of pasteurized egg white in the diagnosis of allergy to fresh raw egg proteins.

Fish allergy in childhood.

Fish and its derived products play an important role in human nutrition, but they may also be a potent food allergen. Fish can be an ingested, contact, and inhalant allergen. Gad c I, a Parvalbumin, the major allergen in codfish, is considered as fish and amphibian pan-allergen. Prevalence of fish allergy appears to depend on the amount of fish eaten in the local diet. In Europe, the highest consumption occurs in Scandinavian countries, Spain and Portugal. In Spain, fish is the third most frequent allergen in children under 2 yr of age after egg and cow's milk. An adverse reaction to fish may be of non-allergic origin, due to food contamination or newly formed toxic products, but the most frequent type of adverse reactions to fish are immunologic-mediated reactions (allergic reactions). Such allergic reactions may be both IgE-mediated and non-IgE-mediated. Most cases are IgE-mediated, due to ingestion or contact with fish or as a result of inhalation of cooking vapors. Some children develop non-IgE-mediated type allergies such as food protein induced enterocolitis syndrome. The clinical symptoms related to IgE-mediated fish allergy are most frequently acute urticaria and angioedema as well as mild oral symptoms, worsening of atopic dermatitis, respiratory symptoms such as rhinitis or asthma, and gastrointestinal symptoms such as nausea and vomiting. Anaphylaxis may also occur. Among all the species studied, those from the Tunidae and Xiphiidae families appear to be the least allergenic.

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits.

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.

Food allergy and cross-reactivity-chickpea as a test case.

Chickpea has become one of the most abundant crops consumed in the Mediterranean and also in western world. Chickpea allergy is reported in specific geographic areas and is associated with lentil and/or pea allergy. We investigated cross-reactivity between chickpea and pea/lentil/soybean/hazelnut. The IgE-binding profiles of chickpea globulin and pea/lentil/soybean/hazelnut extracts were analyzed by immunoblotting and immunoblot-inhibition studies. Inhibition-assay with pea/lentil completely suppressed IgE-binding to chickpea globulin allergens, while not so in the reciprocal inhibition. Pre-absorption of sera with chickpea globulin caused the disappearance of IgE-binding to protein on an immunoblot of soybean/hazelnut protein extract. These results suggest that cross-reactivity exists between chickpea and pea/lentil/soybean/hazelnut. Chickpea allergy is associated with lentil and/or pea allergy, but evidently may not present independently. This, together with the described asymmetric cross-reactivity and phylogenetic aspects, suggest that chickpea allergy is merely an expression of cross-reactivity, caused by pea and/or lentil as the "primary" allergen.

Pectin methylesterases of pollen tissue, a major allergen in olive tree.

Olive tree (Olea europaea) pollen is a main cause of allergy in Mediterranean areas and North America. A novel allergen, Ole e 11, has been detected by proteomic techniques. Protein bands binding IgE from allergic sera were excised from a 2D electrophoresis gel and analysed by Edman degradation and MALDI-TOF MS. Four peptides were sequenced and used for designing primers to clone the cDNA codifying the protein. Ole e 11 consists of a 342 amino acid length polypeptide with a molecular mass of 37.4 kDa and a pI of 7.8. The allergen was identified as a pectin methylesterase and showed low identity with other members of this family from foods such as those from carrot (23%), orange (25%) and tomato (24%), and higher identity with those from Arabidopsis thaliana (57%) and Salsola kali (54%) pollen. The protein was overproduced in Pichia pastoris, purified, and characterized as an active enzyme. CD analysis rendered 3%alpha-helix, 50%beta-sheet and 27%beta-turns for its secondary structure, which is in agreement with other pectin methylesterase structures. The recombinant protein was demonstrated to be immunologically equivalent to the natural form by immunoblotting, indirect ELISA and inhibition experiments, using polyclonal antiserum and sera from olive pollen allergic patients. The prevalence fluctuated between 55.9% and 75.6% in three different allergic populations. The availability of this new olive pollen allergen could improve the component-resolved diagnosis. Its allergenic relevance is stepped up by the biotechnological use of these enzymes to improve organoleptic properties in processing foods and further confirms the need to include it in an accurate diagnosis.

Profilin (Che a 2) and polcalcin (Che a 3) are relevant allergens of Chenopodium album pollen: isolation, amino acid sequences, and immunologic properties.

BACKGROUND: Little is known about the molecular properties of chenopod allergens. Recently, profilin and 2 EF-hand calcium-binding protein (polcalcin) have been shown to play a role in chenopod pollinosis. OBJECTIVE: We sought to analyze these panallergens in chenopod pollen and to evaluate their involvement in the allergy to this biologic source. METHODS: Profilin and polcalcin were purified to homogeneity and characterized by using spectrometric and chemical methods. Immunologic analyses were performed by means of immunoblotting, ELISA, and competitive inhibition assays with olive profilin- and polcalcin-specific rabbit polyclonal antibodies and sera from patients with chenopod allergy. cDNAs encoding these proteins were cloned by means of PCR and sequenced. RESULTS: Purified Che a 2 (profilin) and Che a 3 (polcalcin) exhibited prevalences of 55% and 46%, respectively, in patients (n=104) hypersensitive to chenopod pollen. Both purified allergens individually inhibited the IgE binding to the whole pollen extract and showed strong cross-reactivity with the corresponding olive pollen profilin (Ole e 2) and polcalcin (Ole e 3). Chenopod profilin consists of a 131-amino-acid chain that displays identities of approximately 75% and 82% with pollen and food profilins, respectively. Che a 3 (86 amino acids) displays similarity (65% to 82% identity) with polcalcins from pollens of olive, birch, alder, rapeseed, and timothy. CONCLUSION: Profilin and polcalcin are relevant panallergens in chenopod pollen and good candidates to be involved in IgE cross-reactivity with other pollen sources, thus explaining the highly frequent polysensitization of patients allergic to chenopod.

Len c 1, a major allergen and vicilin from lentil seeds: protein isolation and cDNA cloning.

BACKGROUND: Lentils are among the main plant foods causing allergic reactions in pediatric patients in the Mediterranean area and in many Asian communities. However, very few reports have been devoted to identifying lentil allergens. Seed storage proteins of the vicilin family have been characterized as major allergens in several seed legumes and tree nuts. OBJECTIVE: We sought to evaluate the role of lentil vicilins as food allergens. METHODS: A serum pool and individual sera from 22 patients with lentil allergy were used in different IgE-binding assays. Mature lentil vicilin was isolated by means of cation-exchange chromatography, followed by reverse-phase HPLC, and characterized by means of N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry (MALDI) analysis, complex asparagine-linked glycan detection, specific IgE immunodetection with individual sera, and ELISA inhibition assays. Complete cDNAs encoding lentil vicilin variants were isolated by means of PCR with primers based on the amino acid sequence of the allergen. RESULTS: A major IgE-binding component of approximately 50 kd was detected in lentil extracts. This component was isolated and characterized, showing a single N-terminal amino acid sequence homologous to those of legume vicilins and a broad peak (maximum at 48613 d) in MALDI analysis. The purified allergen was recognized by 77% (17/22) of the individual sera from patients with lentil allergy and reached up to 65% inhibition of the IgE binding to the crude lentil extract. The allergen showed 3 isoforms varying in their degree of N-glycosylation. Two cDNA clones encoding different allergen variants were isolated. The amino acid sequences deduced from both clones (415 and 418 residues; 47.4 and 47.8 kd) showed greater than 50% identity with major peanut (Ara h 1) and soybean (conglutinin subunits) allergens belonging to the vicilin family. Furthermore, these sequences included those of the previously characterized lentil allergen Len c 1.02 (108 amino acid residues of the C-terminal domain) and those of a novel lentil IgE-binding protein of 26 kd. CONCLUSION: The mature 48-kd lentil vicilin, designated Len c 1.01, is a major allergen. Two of its processing fragments, corresponding to subunits of 12 to 16 kd (previously named Len c 1) and 26 kd, are also relevant lentil IgE-binding proteins. The sequence homology of Len c 1.01 to those of major allergens from peanut, soybean, walnut, and cashew can help to investigate potential cross-reactions among these plant foods.