The conformational IgE epitope profile of soya bean allergen Gly m 4.

BACKGROUND: Birch pollen-related soya allergy is mediated by Gly m 4. Conformational IgE epitopes of Gly m 4 are unknown. OBJECTIVE: To identify the IgE epitope profile of Gly m 4 in subjects with birch pollen-related soya allergy utilizing an epitope library presented by Gly m 4-type model proteins. METHODS: Sera from patients with (n = 26) and without (n = 19) allergy to soya as determined by oral provocation tests were studied. Specific IgE (Bet v 1/Gly m 4) was determined by ImmunoCAP. A library of 59 non-allergenic Gly m 4-type model proteins harbouring individual and multiple putative epitopes for IgE was tested in IgE binding assays. Primary, secondary and tertiary protein structures were assessed by mass spectrometry, circular dichroism and nuclear magnetic resonance spectroscopy. RESULTS: All subjects were sensitized to Gly m 4 and Bet v 1. Allergen-specific serum IgE levels ranged from 0.94 to > 100 kUA /L. The avidities of serum IgE were 5.06 ng (allergic) and 1.8 ng (tolerant) as determined by EC50 for IgE binding to Gly m 4. 96% (46/48) of the protein variants bound IgE. Model proteins had Gly m 4-type conformation and individual IgE binding clustered in six major surface areas. Gly m 4-specific IgE binding could be inhibited to up to 80% by model proteins harbouring individual IgE binding sites in an epitope-wise equimolar fashion. Receiver operating curve analysis revealed an area under fitted curve of up to 0.88 for model proteins and 0.66 for Gly m 4. CONCLUSION AND CLINICAL RELEVANCE: Serum levels and avidity of Gly m 4-specific IgE do not correlate with clinical reactivity to soya. Six IgE-binding areas, represented by 23 amino acids, account for more than 80% of total IgE binding capacity of Gly m 4. Model proteins may be used for epitope-resolved diagnosis to differentiate birch-soya allergy from clinical tolerance.

IgE recognition patterns in peanut allergy are age dependent: perspectives of the EuroPrevall study.

BACKGROUND: We tested the hypothesis that specific molecular sensitization patterns correlate with the clinical data/manifestation in a European peanut-allergic population characterized under a common protocol. METHODS: Sixty-eight peanut-allergic subjects and 82 tolerant controls from 11 European countries were included. Allergy to peanut and lowest symptom-eliciting dose was established by double-blind placebo-controlled food challenge in all but anaphylactic subjects. Information of early or late (before or after 14 years of age) onset of peanut allergy was obtained from standardized questionnaires. IgE to peanut allergens rAra h 1-3, 6, 8-9, profilin and CCD was determined using ImmunoCAP. RESULTS: Seventy-eight percent of peanut allergics were sensitized to peanut extract and 90% to at least one peanut component. rAra h 2 was the sole major allergen for the peanut-allergic population. Geographical differences were observed for rAra h 8 and rAra h 9, which were major allergens for central/western and southern Europeans, respectively. Sensitization to rAra h 1 and 2 was exclusively observed in early-onset peanut allergy. Peanut-tolerant subjects were frequently sensitized to rAra h 8 or 9 but not to storage proteins. Sensitization to Ara h 2 ≥ 1.0 kUA /l conferred a 97% probability for a systemic reaction (P = 0.0002). Logistic regression revealed a significant influence of peanut extract sensitization and region on the occurrence of systemic reactions (P = 0.0185 and P = 0.0436, respectively). CONCLUSION: Sensitization to Ara h 1, 2 and 3 is usually acquired in childhood. IgE to Ara h 2 ≥ 1.0 kUA /l is significantly associated with the development of systemic reactions to peanut.

Component-resolved in vitro diagnosis of carrot allergy in three different regions of Europe.

BACKGROUND: Carrot is a frequent cause of food allergy in Europe. The objective of this study was to evaluate a panel of carrot allergens for diagnosis of carrot allergy in Spain, Switzerland and Denmark. METHODS: Forty-nine carrot allergic patients, 71 pollen allergic but carrot-tolerant patients and 63 nonatopic controls were included. Serum IgE to carrot extract, recombinant carrot allergens (rDau c 1.0104; rDau c 1.0201; rDau c 4; the isoflavone reductase-like proteins rDau c IFR 1, rDau c IFR 2; the carrot cyclophilin rDau c Cyc) were analyzed by ImmunoCAP. RESULTS: The sensitivity of the carrot extract-based test was 82%. Use of the recombinant allergens increased the sensitivity to 90%. The Dau c 1 isoforms were major allergens for Swiss and Danish carrot allergic patients, the profilin rDau c 4 for the Spanish patients. The rDau c IFR 1 and rDau c IFR 2 were recognized by 6% and 20% of the carrot allergics, but did not contribute to a further increase of sensitivity. Among pollen allergic controls, 34% had IgE to carrot extract, 18% to each of rDau c 1.0104, rDau c 1.0201 and rDau c 4, 8% to rDau c IFR 1 and 7% to rDau c IFR 2. Sensitization to rDau c Cyc occurred in one carrot allergic patient and one nonatopic control. CONCLUSION: Component-resolved in vitro analyses revealed a significant difference in IgE sensitization pattern between geographical regions and in the prevalence of sensitization to carrot components between carrot allergic and carrot-tolerant but pollen sensitized patients.

Strategy for allergenicity assessment of 'natural novel foods': clinical and molecular investigation of exotic vegetables (water spinach, hyacinth bean and Ethiopian eggplant).

BACKGROUND: Foods not commonly consumed in the European Union must be proven safe before being brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables, namely, water spinach, hyacinth bean, Ethiopian eggplant. METHODS: First, vegetable extracts were analyzed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, nonspecific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Secondly, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects (i) allergic to birch, grass and mugwort pollen, (ii) with food allergy to soy, peanut, tomato, multiple pollen-related foods and (iii) sensitized to LTP. Thirdly, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC). RESULTS: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients. CONCLUSIONS: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods.

Enhancement of hazelnut extract for IgE testing by recombinant allergen spiking.

BACKGROUND: Hazelnuts are a common cause of food allergic reactions. Most hazelnut allergic individuals in central and northern Europe are sensitized to Cor a 1, a member of the PR-10 protein family, while the lipid transfer protein Cor a 8 acts as a major allergen in the south of Europe. Other allergens, including profilin and seed storage proteins, may be important in subgroups of patients. Reliable detection of specific IgE in the clinical diagnosis of food allergy requires allergen reagents with a sufficient representation of all relevant allergen components. Some reported observations suggest that natural hazelnut extract may not be fully adequate in this respect. METHODS: The capacity of immobilized natural hazelnut extract to bind Cor a 1-, Cor a 2- and Cor a 8-specific IgE and IgG antibodies was investigated by serum adsorption and extract dilution experiments and by the use of allergen specific rabbit antisera. All measurements were performed with the ImmunoCAP assay platform. RESULTS: The experimental results revealed an incomplete capacity of immobilized hazelnut extract to capture IgE antibodies directed to the major allergen Cor a 1. Spiking of hazelnut extract with recombinant Cor a 1.04 prior to solid phase coupling gave rise to significantly enhanced IgE antibody binding from Cor a 1 reactive sera. The spiking did not negatively affect the measurement of IgE to extract components other than Cor a 1. CONCLUSION: A hazelnut allergen reagent with enhanced IgE detection capacity can be generated by supplementing the natural food extract with recombinant Cor a 1.04.

[Pruritus in frequent skin diseases and therapeutic options]. / Juckreiz bei häufigen Hauterkrankungen und therapeutische Optionen.

Pruritus is a common symptom in many inflammatory skin conditions. In these cases, itch is typically pruritoceptive and is induced by cutaneous inflammation. The most common skin diseases associated with itch are eczemas, including atopic dermatitis, dry skin or contact dermatitis. Scabies has to be excluded. Psoriasis and lichen planus are other common skin disorders associated with itching. Therapy includes local treatment options including hydration with lotions or creams, topical application of Polidocanol, capsaicin or menthol, phototherapy is an option in many inflammatory skin diseases. Systemic approaches include antihistamines, antidepressants, gabapentin or opiate antagonists.

[Cutaneous symptoms after ingestion of pollen-associated foodstuffs]. / Kutane Symptome nach Genuss pollenassoziierter Nahrungsmittel.

The molecular basis of pollen-related food allergy is the marked similarity in sequence and structure of allergenic proteins in pollens and food plants. In affected patients, specific IgE antibodies are primarily directed against pollen allergens but then recognize homologous allergens in plant food. In Central and Northern Europe up to 80% of birch pollen allergic subjects suffer from a food allergy, in particular to stone- and pip fruits, nuts and vegetables. The main clinical manifestation of pollen-related food allergy is the oral allergy syndrome (OAS), a contact urticaria of the oral mucosa. Other features include contact urticaria of the hands in those handling the foods, as well as generalized urticaria and angioedema following ingestion. The impact of pollen-related food allergy on the severity and course of atopic eczema remain to be elucidated.

Component-resolved in vitro diagnosis in carrot allergy: does the use of recombinant carrot allergens improve the reliability of the diagnostic procedure?

BACKGROUND: In Europe, pollen-related food allergy is the most frequent form of food allergy in adults. Reliability of current diagnostic procedures, however, is poor and therapeutic options are not available. OBJECTIVES: In the present study, we created a panel of recombinant allergens from carrot and evaluated its potential in component-resolved in vitro diagnosis of carrot allergy. METHODS: Recombinant (r) Dau c 1.0104, Dau c 1.0201 and Dau c 4 were cloned by a polymerase chain reaction strategy, expressed in Escherichia coli and purified. Carrot lipid transfer protein (LTP) was expressed in the yeast Pichia pastoris. Sera from 40 carrot-allergic patients were investigated. Twenty-one birch pollen-allergic subjects with negative open provocation to carrot and 20 non-allergic subjects were included as controls. IgE binding to recombinant allergens as well as to cross-reactive carbohydrate determinants (CCD) was measured by ELISA. Cross-reactivity between Dau c 1 isoforms and Bet v 1 was assayed by ELISA inhibition. Biological activity of the recombinant carrot allergens was assessed by histamine release assay and peripheral blood mononuclear cells stimulation. RESULTS: Ninety-eight percent of the carrot-allergic patients were positive to at least one recombinant allergen; 98% reacted to rDau c 1.0104, 65% to rDau c 1.0201, 38% to rDau c 4 and 20% had IgE against CCD. Specificity using the recombinant allergens was high when compared with non-allergic controls, but low compared with birch-sensitized subjects without carrot allergy. Sensitization to Dau c 1.0201, however, proved to be highly specific for clinically relevant sensitization. Inhibition assays indicated the absence of LTP in carrot root extract, and epitope diversity between Dau c 1.0104, Dau c 1.0201 and Bet v 1. CONCLUSIONS: Our panel of recombinant allergens from carrot can provide a standardized tool for in vitro diagnosis of carrot allergy, and for epitope studies.

Roasted hazelnuts--allergenic activity evaluated by double-blind, placebo-controlled food challenge.

BACKGROUND: Allergy to hazelnuts is a common example of birch pollen related food allergy. Symptoms upon ingestion are often confined to the mouth and throat, but severe systemic reactions have been described in some patients. The aim of the study was to evaluate the reduction in allergenicity by roasting of the nuts. METHODS: Double-blind, placebo-controlled food challenges (DBPCFC) with roasted hazelnuts (140 degrees C, 40 min) were performed in 17 birch pollen allergic patients with DBPCFC-confirmed food allergy to raw hazelnuts. The effect of roasting was further evaluated by skin prick test (SPT), histamine release (HR), measurement of specific IgE, and IgE-inhibition experiments. RESULTS: In 5/17 patients the DBPCFC with the roasted nuts were positive. The symptoms were generally mild and included OAS (oral allergy syndrome) in all patients. Roasting of the nuts significantly reduced the allergenic activity evaluated by SPT, HR, specific IgE, and IgE-inhibition. Immunoblotting experiments with recombinant hazelnut allergens showed sensitization against Cor a 1.04 in 16/17 patients and against Cor a 2 in 7/17 patients. None of the patients were sensitized to Cor a 8. Challenge-positive patients did not differ from the rest in IgE-binding pattern. CONCLUSIONS: All the applied methods indicated that roasting of hazelnuts reduces the allergenicity, but since 5/17 birch pollen allergic patients were DBPCFC-positive to the roasted nuts, ingestion of roasted hazelnuts or products containing roasted hazelnuts can not be considered safe for a number of hazelnut allergic consumers. For patients with a history of severe allergic symptoms upon ingestion of hazelnuts, thorough and conscientious food labelling of hazelnuts and hazelnut residues is essential.

Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01.

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

Carrot allergy: double-blinded, placebo-controlled food challenge and identification of allergens.

BACKGROUND: Allergic reactions to carrot affect up to 25% of food-allergic subjects. Clinical manifestations of carrot allergy and IgE responses to carrot proteins, however, have never been studied in subjects with carrot allergy confirmed by means of double-blinded, placebo-controlled food challenge (DBPCFC). OBJECTIVE: The purposes of this investigation were to confirm clinically relevant sensitizations to carrot by means of DBPCFC, to validate current diagnostic methods, and to identify IgE-reactive carrot proteins in patients with true allergy. METHODS: DBPCFCs were performed in 26 subjects with histories of allergic reactions to carrot. Patients underwent skin prick tests with carrot extract, fresh carrot, and various pollen extracts. Specific IgE to carrot, celery, birch, and mugwort pollen and to rBet v 1, rBet v 2, and rBet v 6 were measured through use of the CAP method. Carrot allergens were identified by means of immunoblotting and blotting inhibition. RESULTS: Twenty of 26 patients had positive DBPCFC results. The sensitivity of the determination of carrot-specific IgE antibodies through use of the CAP method (> or =0.7 kU/L) was 90%, the sensitivity for skin prick testing with commercial extracts was 26%, and the sensitivity for prick-to-prick tests with raw carrot was 100%. The Bet v 1--related major carrot allergen Dau c 1 was recognized by IgE from 85% of patients; 45% were sensitized to cross-reactive carbohydrate determinants and 20% to carrot profilin. In 1 subject, a Bet v 6--related carrot allergen was recognized. In 4 patients, IgE binding to Dau c 1 was not inhibited or was weakly inhibited by rBet v 1 or birch pollen extract. CONCLUSION: This study confirmed the allergenicity of carrot by means of DBPCFC. DBPCFC-positive patients had exclusively specific IgE antibodies to birch pollen--related carrot allergens, Dau c 1 being the major allergen. The lack of inhibition of IgE binding to Dau c 1 by birch allergens in a subgroup of patients might indicate an secondary immune response to new epitopes on the food allergen that are not cross-reactive with Bet v 1.

Celery allergy confirmed by double-blind, placebo-controlled food challenge: a clinical study in 32 subjects with a history of adverse reactions to celery root.

BACKGROUND: Celery root is a frequent cause of food allergy in pollen-sensitized patients. Because of problems in blinding challenges with fresh vegetables and the risk of anaphylactic reactions, no double-blind, placebo-controlled, food challenges (DBPCFCs) with celery have been published so far. OBJECTIVE: The aim of the study was to confirm the clinical relevance of celery as a food allergen by DBPCFCs and to evaluate current diagnostic procedures in patients with true allergy. METHODS: DBPCFCs were performed in 32 patients with a history of an allergic reaction to celery. The patients underwent skin prick tests (SPTs) with celery extracts, crude celery, and different pollen extracts. Specific IgE for celery was determined by using the CAP method. RESULTS: Twenty-two of 32 patients had a positive DBPCFC result. Two patients reacted to placebo, and 8 patients did not respond to the challenge. Of the nonresponders, 4 reacted to an open provocation with celery. The sensitivity of CAP determination for specific IgE (> or =0.7 kU/L) to celery in patients with a positive DBPCFC result was 73%, 48% to 86% for SPTs (> or =3 mm) with commercial extracts, and 96% for prick-to-prick tests with crude celery. The positive predictive value of the SPT and CAP tests was between 87% and 96%, whereas the specificity and negative predictive values were poor. CONCLUSION: This study confirms the importance of celery as a food allergen for use in DBPCFCs. The SPT and CAP methods proved to be reliable for the diagnosis of a relevant allergy to celery in regard to sensitivity and positive predictive value but not to specificity and negative predictive value.