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Background: Baker's asthma is a frequent occupational allergic disorder mainly caused by inhalation of cereal flours. Lipid transfer proteins (LTPs) constitute a family of plant food panallergens, but their role as inhalant and wheat allergens is still unclear., Objective: We sought to explore the involvement of wheat LTPs in baker's asthma caused by wheat flour sensitization., Methods: Forty patients with occupational asthma caused by wheat flour inhalation were studied. Wheat LTP, Tri a 14, was purified by using a 2-step chromatographic protocol and characterized by N-terminal amino acid sequencing and 3-dimensional modeling. Its reactivity was confirmed by means of IgE immunodetection, ELISA and ELISA-inhibition assays, and skin prick tests., Results: Specific IgE to Tri a 14 was found in 60% of 40 individual sera from patients with baker's asthma, and the purified allergen elicited positive skin prick test reactions in 62% of 24 of these patients. Tri a 14 and peach LTP, Pru p 3, showed a sequence identity of 45%, but the low cross-reactivity between both allergens detected in several individual sera reflected great differences in their 3-dimensional IgE-binding regions., Conclusions: Wheat LTP is a major inhalant allergen associated with baker's asthma caused by wheat flour sensitization. Poor cross-reactivity with its peach homolog was found in some patients., Clinical Implications: LTPs can be considered relevant inhalant allergens linked to respiratory disorders. LTP from wheat (Tri a 14) can be used as a helpful tool for the diagnosis of baker's asthma.

Background: The lipid transfer protein Pru p 3 has been identified as a major peach fruit allergen. However, the putative peach member of the Bet v 1 family, Pru p 1, has been neither identified nor characterized., Objectives: To determine the distribution and solubility properties of the main peach allergens and to quantify Pru p 3 and Pru p 1 levels in peach and nectarine cultivars., Methods: Peach peel and pulp were extracted using different buffers, and extracts were analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection using polyclonal antibodies against lipid transfer proteins, profilins, and Bet v 1 homologues. Pru p 3 was quantified in peach and nectarine cultivars using a sandwich enzyme-linked immunosorbent assay method. A similar method was developed to quantify Pru p 1., Results: A differential distribution between peel and pulp and different solubility properties were found for Pru p 3, Pru p 1, and peach profilin. Mean Pru p 3 levels were 132.86, 0.61, and 16.92 microg/g of fresh weight of peels, pulps, and whole fruits, respectively. The corresponding mean Pru p 1 levels were 0.62, 0.26, and 0.09 microg/g of fresh weight. Most US cultivars showed higher levels of both allergens than Spanish cultivars., Conclusions: The different distribution and solubility properties of the main peach allergens can determine the quality of fruit extracts used as diagnostic tools. These differences, together with the natural variation of Pru p 3 and Pru p 1 levels among peach and nectarine cultivars, can be exploited to reduce peach allergenicity by means of industrial processing and plant breeding.

Background: Plant profilins are actin-binding proteins that form a well-known panallergen family responsible for cross-sensitization between plant foods and pollens. Melon profilin, Cuc m 2, is the major allergen of this fruit., Objective: We sought to map IgE epitopes on the 3-dimensional structure of Cuc m 2., Methods: IgE binding to synthetic peptides spanning the full Cuc m 2 amino acid sequence was assayed by using a serum pool and individual sera from 10 patients with melon allergy with significant specific IgE levels to this allergen. Three-dimensional modeling and potential epitope location were based on analysis of both solvent exposure and electrostatic properties of the Cuc m 2 surface., Results: Residues included in synthetic peptides that exerted the strongest IgE-binding capacity defined 2 major epitopes (E1, consisting of residues 66-75 and 81-93, and E2, consisting of residues 95-99 and 122-131) that partially overlapped with the actin-binding site of Cuc m 2. Two additional epitopes (E3, including residues 2-10, and E4, including residues 35-45) that should show weaker putative antigen-antibody associations and shared most residues with synthetic peptides with low IgE-binding capacity were predicted on theoretical grounds., Conclusions: Strong and weak IgE epitopes have been uncovered in melon profilin, Cuc m 2., Clinical Implications: The different types of IgE epitopes located in the 3-dimensional structure of melon profilin can constitute the molecular basis to explain the sensitization and cross-reactivity exhibited by this panallergen family.

Background: Chestnut allergy has been almost exclusively considered in the context of the latex-fruit syndrome. Chestnut allergens not linked to latex hypersensitivity have not been studied., Objective: We sought to explore whether differences in sensitization patterns between chestnut allergy with or without associated latex-fruit syndrome can be detected., Methods: Twelve patients sensitized to chestnut but not to latex and 3 control patients with latex-chestnut allergy were analyzed. A major chestnut allergen was purified and characterized. IgE immunoblotting, specific IgE determination, and skin prick tests with 5 isolated allergens involved in food allergy or latex-fruit syndrome were also performed., Results: A major 9-kd allergen was detected in chestnut extract, isolated, and identified as lipid transfer protein (LTP) Cas s 8. Specific IgE to this allergen was found in 91% (by means of IgE immunoblotting) and 58% (by means of ELISA) of sera from patients with chestnut but not latex allergy. Moreover, 66% of these patients had positive skin prick test responses to Cas s 8. Additionally, allergenic LTPs from peach fruit and Artemisia vulgaris pollen were also reactive. In contrast, avocado class I chitinase and latex hevein, allergens associated with the latex-fruit syndrome, showed no reaction. The opposite situation was exhibited by patients with latex-chestnut allergy., Conclusions: Patients with chestnut allergy with or without associated latex hypersensitivity present different patterns of major allergens (LTPs and class I chitinases, respectively)., Clinical Implications: LTPs and class I chitinases can be used as diagnostic tools in patients with chestnut allergy to predict whether an associated latex sensitization and a risk of potential cross-reactivity with other plant foods and pollens exist.

Background: Food IgE-mediated allergy to members of the Brassicaceae family has been increasingly reported., Objective: To characterize cabbage-Brassica oleracea var capitata-allergy and its major allergens., Methods: A prospective study was performed, recruiting 17 patients allergic to cabbage, and control subjects. Skin prick tests and double-blind placebo-controlled food challenges were performed. A major allergen was isolated from cabbage by RP-HPLC and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization mass spectrometry analysis. Specific IgE determinations, IgE immunoblots, and CAP-inhibition assays were also performed., Results: Skin prick test and specific IgE were positive to cabbage in all patients. Five of them referred anaphylactic reactions when eating cabbage, and in another 5 patients, cabbage allergy was further confirmed by double-blind placebo-controlled food challenge. Most of them showed associated sensitizations to mugwort pollen, mustard, and peach. A 9-kd cabbage IgE-binding protein, Bra o 3, was identified as a lipid transfer protein (LTP) with 50% of identity to peach LTP Pru p 3. Skin prick test with Bra o 3 showed positive results in 12 of 14 cases (86%). On CAP inhibition assays, Bra o 3 managed to inhibit significantly the IgE binding to cabbage, mugwort pollen, and peach. Both Bra o 3 and Pru p 3 were recognized by IgE from the patients' sera., Conclusion: Bra o 3, a cabbage LTP, is a major allergen in this food, cross-reacting with mugwort pollen and with other plant foods, such as peach., Clinical Implications: Cabbage IgE-mediated allergy is a potentially severe condition that can present with other plant food and pollen allergies.

Background: Lipid transfer proteins (LTPs) are clinically relevant plant food panallergens and have been proposed as ideal tools to study true food allergy. Pru p 3, the major peach allergen in the Mediterranean area, is among the best-characterized allergenic members of the LTP family. Its diagnostic value for Rosaceae fruit allergy has been demonstrated both in vivo and in vitro., Objective: We sought to locate major IgE-binding epitopes of Pru p 3., Methods: A serum pool and individual sera from patients with peach allergy and positive skin prick test results to Pru p 3 were used. Three-dimensional modeling was achieved by using experimentally available structures of Pru p 3 homologues as templates. Theoretical prediction of potential IgE-binding regions was performed by selecting specific residues on the molecular surface displaying prominent electrostatic potential features. Point mutants of Pru p 3 were constructed by standard polymerase chain reaction procedures with the appropriate primers. Mutants were expressed in P pastoris by means of the pPIC 9 vector and purified from the corresponding supernatants by gel-filtration chromatography followed by RP-HPLC. IgE binding by Pru p 3 mutants was tested by immunodetection and quantified by ELISA and ELISA inhibition assays. Synthetic peptides (10 mer; 5 amino acids overlapping) covering the full Pru p 3 sequence were used to detect IgE epitopes by (125)I-anti-IgE immunodetection., Results: Pru p 3 showed a 3-dimensional structure comprising 4 alpha-helixes and a nonstructured C-terminal coil (residues 73 to 91). Regions around amino acids in positions 23 to 36, 39 to 44, and 80 to 91, particularly residues R39, T40, and R44, K80 and K91, were predicted as potential antibody recognition sites according to their relevant surface and electrostatic properties. Point mutants K80A and K91A were found to have an IgE-binding capacity similar to that of recombinant Pru p 3, but the triple mutant R39A/T40A/R44A showed a substantial decrease (approximately 5 times) of IgE binding. IgE immunodetection of synthetic peptides led to the identification of Pru p 3 sequence regions 11 to 25, 31 to 45, and 71 to 80 as major IgE epitopes., Conclusions: Main IgE-binding regions of the Pru p 3 amino acid sequence were identified. The three major ones comprised the end of an alpha-helix and some residues of the following interhelix loop. These data can help to search for Pru p 3 hypoallergenic forms.