Allergens from birch pollen and pollen of the European chestnut share common epitopes

Type I allergy to pollen of the European chestnut (Castanea sativa) represents a major cause of pollinosis in (sub) Mediterranean areas. Using sera from 14 patients with established allergy to pollen of the European chestnut, 13/14 sera (92%) showed IgE-binding to a 22 kD protein, 2/14 (14%) displayed additional binding to a 14 kD protein and 1/14 (7%) bound only to the 14 kD protein of European chestnut pollen extract. Two monoclonal mouse antibodies, BIP 1 and BIP 4, directed against different epitopes of Bet v I (the major birch pollen allergen), and a rabbit antibody to recombinant birch profilin (rBet v II) were used to characterize the proteins of the European chestnut pollen. The recombinant birch pollen allergens, rBet v I and rBet v II (profilin) were employed to show common allergenic structures on proteins from both birch and European chestnut pollen by IgE-inhibition experiments. Despite the fact that the 22 kD protein displayed a higher molecular weight in comparison to the 17 kD major birch pollen allergen, Bet v I, we could demonstrate reactivity of both monoclonal antibodies, BIP 1 and BIP 4, with this protein. A complete inhibiton of IgE-binding to this 22 kD protein was shown by pre-incubating sera with purified recombinant Bet v I. In addition, the 14 kD protein could be identified by IgE-inhibition studies with recombinant Bet v II and by using a rabbit anti-profilin antibody as the profilin from pollen of the European chestnut.     

Gastrointestinal digestion of Bet v 1-homologous food allergens destroys their mediator-releasing, but not T cell-activating, capacity

BACKGROUND: Food allergy to apples, hazelnuts, and celery is frequent in individuals with birch pollen allergy because IgE antibodies specific for the major birch pollen allergen, Bet v 1, cross-react with structurally related allergens in these foods. In addition, T lymphocytes specific for Bet v 1 also cross-react with these dietary proteins. OBJECTIVE: We sought to evaluate the effects of simulated gastrointestinal degradation of Bet v 1-related food allergens on their mediator-releasing and T cell-activating capacity. METHODS: Recombinant Mal d 1, Cor a 1.04, and Api g 1 were incubated separately with pepsin and trypsin. Binding of IgE was tested in immunoblots. After successive incubation with both enzymes, allergens were tested in mast cell mediator release assays and used to stimulate PBMCs and Bet v 1-specific T-cell lines and clones. Proteolytic fragments of allergens were analyzed and sequenced by means of mass spectrometry. RESULTS: Pepsin completely destroyed IgE binding of all allergens within 1 second, and trypsin completely destroyed IgE binding of all allergens within 15 minutes, except for the major hazelnut allergen, which remained intact for 2 hours of trypsinolysis. Allergens after gastrointestinal digestion did not induce basophil activation but induced proliferation in PBMCs from allergic and nonallergic individuals. Digested Mal d 1 and Cor a 1.04 still activated Bet v 1-specific T cells, whereas digested Api g 1 did not. Different proteolytic fragments of Mal d 1 and Cor a 1.04 matching relevant Bet v 1 T-cell epitopes were found. CONCLUSION: Gastrointestinal degradation of Bet v 1-related food allergens destroys their histamine-releasing, but not T cell-activating, property. Our data emphasize that birch pollen-related foods are relevant activators of pollen-specific T cells.     

Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1- related PR-10 protein in soybean,SAM22

BACKGROUND: Anaphylactic reactions to soy products have been attributed to stable class 1 food allergens. OBJECTIVE: IgE- mediated reactions to a soy-containing dietary food product in patients allergic to birch pollen were investigated. METHODS: Detailed case histories were taken from 20 patients. Their sera were analyzed for IgE (UniCAP) specific for birch, grass, mugwort, the recombinant birch allergens rBet v 1 and rBet v2, and soy protein. Extracts from birch pollen, soy isolate, rBet v 1, and the recombinant PR-10 soy protein rSAM22 were coupled to paper disks or nitrocellulose for IgE measurements (enzyme allergosorbent test) or Western blot analysis. Enzyme allergosorbent testing, Western blot inhibition, and histamine release studies were performed with the same allergens. RESULTS: Most patients (17/20) experienced facial, oropharyngeal, and/or systemic allergic symptoms within 20 minutes after ingesting the soy product for the first time. Birch pollen allergy (16/20) was common, along with oral allergy syndrome to apple (12/20) or hazelnut (11/20). IgE levels to birch and Bet v 1 but not to other inhalants were high in 18 of 20 patients. Significant IgE binding to rSAM22 occurred in 17 of 20 patients. Blot experiments with the soy isolate revealed IgE-binding bands at 17 kd (15/20), 22 kd (1/20), and 35 to 38 kd (2/20); the former was inhibited by preincubation of the sera with rBet v 1 or rSAM22. Birch extract and soy isolate, rBet v 1, and rSAM22 induced dose-dependent histamine release in the nanomolar range. CONCLUSION: Immediate-type allergic symptoms in patients with birch pollen allergy after ingestion of soy protein-containing food items can result from cross-reactivity of Bet v 1 -specific IgE to homologous pathogenesis-related proteins, particularly the PR-10 protein SAM22.     

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.     

Cooking birch pollen-related food: divergent consequences for IgE- and T cell-mediated reactivity in vitro and in vivo

BACKGROUND: The major birch pollen allergen Bet v 1 cross-reacts with homologous food allergens, resulting in IgE-mediated oral allergy syndromes (OASs). To avoid this food, allergy allergologists and guidebooks advise patients to consume birch pollen-related foods after heating. OBJECTIVE: We sought to evaluate whether cooked Bet v 1-related food allergens induce IgE- and T cell-mediated reactions in vitro and in vivo. METHODS: Recombinant Bet v 1, Mal d 1 (apple), Api g 1 (celery), and Dau c 1 (carrot) were incubated at increasing temperatures. Protein structures were determined by means of circular dichroism. Mediator release was tested in basophil activation assays. PBMCs and Bet v 1-specific T-cell lines with known epitope specificity were stimulated with native and cooked food allergens. Patients with birch pollen allergy who experienced OAS and the exacerbation of atopic dermatitis (AD) on ingestion of fresh apple, celery, or carrot were retested in double-blind, placebo-controlled food challenges with the respective foods in cooked form. RESULTS: In vitro, cooked food allergens lost the capacity to bind IgE and to induce mediator release but had the same potency to activate Bet v 1-specific T cells as native proteins. In vivo, ingestion of cooked birch pollen-related foods did not induce OAS but caused atopic eczema to worsen. CONCLUSION: T-cell cross-reactivity between Bet v 1 and related food allergens occurs independently of IgE cross-reactivity in vitro and in vivo. In patients with AD, the resulting immune reaction can even manifest as late eczematous skin reactions. Therefore the view that cooked pollen-related foods can be consumed without allergologic consequences should be reconsidered. CLINICAL IMPLICATIONS: Symptom-free consumed pollen-related food allergens might cause T cell-mediated late-phase skin reactions in patients with pollen allergy and AD.     

Non-anaphylactic surface-exposed peptides of the major birch pollen allergen, Bet v 1, for preventive vaccination

BACKGROUND: Almost 100 million allergic patients are sensitized to the major birch pollen allergen, Bet v 1, a 17 kDa protein containing most of the IgE epitopes present in pollens of trees belonging to the Fagales order and plant-derived food. OBJECTIVE: Our aim was to develop an approach for the rational design of B cell epitope-derived, non-allergenic peptide allergy vaccines. METHODS: According to the three-dimensional (3-D) structure of birch pollen allergen, Bet v 1, six peptides comprising 25-32 preferably solvent-exposed amino acids were synthesized. RESULTS: Because of lack of secondary structure, the peptides showed no allergenic activity in allergic patients. In a mouse model of birch pollen allergy, peptide vaccination induced Bet v 1-specific IgG and prevented IgE-mediated allergic sensitization to Bet v 1. The protective role of peptide-induced blocking antibodies is demonstrated by inhibition of allergic patients IgE binding to the allergen and by blocking of allergen-induced basophil degranulation. CONCLUSION: Our results indicate the mechanistic importance of blocking antibodies for allergy vaccination and present a B cell epitope-based approach for the rational design of safe peptide allergy vaccines whenever the structure of the disease-eliciting allergen is known.     

Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy

BACKGROUND: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. METHODS: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. RESULTS: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. CONCLUSIONS: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.     

Skin test evaluation of genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1: results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season.

BACKGROUND: More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen, Bet v 1, and almost 60% of them are sensitized exclusively to this allergen. OBJECTIVE: The aim of this study was to compare the in vivo biologic activity of genetically engineered hypoallergenic derivatives of Bet v 1 (an equimolar mixture of 2 recombinant [r] Bet v 1 fragments and of rBet v 1 trimer) with that of rBet v 1 wild-type by skin prick and intradermal testing. METHODS: Birch pollen-allergic patients who had not received immunotherapy (n = 23), a group of allergic patients without birch pollen allergy (n = 12), and nonatopic persons (n = 8) from northern Europe (Sweden) underwent skin prick and intradermal testing with different concentrations of the recombinant allergens and commercial birch pollen extract before the birch pollen season. Immediate and late-phase reactions were recorded and allergen-specific IgE and IgG subclass responses were determined by CAP radioallergosorbent test and ELISA, respectively. RESULTS: Atopic persons without birch pollen allergy and nonatopic individuals did not have skin reactions to rBet v 1 wild-type and genetically engineered hypoallergenic derivatives. By intradermal testing, 8 of 23 and 13 of 23 birch pollen-allergic patients did not react with the highest concentration (1 microg/mL) of the rBet v 1 fragment mix and rBet v 1 trimer, respectively, compared with 1 with rBet v 1 wild type. Likewise, the highest concentration (100 microg/mL) of fragment mix or trimer failed to elicit a positive skin prick test in 18 of 23 and 15 of 23 patients in comparison with 0/23 with the monomer. No late reactions were observed. CONCLUSION: The recombinant hypoallergenic birch pollen allergens can probably be used for patient-tailored immunotherapy with a reduced risk to induce anaphylactic reactions.     

Immunologic characterization of isoforms of Car b 1 and Que a 1, the major hornbeam and oak pollen allergens

Background:  Birch pollen allergy is one of the most common causes of spring pollinosis often associated with hypersensitivity reactions to pollen of other Fagales species. Yet, only the major disease eliciting allergens of alder and hazel have been fully characterized. Therefore, the aim of this study was to perform cloning, expression and immunologic characterization of the Bet v 1 homologues from oak (Que a 1) and hornbeam (Car b 1).
Methods:  The isoform pattern of Car b 1 and Que a 1 was analyzed by proteomics using 2D gel electrophoresis and LC ESI-QTOF MS. Isoallergens showing high IgE-binding were cloned and expressed in Escherichia coli . IgE-binding activity of the recombinant proteins was determined by enzyme-linked immunosorbent assay (ELISA) and basophil mediator release assays using serum samples from patients mainly exposed either to oak and hornbeam or to birch pollen. Cross-reactivity of the allergens was further investigated at the T-cell level.
Results:  Dominant isoforms of Car b 1 and Que a 1, identified by mass spectrometry, showed different IgE-binding properties when testing Fagales pollen-allergic patients living in birch-free areas as compared to birch-sensitized individuals.
Conclusion:  Tree pollen-allergic patients who are primarily exposed to Fagales pollen other than birch reacted stronger with rCar b 1 and rQue a 1 than with rBet v 1, as determined by inhibition ELISA and basophil mediator release assays. Thus, rCar b 1 and rQue a 1 allergens should be considered for improving molecule-based diagnosis and therapy of tree pollen allergies manifesting in birch-free areas.     

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.     

Ara h 8, a Bet v 1-homologous allergen from peanut, is a major allergen in patients with combined birch pollen and peanut allergy

BACKGROUND: We recently described patients with soybean allergy mainly mediated by cross-reactivity to birch pollen allergens. A majority of those patients were reported to have peanut allergy. OBJECTIVE: We sought to study the occurrence of peanut allergy in patients allergic to birch pollen and characterized the Bet v 1-homologous peanut allergen Ara h 8. METHODS: Recombinant Ara h 8 was cloned with degenerated primers and expressed in Escherichia coli. Nine Swiss and 11 Dutch patients with peanut and birch pollen allergy and a positive double-blind, placebo-controlled food challenge result to peanut were investigated for IgE reactivity to birch pollen and purified peanut allergens and cross-reactivity between birch and peanut. Ara h 8 stability against digestion and roasting was assessed by means of RAST inhibition. The IgE cross-linking potency of Ara h 8 was tested on the basis of basophil histamine release. RESULTS: During double-blind, placebo-controlled food challenge, all patients experienced symptoms in the oral cavity, progressing to more severe symptoms in 40% of patients. CAP-FEIA detected recombinant (r) Ara h 8-specific IgE in 85%. IgE binding to Ara h 8 was inhibited by Bet v 1 in peanut extract immunoblotting and in RAST inhibition. In EAST inhibition recombinant rAra h 8 inhibited IgE binding to peanut in 4 of 7 tested patient sera. Antipeanut response was dominated by Ara h 8 in 12 of 17 tested patients. Furthermore, our results demonstrate a low stability of Ara h 8 to roasting and no stability to gastric digestion. Basophil histamine release with rAra h 8 was more than 20% in 5 of 7 tested sera. CONCLUSIONS: Peanut allergy might be mediated in a subgroup of our patients by means of cross-reaction of Bet v 1 with the homologous peanut allergen Ara h 8.     

IgE reactivity to profilin in pollen-sensitized subjects with adverse reactions to banana and pineapple

BACKGROUND: The so-called 'latex-fruit syndrome' is a well-documented phenomenon in cross-reactive allergies. By contrast, there is a lack of information about allergy to exotic fruits in patients with a predominant pollen sensitization. Since the ubiquitous protein profilin has been identified as an allergen in natural rubber latex as well as in pollen-related foods, the aim of this study was to investigate the role of profilin in allergy to certain exotic fruits. METHODS: Recombinant profilins from banana and pineapple were cloned by a PCR technique after isolation of total RNA using degenerated profilin-specific primers. The unknown 5' ends of copy DNA (cDNA) were identified by rapid amplification of 5'cDNA ends (5'-RACE) and expression in Escherichia coli BL21(DE3) cells. The recombinant profilins were purified by affinity chromatography using poly-(L)-proline as the solid phase. IgE-binding capabilities were characterized by means of immunoblot and Enzyme Allergosorbent Test (EAST). The cross-reactivity to birch pollen profilin and latex profilin was studied by EAST as well as by immunoblot inhibition experiments. RESULTS: Both banana and pineapple profilin were found to consist of 131 amino acid residues with high amino acid sequence identity to known allergenic pollen and food profilins (71-84%). IgE binding to the recombinant profilins was observed in 7/16 sera from subjects with suspected banana allergy (44%) and in 8/19 sera from subjects with suspected pineapple allergy (42%). Inhibition experiments indicated similar IgE reactivity of natural and recombinant allergens. In addition, high cross-reactivity to birch pollen profilin Bet v 2 and latex profilin Hev b 8 was demonstrated by immunoblot inhibition as well as EAST inhibition experiments. CONCLUSIONS: Since a high IgE-binding prevalence of about 40% was obtained in both banana and pineapple allergy, we conclude that profilin is an important mediator of IgE cross-reactivity between pollen and exotic fruits.     

Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1

BACKGROUND: We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity. OBJECTIVE: The aim of this study was the in vivo characterization of potential allergy vaccines based on Al(OH)3-adsorbed genetically modified rBet v 1 derivatives in mice. METHODS: BALB/c mice were immunized either with courses of nine injections of increasing doses of Al(OH)3-adsorbed rBet v 1 wild-type, rBet v 1 fragments, rBet v 1 trimer or Al(OH)3 alone in weekly intervals or with three high-dose injections applied in intervals of 3 weeks. Humoral immune responses to rBet v 1 wild-type and homologous plant allergens were measured by ELISA and Western blotting, and the ability of mouse antibodies to inhibit the binding of allergic patients IgE to Bet v 1 was studied by ELISA competition experiments. RESULTS: In both schemes, hypoallergenic rBet v 1 derivatives induced low IgE but high IgG1 responses against rBet v 1 wild-type. The IgG1 antibodies induced by genetically modified rBet v 1 derivatives cross-reacted with natural Bet v 1 and its homologues from alder (Aln g 1) as well as hazel (Cor a 1) and strongly inhibited the binding of birch pollen allergic patients' IgE to Bet v 1 wild-type. CONCLUSION: Genetically modified hypoallergenic rBet v 1 derivatives induce blocking antibodies in vivo. Their safety and efficacy for the treatment of birch pollen and associated plant allergies can now be evaluated in clinical immunotherapy studies.     

Altered IgE epitope presentation: A model for hypoallergenic activity revealed for Bet v 1 trimer

In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.     

Oral allergy syndrome to chicory associated with birch pollen allergy

BACKGROUND: A few cases of IgE-mediated chicory allergy with oral, cutaneous, and/or respiratory symptoms are reported. We present 4 patients with inhalant birch pollen allergy and oral allergy syndrome to chicory. IgE-binding proteins in chicory and cross-reactivity with birch pollen were studied. METHODS: Chicory extract was prepared and immunoblotting was used to study IgE reactivity and cross-reactions with birch pollen. RESULTS: The pattern of IgE binding to chicory was variable among the patients, with protein bands recognized at 18, 21, 40, 52 and 71 kD. Bet v 1-like proteins were detected in chicory by monoclonal antibody binding. Chicory-birch pollen cross-reactivity, as studied in 2 patients from whom enough serum was available, could be demonstrated but did not involve the Bet v 1 protein family. In one of these cases, a 51-kD protein of birch pollen was found to be responsible for cross-reactivity. CONCLUSIONS: Chicory should be added to the list of foods that can cross-react with birch pollen and cause the birch pollen-associated oral allergy syndrome.     

Characterization of api g 1.0201, a new member of the Api g 1 family of celery allergens

BACKGROUND: The association of pollinosis with allergy to plant foods occurs in up to 70% of tree pollen-allergic patients. In recent years, some of the relevant cross-reacting proteins have been characterized at the molecular and immunological level. Api g 1 has been identified as the celery homologue of the major birch pollen allergen, Bet v 1. Although a number of Bet v 1 isoforms have been characterized from birch pollen, little is known about isoforms of food allergens and their allergenic features. METHODS: Api g 1.0201, an isoform of Api g 1, was isolated from a cDNA library, cloned and sequenced. The cDNA was expressed in Escherichia coli and the purified recombinant protein was tested in immunoblots. RESULTS: Api g 1.0201 displays 72% sequence similarity to the previously identified Api g 1.0101 and consists of 159 amino acid residues. The sequence of Api g 1.0201 has five additional amino acid residues at the carboxy-terminus as compared to Api g 1.0101. Purified recombinant Api g 1.0201 is recognized by IgE from the sera of celery-allergic patients, as well as by the murine monoclonal anti-Bet v 1 antibody. In general, this isoform displays a weaker IgE-binding capacity than Api g 1.0101, as concluded from immunoblotting experiments. Results from inhibition assays revealed that IgE-binding to Api g 1.0201 is only slightly reduced by preincubation with either purified recombinant Api g 1.0101 or purified recombinant Bet v 1a. Total inhibition was only achieved when using purified natural Bet v 1. CONCLUSIONS: At present, little is known about the IgE-binding capacity of isoforms of Bet v 1 homologues of food allergens. Identification and characterization of such isoforms may help to contribute to a better understanding of food allergy and the observed cross-reactivity to pollen allergy.     

Soybean allergy in patients allergic to birch pollen: clinical investigation and molecular characterization of allergens

BACKGROUND: Allergic reactions to legumes are generally thought to be acquired by means of primary sensitization through the gastrointestinal tract. Recently, Gly m 4 (starvation-associated message 22), a Bet v 1-related pathogenesis-related protein 10 from soy, was suggested to be an allergen in patients with allergic reactions to a dietary product containing a soy protein isolate. OBJECTIVE: We sought to evaluate the clinical relevance of Gly m 4 in subjects allergic to birch pollen with soy allergy and to assess the risk for subjects allergic to birch pollen to acquire soy allergy. METHODS: Twenty-two patients allergic to birch pollen with soy allergy confirmed by means of positive double-blind, placebo-controlled food challenge results (n = 16) or a convincing history (n = 6) were investigated for IgE reactivity to birch pollen and soy allergens by using the Pharmacia CAP system and immunoblot analysis. Cross-reactivity was assessed by means of enzyme allergosorbent test inhibition. Ninety-four patients with birch pollen allergy were interviewed to assess soy tolerance and screened for IgE reactivity to Gly m 4 by means of immunoblotting. The Gly m 4 content in soy foods and soybean varieties was investigated by means of quantitative evaluation of immunoblots. RESULTS: During double-blind, placebo-controlled food challenge, 10 patients experienced symptoms localized to the oral cavity, and 6 patients had a more severe reaction. CAP analysis revealed Gly m 4-specific IgE in 96% (21/22) of the patients. All patients had Bet v 1-specific IgE antibodies, and 23% (5/22) had positive Bet v 2 results. In IgE immunoblotting 25% (6/22) of the patients recognized soy profilin (Gly m 3), and 64% (14/22) recognized other soy proteins. IgE binding to soy was at least 80% inhibited by birch pollen and 60% inhibited by rGly m 4 in 9 of 11 sera tested. Seventy-one percent (67/94) of highly Bet v 1-sensitized patients with birch pollen allergy were sensitized to Gly m 4, and 9 (9.6%) of those patients reported soy allergy. The Gly m 4 content in soy products ranged between 0 and 70 ppm (milligrams per kilogram). CONCLUSIONS: Our results confirm that soybean is another birch pollen-related allergenic food. Gly m 4 is the major soy allergen for patients allergic to birch pollen with soy allergy. The content of Gly m 4 in soy food products strongly depends on the degree of food processing.     

Characterization of allergens in Apiaceae spices: anise, fennel, coriander and cumin

Background Symptoms elicited by IgEmediated 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 60kDa. 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 bo elicited by other types of hypersensitivity (Type II. IIII, 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.     

Identification of oilseed rape (Brassica napus) pollen profilin as a cross-reactive allergen

BACKGROUND: Major allergens of oilseed rape (OSR) pollen with molecular weights of 6/8, 14 and between 27 and 69 kD have been described. The aim of the present study was to further characterize the 14-kD allergen. METHODS: The 14-kD protein was purified from OSR pollen extracts by poly-(L-proline) (PLP)-Sepharose affinity chromatography and characterized immunologically by means of allergic patients' IgE antibodies, profilin-specific rabbit antisera, Western blot and ELISA inhibition using recombinant birch profilin (rBet v 2), and skin prick testing. RESULTS: By PLP affinity chromatography, OSR pollen profilin was purified as a single protein of 14.5 kD and further identified as a profilin by three polyclonal rabbit antisera raised against ragweed and tobacco pollen profilin and the C-terminus of birch profilin. IgE binding of a human serum pool (n = 15) and four profilin-reactive sera to nitrocellulose-blotted OSR profilin was completely inhibited by 1 microg/ml rBet v 2 (birch profilin). Reciprocal ELISA inhibition using increasing concentrations of rBet v 2 and purified OSR profilin, respectively, showed that rBet v 2 strongly inhibits antibody binding to OSR profilin, whereas almost 100 times the amount of OSR profilin was needed to inhibit IgE binding to rBet v 2. Skin prick tests were positive (wheal >/=3 mm) with 5 microg/ml rBet v 2 in all three patients tested, and with OSR profilin in two patients at a concentration of 50 microg/ml. CONCLUSIONS: OSR pollen profilin shares IgE and IgG epitopes with Bet v 2 and other plant profilins and may represent a potentially relevant allergen for profilin-sensitized patients.     

Inhibition of rBet v 1-induced basophil histamine release with specific immunotherapy -induced serum immunoglobulin G: no evidence that FcγRIIB signalling is important

BACKGROUND: Human basophils and mast cells express the low-affinity immunoglobulin (Ig)G receptor FcgammaRIIB. It has previously been shown in artificial model systems that cross-linking of the high-affinity IgE receptor FcepsilonRI and FcgammaRIIB leads to inhibition of FcepsilonRI signalling. OBJECTIVE: The aim of the present study was to investigate whether cross-linking of FcepsilonRI and FcgammaRIIB contributes to IgG-mediated inhibition of histamine release in human basophils in a system using the sera from specific immunotherapy (SIT) patients and the major allergen from birch pollen, Bet v 1. As IgG4 furthermore has been proposed to have special blocking properties, we investigated the significance of IgG subclass specificity for this inhibition. METHODS: Binding of recombinant Bet v 1-IgG complexes to FcgammaRII and IgG-binding activities in the sera from 25 birch pollen-allergic patients treated with SIT were measured using (125)I-rBet v 1. Inhibition of basophil histamine release was assessed by incubating washed leucocytes with complexes of rBet v 1-IgG with or without blocking of FcgammaRII. RESULTS: We observed low binding of rBet v 1-IgG complexes to FcgammaRII, which was negatively correlated with the relative IgG4-binding activities. Blocking of FcgammaRII did not reverse the SIT-IgG-induced inhibition of basophil histamine release. However, IgG-binding activities correlated significantly with the ability of the SIT sera to inhibit basophil histamine release. CONCLUSION: We suggest that at least in birch pollen SIT, the contribution of FcgammaRIIB-mediated inhibitory signalling to SIT-IgG-induced inhibition of human basophil histamine release is of minor importance. The main contributor to the inhibitory effect of SIT-induced IgG seems to be blocking of the allergen-IgE interaction.