Les fruits à coque : monographies

[1] - Codreanu F. L'activité du réseau Allergo-Vigilance: allergies alimentaires. Alim'Inter 2007;12:239-242

[2] - Tawde P, Venkatesh YP, Wang F, Teuber SS, Sathe SK, Roux KH. Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen. J Allergy Clin Immunol 2006;118:915-922

BACKGROUND: The identity of allergenic almond proteins is incomplete . OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen . METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin . RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins . CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

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[3] - Roux KH, Teuber SS, Sathe SK. Tree Nut Allergens. Int Arch Allergy Immunol 2003;131:234-244

Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.

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[4] - Sathe SK, Wolf WJ, Roux KH, Teuber SS, Venkatachalam M, Sze-Tao KW. Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.). J Agric Food Chem 2002;50:4333-4341

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.

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[5] - Sathe SK, Wolf WJ, Roux KH, Teuber SS, Venkatachalam M, Sze-Tao KW. Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.). J Agric Food Chem 2002;50:4333-4341

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.

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[6] - Roux KH, Teuber SS, Robotham JM, Sathe SK. Detection and stability of the major almond allergen in foods. J Agric Food Chem 2001;49:2131-2136

Almond major protein (AMP or amandin), the primary storage protein in almonds, is the major allergen recognized by almond-allergic patients. A rabbit antibody-based inhibition ELISA assay for detecting and quantifying AMP in commercial foods has been developed, and this assay, in conjunction with Western blotting analyses, has been applied to the investigation of the antigenic stability of AMP to harsh food-processing conditions. The ELISA assay detects purified AMP at levels as low as 87 +/-16 ng/mL and can detect almond at between 5 and 37 ppm in the tested foods. The assay was used to quantify AMP in aqueous extracts of various foods that were defatted and spiked with known amounts of purified AMP or almond flour. In addition, AMP was quantified in commercially prepared and processed almond-containing foods. Neither blanching, roasting, nor autoclaving of almonds markedly decreased the detectability of AMP in subsequent aqueous extracts of almonds. Western blots using both rabbit antisera and sera from human almond-allergic patients confirm a general stability of the various peptides that comprise this complex molecule and show that the rabbit antibody-based assay recognizes substantially the same set of peptides as does the IgE in sera from almond-allergic patients.

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[7] - Bargman TJ, Rupnow JH, Taylor SL IgE-binding proteins in almonds (Prunus amygdalus L.): identification by immunoblotting with sera from almond-allergic adults. J Food Sci 1992;57:717-720

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[8] - Roux KH, Teuber SS, Sathe SK. Tree Nut Allergens. Int Arch Allergy Immunol 2003;131:234-244

Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.

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[9] - Poltronieri P, Cappello MS, Dohmae N, Conti A, Fortunato D, Pastorello EA, et al. Identification and Characterisation of the IgE-Binding Proteins 2S Albumin and Conglutin gamma in Almond (Prunus dulcis) Seeds. Int Arch Allergy Immunol 2002;128:97-104

Background: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. Methods: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. Results: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. Conclusions: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.

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[10] - Tawde P, Venkatesh YP, Wang F, Teuber SS, Sathe SK, Roux KH. Cloning and characterization of profilin (Pru du 4), a cross-reactive almond (Prunus dulcis) allergen. J Allergy Clin Immunol 2006;118:915-922

BACKGROUND: The identity of allergenic almond proteins is incomplete . OBJECTIVE: Our objective was to characterize patient IgE reactivity to a recombinant and corresponding native almond allergen . METHODS: An almond cDNA library was screened with sera from patients with allergy for IgE binding proteins. Two reactive clones were sequenced, and 1 was expressed. The expressed recombinant allergen and its native counterpart (purified from unprocessed almond flour) were assayed by 1-dimensional and 2-dimensional gel electrophoresis, dot blot, and ELISA, and screened for cross-reactivity with grass profilin . RESULTS: The 2 selected clones encoded profilin (designated Pru du 4) sequences that differed by 2 silent mutations. By dot-blot analyses, 6 of 18 patient sera (33%) reacted with the recombinant Pru du 4 protein, and 8 of 18 (44%) reacted with the native form. ELISA results were similar. Almond and ryegrass profilins were mutually inhibitable. Two-dimensional immunoblotting revealed the presence of more than 1 native almond profilin isoform. The strength of reactivity of some patients' serum IgE differed markedly between assays and between native and recombinant profilins . CONCLUSION: Almond nut profilin is an IgE-binding food protein that is cross-reactive with grass pollen profilin and is susceptible to denaturation, resulting in variable reactivity between assay types and between patients. CLINICAL IMPLICATIONS: Serum IgE of nearly half of the tested patients with almond allergy reacts with almond nut profilin. Because most patients also had pollinosis, the well-known cross-reactivity between pollen and food profilins could account for this pattern of reactivity.

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[11] - Poltronieri P, Cappello MS, Dohmae N, Conti A, Fortunato D, Pastorello EA, et al. Identification and Characterisation of the IgE-Binding Proteins 2S Albumin and Conglutin gamma in Almond (Prunus dulcis) Seeds. Int Arch Allergy Immunol 2002;128:97-104

Background: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. Methods: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. Results: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. Conclusions: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.

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[12] - Besler M, Rodriguez J, Taylor SL. Allergen Data Collection: almond (Prunus dulcis). Internet Symp Food Allergens 2001;3(2):71-85

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[13] - Pasini G, Simonato B, Giannattasio M, Gemignani C, Curioni A. IgE binding to almond proteins in two CAP-FEIA-negative patients with allergic symtoms to almond as compared to three CAP-FEIA-false-positive subjects. Allergy 2000;55:955-958

BACKGROUND: Allergy to almonds has been frequently reported, but data on the identification of the almond allergens, as well as on the reliability of the methods for in vitro detection of specific IgE for these allergens, are scant. This study aimed to identify the almond allergens and to evaluate the reliability of the CAP-FEIA as the standard system for detection of almond-specific IgE with clinical significance . METHODS: Immunoblotting performed with an almond-protein extract was carried out on the sera of five patients who had previously been tested by the CAP-FEIA system; two of these patients had tested negative with the CAP-FEIA system but suffered life-threatening laryngeal edema after eating almonds, whereas the other three subjects, who had tested positive with CAP-FEIA, did not present any symptoms subsequent to almond ingestion . RESULTS: The sera of the two symptomatic CAP-FEIA-negative patients had IgE that bound only to a 37-kDa protein in immunoblotting. On the contrary, the sera of the three asymptomatic subjects all showed IgE binding to two almond proteins of 62 and 50 kDa, corresponding to the glycosylated components of the extract . CONCLUSIONS: The results here presented suggest that, at least for the examined subjects, the positivity to almond, as measured with a standard laboratory method, is due to the presence of the 62/50-kDa glycoproteins with little or no immunologic significance, and not to the binding to the 37-kDa polypeptide, which appears to be a true almond allergen.

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[14] - Lüttkopf D, Müller U, Skov PS, Ballmer-Weber BK, Wüthrich B, Skamstrup Hansen K, et al. 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. Mol Immunol 2001;38:515-525

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.

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[15] - Breiteneder H, Ferreira F, Hoffmann-Sommergruber K, Ebner C, Breitenbach M, Rumpold H, et al. Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-binding properties. Eur J Biochem 1993;212:355-362

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding to these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3'-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3'-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96-99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71-73% (80.5-83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Car b I and isoforms, the major allergen from hornbeam, 75.5-76.7% identity (83.6-85% similarity) and 83.6-89.9% sequence identity (89.3-95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor a I. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.

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[16] - Lüttkopf D, Müller U, Skov PS, Ballmer-Weber BK, Wüthrich B, Skamstrup Hansen K, et al. 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. Mol Immunol 2001;38:515-525

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.

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[17] - Skamstrup Hansen K, Ballmer-Weber B, Sastre J, Dominguez J, Vieths S, Lidholm J, et al. Hazelnut allergy: sensitisation pattern in three European regions. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°3

Background: Tree nuts are common causes of food allergy and occasionally of severe systemic reactions. Hazelnut allergy occurs both with and without concomitant pollen allergy. The aim of this study was to evaluate a panel of recombinant hazelnut allergens for IgE-analyses in 3 subsets of patients: hazelnut allergic patients, birch or olive pollen allergic patients without allergy to hazelnut and healthy controls from 3 European regions. Methods: In all, 119 allergic patients and 63 non-atopic controls from Spain (ES), Switzerland (CH) and Denmark (DK) were included. Hazelnut allergy was confirmed in 57 patients by double-blind, placebo-controlled food challenge (DBPCFC). The 62 allergic controls had a history of pollinosis confirmed by skin prick test to birch or olive pollen. Absence of hazelnut allergy was ensured by open food challenge in the 2 control groups. Serum IgE to hazelnut extract and recombinant (r) hazelnut allergens was analysed by experimental ImmunoCAP tests using rCor a 1.04, rCor a 2, rCor a 8 and rCor a 11 expressed in E. coli. Results: In the DBPCFC-positive population, 91% (CH/ES 100%, DK 75%) had IgE to hazelnut extract, 75% to rCor a 1.04, 42% to rCor a 2, 28% to rCor a 8, and 2% to rCor a 11. The highest rate of sensitisation to the Bet v 1-homologue Cor a 1.04 was found in the northern regions (CH/DK 100%, ES 18%), while IgE to the lipid transfer protein (LTP) rCor a 8 prevailed in ES (ES 71%, CH 15%, DK 5%). IgE specific for profilin rCor a 2 was equally distributed (40-43%). Among pollen allergic controls, 61% had IgE to hazelnut extract, 69% to rCor a 1.04, 34% to rCor a 2, 10% to rCor a 8, and 6% to rCor a 11. In this group, IgE to rCor a 8 was restricted to ES and the rCor a 2-profile was different (DK 14%, CH/ES 45%). Conclusion: Component-resolved in vitro analyses confirmed differences in IgE-profile between southern, central and northern parts of Europe and between hazelnut allergic patients and hazelnut-tolerant patients with pollen allergy. Most hazelnut allergic patients from Spain were LTP-positive. In CH and DK, IgE directed against Cor a 1.04 clearly dominated whereas IgE to hazelnut LTP was rare and exclusively seen in symptomatic patients. Profilin and the vicilin-like protein rCor a 11 were of minor importance in this population. Diversity in IgE-pattern between the studied regions suggests the need for a geographically differentiated use of allergens for diagnosis in plant food allergy.

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[18] - Skamstrup Hansen K, Ballmer-Weber BK, Lüttkopf D, Skov PS, Wüthrich B, Bindslev-Jensen C, et al. Roasted hazelnuts – allergenic activity evaluated by double-blind, placebo-controlled food challenge. Allergy 2003;58:132-138

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.

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[19] - Breiteneder H, Ferreira F, Hoffmann-Sommergruber K, Ebner C, Breitenbach M, Rumpold H, et al. Four recombinant isoforms of Cor a I, the major allergen of hazel pollen, show different IgE-binding properties. Eur J Biochem 1993;212:355-362

Previous studies showed that pollens from trees of the order Fagales (e.g. birch, alder, hazel and hornbeam) all contain one major allergen. These proteins are cross-reactive between these tree species, and approximately 95% of tree-pollen-allergic patients display IgE binding to these allergens. Using the reported N-terminal amino acid sequence of the hazel pollen allergen Cor a I, it was possible to amplify Cor-a-I cDNA by use of the polymerase chain reaction. Four clones with cDNA inserts were isolated. All four clones contained an open reading frame of 477 nucleotides (159 amino acids) but differed in length of their 3'-non-coding regions. Within the overlapping regions, the nucleotide sequence of the 3'-non-coding regions of the four clones were nearly identical. The open reading frames coded for different isoforms of the major hazel pollen allergen, Cor a I. The clones were designated Cor a I/5, 6, 11 and 16, respectively. Comparison of the deduced amino acid sequences of these Cor a I isoforms revealed identities of 96-99%. The sequence identities between the Cor a I isoforms and Bet v I, the major birch pollen allergen, were 71-73% (80.5-83% similarity). Comparing amino acid sequences of Cor a I isoforms with the published sequences of Aln g I, the major allergen from alder, and Car b I and isoforms, the major allergen from hornbeam, 75.5-76.7% identity (83.6-85% similarity) and 83.6-89.9% sequence identity (89.3-95% similarity), respectively, was found. The four Cor a I cDNAs were subcloned into plasmid pKK223-3 and expressed in Escherichia coli as non-fusion proteins; their capacity to bind serum IgE from tree-pollen-allergic patients was investigated. The four cloned isoforms showed an apparent molecular mass of 17 kDa in SDS/PAGE, identical to the natural, pollen-derived Cor a I. IgE antibodies from tree-pollen-allergic patients reacted with all four recombinant isoforms. However, we noted marked differences in the IgE-binding patterns of the distinct isoforms. Furthermore, Cor a I/11 was the only isoform recognized by the anti-(Bet v I) mAb, BIP 1. Our results demonstrate that Cor a I isoforms display different antigenic and allergenic properties, very likely due to few but significant changes in their amino acid sequences. These findings have implications for the development of reagents for diagnosis and immunotherapy of type I allergies.

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[20] - Hirschwehr R, Valenta R, Ebner C, Ferreira F, Sperr WR, Valent P, et al. Identification of common allergenic structures in hazel pollen and hazelnuts: a possible explanation for sensitivity to hazelnuts in patients allergic to tree pollen. J Allergy Clin Immunol 1992;90:927-936

It is known that most patients with type I allergy to tree pollens also suffer from intolerance to nuts. To identify allergenic structures common to hazel pollen and hazelnuts, cross-reactivity of patients' IgE was investigated. With use of immunoblotting,.serum IgE from 25 patients displaying type I allergic reactions to tree pollens and intolerance to hazelnuts (group I) bound to the 17 kd major hazel pollen allergen Cor a I (100%) and to the 14 kd hazel pollen profilin (16%). IgE binding to proteins of comparable molecular weights in hazelnut extracts was found (18 kd and 14 kd), suggesting that proteins similar to Cor a I and hazel profilin might be also expressed in hazelnuts. In contrast, only four sera (22%) from 18 patients (group II) with tree pollen allergy but without any case history of nut hypersensitivity showed IgE binding to the 18 kd protein of hazelnut extract, and none of these sera exhibited IgE reactivity to the hazelnut profilin. To characterize the hazel pollen and hazelnut allergens, purified recombinant Bet v I (major birch pollen allergen) and purified recombinant Bet v II (birch profilin), respectively, were used for IgE-inhibition experiments. Binding of IgE from patients (with nut allergy) to the blotted hazelnut allergens could be blocked by preincubation of patients' sera with the recombinant proteins. Furthermore, the 18 kd protein of hazelnut extract was purified and induced specific release of histamine from basophils of a patient suffering nut hypersensitivity but not from a healthy control donor. A rabbit antibody raised against celery profilin identified the 14 kd proteins in hazel pollen and hazelnuts as profilin. Our experiments suggest a protein with IgE binding properties similar to the major allergens from pollens of hazel, Cor a I, and of birch, Bet v I, as predominant allergens in hazelnuts, and show that the plant pan-allergen profilin can be detected in both hazel pollen and hazelnut extracts.

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[21] - Müller U, Lüttkopf D, Hoffmann A, Petersen A, Becker WM, Schocker F, et al. Allergens in raw and roasted hazelnuts (Corylus avellana) and their cross-reactivity to pollen. Eur Food Res Technol 2000;212:2-12

Hazelnuts provoke one of the most frequent pollen-associated food allergies. In this study, hazelnut allergens were investigated and immunologically characterized, focussing on their heat stability and cross-reactivity with known allergenic structures. 27 sera from hazelnut-allergic patients and 28 sera from children with positive CAP classes for hazelnut and birch pollen were submitted to immunoblot and immunoblot inhibition. The major hazelnut allergen was found to present a mol. wt. of approx. 17-18 kDa, and to share IgE epitopes with Bet v 1, the major birch pollen allergen. This allergen was recognized by IgE of 93% of hazelnut-allergic patients and by 79% of sensitized children. A 48 kDa glycoprotein was identified as a minor hazelnut allergen with cross-reactive carbohydrate determinants. The major N-glycan species was determined by matrix-assisted laser desorption MS to be Man3XylGlcNAc2. IgE binding to this protein was detected in sera of 41% of the allergic patients and 61% of the sensitized children. Partial N-terminal sequencing demonstrated similarity to legume storage proteins. The IgE reactivity of this structure was partially resistant to heating. The rat basophil leukaemia cell mediator release assay was used for estimation of cross-sensitization between hazelnut and birch pollen, confirming the finding that hazelnuts contain a heat-resistant allergenicity without cross-reactivity to birch pollen allergens

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[22] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334

In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

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[23] - Akkerdaas JH, Wensing M, van Leeuwen A, Schilte P, Hefle SL, Aalberse RC, et al. IgE Profiles in Dutch Hazelnut Allergic Patients: Non-pollen Related Recognition of Cor a 1 and Pepsin-induced Neo-epitopes. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°801

Rationale Dutch hazelnut allergic patients are typically sensitized to Cor a 1 and profilin on the basis of primary sensitization to birch pollen Bet v 1 and Bet v 2. These allergens are extremely pepsin-sensitive, thus limiting clinical allergy to OAS. The aim of this study was to investigate whether other (potentially stable) allergens play a role. Method s : Sera were selected from patients (n=60) with history- and/or DBPCFC-proven hazelnut allergy or from subjects (n=18) with high IgE titers to hazelnut but low or absent to apple and birch. Extracts of crude, roasted and pepsin-digested hazelnut were used to assess the stability of IgE epitopes. Extraction of hazelnut at pH2.5 resulted in enrichment for 2S albumin. These extracts and affinity-purified Cor a 1 were used in RAST and immunoblot analyses. Result s : As expected, in the group of patients with proven hazelnut allergy, IgE recognition of Cor a 1 dominated (58/60 with >0.3 IU/ml). In accordance with the lability of Cor a 1, recognition of roasted and pepsin-digested hazelnut was much less frequent: 36% and 12% positives, respectively. Two sera contained IgE antibodies that preferentially recognized hazelnut extract after pepsin-digestion, thus pointing towards neo-epitopes. Among the sera selected on low reactivity to birch/apple, 5/18 recognized hazelnut Cor a 1 but not pollen Cor a 1 or Bet v 1. Conclusions : Cor a 1 recognition independent from pollen sensitization exists, raising questions about its stability. Pepsin-digestion supports the existence of neo-epitopes.

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[24] - Flinterman AE, Akkerdaas JH, den Hartog Jager CF, Rigby NM, Fernandez-Rivas M, Hoekstra MO, et al. Lipid transfer protein–linked hazelnut allergy in children from a non-Mediterranean birch-endemic area. J Allergy Clin Immunol 2008;121:423-428

BACKGROUND: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8 . OBJECTIVE: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area . METHODS: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed . RESULTS: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1 . CONCLUSION: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.

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[25] - Roux KH, Teuber SS, Sathe SK. Tree Nut Allergens. Int Arch Allergy Immunol 2003;131:234-244

Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.

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[26] - Hirschwehr R, Valenta R, Ebner C, Ferreira F, Sperr WR, Valent P, et al. Identification of common allergenic structures in hazel pollen and hazelnuts: a possible explanation for sensitivity to hazelnuts in patients allergic to tree pollen. J Allergy Clin Immunol 1992;90:927-936

It is known that most patients with type I allergy to tree pollens also suffer from intolerance to nuts. To identify allergenic structures common to hazel pollen and hazelnuts, cross-reactivity of patients' IgE was investigated. With use of immunoblotting,.serum IgE from 25 patients displaying type I allergic reactions to tree pollens and intolerance to hazelnuts (group I) bound to the 17 kd major hazel pollen allergen Cor a I (100%) and to the 14 kd hazel pollen profilin (16%). IgE binding to proteins of comparable molecular weights in hazelnut extracts was found (18 kd and 14 kd), suggesting that proteins similar to Cor a I and hazel profilin might be also expressed in hazelnuts. In contrast, only four sera (22%) from 18 patients (group II) with tree pollen allergy but without any case history of nut hypersensitivity showed IgE binding to the 18 kd protein of hazelnut extract, and none of these sera exhibited IgE reactivity to the hazelnut profilin. To characterize the hazel pollen and hazelnut allergens, purified recombinant Bet v I (major birch pollen allergen) and purified recombinant Bet v II (birch profilin), respectively, were used for IgE-inhibition experiments. Binding of IgE from patients (with nut allergy) to the blotted hazelnut allergens could be blocked by preincubation of patients' sera with the recombinant proteins. Furthermore, the 18 kd protein of hazelnut extract was purified and induced specific release of histamine from basophils of a patient suffering nut hypersensitivity but not from a healthy control donor. A rabbit antibody raised against celery profilin identified the 14 kd proteins in hazel pollen and hazelnuts as profilin. Our experiments suggest a protein with IgE binding properties similar to the major allergens from pollens of hazel, Cor a I, and of birch, Bet v I, as predominant allergens in hazelnuts, and show that the plant pan-allergen profilin can be detected in both hazel pollen and hazelnut extracts.

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[27] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334

In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

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[28] - Skamstrup Hansen K, Ballmer-Weber BK, Lüttkopf D, Skov PS, Wüthrich B, Bindslev-Jensen C, et al. Roasted hazelnuts – allergenic activity evaluated by double-blind, placebo-controlled food challenge. Allergy 2003;58:132-138

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.

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[29] - Flinterman AE, Akkerdaas JH, den Hartog Jager CF, Rigby NM, Fernandez-Rivas M, Hoekstra MO, et al. Lipid transfer protein–linked hazelnut allergy in children from a non-Mediterranean birch-endemic area. J Allergy Clin Immunol 2008;121:423-428

BACKGROUND: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8 . OBJECTIVE: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area . METHODS: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed . RESULTS: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1 . CONCLUSION: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.

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[30] - Schocker F, Lüttkopf D, Scheurer S, Petersen A, Vieths S, Becker WM. Cloning and sequencing of the lipid transfer protein from hazelnut (Corylus avellana). 8th International Symposium on Problems of Food Allergy, Venice 2001, March 11-13

Purpose: Hazelnut allergy in patients allergic to tree pollen is well documented but very little is known about hazelnut allergy provoked independently of pollinosis. Therefore, we focus on the lipid transfer protein, a non pollen-associated allergen of hazelnuts. Methods: Total RNA was isolated from immature hazelnuts and transcribed into cDNA. By means of a degenerated primer designed on the basis of known homologous sequences of the lipid transfer protein from cherry the full length coding cDNA was amplified. It was ligated into pGEM-T vector and sequenced. Clinical cases: Two patients with more severe allergic reactions to hazelnuts show IgE reactivity to a 9 kDa allergen in immunoblot experiments with hazelnut extract. Results: The coding region of the cDNA of the lipid transfer protein was identified as a 345 bp open reading frame coding for a protein of 92 amino acids and a signal peptide of 23 amino acids. The mature lipid transfer protein has a calculated molecular weight of 9,4 kDa and shows amino acid identities with allergenic LTPs from almond (62%), peach (59%) and cherry (59%). The N-terminus of the mature lipid transfer protein was confirmed on amino acid level by N-terminal sequencing of a 9 kDa protein fraction enriched from hazelnut extract by ion exchange chromatography. Conclusions: Lipid transfer protein from hazelnuts was identified as a potential new allergen which is not birch pollen-related. The sequence was submitted to NCBIGenBank database

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[31] - Schocker F, Lüttkopf D, Scheurer S, Petersen A, Cisteró-Bahima A, Enrique E, et al. Recombinant lipid transfer protein Cor a 8 from hazelnut: A new tool for in vitro diagnosis of potentially severe hazelnut allergy. J Allergy Clin Immunol 2004;113:141-147

BACKGROUND: Cor a 1.04 has been identified as the major hazelnut allergen in 65 European patients with positive double-blind, placebo-controlled food challenge results to hazelnut. Recently, the 11S globulin Cor a 9 was shown to be a pollen-independent hazelnut allergen in the United States, whereas preliminary data suggest the lipid transfer protein (LTP) as an important birch pollen-unrelated hazelnut allergen in Europe . OBJECTIVE: We sought to recruit a group of European patients allergic to hazelnut without birch pollen allergy and to identify and clone the major food allergen(s) in this study population . METHODS: We recruited 26 such Spanish patients, including 10 patients with anaphylaxis. IgE immunoblotting was performed with hazelnut extract. Hazelnut LTP Cor a 8 was cloned by using a PCR strategy, purified, and subjected to IgE immunoblotting. Recombinant Cor a 8, rCor a 1.0401, and rCor a 2 (profilin) were further investigated by means of enzyme allergosorbent test. Immunoblot inhibition experiments were used to compare the immunologic properties of natural and recombinant LTP . RESULTS: A 9-kd major allergen was identified in hazelnut extract. Cloning, sequencing, heterologous expression, and inhibition experiments identified it as an LTP. The prevalence of specific IgE antibody reactivity to LTP was 62% in hazelnut extract and 77% when recombinant LTP was tested by means of immunoblotting. IgE immunoblot inhibition with hazelnut extract showed that natural Cor a 8 and rCor a 8 shared identical epitopes. Only one patient had positive reactivity to Cor a 1.04, and no patients had positive reactivity to Cor a 2. Two sera bound to high-molecular-weight allergens. The LTP was denominated as Cor a 8 and submitted to the allergen database of the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Subcommittee . CONCLUSIONS: Cor a 8 is a relevant allergen for a majority of Spanish patients with hazelnut allergy that can cause severe allergic reactions.

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[32] - Schocker F, Lüttkopf D, Müller U, Thomas P, Vieths S, Becker WM. IgE binding to unique hazelnut allergens: identification of non pollen-related and heat-stable hazelnut allergens eliciting severe allergic reactions. Eur J Nutr 2000;39:172-180

BACKGROUND: Usually hazelnut allergic patients suffer from the tree pollen associated oral allergy syndrome (OAS) caused by cross-reactive structures. Anaphylactic reactions elicited by hazelnuts happen rarely but are of high clinical significance. Considering that hazelnuts are ingredients in processed foods, hazelnuts may play an important role as hidden allergens for these high risk patients. Therefore, we analyzed the IgE reactivity of a young woman with severe allergic reactions after ingestion of hazelnuts without any association to tree pollen allergy. AIM OF THE STUDY: The aim of this study was to identify and characterize these potent hazelnut-specific allergens. We compared these allergens to structures displayed by sera from patients with a completely or partially non pollen-related hazelnut allergy and with birch pollen-related hazelnut allergy. None of the sera had a clinical history of anaphylaxis. Special emphasis was placed on the heat stability and cross-reactivity of these allergens. METHODS/RESULTS: Using Western blotting with extract from birch pollen and EAST inhibition techniques we were able to show that the allergens in the serum sample of the young woman were not cross-reactive with birch pollen. Immunoblot experiments with extracts from native and heated hazelnuts and EAST inhibition tests further characterized these allergens to be heat-stable. Unlike the IgE binding pattern of the sera from the patients with pollen-related hazelnut allergy, low molecular weight proteins below 10 kDa were identified by the sera from the patients without pollinosis. CONCLUSIONS: Since the binding pattern of the serum sample of the young woman was different from that of the sera from patients without pollen allergy but less severe symptoms, we assume an association between single non pollen-dependent hazelnut allergens in the low molecular range and severe allergic reactions. These results enable us to approach a subgroup of hazelnut allergens which we believe to be responsible for anaphylactic reactions in hazelnut allergic patients after ingestion of heat-stable hazelnut structures in processed food stuff, independent of pollinosis.

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[33] - Skamstrup Hansen K, Ballmer-Weber B, Sastre J, Dominguez J, Vieths S, Lidholm J, et al. Hazelnut allergy: sensitisation pattern in three European regions. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°3

Background: Tree nuts are common causes of food allergy and occasionally of severe systemic reactions. Hazelnut allergy occurs both with and without concomitant pollen allergy. The aim of this study was to evaluate a panel of recombinant hazelnut allergens for IgE-analyses in 3 subsets of patients: hazelnut allergic patients, birch or olive pollen allergic patients without allergy to hazelnut and healthy controls from 3 European regions. Methods: In all, 119 allergic patients and 63 non-atopic controls from Spain (ES), Switzerland (CH) and Denmark (DK) were included. Hazelnut allergy was confirmed in 57 patients by double-blind, placebo-controlled food challenge (DBPCFC). The 62 allergic controls had a history of pollinosis confirmed by skin prick test to birch or olive pollen. Absence of hazelnut allergy was ensured by open food challenge in the 2 control groups. Serum IgE to hazelnut extract and recombinant (r) hazelnut allergens was analysed by experimental ImmunoCAP tests using rCor a 1.04, rCor a 2, rCor a 8 and rCor a 11 expressed in E. coli. Results: In the DBPCFC-positive population, 91% (CH/ES 100%, DK 75%) had IgE to hazelnut extract, 75% to rCor a 1.04, 42% to rCor a 2, 28% to rCor a 8, and 2% to rCor a 11. The highest rate of sensitisation to the Bet v 1-homologue Cor a 1.04 was found in the northern regions (CH/DK 100%, ES 18%), while IgE to the lipid transfer protein (LTP) rCor a 8 prevailed in ES (ES 71%, CH 15%, DK 5%). IgE specific for profilin rCor a 2 was equally distributed (40-43%). Among pollen allergic controls, 61% had IgE to hazelnut extract, 69% to rCor a 1.04, 34% to rCor a 2, 10% to rCor a 8, and 6% to rCor a 11. In this group, IgE to rCor a 8 was restricted to ES and the rCor a 2-profile was different (DK 14%, CH/ES 45%). Conclusion: Component-resolved in vitro analyses confirmed differences in IgE-profile between southern, central and northern parts of Europe and between hazelnut allergic patients and hazelnut-tolerant patients with pollen allergy. Most hazelnut allergic patients from Spain were LTP-positive. In CH and DK, IgE directed against Cor a 1.04 clearly dominated whereas IgE to hazelnut LTP was rare and exclusively seen in symptomatic patients. Profilin and the vicilin-like protein rCor a 11 were of minor importance in this population. Diversity in IgE-pattern between the studied regions suggests the need for a geographically differentiated use of allergens for diagnosis in plant food allergy.

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[34] - Schocker F, Lüttkopf D, Cisteró-BahímaA, Enrique E, Akkerdaas JH, van Ree R, et al. Recombinant Hazelnut LTP, Cor a 8: A Useful Tool in the in vitro Diagnosis of Hazelnut Allergy. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°723

RATIONALE: Lipid transfer proteins (LTP) are known as food allergens which can elicit severe allergenic reactions mainly in the Mediterranean area. Recently, we were able to clone the full length cDNA of hazelnut LTP and express its recombinant form (denominated as Cor a 8 according to the WHO/IUIS Allergen Nomenclature Subcommittee). The purpose of this study was to investigate the IgE-reactivity to the recombinant Cor a 8 in comparison to hazelnut extract. METHODS: Cor a 8 was expressed in E. coli and purified by His-tag chromatography. Sera from 26 Spanish patients with hazelnut allergy were tested by performing immunoblots with r Cor a 8 and hazelnut extract. Immunoblot inhibition was performed to investigate immunological properties of natural and recombinant Cor a 8. RESULTS: Eighteen out of 26 Spanish sera showed IgE-reactivity to n Cor a 8 in hazelnut extract of which 13 patients were monosensitized to Cor a 8. These results could be fully confirmed by using the recombinant allergen. Immunoblot inhibition demonstrated epitope similarity of natural and recombinant Cor a 8. CONCLUSIONS: LTP is a major allergen in Spanish patients with hazelnut allergy. As demonstrated by means of immunoblot and immunoblot inhibition recombinant hazelnut LTP has the same biologic activity as natural LTP. Since quality problems with allergen extracts from foods still exist and LTPs are known as potentially severe food allergens, the use of r Cor a 8 may improve the in vitro diagnosis of food allergy.

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[35] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334

In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

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[36] - Skamstrup Hansen K, Ballmer-Weber BK, Lüttkopf D, Skov PS, Wüthrich B, Bindslev-Jensen C, et al. Roasted hazelnuts – allergenic activity evaluated by double-blind, placebo-controlled food challenge. Allergy 2003;58:132-138

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.

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[37] - Flinterman AE, Akkerdaas JH, den Hartog Jager CF, Rigby NM, Fernandez-Rivas M, Hoekstra MO, et al. Lipid transfer protein–linked hazelnut allergy in children from a non-Mediterranean birch-endemic area. J Allergy Clin Immunol 2008;121:423-428

BACKGROUND: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8 . OBJECTIVE: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area . METHODS: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed . RESULTS: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1 . CONCLUSION: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.

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[38] - Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-570

Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd.

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[39] - Beyer K, Grishina G, Bardina L, Grishin A, Sampson HA. Identification of an 11S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions. J Allergy Clin Immunol 2002;110:517-523

BACKGROUND: Hazelnuts are a common cause of food allergy. Allergic reactions to hazelnuts range from oral allergy syndrome caused by cross-reactivity between tree pollen and hazelnut proteins to severe anaphylactic reactions. Little information is available regarding the identification of pollen-independent hazelnut allergens. OBJECTIVE: The aim of the study was to identify these pollen-independent allergens in patients with hazelnut allergy with systemic reactions. METHODS: Extracted hazelnut proteins were separated by means of 2-dimensional PAGE, and immunolabeling was performed with individual sera from 14 patients with hazelnut-induced systemic reactions. Edman sequencing was performed on a 40-kd protein identified as an allergen. In parallel, RNA isolated from hazelnuts was used to construct a cDNA library. By using the peptide sequence data, oligonucleotide primers were synthesized and used to screen the library. Full-length cDNA clones were isolated, sequenced, expressed, and screened with patient sera. RESULTS: By using 2-dimensional proteomics, a protein fraction at 40 kd was recognized by serum IgE from 86% (12/14) of the patients with hazelnut allergy with systemic reactions. Two internal amino acid sequences were determined by means of Edman sequencing. Screening of the prepared hazelnut cDNA library with oligonucleotides based on these internal peptide sequences resulted in isolation of a novel protein cDNA. The new protein, named Cor a 9, belongs to the 11S globulin seed storage protein family. This family comprises known food allergens in peanut (Ara h 3) and soybean (glycine max). The pairwise homology among these 3 proteins ranges from 45% to 50%. Interestingly, one known IgE-binding epitope of Ara h 3 shares 67% of homologous amino acid residues with the corresponding area of Cor a 9. The amino acids that differ were previously shown not to be critical for IgE binding in Ara h 3. CONCLUSION: Cor a 9 is the first tree pollen-unrelated hazelnut allergen isolated, sequenced, and cloned. The identification of food allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE epitopes in common food allergens, such as seed storage proteins, might be a useful tool for predicting cross-reactivity to certain foods.

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[40] - Beyer K, Grishina G, Bardina L, Grishin A, Sampson HA. Identification of an 11S globulin as a major hazelnut food allergen in hazelnut-induced systemic reactions. J Allergy Clin Immunol 2002;110:517-523

BACKGROUND: Hazelnuts are a common cause of food allergy. Allergic reactions to hazelnuts range from oral allergy syndrome caused by cross-reactivity between tree pollen and hazelnut proteins to severe anaphylactic reactions. Little information is available regarding the identification of pollen-independent hazelnut allergens. OBJECTIVE: The aim of the study was to identify these pollen-independent allergens in patients with hazelnut allergy with systemic reactions. METHODS: Extracted hazelnut proteins were separated by means of 2-dimensional PAGE, and immunolabeling was performed with individual sera from 14 patients with hazelnut-induced systemic reactions. Edman sequencing was performed on a 40-kd protein identified as an allergen. In parallel, RNA isolated from hazelnuts was used to construct a cDNA library. By using the peptide sequence data, oligonucleotide primers were synthesized and used to screen the library. Full-length cDNA clones were isolated, sequenced, expressed, and screened with patient sera. RESULTS: By using 2-dimensional proteomics, a protein fraction at 40 kd was recognized by serum IgE from 86% (12/14) of the patients with hazelnut allergy with systemic reactions. Two internal amino acid sequences were determined by means of Edman sequencing. Screening of the prepared hazelnut cDNA library with oligonucleotides based on these internal peptide sequences resulted in isolation of a novel protein cDNA. The new protein, named Cor a 9, belongs to the 11S globulin seed storage protein family. This family comprises known food allergens in peanut (Ara h 3) and soybean (glycine max). The pairwise homology among these 3 proteins ranges from 45% to 50%. Interestingly, one known IgE-binding epitope of Ara h 3 shares 67% of homologous amino acid residues with the corresponding area of Cor a 9. The amino acids that differ were previously shown not to be critical for IgE binding in Ara h 3. CONCLUSION: Cor a 9 is the first tree pollen-unrelated hazelnut allergen isolated, sequenced, and cloned. The identification of food allergens is the first step toward generating recombinant allergens for use in future immunotherapeutic approaches. In addition, the detection of conserved IgE epitopes in common food allergens, such as seed storage proteins, might be a useful tool for predicting cross-reactivity to certain foods.

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[41] - Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-570

Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd.

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[42] - Lauer I, Mueller U, Westphal S, Haustein D, Vieths S, Scheurer S. Characterisation and Cloning of a 48 kDa Glycoprotein, From Hazelnut (Corylus Avellana) as Minor Allergen. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°927

Hazelnut (HN), widely distributed in sweets, provokes one of the most common food allergies similar to peanut and walnut. In Central Europe, 90% of the hazelnut-allergic patients suffer from pollen-associated HN-allergy, recognizable by symptoms such as oral allergy syndrome, urticaxia, angioedema and respiratory symptoms. Rarely, anaphylactic reactions have been reported. The aim of this study was the cloning and the immunological characterization of a 48-kDa glycoprotein described as putative IgE-reactive vicilin by N-terminal sequencing in a previous study. Furthermore, the reactivity of the hazelnut-allergic patients`IgE towards N-linked glycanstructures of this allergen was analysed. Recently, cloning of full length cDNA was performed by using degenerate oligonucleotides based on the known N-terminal sequence of the 48 kDa-HN-protein. This vicilin c-DNA was cloned into the pET100 vector, and the recombinant protein was expressed in E.coli and purified via His-tag chromatography. The sequence analysis shows high degrees of protein aminio acid (aa) identities with legume storage proteins belonging to the vicilin family, such as Jug r 2 from walnut (47.3% ID), P54 from pea (45.8% ID), a vicilin-precursor of the macadamia nut (43.6% ID) and Ara h 1 of the peanut (29.3% ID). In addition, the peptide chain includes two possible N-glycosylation sites. The recombinant, non glycosylated protein showed no IgE reactivity in immunoblot analysis, whereas the partially by HPLC purified native 48-kDa glycoprotein was recognized by IgE of 6 of our 35 HN-allergic patients (21%) in Western blots („Minor allergen‰). In our previous findings, Nglycan analysis (Maldi-TOF) of the 48-kDa glycoprotein showed 74% MMX- and 3% MOXF-structures. The IgE-immunoblot inhibition with the glycoproteins bromelain and horseradish-peroxidase indicates a high cross-reactivity with the MOXF3 structure. In conclusion, these results strongly suggest an important role of complex N-glycan structures in the epitope that is recognized by the hazelnut allergic patients`IgE. In particular, xylose and, possibly, fucose may be very important

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[43] - Müller U, Lüttkopf D, Hoffmann A, Petersen A, Becker WM, Schocker F, et al. Allergens in raw and roasted hazelnuts (Corylus avellana) and their cross-reactivity to pollen. Eur Food Res Technol 2000;212:2-12

Hazelnuts provoke one of the most frequent pollen-associated food allergies. In this study, hazelnut allergens were investigated and immunologically characterized, focussing on their heat stability and cross-reactivity with known allergenic structures. 27 sera from hazelnut-allergic patients and 28 sera from children with positive CAP classes for hazelnut and birch pollen were submitted to immunoblot and immunoblot inhibition. The major hazelnut allergen was found to present a mol. wt. of approx. 17-18 kDa, and to share IgE epitopes with Bet v 1, the major birch pollen allergen. This allergen was recognized by IgE of 93% of hazelnut-allergic patients and by 79% of sensitized children. A 48 kDa glycoprotein was identified as a minor hazelnut allergen with cross-reactive carbohydrate determinants. The major N-glycan species was determined by matrix-assisted laser desorption MS to be Man3XylGlcNAc2. IgE binding to this protein was detected in sera of 41% of the allergic patients and 61% of the sensitized children. Partial N-terminal sequencing demonstrated similarity to legume storage proteins. The IgE reactivity of this structure was partially resistant to heating. The rat basophil leukaemia cell mediator release assay was used for estimation of cross-sensitization between hazelnut and birch pollen, confirming the finding that hazelnuts contain a heat-resistant allergenicity without cross-reactivity to birch pollen allergens

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[44] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334

In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

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[45] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334

In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

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[46] - Lauer I, Mueller U, Westphal S, Haustein D, Vieths S, Scheurer S. Characterisation and Cloning of a 48 kDa Glycoprotein, From Hazelnut (Corylus Avellana) as Minor Allergen. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°927

Hazelnut (HN), widely distributed in sweets, provokes one of the most common food allergies similar to peanut and walnut. In Central Europe, 90% of the hazelnut-allergic patients suffer from pollen-associated HN-allergy, recognizable by symptoms such as oral allergy syndrome, urticaxia, angioedema and respiratory symptoms. Rarely, anaphylactic reactions have been reported. The aim of this study was the cloning and the immunological characterization of a 48-kDa glycoprotein described as putative IgE-reactive vicilin by N-terminal sequencing in a previous study. Furthermore, the reactivity of the hazelnut-allergic patients`IgE towards N-linked glycanstructures of this allergen was analysed. Recently, cloning of full length cDNA was performed by using degenerate oligonucleotides based on the known N-terminal sequence of the 48 kDa-HN-protein. This vicilin c-DNA was cloned into the pET100 vector, and the recombinant protein was expressed in E.coli and purified via His-tag chromatography. The sequence analysis shows high degrees of protein aminio acid (aa) identities with legume storage proteins belonging to the vicilin family, such as Jug r 2 from walnut (47.3% ID), P54 from pea (45.8% ID), a vicilin-precursor of the macadamia nut (43.6% ID) and Ara h 1 of the peanut (29.3% ID). In addition, the peptide chain includes two possible N-glycosylation sites. The recombinant, non glycosylated protein showed no IgE reactivity in immunoblot analysis, whereas the partially by HPLC purified native 48-kDa glycoprotein was recognized by IgE of 6 of our 35 HN-allergic patients (21%) in Western blots („Minor allergen‰). In our previous findings, Nglycan analysis (Maldi-TOF) of the 48-kDa glycoprotein showed 74% MMX- and 3% MOXF-structures. The IgE-immunoblot inhibition with the glycoproteins bromelain and horseradish-peroxidase indicates a high cross-reactivity with the MOXF3 structure. In conclusion, these results strongly suggest an important role of complex N-glycan structures in the epitope that is recognized by the hazelnut allergic patients`IgE. In particular, xylose and, possibly, fucose may be very important

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[47] - Lauer I, Fötisch K, Kolarich D, Ballmer-Weber BK, Conti A, Altmann F, et al. Hazelnut Vicilin Cor a 11: Molecular characterisation of a glycoprotein and its allergenic activity. Biochem J 2004;383:327-334

In Europe, hazelnuts are a frequent cause of food allergies. Several important hazelnut allergens have been previously identified and characterised. Specific N-glycans are known to induce strong IgE responses of uncertain clinical relevance, but so far the allergenic potential of glycoproteins from hazelnut has not been investigated. The aim of the study was the molecular characterisation of the glycosylated vicilin Cor a 11 from hazelnut and the analysis of its allergenic activity. Whereas MALDI-TOF mass spectrometry, one of two potential glycosylation sites of Cor a 11 was found to be glycosylated, Circular Dichroism spectroscopy indicated that recombinant and natural Cor a 11 share similar secondary structures. Thus, to analyse the impact of the glycan residues of Cor a 11 on IgE-binding, the allergenic activity of natural glycosylated Cor a 11 and recombinant Cor a 11 was compared. In addition, the IgE sensitisation pattern to recombinant Cor a 11, Cor a 1, Cor a 2 and Cor a 8 of 65 hazelnut allergic patients was determined in vitro. The prevalence of IgE-reactivity to hazelnut vicilin Cor a 11 was below 50%. Basophil histamine release assays were used to determine the allergenic activity of both, natural and recombinant Cor a 11 in comparison to Cor a 1, a birch pollen-related major hazelnut allergen. Both forms of Cor a 11 induced mediator release from basophils to a similar extent, indicating that the hazelnut allergic patients had cross-linking IgE-antibodies binding to the protein backbone and not to carbohydrate structures. In comparison to Cor a 1, a 10000-fold higher concentration of Cor a 11 was required to induce similar basophil mediator release. In conclusion, the hazelnut vicilin Cor a 11 is a minor allergen both in regard to prevalence and allergenic potency, whereas its glycan does not contribute to its allergenic activity.

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[48] - Akkerdaas JH, Schocker F, Vieths S, Versteeg S, Zuidmeer L, Hefle SL, et al. Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library. Mol Nutr Food Res 2006;50:18-23

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.

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[49] - Beyer K, Bardina L, Grishina G, Grishin A, Sampson HA. Identification of a Heat Shock Protein as a Major Hazelnut Food Allergen in Hazelnut Induced Systemic Reactions. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°724

RATIONALE: Hazelnuts are a common cause of food allergy. We recently identified Cor a 9, a seed storage protein as a pollen-independent hazelnut allergen. Using the same patient population, a second major hazelnut food allergen was isolated. METHODS: Extracted hazelnut proteins were separated by 2D-PAGE, and immunolabelling was performed with individual sera from 14 patients with hazelnut-induced systemic reactions. Edman sequencing was performed on a 17 kD protein identified as an allergen. In parallel, RNA isolated from hazelnuts was used to construct a cDNA library. Using the peptide sequence data, oligonucleotide primers were synthesized and used to screen the library. Full-length cDNA clones were isolated, sequenced, expressed and screened with patient sera. RESULTS: Using 2D-Proteomics, a protein fraction at 17 kD was recognized by serum IgE from 10/14 [71%] of the hazelnut allergic patients with systemic reactions. Amino acid sequences of two peptides were determined by internal Edman sequencing. Screening the constructed hazelnut cDNA library with oligonucleotides based on theses internal peptide sequences resulted in isolation of a novel protein cDNA. The new protein showed homology to class I heat shock proteins. CONCLUSIONS: A small molecular weight heat shock protein, a protective protein in hazelnuts, was isolated, sequenced and cloned and identified as a tree-pollen unrelated hazelnut allergen. In addition to seed storage proteins, protective proteins are believed to account for the majority of plant food allergens. The identification of food allergens is the first step towards generating recombinant allergens for use in future immunotherapeutic approaches.

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[50] - Pastorello EA, Vieths S, Pravettoni V, Farioli L, Trambaioli C, Fortunato D, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol 2002;109:563-570

Background: The hazelnut major allergens identified to date are an 18-kd protein homologous to Bet v 1 and a 14-kd allergen homologous to Bet v 2. No studies have reported hazelnut allergens recognized in patients with positive double-blind, placebo-controlled food challenge (DBPCFC) results or in patients allergic to hazelnut but not to birch. Objective: We characterized the hazelnut allergens by studying the IgE reactivity of 65 patients with positive DBPCFC results and 7 patients with severe anaphylaxis to hazelnut. Methods: Hazelnut allergens were identified by means of SDS-PAGE and IgE immunoblotting. Further characterization was done with amino acid sequencing, evaluation of the IgE-binding properties of raw and roasted hazelnut with enzyme allergosorbent test inhibition, assessment of cross-reactivity with different allergens by means of immunoblotting inhibition, and purification by means of HPLC. Results: All the sera from the patients with positive DBPCFC results recognized an 18- and a 47-kd allergen; other major allergens were at molecular weights of 32 and 35 kd. Binding to the 18-kd band was inhibited by birch extract, indicating its homology with the birch major allergen, and abolished in roasted hazelnut. The 47-kd allergen is a sucrose-binding protein, the 35-kd allergen is a legumin, and the 32-kd allergen is a 2S albumin. Patients with severe anaphylactic reactions to hazelnut showed specific IgE reactivity to a 9-kd allergen, totally inhibited by purified peach lipid-transfer protein (LTP), which was heat stable and, when purified, corresponded to an LTP. Conclusions: The major allergen of hazelnut is an 18-kd protein homologous to Bet v 1, and the 9-kd allergen is presumably an LTP. Other major allergens have molecular weights of 47, 32, and 35 kd.

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[51] - Wensing M, Akkerdaas J, Hefle SL, Penninks AH, Bruijnzeel-Koomen CA, Knulst AC, et al. Allergen Recognition Patterns in a Dutch Hazelnut Allergic Population in Relation to Reactivity Patterns as Observed During Double Blind Placebo Controlled Food Challenges (DBPCFC). AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°924

BACKGROUND: Hazelnut allergic patients in Northern Europe mostly are sensitized to Cor a 1, a hazelnut allergen with high homology to the major birch pollen allergen Bet v 1, and most frequently suffer from the oral allergy syndrome (OAS). Other hazelnut allergens like LTPs and 2S albumins are thought to be associated with more severe symptoms. In this study, allergen recognition patterns of a Dutch hazelnut allergic population (n=29) were revealed and correlated with the severity of symptoms and minimum provoking doses (MPDs) as observed during DBPCFCs. METHODS: Sera were obtained from 29 patients with positive DBPCFCs to hazelnut. The presence of specific IgE to hazelnut was determined using CAP system FEIA and RAST. Immunoblotting was performed using raw hazelnut extract and a pH2.5 hazelnut extract, mainly representing low molecular weight allergens (Akkerdaas et al. Poster no 71. Symposium on Immunological, Chemical and Clinical Problems of Food Allergy, March 2001, Venice). RESULTS: Specific IgE to hazelnut was found in all but two patients using RAST (> 0.3 IE/ml) in contrast to 17 out of 29 using CAP system FEIA (> 0.35 kU/ml). Immunoblotting using raw hazelnut extract demonstrated IgE binding to Cor a 1 in 21 sera, of whom 3 had additional IgE-binding to a 30-32 kD protein. In two sera exclusive IgE binding to high molecular weight proteins was demonstrated. Immunoblotting using pH2.5 hazelnut extract revealed additional IgE binding to proteins of < 17 kD in 3 sera. Four sera did not display IgE binding on immunoblots. No significant differences in MPDs and severity of symptoms could be revealed among patients with Cor a 1- or Œother' allergen recognition profiles. However, in 4 out of 8 patients (50 %) with Œother' recognition patterns, the challenge had to be discontinued because of longstanding gastro-intestinal symptoms or OAS in contrast to 4 out of 17 purely Cor a 1-sensitized patients (24 %). Besides, objective symptoms occurred only in patients who did not recognize Cor a 1 on immunoblots. CONCLUSION: Most hazelnut allergic patients in this study are sensitized to Cor a 1. A minority of patients appeared to have IgE directed to proteins of <17, 30-32 kD or with high molecular weights. Low MPDs and more severe symptoms tended to be more prominent in patients who did not recognize Cor a 1 on immunoblot, but only non-significant differences could be revealed among patients with different allergen recognition profiles. This study was financially supported by the International Life Sciences Institute.

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[52] - Comstock SS, McGranahan G, Peterson WR, Teuber SS. IgE Binding to and Cross-Reactivity Among English Walnut and Other Walnut (Juglans) Species. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°851

Rationale English walnuts are frequently associated with severe allergic reactions. We sought English walnut cultivars or other walnut species for which IgE binding to walnut seed storage proteins was decreased or the ability to inhibit IgE binding to English walnut extract was incomplete, thereby identifying possible germplasm sources of hypoallergenic walnut seed storage proteins. Method s : Sera from patients who have had life-threatening systemic reactions to English walnut were used in immunoblots to identify IgE-reactive proteins in the walnut species and cultivars. In addition, the same sera were used in immunoblot inhibition with several species to determine in vitro cross-reactivity of recombinant and native Jug r 1 and Jug r 2, two important walnut allergens. Result s : Patient IgE showed similar binding to all walnut extracts with some minor differences between the English and black walnut species. Inhibition with the various walnut species eliminated nearly all IgE binding to the proteins in both the English walnut Chandler cultivar and the Black Walnut Thomas cultivar. Absorption with all walnut species eliminated IgE binding to the recombinant Jug r 2 and decreased IgE binding to the recombinant Jug r 1. Conclusions : It is unlikely a hypoallergenic walnut could be bred from available germplasm. Furthermore, based on the extensive cross-reactivity seen in vitro, patients with severe English walnut allergy will likely react to all commercial English walnut cultivars and other closely-related walnut species.

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[53] - Sicherer SH, Furlong TJ, Muñoz-Furlong A, Burks AW, Sampson HA. A voluntary registry for peanut and tree nut allergy: Characteristics of the first 5149 registrants. J Allergy Clin Immunol 2001;108:128-132

Background: A voluntary registry of individuals with peanut and/or tree nut allergy was established in 1997 to learn more about these food allergies. OBJECTIVE: The purpose of this study was to elucidate a variety of features of peanut and tree nut allergy among the first 5149 registry participants. METHODS: The registry was established through use of a structured questionnaire distributed to all members of the Food Allergy and Anaphylaxis Network and to patients by allergists. Parental surrogates completed the forms for children under the age of 18 years. RESULTS: Registrants were primarily children (89% of registrants were younger than 18 years of age; the median age was 5 years), reflecting the membership of the Network. Isolated peanut allergy was reported by 3482 registrants (68%), isolated tree nut allergy by 464 (9%), and allergy to both foods by 1203 (23%). Registrants were more likely to have been born in October, November, or December (odds ratio, 1.2; 95% CI, 1.18-1.23; P < .0001). The median age of reaction to peanut was 14 months, and the median age of reaction to tree nuts was 36 months; these represented the first known exposure for 74% and 68% of registrants, respectively. One half of the reactions involved more than 1 organ system, and more than 75% required treatment, frequently from medical personnel. Registrants with asthma were more likely than those without asthma to have severe reactions (33% vs 21%; P < .0001). In comparison with initial reactions, subsequent reactions due to accidental ingestion were more severe, more common outside the home, and more likely to be treated with epinephrine. CONCLUSIONS: Allergic reactions to peanut and tree nut are frequently severe, often occur on the first known exposure, and can become more severe over time.

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[54] - Sicherer SH, Burks AW, Sampson HA. Clinical features of acute allergic reactions to peanut and tree nuts in children. Pediatrics 1998;102:131-132

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[55] - Codreanu F. L'activité du réseau Allergo-Vigilance: allergies alimentaires. Alim'Inter 2007;12:239-242

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[56] - Bannon GA, Ling M, Cockrell G, Sampson HA, Burks AW. Cloning, expression and characterization of two major allergens, Jug n 1 and Jug n 2, from the black walnut, Juglans niger. AAAAI 57th Annual Meeting, New Orleans, 16-21 March, 2001, Poster n°463

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[57] - Ling M, Ye J, Beyer K, Cockrell G, Bannon GA, Stanley JS, et al. Cloning, Identification, and Epitope-Mapping Two Black Walnut (Juglans Niger) Allergens. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°722

RATIONALE: 2D-proteomic mapping, immunoblotting and MS/MS analysis combined with sera of walnut allergic individuals were used to identify walnut proteins that bound IgE. METHODS: Utilizing amino acid sequence information from MS/MS analysis, specific degenerate primers were synthesized and PCR were used to amplify several gene fragments from a cDNA black walnut library. PCR products were ligated into the TA-vector and sequenced. The sequence of these fragments was used to design additional non-degenerated primers to obtain full length coding regions. RESULTS: A cDNA clone (749 bp), which codes a 234 amino acid protein revealed 83%, 82% and 81% protein sequence homology with rubber tree manganese superdoxide dismumtase (MnSOD), rice MnSOD and tobacco MnSOD, respectively. A second clone (1317 bp), which code a 338 amino acid protein, revealed 84% and 83% protein sequence homology with Pea and Tobacco glyceraldehydes (GAPDH), respectively. The coding sequences were put into pET 24a vector with His-tail and recombinant proteins expressed as single proteins. Using black walnut positive sera and SDS-PAGE/immunoblotting, 5/16 sera bound to SOD and 4/16 to GAPDH. Epitope mappings of these two allergens performed by the SPOTS techniques revealed four IgE-binding epitopes in SOD and four in GAPDH respectively. CONCLUSIONS: These results indicate that IgE from walnut allergic patients recognize walnut SOD and GAPDH in addition of Jug n1 and Jug n2.

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[58] - Teuber SS, Dandekar AM, Peterson WR, Sellers CL. Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia), a major food allergen. J Allergy Clin Immunol 1998;101:807-814

Walnuts rank third in per capita consumption of tree nuts in the United States and can be associated with systemic IgE-mediated reactions in some individuals. OBJECTIVE: The objectives of the study were to clone a gene encoding one of the major food allergens in the walnut kernel and to characterize the recombinant allergen. METHODS: A cDNA expression library in the lambda vector Uni-ZAP, which was prepared from walnut somatic embryos, was screened by using a patient's sera that reacted with multiple protein bands on immunoblotting. RESULTS: A cDNA clone containing an insert of 663 bp was identified and named Jug r 1. DNA sequence analysis of this clone revealed that it encoded a protein 142 amino acids in length. Comparison of the encoded protein sequences with protein databases revealed that this clone exhibits a 46.1% identity with the Brazil nut (Bertholletia excelsa) methionine-rich 2S albumin seed storage protein precursor, Ber e 1. Jug r 1 appears to be an important walnut food allergen; 12 of 16 sera from patients allergic to walnuts demonstrated IgE binding to the 2S albumin seed storage protein precursor fusion protein. An IgE-binding inhibition study suggests that the walnut 2S protein precursor undergoes posttranslational modification into a large and small subunit that is similar to castor seed, cottonseed, mustard seed, and Brazil nut 2S seed storage protein allergens. Interestingly, the gene encoding this allergenic protein in Brazil nuts has recently gained notoriety because of its experimental use as a transgene to enhance the nutritional quality of legumes. CONCLUSION: This is now the sixth definitive 2S albumin seed storage protein demonstrated to bind IgE, suggesting that this class of proteins is inherently allergenic.

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[59] - Teuber SS, Jarvis KC, Dandekar AM, Peterson WR, Ansari AA. Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, Jug r 2, from english walnut kernel (Juglans regia), a major food allergen. J Allergy Clin Immunol 1999;104:1311-1320

Walnuts and other tree nuts are important food-allergen sources that have the potential to be associated with life-threatening, IgE-mediated systemic reactions in some individuals. OBJECTIVE: The purpose of this study was to characterize a complementary (c)DNA clone encoding one of the walnut food allergens. METHODS: A cDNA expression library prepared from walnut somatic embryo was screened for IgE reactivity with patient serum. A reactive clone of 2060 bp, which encoded a protein of 593 amino acids in length, was subcloned by excision into the pGEX expression vector. IgE-binding inhibition experiments were performed. RESULTS: A recombinant fusion protein was induced and shown to bind serum IgE from 9 of 15 patients tested, thus identifying a major allergen. This clone, named Jug r 2, exhibited significant homology with genes encoding the vicilin group of seed proteins. An IgE-binding inhibition experiment suggested that the encoded protein undergoes posttranslational modification into at least one major polypeptide (47 kd) and possibly several others, which is similar to the vicilin-like proteins characterized in cocoa bean (Theobroma cacao) and cottonseed (Gossypium hirsutum). N-terminal sequencing of the 47-kd band, Jug r 2, identified it as a mature protein obtained from the precursor. A second IgE-binding inhibition experiment showed that there is minimal or no cross-reactivity between Jug r 2 and pea vicilin, peanut proteins, or cacao proteins. CONCLUSION: Jug r 2 is the third vicilin food allergen identified in addition to vicilins from soy and peanut. The availability of recombinant food allergens should help advance studies on the immunopathogenesis and possible treatment of IgE-mediated food hypersensitivity.

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[60] - Teuber SS, Comstock SS, Sathe SK, Roux KH. Tree nut allergy. Curr Allergy Asthma Rep 2003;3:54-61

Tree nuts are clinically associated with severe immunoglobulin E-mediated systemic allergic reactions independent of pollen allergy and with reactions that are usually confined to the oral mucosa in patients with immunoglobulin E directed toward cross-reacting pollen allergens. The latter reactions can progress to severe and life-threatening episodes in some patients. Many patients with severe tree nut allergy are co-sensitized to peanut. Clinical studies on cross-reactivity between the tree nuts are few in number, but based on reports to date, avoidance of the other tree nuts once sensitivity is diagnosed appears prudent unless specific challenges are performed to ensure clinical tolerance. Even then, great care must be taken to avoid cross-contamination. As with other severe food allergies, a recurrent problem in clinical management is the failure of physicians to prescribe self-injectable epinephrine to patients who are at risk of anaphylaxis.

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[61] - Teuber SS, Peterson WR, Uratsu S, Dandekar A, Roux KH, Sathe SK. Identification and Cloning of Jug r 4, a Major Food Allergen from English Walnut Belonging to the Legumin Group. AAAAI 59th Annual Meeting, Denver, 7-12 March, 2003, Poster n°720

Food allergens in English walnut (Juglans regia) kernels are still incompletely characterized. We have found that sera from a majority of patients with a history of life-threatening systemic allergic reactions to walnut show IgE binding to legumin group seed storage proteins. We sought to clone one of the genes encoding a legumin group protein, express the fusion protein, and screen with sera from food-allergic patients with a history of severe, life-threatening reactions to walnut. Degenerate probes were used to obtain the cDNA by 5´- and 3´-RACE-PCR from walnut mRNA. The cDNA was subcloned into the pMAL vector and the recombinant fusion protein expressed in E. coli. The 1.5 kB insert encodes a polypeptide precursor protein with a predicted MW of approximately 56 kD. Excellent expression of the fusion protein was only obtained after deleting 96 bases of the 5´ end, which corresponds to removing 32 amino acids from the hydrophobic N-terminus, and subcloning and expressing this fragment. 15 of 23 (65%) sera from patients with life-threatening reactions to walnut showed igE binding by immunoblot to the fusion protein. In conclusion, a gene encoding a walnut legumin precursor has been identified and validated as a major food allergen via analysis of the fusion protein and has tentatively been named Jug r 4.

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[62] - Wallowitz M, Peterson WR, Uratsu S, Comstock SS, Dandekar AM, Teuber SS. Jug r 4, a Legumin Group Food Allergen from Walnut (Juglans regia Cv. Chandler). J Agric Food Chem 2006;54:8369-8375

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.

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[63] - Wallowitz ML, Teuber S, Beyer K, Sampson HA, Roux KH, Sathe SK, et al. Cross-Reactivity of Walnut, Cashew, and Hazelnut Legumin Proteins in Tree Nut Allergic Patients. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°524

Rationale Walnut, cashew, and hazelnut contain similar legumin proteins identified as allergens. The aim of this study was to determine if cross-reactivity of IgE specific for the legumin proteins from walnut (Jug r 4), cashew (Ana o 2), and hazelnut (Cor a 9) in sera of tree nut allergic patients could be responsible for multiple tree nut allergies. Method s : IgE immunoblotting was performed using sera from three adult subjects with a history of systemic allergic reactions to walnut, cashew and hazelnut to assess IgE reactivity to both recombinant legumin proteins and tree nut extracts. Inhibition blots were conducted to assess the presence ofIgE cross-reactivity between the recombinants and extracts. Result s : Immunoblot results showed IgE reactivity to both the recombinant legumins and extracts. Inhibition blots revealed pre-incubation of sera with walnut, cashew, or hazelnut extracts resulted in a significant decrease in IgE binding to the recombinant proteins, indicating allergenic cross-reactivity. Pre-incubation of sera with the recombinant legumin proteins was not as effective as the extracts in absorbing out IgE in sera, which completely abolished IgE binding to Ana o 2 and Jug r 4 recombinants in crossed inhibitions for two of the three sera used. Conclusions : These results support the hypothesis that multiple tree nut sensitivities observed in allergic subjects may be due to cross-reactive IgE epitopes present in homologous tree nut allergens.

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[64] - Wallowitz M, Peterson WR, Uratsu S, Comstock SS, Dandekar AM, Teuber SS. Jug r 4, a Legumin Group Food Allergen from Walnut (Juglans regia Cv. Chandler). J Agric Food Chem 2006;54:8369-8375

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.

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[65] - Wallowitz ML, Chen RJ, Tzen JT, Teuber SS. Ses i 6, the sesame 11S globulin, can activate basophils and shows cross-reactivity with walnut in vitro. Clin Exp Allergy 2007;37:929-938

Background Sesame allergy is increasingly being reported, and multi-sensitization to peanut and tree nuts has been described. The clinical relevance and cross-reactivity of many sesame proteins, such as Ses i 6, are unknown. Objective The aims of this study were to perform a preliminary examination of the cross-reactivity of Ses i 6 in vitro, examine the ability of Ses i 6 to activate basophils in a modified basophil activation test (mBAT), and assess whether such an assay may help to distinguish between potentially relevant and irrelevant IgE reactivity towards 11S globulin proteins. Methods Inhibition immunoblotting and chicken anti-rJug r 4 antibodies were used to determine the cross-reactivity of rSes i 6. Basophils from atopic donors were stripped of resident IgE before passive sensitization with food-allergic sera and challenged with protein extracts or recombinant protein. Basophil activation was measured using two activation markers, CD203c and CD63, via flow cytometry. Results IgE immunoblotting showed cross-reactivity between rJug r 4 and rSes i 6 using sera from two human donors and chicken IgY. Additionally, rSes i 6 activated basophils passively sensitized with sesame-allergic sera. Cross-reactive serum from a sesame-allergic but walnut-tolerant donor was not able to activate basophils when challenged by walnut extract despite IgE reactivity to walnut determined by immunoblotting. Conclusions The sesame 11S globulin shows partial immunological cross-reactivity with walnut, and although it is classified as a minor allergen, activated basophils sensitized with serum from seven out of eleven sesame-allergic donors. Additionally, the mBAT may help distinguish between clinically relevant and irrelevant in vitro IgE cross-reactivity of seed storage proteins in nuts and seeds and thus warrants use in further studies.

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[66] - Wallowitz M, Peterson WR, Uratsu S, Comstock SS, Dandekar AM, Teuber SS. Jug r 4, a Legumin Group Food Allergen from Walnut (Juglans regia Cv. Chandler). J Agric Food Chem 2006;54:8369-8375

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.

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[67] - Leshchinsky TV, Teuber SS, Sathe SK, Roux KH, Sze-Tao K. The Glutelin Fraction of Walnut Kernels (Juglans Regia) Contains Unique Allergens Not Visible in the Salt Soluble Fraction. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°930

Most studies characterizing allergens from nuts and seeds have previously focused on identification and cloning of allergens detected in water- or salt-soluble fractions. Walnut, one of the tree nuts most frequently associated with food-induced allergic reactions, is unusual in that the majority of proteins are poorly soluble and are glutelins as classically defined by solubility. We sought to determine if the glutelin fraction contained allergens that were not previously detected in water or salt extractions. Walnuts were defatted with acetone and the dried flour was then extracted sequentially with 1.0 M NaCl (albumin + globulin fraction), then 70% ethanol (prolamin fraction) and finally, 0.1 M NaOH (glutelin fraction). The fractions were then dialyzed against water and lyophilized. IgE immunoblotting was performed against the fractions using sera from 25 adult patients with life-threatening systemic reactions to walnuts, 16 adult patients with allergic rhinitis and self-reported mild walnut food reactions involving itching, hives or mild angioedema confined to the mouth and face, and 16 patients with pollinosis, peanut allergy or latex allergy who were not tree nut allergic. Twenty of 25 sera (80%) from severely allergic subjects showed IgE binding to a doublet at approximately12 kDa present in the non-reduced glutelin sample that was unaffected by reduction of the sample with dithiothreitol. No sera from patients with mild walnut allergy and only one sera from a patient with pollinosis and latex allergy had IgE binding to this doublet. This doublet was not represented in the albumin, globulin or prolamin fractions. In conclusion, we have detected a major food allergen in walnut kernel that was previously unknown and may not be represented in commercial walnut extracts available for skin testing or used for in vitro specific IgE assays, unless there are similar proteins (of different molecular weight) soluble in salt solutions. Such possibilities are being explored.

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[68] - Pastorello EA, Farioli L, Pravettoni V, Robino AM, Scibilia J, Fortunato D, et al. Lipid transfer protein and vicilin are important walnut allergens in patients not allergic to pollen. J Allergy Clin Immunol 2004;114:908-914

Background Walnut is the most common cause of allergic reactions to tree nuts, as reported by large population studies. Two major allergens of walnut have been identified up until now: a 2S albumin and a vicilin-like protein. Objective : This study was designed to identify the walnut major allergens in the Italian population and to compare the walnut IgE-binding profile in patients with or without pollen allergy. Method s : We selected 46 patients either with oral allergy syndrome confirmed by open oral challenge or with systemic symptoms after ingestion of walnut. These patients' sera were used for the immunoblotting of walnut extract; the identified allergens were purified by HPLC and sequenced. A peach-walnut cross-inhibition study was then performed. Result s : The only major allergen recognized by our study population was a 9-kd lipid transfer protein (LTP), recognized by 37 patients. Two other minor allergens of approximately 9-kd molecular weight, both belonging to the vicilin family, were recognized by 10 patients. IgE binding to walnut LTP was completely inhibited by peach LTP. Conclusion : In Italian patients with walnut allergy confirmed by documented history of severe systemic reactions or by open oral food challenge, the major allergen is an LTP. The sensitization to this protein seems to be secondary to the sensitization to peach LTP, which acts as the primary sensitizer. LTP and vicilins were able to sensitize patients not allergic to pollen.

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[69] - Rancé F, Bidat E, Bourrier T, Sabouraud D. Cashew allergy: observations of 42 children without associated peanut allergy. Allergy 2003;58:1311-1314

BACKGROUND: Cashew allergy seems to be increasingly frequent. The goal of the present study was to analyse the clinical features and results of investigations of 42 children with cashew allergy . METHODS: The clinical features and results of skin prick tests, specific IgE assays, and food challenges were analysed . RESULTS: The mean age at first allergic reaction was 2 years and the mean age at diagnosis of cashew allergy was 2.7 years. One in five children (12%) had a prior history of exposure to cashew nuts. Fifty-six per cent had skin symptoms, 25% had respiratory signs and 17% had digestive signs. Eighteen children had proven, associated food allergies (pistachio, seven; egg, five; mustard, three; shrimp, two; cow milk, one). The mean wheal diameter of the skin prick tests was 7 mm (3-16 mm) and the mean specific IgE level was 3.1 kUA/L (<0.35->100 kUA/L). Eight children had positive food challenges . CONCLUSION: The increase in cashew allergy is worrying because it affects young children who may have a reaction without ever having been exposed to cashews. Almost one-third of children are allergic to pistachios, which belong to the same botanical family as cashews. Clinical history is generally and sufficiently suggestive to diagnose cashew allergy without recourse to food challenges.

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[70] - Giovannini-Chami L, Bourrier T, Berthier F, Vivinus M, Albertini M. Une immunoréactivité croisée probable entre noix de cajou et arachide. Rev Fr Allergol Immunol Clin 2007;47:283

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[71] - Roux KH, Teuber SS, Sathe SK. Tree Nut Allergens. Int Arch Allergy Immunol 2003;131:234-244

Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.

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[72] - Hourihane JO, Harris H, Langton-Hewer S, Kilburn SA, Warner JO. Clinical features of cashew allergy. Allergy 2001;56:252-253

This study reports on the findings of cashew allergy in 29 patients. 48% reacted to minimal contact with cashew, i.e., smelling, touching, or tasting, but not eating cashew. The authors point out that severe reactions are as severe as those from peanut allergy.

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[73] - Wang F, Robotham JM, Teuber S, Sathe SK, Roux KH. Ana o 2, a Major Cashew (Anacardium occidentale L.) Nut Allergen of the Legumin Family. Int Arch Allergy Immunol 2003;132:27-39

BACKGROUND: We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen . METHODS: A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology . RESULTS: Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes . CONCLUSIONS: We conclude that this legumin-like protein is a major allergen in cashew nut.

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[74] - Rancé F, Bidat E, Bourrier T, Sabouraud D. Cashew allergy: observations of 42 children without associated peanut allergy. Allergy 2003;58:1311-1314

BACKGROUND: Cashew allergy seems to be increasingly frequent. The goal of the present study was to analyse the clinical features and results of investigations of 42 children with cashew allergy . METHODS: The clinical features and results of skin prick tests, specific IgE assays, and food challenges were analysed . RESULTS: The mean age at first allergic reaction was 2 years and the mean age at diagnosis of cashew allergy was 2.7 years. One in five children (12%) had a prior history of exposure to cashew nuts. Fifty-six per cent had skin symptoms, 25% had respiratory signs and 17% had digestive signs. Eighteen children had proven, associated food allergies (pistachio, seven; egg, five; mustard, three; shrimp, two; cow milk, one). The mean wheal diameter of the skin prick tests was 7 mm (3-16 mm) and the mean specific IgE level was 3.1 kUA/L (<0.35->100 kUA/L). Eight children had positive food challenges . CONCLUSION: The increase in cashew allergy is worrying because it affects young children who may have a reaction without ever having been exposed to cashews. Almost one-third of children are allergic to pistachios, which belong to the same botanical family as cashews. Clinical history is generally and sufficiently suggestive to diagnose cashew allergy without recourse to food challenges.

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[75] - Robotham JM, Wang F, Seamon V, Teuber SS, Sathe SK, Sampson HA, et al. Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family. J Allergy Clin Immunol 2005;115:1284-1290

BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins . OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin . METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes . RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology . CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.

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[76] - Giovannini-Chami L, Bourrier T, Berthier F, Vivinus M, Albertini M. Une immunoréactivité croisée probable entre noix de cajou et arachide. Rev Fr Allergol Immunol Clin 2007;47:283

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[77] - Wang F, Robotham JM, Teuber SS, Tawde P, Sathe SK, Roux KH. Ana o 1, a cashew (Anacardium occidental) allergen of the vicilin seed storage protein family. J Allergy Clin Immunol 2002;110:160-166

BACKGROUND: The allergens responsible for cashew food allergy have not been well characterized . OBJECTIVES: We initiated a study to clone cDNAs encoding cashew food allergens . METHODS: A cashew cDNA library was screened with human serum for IgE-reactive clones and rabbit IgG anti-cashew extract antisera. Reactive clones were sequenced and expressed, and linear epitopes were identified by means of solid-phase overlapping peptide analysis. Immunoblot inhibition was used to identify the native peptide in cashew extract . RESULTS: Four closely related clones reactive with both human and rabbit antisera were sequenced. Sequence analysis showed that these encode members of the vicilin/sucrose-binding protein family of plant seed storage proteins. Screening of the recombinant protein with sera from 20 patients with cashew allergy and 8 cashew-tolerant patients with allergies to other tree nuts showed that 50% and 25% of sera from patients with cashew allergy and cashew-tolerant subjects, respectively, bound the recombinant protein. The corresponding native allergen protein, designated Ana o 1, was located at approximately 50 kd. Epitope mapping revealed 11 linear IgE-binding epitopes, of which 3 appear to be immunodominant. None of the epitopes were shared in common with those of the peanut vicilin allergen Ara h 1 . CONCLUSION: Ana o 1, a vicilin-like protein, is a major food allergen in cashews. Cashew and peanut vicilins do not share linear epitopes.

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[78] - Wang F, Robotham JM, Teuber S, Sathe SK, Roux KH. Ana o 2, a Major Cashew (Anacardium occidentale L.) Nut Allergen of the Legumin Family. Int Arch Allergy Immunol 2003;132:27-39

BACKGROUND: We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen . METHODS: A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology . RESULTS: Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes . CONCLUSIONS: We conclude that this legumin-like protein is a major allergen in cashew nut.

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[79] - Garcia F, Moneo I, Fernandez B, Garcia-Menaya JM, Blanco J, Juste S, et al. Allergy to Anacardiaceae: description of cashew and pistachio nut allergens. J Investig Allergol Clin Immunol 2000;10:173-177

Some members of the Anacardiaceae family, such as cashew nut, pistachio nut and mango, have been reported to cause immediate allergic reactions. We report three cases of anaphylaxis to cashew nuts. With the aim of describing the allergens existing in cashew and pistachio nuts, patients were prick tested with cashew and pistachio extracts. Specific IgE against both nuts was studied by CAP and SDS-PAGE/immunoblotting. It was found that skin tests and specific IgE to cashew and pistachio nuts were positive in the three patients. Both nuts showed several protein bands in SDS-PAGE. The strongest IgE-binding bands had similar molecular weights (15, 30 and 60 kDa) in cashew and pistachio nuts. These main bands were found to be sensitive to reducing agents. It was concluded that these three patients suffered immediate reactions to cashew nut due to an IgE-mediated mechanism.

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[80] - Teuber SS, Roux KH, Sathe SK, Robotham JM, Wang F. Cashew nut allergenicity and study of cross-reactivity. Alim'Inter 2003;8:51-53

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[81] - Wang F, Robotham JM, Teuber S, Sathe SK, Roux KH. Ana o 2, a Major Cashew (Anacardium occidentale L.) Nut Allergen of the Legumin Family. Int Arch Allergy Immunol 2003;132:27-39

BACKGROUND: We recently cloned and described a vicilin and showed it to be a major cashew allergen. Additional IgE-reactive cashew peptides of the legumin group and 2S albumin families have also been reported. Here, we attempt to clone, express and characterize a second major cashew allergen . METHODS: A cashew cDNA library was screened with human IgE and rabbit IgG anti-cashew extract antisera, and a reactive nonvicilin clone was sequenced and expressed as a fusion protein in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition of immunoblotting was used to identify the corresponding native peptides in cashew nut extract. The identified allergen was subjected to linear epitope mapping using SPOTs solid-phase synthetic peptide technology . RESULTS: Sequence analysis showed the selected clone, designated Ana o 2, to encode for a member of the legumin family (an 11S globulin) of seed storage proteins. By IgE immunoblotting, 13 of 21 sera (62%) from cashew-allergic patients were reactive. Immunoblot inhibition data showed that the native Ana o 2 constitutes a major band at approximately 33 kD and a minor band at approximately 53 kD. Probing of overlapping synthetic peptides with pooled human cashew-allergic sera identified 22 reactive peptides, 7 of which gave strong signals. Several Ana o 2 epitopes were shown to overlap those of the peanut legumin group allergen, Ara h 3, in position but with little sequence similarity. Greater positional overlap and identity was observed between Ana o 2 and soybean glycinin epitopes . CONCLUSIONS: We conclude that this legumin-like protein is a major allergen in cashew nut.

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[82] - Teuber SS, Sathe SK, Peterson WR, Roux KH. Characterization of the Soluble Allergenic Proteins of Cashew Nut (Anacardium occidentale L.). J Agric Food Chem 2002;50:6543-6549

The allergens associated with cashew food allergy have not been well-characterized. We sought to identify the major allergens in cashew nut by performing IgE immunoblots to dissociated and reduced or nonreduced cashew protein extracts, followed by sequencing of the peptides of interest. Sera from 15 subjects with life-threatening reactions to cashews and 8 subjects who tolerate cashews but have life-threatening reactions to other tree nuts were compared. An aqueous cashew protein extract containing albumin/globulin was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to IgE immunoblotting using patient sera. Selected IgE reactive bands were subjected to N-terminal amino acid sequencing. Each of the 15 sera from cashew-allergic subjects showed IgE binding to the cashew protein extract. The dominant IgE-binding antigens in the reduced preparations included peptides in the 31-35 kD range, consistent with the large subunits of the major storage 13S globulin (legumin-like protein). Low-molecular-weight polypeptides of the 2S albumin family, with similarity to the major walnut allergen Jug r 1, also bound IgE. The sera from eight patients who tolerate cashew but displayed allergies to other tree nuts showed only minimal or no IgE binding to cashew. Cashew food allergy is associated with the presence of IgE directed against the major seed storage proteins in cashew, including the 13S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Thus, the legumin-group proteins and 2S albumins are again identified as major food allergens, which will help further research into seed protein allergenicity.

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[83] - Robotham JM, Wang F, Vest VM, Teuber SS, Sathe SK, Beyer K, et al. Ana o 3, an Important Cashew Nut Allergen of the 2S Albumin Family. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°840

Rationale Cashew nut allergy is the second most commonly reported tree nut allergy in the US. Method s : We have identified a third recombinant cashew allergen from a cashew cDNA library by PCR with degenerate primers. This allergen was subjected to linear epitope mapping using the SPOTs synthetic peptide technology. Result s : Immunoblotting, using the recombinant protein, designated Ana o 3, was used to screen for reactivity with cashew-allergic patient sera (17 of 25, 62%, were reactive) and inhibition of immunoblotting was used to identify the native protein in cashew extract. The sequence of Ana o 3 shows it encodes a member of the 2S albumin protein family. Reactive epitopes were identified by probing SPOTs membranes displaying the sequence of Ana o 3 in the form of 36, 12-aa peptides, each offset by 3 residues. Probing with pooled human cashew-allergic sera identified 16 reactive peptides, 4 of which gave strong signals. One strongly reactive Ana o 3 epitope overlapped in aa position with the lone linear epitope of the English walnut allergenic 2S albumin, Jug r 1, and the epitopes shared considerable sequence homology (81% similarity). Significantly, all 5 amino acids found to be critical or influential for IgE binding to Jug r 1 were conserved in Ana o 3 (100% similarity), perhaps explaining why patients allergic to cashew are frequently also allergic to walnut in our patient population. Conclusions : Cashew 2S albumin is a significant allergen with sequence similarity to other 2S allergens, including shared linear epitope homology with English walnut 2S albumin.

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[84] - Robotham JM, Wang F, Seamon V, Teuber SS, Sathe SK, Sampson HA, et al. Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family. J Allergy Clin Immunol 2005;115:1284-1290

BACKGROUND: Cashew nut allergy is the second most commonly reported tree nut allergy in the United States. We have previously cloned and characterized major cashew allergens belonging to the vicilin and legumin families of seed storage proteins . OBJECTIVE: Here we set out to describe a third major cashew allergen, a 2S albumin . METHODS: The recombinant cashew 2S albumin was amplified from a cDNA library by means of PCR, sequenced, and expressed in Escherichia coli. Immunoblotting was used to screen for reactivity with patients' sera, and inhibition immunoblotting was used to identify the corresponding native cashew nut proteins. The mass of affinity-purified native allergen was determined by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectroscopy. Patients' sera were used to probe solid-phase 2S albumin peptides to identify linear epitopes . RESULTS: The cloned allergen, designated Ana o 3, was identified as 2S albumin. MALDI-TOF mass spectroscopy of native Ana o 3 yielded a molecular mass of 12,598 d. Immunoblot analysis showed 21 (81%) of 26 sera from patients with cashew allergy were reactive. Three native Ana o 3 large-subunit isoforms with molecular weights ranging from approximately 6 to 10 kd were identified. Probing of overlapping synthetic Ana o 3 peptides with patients' sera identified 16 reactive peptides, 4 of which gave strong signals and one of which positionally overlaps linear epitopes in mustard and walnut allergenic 2S albumins. The overlapping cashew and walnut epitopes also share considerable homology . CONCLUSIONS: We conclude that this 2S albumin protein is a major allergen in cashew nut and demonstrates a possible basis for cross-reactivity with walnut 2S albumin.

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[85] - Rasanen L, Makinen-Kiljunen S, Harvima RJ. Pectin and cashew nut allergy: cross-reacting allergens ? Allergy 1998;53:626-628

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[86] - Ferdman RM, Ong PY, Church JA. Pectin anaphylaxis and possible association with cashew allergy. Ann Allergy Asthma Immunol 2006;97:759-760

BACKGROUND: Inhalation of pectin has been identified as a cause of occupational asthma. However, allergic reactions to orally ingested pectin have not been reported. OBJECTIVES: To describe a child with pectin-induced food anaphylaxis and to discuss its possible relationship to cashew allergy. METHODS: A 3 1/2-year-old boy developed anaphylaxis once after eating cashews and later after eating a pectin-containing fruit "smoothie." He also has a history of generalized pruritus after eating grapefruit. Skin tests or radioallergosorbent tests (RASTs) were performed to pectin and other suspected food allergens. RESULTS: The child had a positive skin prick test reaction to pectin and a high RAST reaction to cashew and pistachio. He had a low-level positive RAST reaction to grapefruit. Results of allergy tests for the other potential food allergens were negative. The pectin in the smoothie was confirmed to be of citrus origin. Review of previous case reports of pectin-induced occupational asthma revealed several patients with allergies to and cross-reactivity with cashew. CONCLUSIONS: Ingestion, not only inhalation, of pectin can cause hypersensitivity reactions. Cashew, and possibly pistachio, allergy may be associated with pectin allergy, and the possibility of pectin allergy should be considered in cashew- or pistachio-allergic patients who have unexplained allergic reactions.

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[87] - Bidat E, Baranes T, Feuillet-Dassonval C, Rossignol B. De l'importance de lire attentivement les étiquettes avant de consommer… des spaghettis. Rev Fr Allergol Immunol Clin 2006;46:413-415

Trois enfants allergiques à l'arachide, connaissant et gérant bien leur allergie, ont présenté des réactions allergiques immédiates après avoir consommé des pâtes alimentaires accompagnées de sauce tomate. Ces observations permettent de discuter l'attitude vis-à-vis des fruits à coque chez l'allergique à l'arachide, les réglementations européennes actuelles concernant l'étiquetage, et de rappeler l'absolue nécessité d'une lecture des étiquettes de tout produit consommé par un sujet présentant une allergie alimentaire, même s'il s'agit d'un produit composé d'ingrédients auxquels le sujet n'est pas a priori allergique.

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[88] - Moneret-Vautrin DA, Lemerdy P. Allergie alimentaire et sensibilisation à la noix de coco. Alim'Inter 2004;9:131-133

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[89] - Codreanu F. L'activité du réseau Allergo-Vigilance: allergies alimentaires. Alim'Inter 2007;12:239-242

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[90] - Moneret-Vautrin DA, Lemerdy P. Allergie alimentaire et sensibilisation à la noix de coco. Alim'Inter 2004;9:131-133

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[91] - Martin E, Tornero P, De Barrio M, Pérez CI, Beitia JM, Baeza ML. Monosensitization to Coconut. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°1160

Rationale Allergy to coconut (Coccus Nucifera, Arecaceae's family) it is a very uncommon entity. A few published reports sign out immunologic cross-reactivity between coconut and nuts, soybean and legumes. Method s : A 64 year-old woman, with hypertension and autoimmune thyroiditis, referred a few minutes after eating a coconut biscuit, a generalized urticaria, facial and uvula edema, dysphagia and dyspnea. A similar second episode was followed by bronchoconstriction, hypotension (100/60) and hypoxemia (PaO2:58) minutes after eating a Spanish sweet containing coconut, almond, sugar, milk, cocoa and yolk. It subsided in a few hours after symptomatic therapy in the emergency room. Next, she tolerated almond, milk, egg, sugar, cocoa, date, other nuts and legumes. Result s : Skin prick tests (SPT) were strongly positive to coconut pulp (19◊10 mm) and coconut milk (13◊10 mm), with a late skin reaction 4-6 hours later (edema and erithema). SPT were negative to palmate, date, palm pollen, peanut, almond, pistachio nut, walnut, hazelnut, sweet chestnut, soybean, cocoa, chickpea, lentil, mustard and latex. Specific Ig E (CAP Pharmacia˙) to coconut measured 20,8 KU/L and was negative to nuts, legumes and soybean. Multiple specific Ig E reacting bands to coconut milk and coconut pulp as well as to dry commercial coconut powder were revealed by immunoblotting. The most prominent had an apparent molecular weight of 18, 22 and 28 kD. Conclusions : We report an IgE-mediated anaphylaxis to coconut, demonstrated by SPT, specific Ig E and immunoblotting. No clinical or immunological cross-reactivity was found with other related fruits. The main allergens measured 18, 22, and 28 kD.

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[92] - Nguyen SA, More DR, Whisman BA, Hagan LL. Cross-reactivity between coconut and hazelnut proteins in a patient with coconut anaphylaxis. Ann Allergy Asthma Immunol 2004;92:281-284

BACKGROUND: The medical literature reports few cases of severe allergic reactions to coconut. We encountered a patient with anaphylaxis to coconut and oral symptoms to tree nuts . OBJECTIVE: To identify cross-reactive antibodies between coconut and other tree nuts . METHODS: We performed commercial radioallergosorbent tests to coconut and various tree nuts using the patient's serum. Skin prick testing was performed to fresh coconut and commercial extracts of coconut, almond, Brazil nut, cashew, pecan, walnut, and hazelnut. Proteins from fresh coconut, commercial coconut extract, and tree nuts were extracted. Immunoblot and inhibition assays were performed to evaluate for cross-reacting IgE antibodies between similar-sized allergens in coconut and hazelnut . RESULTS: Positive skin test results occurred to the coconut and multiple tree nut extracts. In vitro serum specific IgE was present for coconut, hazelnut, Brazil nut, and cashew. Immunoblots demonstrated IgE binding to 35- and 50-kDa protein bands in the coconut and hazelnut extracts. Inhibition assays using coconut demonstrated complete inhibition of hazelnut specific IgE, but inhibition assays using hazelnut showed only partial inhibition of coconut specific IgE . CONCLUSIONS: Our study demonstrates the presence of cross-reactive allergens between hazelnut (a tree nut) and coconut (a distantly related palm family member). Because there are many potentially cross-reactive allergens among the tree nuts, we recommend patients with coconut hypersensitivity be investigated for further tree nut allergies.

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[93] - Kidon MI, Lim S, Loo A, Wee A, Ling S, Liew W, et al. Coconut Allergy in Two Children of Asian Origins and Identification of the IgE Binding Components. J Allergy Clin Immunol 2005;115(2 suppl.):S3

RATIONALE: To describe two children who developed primary coconut (Cocus nucifera) hypersensitivity and characterize IgE binding components METHODS: Two children with systemic allergic reactions to coconut (fresh and cooked) without previous tree nut exposure were subjected to prick-prick test with fresh coconut endosperm and routine panel (food and aero-allergens). Serum from each patient was tested for specific IgE Western blotting and mass spectrometric analysis were performed to identify IgE binding components RESULTS: Patient 1 tested positive for egg white, Dematophagoides mix and Blomia tropicalis. Patient 2 tested negative to all allergen extracts With the fresh endosperm, Patient 1 exhibited a huge reaction, with the wheal measuring 24.5 mm in excess of negative. Patient 2 demonstrated a wheal measuring 20mm and pseudopods were observed extending from forearm to axilla. IgE from sera of both patients were found to bind the endosperm and coconut milk (santan) but not the coconut water. Western blotting (patient 2) showed at least 8 IgE binding components with molecular weights ranging from 15kD to above 80kD (major band at 25kD) Sera from patient 1 bind only to three components between 20-25kD Mass spectrometric analysis of the trypsin-digested 25kD major band suggests it is a glutelin-like allergen CONCLUSIONS: We report 2 Asian patients with primary coconut allergy, with no previous sensitization to other tree nuts. Specific anti-IgE to eight new coconut antigens was also detected

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[94] - Dutau G, Dutau H, Rancé F, Juchet A, Rittié JL, Brémont F, et al. Allergie IgE-dépendante à la noix de coco: à propos d'un cas avec syndrome oral et manifestations cutanées. Sem Hop Paris 1998;74:650-652

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[95] - Martin E, Tornero P, De Barrio M, Pérez CI, Beitia JM, Baeza ML. Monosensitization to Coconut. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°1160

Rationale Allergy to coconut (Coccus Nucifera, Arecaceae's family) it is a very uncommon entity. A few published reports sign out immunologic cross-reactivity between coconut and nuts, soybean and legumes. Method s : A 64 year-old woman, with hypertension and autoimmune thyroiditis, referred a few minutes after eating a coconut biscuit, a generalized urticaria, facial and uvula edema, dysphagia and dyspnea. A similar second episode was followed by bronchoconstriction, hypotension (100/60) and hypoxemia (PaO2:58) minutes after eating a Spanish sweet containing coconut, almond, sugar, milk, cocoa and yolk. It subsided in a few hours after symptomatic therapy in the emergency room. Next, she tolerated almond, milk, egg, sugar, cocoa, date, other nuts and legumes. Result s : Skin prick tests (SPT) were strongly positive to coconut pulp (19◊10 mm) and coconut milk (13◊10 mm), with a late skin reaction 4-6 hours later (edema and erithema). SPT were negative to palmate, date, palm pollen, peanut, almond, pistachio nut, walnut, hazelnut, sweet chestnut, soybean, cocoa, chickpea, lentil, mustard and latex. Specific Ig E (CAP Pharmacia˙) to coconut measured 20,8 KU/L and was negative to nuts, legumes and soybean. Multiple specific Ig E reacting bands to coconut milk and coconut pulp as well as to dry commercial coconut powder were revealed by immunoblotting. The most prominent had an apparent molecular weight of 18, 22 and 28 kD. Conclusions : We report an IgE-mediated anaphylaxis to coconut, demonstrated by SPT, specific Ig E and immunoblotting. No clinical or immunological cross-reactivity was found with other related fruits. The main allergens measured 18, 22, and 28 kD.

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[96] - Tella R, Gaig P, Lombardero M, Paniagua MJ, Garcia-Ortega P, Richart C. A case of coconut allergy. Allergy 2003;58:825-826

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[97] - Teuber SS, Peterson WR. Systemic allergic reaction to coconut (Cocos nucifera) in 2 subjects with hypersensitivity to tree nut and demonstration of cross-reactivity to legumin-seed storage proteins. J Allergy Clin Immunol 1999;103:1180-1185

BACKGROUND: Two patients with tree nut allergy manifested by life-threatening systemic reactions reported the subsequent onset of systemic reactions after the consumption of coconut. OBJECTIVE: Herein, the IgE-binding proteins from coconut are described, and in vitro cross-reactivity with other nuts is investigated. METHODS: The IgE-binding profile of coconut endosperm tissue extract was analyzed by SDS-PAGE followed by immunoblotting. Immunoblot inhibition studies with walnut, almond, peanut, and coconut were performed. RESULTS: Sera IgE from both patients recognized reduced coconut allergens with molecular weights of 35 and 36.5 kd. IgE from 1 patient also bound a 55-kd antigen. Preabsorption of sera with nut extracts suppressed IgE binding to coconut proteins. Preabsorption of sera with coconut caused the disappearance of IgE binding to protein bands at 35 and 36 kd on a reduced immunoblot of walnut protein extract in 1 patient and suppression of IgE binding to a protein at 36 kd in the other patient. CONCLUSION: The reduced coconut protein at 35 kd was previously shown to be immunologically similar to soy glycinin (legumin group of seed storage proteins). The clinical reactivity in these 2 patients is likely due to cross-reacting IgE antibodies primarily directed against walnut, the original clinical allergy reported, and most likely to a walnut legumin-like protein. Coconut allergy in patients with tree nut allergy is rare; these are the first 2 patients ever reported, and therefore there is no general indication to advise patients with tree nut allergy to avoid coconut

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[98] - Rosado A, Fernández-Rivas M, González-Mancebo E, Léon F, Campos C, Tejedor MA. Anaphylaxis to coconut. Allergy 2002;57:182-183

Allergic reactions to coconut are very infrequent. To the best of our knowledge, allergy to coconut has been reported in two patients allergic to walnut and almond. (1). A 28 years-old atopic man presented an immediate reaction after the ingestion of a coconut ice cream consisted on oral pruritus, vomiting, and intense angioedema of lips and uvula, which subsided in 24 hours with symptomatic treatment at the emergency room. He had previously noticed oral pruritus with walnuts, but tolerated the ingestion of other nuts. He denied adverse reactions with other foodstuffs, and tolerated contact with latex materials. The patient gave written consent to the following study. Prick-prick tests with coconut ice cream and fresh coconut was positive (9 and 13 mm, respectively). Specific IgE to coconut (CAP System, Pharmacia) was 18.30 kU/L. Total IgE was 572 KU/L. Defatted homogenized coconut was extracted in 0.9 % saline. After filtration and centrifugation, the supernant was dialysed against saline solution 0.9% overnight at 4°C, and finally passed through a 0.45 µm Millipore filter. The protein concentration was 3.6 mg/mL (turbidimetry). A prick test performed with this extract elicited a strongly positive immediate reaction (20 mm) with an associated late response at 6 hours (30 mm). This test was negative in five atopic controls. The SDS-PAGE of coconut extract showed bands of molecular weights (MW) of 15, 20, 35, 45 and 200 kDa. In the IgE-Immunoblotting intense reactivity to a protein of 78 kDa was observed, as well as weaker bands of 15-20, 22 and 30 kDa. Bands of 20, 35, 200 kDa could not be considered specific since they also appeared with the control serum. Allergy to walnut was ruled out by prick-prick test and CAP. An open oral challenge with walnut was negative. SPT and CAP to latex were both negative. Another nuts, banana and compounds of coconut ice cream were also tolerated. In conclusion, we present an exceptional case of anaphylaxis induced by the ingestion of a coconut ice cream due to an IgE-mediated reaction to coconut, which was demonstrated by both in vivo (skin tests) and in vitro tests. In the two previous reported cases of combined allergy to nuts and coconut the main allergens involved had MW of 35-36 kDa (1). A different allergen pattern was identified in our patient probably because he was monosensitized to coconut. REFERENCES 1. Teuber SS, Peterson WR. Systemic allergic reaction to coconut (cocos nucifera) in 2 subjects with tree nut and demonstration of cross-reactivity to legumin-like seed store coconut and walnut food allergens. J Allergy Clin Immunol. 1999; 103:1180-5.]

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[99] - Benito C, González-Mancebo E, de Durana MD, Tolón RM, Fernández-Rivas M. Identification of a 7S globulin as a novel coconut allergen. Ann Allergy Asthma Immunol 2007;98:580-584

BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.

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[100] - Garcia RN, Arocena RV, Laurena AC, Tecson-Mendoza EM. 11S and 7S globulins of coconut (Cocos nucifera L.): purification and characterization. J Agric Food Chem 2005;53:1734-1739

Total globulins extracted with 0.4 M NaCl in buffer from coconut endosperm separated into two peaks on gel filtration: peak I corresponding to 11S globulin or cocosin and peak II to 7S globulin with native molecular weights of 326 000 and 156 000, respectively. The percent composition of total globulins was estimated to be 11S, 86% and 7S, 14%. On SDS-PAGE, cocosin resolved into two closely migrating bands at approximately 34 000 (acidic polypeptide) and another set of 2 bands at 24 000 (basic polypeptide). Each set consisted of one darkly stained band and one lightly stained band. The 7S globulin consisted of three bands of 16 000, 22 000, and 24 000. Three isoforms of cocosin were identified after anion exchange chromatography. Cocosin, but not the 7S, was found to have disulfide bonds. Using periodic acid-Schiff's reagent, all of the bands of cocosin on SDS-PAGE were positive for carbohydrate. However, when con A-peroxidase was used, only the basic polypeptide stained positively for carbohydrate. For the 7S globulin, no carbohydrate group was detected using the PAS and con A-peroxidase tests. The 7S globulin was easily extracted with 0.10-0.15 M NaCl, whereas cocosin was extracted with 0.35 M NaCl. The N-terminal amino acid sequences of the 34 k band and 24 k band of cocosin were SVRSVNEFRXE and GLEETQ, respectively, and that of the 7S was EQEDPELQK.

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[101] - Kidon MI, Lim S, Loo A, Wee A, Ling S, Liew W, et al. Coconut Allergy in Two Children of Asian Origins and Identification of the IgE Binding Components. J Allergy Clin Immunol 2005;115(2 suppl.):S3

RATIONALE: To describe two children who developed primary coconut (Cocus nucifera) hypersensitivity and characterize IgE binding components METHODS: Two children with systemic allergic reactions to coconut (fresh and cooked) without previous tree nut exposure were subjected to prick-prick test with fresh coconut endosperm and routine panel (food and aero-allergens). Serum from each patient was tested for specific IgE Western blotting and mass spectrometric analysis were performed to identify IgE binding components RESULTS: Patient 1 tested positive for egg white, Dematophagoides mix and Blomia tropicalis. Patient 2 tested negative to all allergen extracts With the fresh endosperm, Patient 1 exhibited a huge reaction, with the wheal measuring 24.5 mm in excess of negative. Patient 2 demonstrated a wheal measuring 20mm and pseudopods were observed extending from forearm to axilla. IgE from sera of both patients were found to bind the endosperm and coconut milk (santan) but not the coconut water. Western blotting (patient 2) showed at least 8 IgE binding components with molecular weights ranging from 15kD to above 80kD (major band at 25kD) Sera from patient 1 bind only to three components between 20-25kD Mass spectrometric analysis of the trypsin-digested 25kD major band suggests it is a glutelin-like allergen CONCLUSIONS: We report 2 Asian patients with primary coconut allergy, with no previous sensitization to other tree nuts. Specific anti-IgE to eight new coconut antigens was also detected

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[102] - Benito C, González-Mancebo E, de Durana MD, Tolón RM, Fernández-Rivas M. Identification of a 7S globulin as a novel coconut allergen. Ann Allergy Asthma Immunol 2007;98:580-584

BACKGROUND: Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. OBJECTIVE: To identify coconut allergens in 2 patients allergic to this food. METHODS: We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. RESULTS: An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. CONCLUSION: We have identified a 7S storage protein as a novel coconut allergen.

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[103] - Teuber SS, Peterson WR. Systemic allergic reaction to coconut (Cocos nucifera) in 2 subjects with hypersensitivity to tree nut and demonstration of cross-reactivity to legumin-seed storage proteins. J Allergy Clin Immunol 1999;103:1180-1185

BACKGROUND: Two patients with tree nut allergy manifested by life-threatening systemic reactions reported the subsequent onset of systemic reactions after the consumption of coconut. OBJECTIVE: Herein, the IgE-binding proteins from coconut are described, and in vitro cross-reactivity with other nuts is investigated. METHODS: The IgE-binding profile of coconut endosperm tissue extract was analyzed by SDS-PAGE followed by immunoblotting. Immunoblot inhibition studies with walnut, almond, peanut, and coconut were performed. RESULTS: Sera IgE from both patients recognized reduced coconut allergens with molecular weights of 35 and 36.5 kd. IgE from 1 patient also bound a 55-kd antigen. Preabsorption of sera with nut extracts suppressed IgE binding to coconut proteins. Preabsorption of sera with coconut caused the disappearance of IgE binding to protein bands at 35 and 36 kd on a reduced immunoblot of walnut protein extract in 1 patient and suppression of IgE binding to a protein at 36 kd in the other patient. CONCLUSION: The reduced coconut protein at 35 kd was previously shown to be immunologically similar to soy glycinin (legumin group of seed storage proteins). The clinical reactivity in these 2 patients is likely due to cross-reacting IgE antibodies primarily directed against walnut, the original clinical allergy reported, and most likely to a walnut legumin-like protein. Coconut allergy in patients with tree nut allergy is rare; these are the first 2 patients ever reported, and therefore there is no general indication to advise patients with tree nut allergy to avoid coconut

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[104] - Lerch M, Egger C, Bircher AJ. Allergic reactions to macadamia nut. Allergy 2005;60:130-131

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[105] - Sutherland MF, Ohehir RE, Czarny D, Suphioglu C. Macadamia nut anaphylaxis: demonstration of specific IgE reactivity and partial cross-reactivity with hazelnut. J Allergy Clin Immunol 1999;104:889-890

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[106] - Pallares DE. Allergy to macadamia nut. Ann Allergy Asthma Immunol 2000;85:385-386

Nuts are one of the most common foods causing allergic reactions in children and adults although an allergic reaction to macadamia nut has been reported only once previously. OBJECTIVE: To report a case of macadamia nut-induced allergic reaction. METHODS: Skin prick test with macadamia nut was performed in a private practice office. RESULTS: The results demonstrated type-I hypersensitivity reaction with macadamia nut. CONCLUSION: Macadamia nut ingestion can cause immediate-type allergic reactions.

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[107] - Falk M, Katelaris CH, Fulcher DA. Macadamia allergy. Allergy Clin Immunol Int 2000;12:240-242

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[108] - Teuber SS, Brown RL, Haapanen LA. Allergenicity of gourmet nut oils processed by different methods. J Allergy Clin Immunol 1997;99:502-506

BACKGROUND: No information is available on allergenicity of tree nut oils, and information on peanut oils has been conflicting. Many of the nut oils now on the market undergo minimal processing and may contain residual antigen. OBJECTIVE: This study was carried out to determine whether several of the new "gourmet" tree nut oils, as well as peanut oils, contain residual proteins that could bind IgE from sera of patients with allergy. METHODS: Several brands of walnut, almond, hazelnut, pistachio, and macadamia nut oils were examined. Peanut oils, both unrefined oils (which have been shown to contain allergenic proteins) and refined oils (without previously demonstrable allergens), were also examined to confirm reproducibility of immunoreactivity as reported by other investigators. Oils were extracted with 0.2 mol/L ammonium bicarbonate, and protein concentrations in the aqueous extracts were measured. IgE binding was assayed by slot-blot and Western immunoblotting. Pooled sera from patients with a history of systemic reactions to various tree nuts or peanuts were used as appropriate. RESULTS: The oil extracts known to be from oils that had undergone less processing at lower temperatures tended to demonstrate qualitatively greater IgE binding and higher protein concentrations. CONCLUSIONS: Tree nut and peanut oils may pose a threat to patients with allergy, depending on the method of manufacture and processing.

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[109] - Sutherland MF, Ohehir RE, Czarny D, Suphioglu C. Macadamia nut anaphylaxis: demonstration of specific IgE reactivity and partial cross-reactivity with hazelnut. J Allergy Clin Immunol 1999;104:889-890

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[110] - Frémont S, Moneret-Vautrin DA, Nicolas JP. Allergenicity of the nan-gai nut. Allergy 2001;56:581

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[111] - Sten E, Stahl Skov P, Andersen SB, Torp AM, Olesen A, Bindslev-Jensen U, et al. Allergenic components of a novel food, Micronesian nut Nangai (Canarium indicum), show IgE cross-reactivity in pollen allergic patients. Allergy 2002;57:398-404

Background:New foods may present a risk for food hypersensitive patients. Several examples exist of allergic reactions caused by cross-reactive plant-derived foods, and new foods should be scrutinised before introducing them to the market. We have evaluated the clinical and serological relevance of cross-reactivity between Nangai and pollen allergens. Methods:Cross-reactivity was examined with Maxisorp RAST (radioallergosorbent test), RAST inhibition and Western blot, using sera from patients allergic to grass, birch and mugwort pollen. None of the patients reported having seen or eaten Nangai previously. To determine the biological and clinical relevance of the cross-reactivity, histamine release (HR) test, skin prick test (SPT) and food challenge were used. Results:There was prevalence for reactivity against Nangai in the group of pollen allergic patients. This cross-reactivity seems to be related - at least in part - to carbohydrate epitopes. Three out of 12 patients tested with Nangai were positive upon open challenge, but using double blind placebo controlled food challenge (DBPCFC) this could not be confirmed in two patients. The biological effects of Nangai on allergic patients were confirmed using HR and SPT. Conclusion:The Nangai specific IgE found among pollen allergic patients addresses the need for control of new or changed foods before introduction to the market.

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[112] - Wallowitz M, Peterson WR, Uratsu S, Comstock SS, Dandekar AM, Teuber SS. Jug r 4, a Legumin Group Food Allergen from Walnut (Juglans regia Cv. Chandler). J Agric Food Chem 2006;54:8369-8375

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.

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[113] - Ewan PW. Clinical study of peanut and nut allergy in 62 consecutive patients: new features and associations. Br Med J 1996;312:1074-1078

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[114] - Sampson HA, Mendelson L, Rosen JP. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N Engl J Med 1992;327:380-384

BACKGROUND AND METHODS. Reports of fatal or near-fatal anaphylactic reactions to foods in children and adolescents are rare. We identified six children and adolescents who died of anaphylactic reactions to foods and seven others who nearly died and required intubation. All the cases but one occurred in one of three metropolitan areas over a period of 14 months. Our investigations included a review of emergency medical care reports, medical records, and depositions by witnesses to the events, as well as interviews with parents (and some patients). RESULTS. Of the 13 children and adolescents (age range, 2 to 17 years), 12 had asthma that was well controlled. All had known food allergies, but had unknowingly ingested the foods responsible for the reactions. The reactions were to peanuts (four patients), nuts (six patients), eggs (one patient), and milk (two patients), all of which were contained in foods such as candy, cookies, and pastry. The six patients who died had symptoms within 3 to 30 minutes of the ingestion of the allergen, but only two received epinephrine in the first hour. All the patients who survived had symptoms within 5 minutes of allergen ingestion, and all but one received epinephrine within 30 minutes. The course of anaphylaxis was rapidly progressive and uniphasic in seven patients; biphasic, with a relatively symptom-free interval in three; and protracted in three, requiring intubation for 3 to 21 days. CONCLUSIONS. Dangerous anaphylactic reactions to food occur in children and adolescents. The failure to recognize the severity of these reactions and to administer epinephrine promptly increases the risk of a fatal outcome.

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[115] - Asero R, Mistrello G, Roncarolo D, Amato S. Allergy to minor allergens of Brazil nut. Allergy 2002;57:1080-1081

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[116] - Arshad SH, Malmberg E, Krapf K, Hide DW. Clinical and immunological characteristics of Brazil nut allergy. Clin Exp Allergy 1991;21:373-376

Twelve cases of allergy to Brazil nut have been recorded in the last 8 years. The reaction is usually IgE mediated and most patients have other atopic diseases. There appear to be several proteins with potent antigenic components in Brazil nut.

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[117] - Pastorello EA, Farioli L, Pravettoni V, Ispano M, Conti A, Ansaloni R, et al. Sensitization to the major allergen of Brazil nut is correlated with the clinical expression of allergy. J Allergy Clin Immunol 1998;102:1021-1027

BACKGROUND: Only a few studies have investigated the clinical role of food allergens, especially the relationship between sensitization to a given allergen and occurrence of adverse reactions when eating the relevant food item. OBJECTIVE: This study evaluated the clinical role of the allergens of Brazil nut by comparing the patterns of IgE binding in sera from 11 patients with anaphylaxis after eating Brazil nuts with those from 10 subjects with no symptoms to this food item. Both groups had specific IgE to Brazil nut. METHODS: Allergens in the in-house extract of Brazil nut were identified by SDS-PAGE/immunoblotting, the major allergen was purified by HPLC, and its N-terminal sequence was determined by a protein sequencer. RESULTS: SDS-PAGE/immunoblotting detected a number of allergenic components with molecular weights ranging from 4 to 58 kd. All sera from symptomatic patients recognized a 9-kd allergen corresponding (as established by amino acid sequencing) to a 2S albumin already described as a major allergen of Brazil nut, whereas the other allergens each bound IgE from less than 50% of sera. No sera from asymptomatic subjects showed IgE binding to the 9-kd allergen, but they did recognize components from 25 to 58 kd, which are minor allergens. CONCLUSIONS: These findings indicate that the allergen underlying clinical reactions to Brazil nut is a 2S albumin of 9 kd and that in vitro reactivity to this allergen identifies subjects who react in vivo to ingestion of this food.

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[118] - Borja JM, Bartolome B, Gomez E, Galindo PA, Feo F. Anaphylaxis from Brazil nut. Allergy 1999;54:1007-1008

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[119] - Codreanu F. L'activité du réseau Allergo-Vigilance: allergies alimentaires. Alim'Inter 2007;12:239-242

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[120] - Bourrier T, Villevieille L, Albertini M, Boutte P. Allergie alimentaire à la noix du Brésil: à propos de cinq cas chez l'enfant d'âge préscolaire. Rev Fr Allergol Immunol Clin 2001;41:401-406

Les auteurs rapportent cinq observations d'allergie alimentaire à la noix du Brésil chez des enfants âgés de moins de six ans. Tous les enfants ont un terrain atopique. Les symptômes, survenant en quelques minutes, sont violents, de type anaphylaxie aiguë. La mise en évidence de cette allergie alimentaire conduit à rechercher systématiquement une sensibilisation aux autres noix et à l'arachide : une fois pour la noix et deux fois pour la noisette en l'absence de sensibilisation à l'arachide.

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[121] - Senna G, Bonadonna P, Crivellaro M, Schiappoli M, Passalacqua G. Anaphylaxis due to Brazil nut skin testing in a walnut-allergic subject. J Investig Allergol Clin Immunol 2005;15:225-227

The diagnosis and management of nut allergy can be difficult because of the possible severity of the clinical manifestations and the cross reactivity between different species. We report a case of anaphylaxis due to skin testing in a young adult with clinically ascertained walnut allergy. After an episode of anaphylaxis due to walnut ingestion, a routine diagnostic workup was carried out, involving skin prick test with commercial extracts, prick by prick with fresh food and CAP-RAST assay for different nuts. Immediately after pricking with fresh Brazil nut, a severe episode of anaphylaxis occurred, that required epinephrine and intravenous steroids. The subject had never eaten Brazil nut before. Therefore we hypothesize a cross reactivity effect, since this phenomenon is well known for tree nuts. Our case suggests that in vivo diagnosis, especially if fresh nuts are used, should be performed only if adequate equipment to treat anaphylaxis is available.

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[122] - Asero R, Mistrello G, Roncarolo D, Amato S. Allergy to minor allergens of Brazil nut. Allergy 2002;57:1080-1081

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[123] - Borja JM, Bartolome B, Gomez E, Galindo PA, Feo F. Anaphylaxis from Brazil nut. Allergy 1999;54:1007-1008

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[124] - Visitsunthorn N, Tiranathanakul A, Netrakul R, Vichyanond P. Evaluation of consistency between local and imported seafood allergen extracts. J Med Assoc Thai 2002;85(Suppl. 2):S593-S598

BACKGROUND: Seafood is a common cause of food allergy in Thai adults and children. Skin prick test is a safe and convenient way to screen seafood allergy. To date, the Allergy Unit, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University uses imported extracts for seafood skin prick test. The extracts are expensive and may not be the same species as seafood in Thailand. OBJECTIVES: To compare the consistency between local seafood allergen extracts prepared by the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University and imported seafood allergen extracts. METHODS: The study was a prospective comparative trial performed in children who attended the Pediatric Allergy Clinic, Siriraj Hospital from March 1999 to October 2000. The skin prick test was performed with the local seafood allergen extracts prepared by a pharmacist from the Department of Pharmacology and the imported seafood allergen extracts included shrimp, fish and crab. Histamine and normal saline were used as positive and negative control respectively. The positive result was recorded when wheal reaction was > or = 3 mm larger than negative control. RESULTS: Eighty eight patients (57 boys and 31 girls) were included in this study. The average age was 7.7 years (1-15 years). Half of the patients had a history of seafood allergy. The study showed probable consistency between imported and local skin prick test of shrimp and crab extracts (kappa = 5-7) but no consistency between imported and local skin prick test of fish extracts (kappa < 5). The study also showed no consistency between history of seafood and skin prick test result. CONCLUSION: Local seafood allergen extracts from the Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University are safe but they cannot replace the imported seafood allergen extracts. Further investigations about sensitivity and specificity of both kinds of allergen extracts are necessary.

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[125] - Beyer K, Bardina L, Grishina G, Ashraf A, Teuber S, Niggemann B, et al. Identification Of A New Brazil Nut Allergen - Ber e 2. J Allergy Clin Immunol 2008;121:S247

RATIONALE: Allergic reactions to Brazil nut have been identified as one of the most common causes of tree nut allergies. Ber e 1 has been identified as the major Brazil nut allergen. The purpose of this study was to determine whether additional proteins in Brazil nut also have allergenic potential. METHODS: Proteins from Brazil nut were extracted and separated by SDS-Page. Immunolabeling was performed with serum from 27 Brazil nutsensitized patients. Proteins of interest were further analyzed by Edman sequencing. RNA isolated from Brazil nut was used to construct a cDNA library. The library was screened using primers designed on the basis of amino acid sequences identified through Edman sequencing. Full-length cDNA clones were isolated, sequenced, expressed and screened with individual patient sera from all patients. RESULTS: An IgE-binding protein that was recognized by 56% of the patients was identified at 29 kD. Analysis by Edman sequencing revealed that this protein was homologue to 11S globulins. Screening the Brazil nut cDNA library the protein was isolated. UsingWestern blot analysis, 12 out of the 27 Brazil nut-sensitized patients showed IgE-binding to the expressed recombinant protein. Therefore, this 11S globulin was named Ber e 2 by the allergen nomenclature sub-committee. CONCLUSIONS: The present abstract reports a new Brazil nut allergen, Ber e 2, an 11S globulin that belongs to the family of seed storage proteins. The identification of new food allergens is the first step towards the use of these recombinant allergens in future diagnostic and therapeutic approaches.

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[126] - Bartolome B, Mendez JD, Armentia A, Vallverdu A, Palacios R. Allergens from Brazil nut: immunochemical characterization. Allergol Immunopathol (Madr) 1997;25:135-144

The increase in the consumption of tropical nuts in the Northern Hemisphere during the last years, has evolved in a simultaneous enhancement of allergic IgE mediated (Hypersensitivity type 1) reported cases produced by this kind of food. The Brazil nut is the seed of the Bertholletia excelsa tree (Family Lecythidaceae) and, as in other seeds, proteins represent one of its major components making up 15-17% of its fresh weight and 50% of defatted flour. Of these, storage proteins are the most important ones, and the 12 S globulin legumin-like protein and the 2 S albumin have been described as the most representative. The 2 S protein, due to its high sulfur-rich amino acid content (3% cysteine and 18% methionine), is being studied, cloned and expressed in some important agronomic seeds (soybean, bean, oilseed rape) in order to enrich the nutritional quality of them. The case of a patient with serious clinical allergic symptoms (vomiting, diarrhoea and loss of consciousness) caused by oral contact with the Brazil nut, is presented. The patient gave a positive Skin Prick Test response to Brazil nut, kiwi and hazelnut extracts, and negative to regionally specific aeroallergens and other food extracts. The patient serum showed a high level of specific IgE by RAST to Brazil nut (> 17.5 PRU/ml, Class 4), and significative levels to hazelnut, and mustard. In vitro immunological studies (SDS-Immunoblotting and IEF-Immunoblotting) revealed IgE-binding proteins present in the extract. It was shown that not only the heavy (Mr 9) and light (Mr 4) subunits of the known allergenic 2 S albumin but also the alpha-subunits (Mr approximately 33.5 and 32) and at least one of the beta-subunits (Mr approximately 21) of the 12 S Brazil nut globulin, hitherto never involved in allergic problems, showed a strong IgE-binding capacity.

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[127] - Pastorello EA, Farioli L, Pravettoni V, Ispano M, Conti A, Ansaloni R, et al. Sensitization to the major allergen of Brazil nut is correlated with the clinical expression of allergy. J Allergy Clin Immunol 1998;102:1021-1027

BACKGROUND: Only a few studies have investigated the clinical role of food allergens, especially the relationship between sensitization to a given allergen and occurrence of adverse reactions when eating the relevant food item. OBJECTIVE: This study evaluated the clinical role of the allergens of Brazil nut by comparing the patterns of IgE binding in sera from 11 patients with anaphylaxis after eating Brazil nuts with those from 10 subjects with no symptoms to this food item. Both groups had specific IgE to Brazil nut. METHODS: Allergens in the in-house extract of Brazil nut were identified by SDS-PAGE/immunoblotting, the major allergen was purified by HPLC, and its N-terminal sequence was determined by a protein sequencer. RESULTS: SDS-PAGE/immunoblotting detected a number of allergenic components with molecular weights ranging from 4 to 58 kd. All sera from symptomatic patients recognized a 9-kd allergen corresponding (as established by amino acid sequencing) to a 2S albumin already described as a major allergen of Brazil nut, whereas the other allergens each bound IgE from less than 50% of sera. No sera from asymptomatic subjects showed IgE binding to the 9-kd allergen, but they did recognize components from 25 to 58 kd, which are minor allergens. CONCLUSIONS: These findings indicate that the allergen underlying clinical reactions to Brazil nut is a 2S albumin of 9 kd and that in vitro reactivity to this allergen identifies subjects who react in vivo to ingestion of this food.

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[128] - de Leon MP, Drew AC, Glaspole IN, Suphioglu C, O'hehir RE, Rolland JM. IgE cross-reactivity between the major peanut allergen Ara h 2 and tree nut allergens. Mol Immunol 2007;44:463-471

Allergy to peanut and tree nuts is characterised by a high frequency of life-threatening anaphylactic reactions and typically lifelong persistence. Although peanut is the most common cause of nut allergy, peanut allergic patients are frequently also sensitive to tree nuts. It is not known if this is due to cross-reactivity between peanut and tree nut allergens. In this study, the major peanut allergen Ara h 2 was cloned from peanut cDNA, expressed in E. coli cells as a His-tag fusion protein and purified using a Ni-NTA column. Immunoblotting, ELISA and basophil activation indicated by CD63 expression all confirmed the IgE reactivity and biological activity of rAra h 2. To determine whether or not this allergen plays a role in IgE cross-reactivity between peanut and tree nuts, inhibition ELISA was performed. Pre-incubation of serum from peanut allergic patients with increasing concentrations of almond or Brazil nut extract inhibited IgE binding to rAra h 2. Purified rAra h 2-specific serum IgE antibodies also bound to proteins present in almond and Brazil nut extracts by immunoblotting. This indicates that the major peanut allergen, Ara h 2, shares common IgE-binding epitopes with almond and Brazil nut allergens, which may contribute to the high incidence of tree nut sensitisation in peanut allergic individuals.

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[129] - Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. Identification of a Brazil-nut allergen in transgenic soybeans. N Engl J Med 1996;334:688-692

The nutritional quality of soybeans (Glycine max) is compromised by a relative deficiency of methionine in the protein fraction of the seeds. To improve the nutritional quality, methionine-rich 2S albumin from the Brazil nut (Betholletia excelsa) has been introduced into transgenic soybeans. Since the Brazil nut is a known allergenic food, we assessed the allergenicity of the 2S albumin. METHODS. The ability of proteins in transgenic and non-transgenic soybeans, Brazil nuts, and purified 2S albumin to bind to IgE in serum from subjects allergic to Brazil nuts was determined by radioallergosorbent tests (4 subjects) and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (9 subjects) with immunoblotting and autoradiography. Three subjects also underwent skin-prick testing with extracts of soybean, transgenic soybean, and Brazil nut. RESULTS. On radioallergosorbent testing of pooled serum from four subjects allergic to Brazil nuts, protein extracts of transgenic soybean inhibited binding of IgE to Brazil-nut proteins. On immunoblotting, serum IgE from eight of nine subjects bound to purified 2S albumin from the Brazil nut and the transgenic soybean. On skin-prick testing, three subjects had positive reactions to extracts of Brazil nut and transgenic soybean and negative reactions to soybean extract CONCLUSIONS. The 2S albumin is probably a major Brazil-nut allergen, and the transgenic soybeans analyzed in this study contain this protein. Our study show that an allergen from a food known to be allergenic can be transferred into another food by genetic engineering.

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[130] - Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. Identification of a Brazil-nut allergen in transgenic soybeans. N Engl J Med 1996;334:688-692

The nutritional quality of soybeans (Glycine max) is compromised by a relative deficiency of methionine in the protein fraction of the seeds. To improve the nutritional quality, methionine-rich 2S albumin from the Brazil nut (Betholletia excelsa) has been introduced into transgenic soybeans. Since the Brazil nut is a known allergenic food, we assessed the allergenicity of the 2S albumin. METHODS. The ability of proteins in transgenic and non-transgenic soybeans, Brazil nuts, and purified 2S albumin to bind to IgE in serum from subjects allergic to Brazil nuts was determined by radioallergosorbent tests (4 subjects) and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (9 subjects) with immunoblotting and autoradiography. Three subjects also underwent skin-prick testing with extracts of soybean, transgenic soybean, and Brazil nut. RESULTS. On radioallergosorbent testing of pooled serum from four subjects allergic to Brazil nuts, protein extracts of transgenic soybean inhibited binding of IgE to Brazil-nut proteins. On immunoblotting, serum IgE from eight of nine subjects bound to purified 2S albumin from the Brazil nut and the transgenic soybean. On skin-prick testing, three subjects had positive reactions to extracts of Brazil nut and transgenic soybean and negative reactions to soybean extract CONCLUSIONS. The 2S albumin is probably a major Brazil-nut allergen, and the transgenic soybeans analyzed in this study contain this protein. Our study show that an allergen from a food known to be allergenic can be transferred into another food by genetic engineering.

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[131] - Dearman RJ, Alcocer MJC, Archer DA, Kimber I. Influence of plant lipids on IgE antibody responses to Ber e 1. Allergy Clin Immunol Int 2005;17(Suppl. 1):343

Background: It has been demonstrated previously that lipids, particularly bacterial lipopolysaccharide (LPS), can impact on immune responses to proteins, with low doses of LPS enhancing type 2 responses including IgE antibody production and airway hyperreactivity. In the current experiments we have examined the influence of extracts of native plant lipids on antibody responses provoked by a preparation of recombinant Ber e 1, the major allergen in brazil nuts. Methods: Recombinant Ber e l (correctly folded) was produced in Pichia pastoris, purified by ion exchange/reverse phase chromatography and supplemented with various lipid fractions isolated from native brazil nuts (stoichiometry 1:1 with respect to phosphatidyl choline content of mixture). BALB/c strain mice (n=5) received 2.5 mg of recombinant Ber e 1 alone or supplemented with lipid fractions by intraperitoneal injection on days 0 and 7. Seven days later, serum samples were analyzed for specific IgE antibody by homologous passive cutaneous anaphylaxis assay (PCA) and for IgG1 by enzyme-linked immunosorbant assay (ELISA). Results: Exposure to recombinant Ber e 1 alone failed to induce detectable IgG or IgE antibody. Administration of Ber e 1 in the presence of the total lipid fraction isolated from native brazil nuts resulted in relatively high titre IgG antibody and 3 of 5 animals were IgE responders. Co-administration of the complex lipid fraction also resulted in a similar marked adjuvant effect on IgG and IgE antibody. The steroid and low molecular weight lipid fractions induced low level IgG antibody and had little impact on IgE antibody production. Native Ber e 1 containing endogenous lipids also provoked IgG and IgE antibody responses. Identical IgE and IgG antibody responses were detected regardless of whether native or recombinant Ber e 1 was used as substrate in the PCA or ELISA. Conclusion: These data suggest that endogenous brazil nut lipids are required for the induction of optimal antibody responses to Ber e 1 in the BALB/c strain mouse. The observation that the appropriate antibody binding sites are present on both native and recombinant forms of Ber e 1 suggests that the impact of lipid is at the induction phase, and is possibly required for efficient antigen presentation.

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[132] - Malandain H. Les OGM : risques réels ou imaginaires ? Vaisseaux Cœur Poumon 2003, n° spécial « 3èmes Rencontres Internationales de Formation Continue en Allergologie », Bruxelles 16-18/10/2003, pp 7-9

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[133] - Goodman RE, Vieths S, Sampson HA, Hill D, Ebisawa M, Taylor SL, et al. Allergenicity assessment of genetically modified crops—what makes sense ? Nat Biotechnol 2008;26:73-81

GM crops have great potential to improve food quality, increase harvest yields and decrease dependency on certain chemical pesticides. Before entering the market their safety needs to be scrutinized. This includes a detailed analysis of allergenic risks, as the safety of allergic consumers has high priority. However, not all tests currently being applied to assessing allergenicity have a sound scientific basis. Recent events with transgenic crops reveal the fallacy of applying such tests to GM crops.

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[134] - Prescott VE, Hogan SP. Genetically modified plants and food hypersensitivity diseases: Usage and implications of experimental models for risk assessment. Pharmacol Ther 2006;111:374-383

The recent advances in biotechnology in the plant industry have led to increasing crop production and yield that in turn has increased the usage of genetically modified (GM) food in the human food chain. The usage of GM foods for human consumption has raised a number of fundamental questions including the ability of GM foods to elicit potentially harmful immunological responses, including allergic hypersensitivity. To assess the safety of foods derived from GM plants including allergenic potential, the US FDA, Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO), and the EU have developed approaches for evaluation assessment. One assessment approach that has been a very active area of research and debate is the development and usage of animal models to assess the potential allergenicity of GM foods. A number of specific animal models employing rodents, pigs, and dogs have been developed for allergenicity assessment. However, validation of these models is needed and consideration of the criteria for an appropriate animal model for the assessment of allergenicity in GM plants is required. We have recently employed a BALB/c mouse model to assess the potential allergenicity of GM plants. We have been able to demonstrate that this model is able to detect differences in antigenicity and identify aspects of protein post-translational modifications that can alter antigenicity. Furthermore, this model has also enabled us to examine the usage of GM plants as a therapeutic approach for the treatment of allergic diseases. This review discusses the current approaches to assess the allergenic potential of GM food and particularly focusing on the usage of animal models to determine the potential allergenicity of GM foods and gives an overview of our recent findings and implications of these studies.

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[135] - Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK. Identification of a Brazil-nut allergen in transgenic soybeans. N Engl J Med 1996;334:688-692

The nutritional quality of soybeans (Glycine max) is compromised by a relative deficiency of methionine in the protein fraction of the seeds. To improve the nutritional quality, methionine-rich 2S albumin from the Brazil nut (Betholletia excelsa) has been introduced into transgenic soybeans. Since the Brazil nut is a known allergenic food, we assessed the allergenicity of the 2S albumin. METHODS. The ability of proteins in transgenic and non-transgenic soybeans, Brazil nuts, and purified 2S albumin to bind to IgE in serum from subjects allergic to Brazil nuts was determined by radioallergosorbent tests (4 subjects) and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis (9 subjects) with immunoblotting and autoradiography. Three subjects also underwent skin-prick testing with extracts of soybean, transgenic soybean, and Brazil nut. RESULTS. On radioallergosorbent testing of pooled serum from four subjects allergic to Brazil nuts, protein extracts of transgenic soybean inhibited binding of IgE to Brazil-nut proteins. On immunoblotting, serum IgE from eight of nine subjects bound to purified 2S albumin from the Brazil nut and the transgenic soybean. On skin-prick testing, three subjects had positive reactions to extracts of Brazil nut and transgenic soybean and negative reactions to soybean extract CONCLUSIONS. The 2S albumin is probably a major Brazil-nut allergen, and the transgenic soybeans analyzed in this study contain this protein. Our study show that an allergen from a food known to be allergenic can be transferred into another food by genetic engineering.

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[136] - Tawde PD, Teuber SS, Sathe SK, Roux KH. Identification of Allergenic Cross-Reactive Proteins in Cashew and Pistachio Nut. J Allergy Clin Immunol 2005;115(2 suppl.):S93

RATIONALE: Cashew and pistachio belong to the Anacardiaceae family and strong allergenic cross-reactivity between them has been reported Our aim was to identify these cross-reactive allergenic proteins from cashew and pistachio METHODS: Pooled sera from cashew-allergic patients were tested for IgE reactivity to soluble cashew and pistachio proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis The IgE-reactive spots from the immunoblots were further analyzed by Nterminal amino acid sequencing. ELISAs were performed using individual sera from cashew and tree nut allergic patients to assess IgE reactivity to cashew and pistachio. Inhibition ELISAs assessed the degree of allergenic cross-reactivity between cashew and pistachio nuts RESULTS: IgE immunoblots of cashew and pistachio proteins with cashew-allergic sera identified reactive proteins of 35kDa, 22kDa, and 7- 9kDa. N-terminal amino acid sequencing of the IgE-reactive proteins from the pistachio immunoblot identified them as the acidic and basic subunits of 11S globulin and the 2S albumin seed storage proteins, respectively and have also been identified as allergens in cashew. ELISA results with ten individual cashew-allergic sera (2 of 10 patients report pistachio allergy, the remaining 8 report never having eaten pistachio) showed IgE reactivity to both cashew and pistachio. Inhibition ELISA demonstrated that pre-incubation of the patient sera with pistachio extract resulted in a marked decrease in IgE binding to cashew extract and vice versa indicating allergenic cross-reactivity CONCLUSIONS: The results demonstrate the presence of considerable cross-reactive B-cell epitopes on cashew and pistachio nut allergens as would be expected based on their plant taxonomic classification

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[137] - Parra FM, Cuevas M, Lezaun A, Alonso MD, Beristain AM, Losada E. Pistachio nut hypersensitivity: identification of pistachio nut allergens. Clin Exp Allergy 1993;23:996-1001

Type I hypersensitivity to pistachio nut antigens was demonstrated in three patients by means of immediate skin-test reactivity, specific IgE determination by a fluoroimmunoassay (CAP), CAP-inhibition and leucocyte histamine release. Sensitization to other dried fruits and pollens was observed in the patients. The CAP-inhibition studies revealed significant crossreactivity between pistachio and cashew nut belonging to the Anacardiaceae family, and between pistachio nut and other dried fruits belonging to taxonomically unrelated botanical families. No relevant crossallergenicity was observed between pistachio nut and Lolium and Olea pollens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a pistachio nut extract followed by immunoblotting analysis identified four IgE-binding bands with molecular weights of 34, 41, 52 and 60 kD.

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[138] - Fernandez C, Fiandor A, Martinez-Garate A, Martinez Quesada J. Allergy to pistachio: crossreactivity between pistachio nut and other Anacardiaceae. Clin Exp Allergy 1995;25:1254-1259

BACKGROUND: Anaphylaxis against Anacardiaceae nuts is uncommon and the allergens involved still poorly characterized. For this reason two patients with allergy towards pistachio nut (a member of the Anacardiaceae family) have been studied. OBJECTIVE: Identification of immunoallergens present in pistachio nut and analysis of crossreactive antigens in other members of the same plant family, specifically cashew and mango. METHODS: Presence of specific IgE for pistachio and cashew nut and for mango seed and pulp was determined by skin tests and radioallergosorbent assay (RAST). The allergenic profile of pistachio and cashew was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Crossreactivity between pistachio and the other Anacardiaceae was studied by RAST inhibition. RESULTS: Skin tests were positive for pistachio and cashew in the two children and for mango seed in one. RAST was positive for pistachio and cashew in both patients. On immunoblotting, serum from both patients recognized several pistachio and cashew allergens with a molecular weight ranging from < 14.2-70 kDa. RAST inhibition demonstrated common antigenic determinants between pistachio and cashew nut. Crossreactivity was also found between pistachio nut and mango seed but not with mango pulp. CONCLUSION: Pistachio nut contains several protein allergens able to trigger type I hypersensitivity reactions. These allergens can be found also in cashew nut and mango seed but not in mango pulp.

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[139] - Tawde PD, Teuber SS, Sathe SK, Roux KH. Identification of Allergenic Cross-Reactive Proteins in Cashew and Pistachio Nut. J Allergy Clin Immunol 2005;115(2 suppl.):S93

RATIONALE: Cashew and pistachio belong to the Anacardiaceae family and strong allergenic cross-reactivity between them has been reported Our aim was to identify these cross-reactive allergenic proteins from cashew and pistachio METHODS: Pooled sera from cashew-allergic patients were tested for IgE reactivity to soluble cashew and pistachio proteins by IgE immunoblotting after one-dimensional (1-D) and 2-D electrophoresis The IgE-reactive spots from the immunoblots were further analyzed by Nterminal amino acid sequencing. ELISAs were performed using individual sera from cashew and tree nut allergic patients to assess IgE reactivity to cashew and pistachio. Inhibition ELISAs assessed the degree of allergenic cross-reactivity between cashew and pistachio nuts RESULTS: IgE immunoblots of cashew and pistachio proteins with cashew-allergic sera identified reactive proteins of 35kDa, 22kDa, and 7- 9kDa. N-terminal amino acid sequencing of the IgE-reactive proteins from the pistachio immunoblot identified them as the acidic and basic subunits of 11S globulin and the 2S albumin seed storage proteins, respectively and have also been identified as allergens in cashew. ELISA results with ten individual cashew-allergic sera (2 of 10 patients report pistachio allergy, the remaining 8 report never having eaten pistachio) showed IgE reactivity to both cashew and pistachio. Inhibition ELISA demonstrated that pre-incubation of the patient sera with pistachio extract resulted in a marked decrease in IgE binding to cashew extract and vice versa indicating allergenic cross-reactivity CONCLUSIONS: The results demonstrate the presence of considerable cross-reactive B-cell epitopes on cashew and pistachio nut allergens as would be expected based on their plant taxonomic classification

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[140] - Ayuso R, Grishina G, Ahn K, Bardina L, Beyer K, Sampson H. Identification of a MnSOD-like Protein as a New Major Pistachio Allergen. J Allergy Clin Immunol 2007;119(1 suppl):S115

RATIONALE: Among tree nuts, Pistachio is frequently responsible for food-induced allergic reactions in allergic individuals. Pistachio (Pistacia vera) belongs to the Anacardiacea family, which also includes mango and cashew. In contrast to other tree nuts, little is know about the allergenic proteins in pistachio. AIM: Identify and characterize new pistachio allergens. METHODS: 1D- and 2D-electrophoresis followed by immunoblotting for IgE detection were done using sera from 27 well-characterized pistachio-allergic subjects. Each patient had history of an immediate allergic reaction following ingestion of pistachio and pistachio-specific IgE by CAP-RAST. Protein identification was done from a database using LC-MS/MS (nano-capillary HPLC ion trap mass spectrometry) of 2D in-gel tryptic digest. RESULTS: Immunolabeling of 1D and 2D membranes demonstrated IgE-binding to a 23 kD pistachio protein by 16 out of 27 sera (59%). 2D electrophoresis revealed that this protein had an isoelectric point of 6. Tryptic digestion of the 23 kD band followed by LC-MS/MS analysis of the peptides showed high homology with sequences present in Hevea braziliensis (latex) IgE-binding MnSOD (manganese superoxide dismutase), indicating that this 23 kD pistachio protein is a new allergenic MnSOD-like molecule. CONCLUSIONS: A MnSOD-like protein appears to be a novel major pistachio allergen, which may be responsible for IgE cross-reactivity with latex.

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[141] - Asero R, Mistrello G, Roncarolo D, Amato S, Caldironi G, Barocci F, et al. Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study. Allergy 2002;57:900-906

BACKGROUND: Lipid transfer proteins (LTP) are highly conserved and widely distributed throughout the plant kingdom. Recent studies demonstrated immunological cross-reactivity between LTP from many botanically unrelated fruits and vegetables and concluded that LTP are pan-allergens. This study aimed to evaluate the clinical relevance of such cross-reactivity in a group of subjects monosensitized to LTP . METHODS: Twenty LTP-hypersensitive patients were selected from a population of about 600 subjects with history of Rosaceae allergy by means of: 1) negative skin prick test (SPT) with a commercial birch pollen extract; 2) positive SPT with a commercial plum extract, rich in LTP but virtually lacking both Bet v 1-like proteins and profilin; 3) in-vitro IgE reactivity to the 9-10 kDa fraction of peach peel or immunoblot with peach peel showing a single band at 10 kDa; and 4) total inhibition of reactivity to whole peach extract (containing Bet v 1-related allergen, profilin, and LTP) by purified peach LTP on enzyme-linked immunoassay (ELISA). Allergy to foods other than Rosaceae was ascertained by careful interview and analysis of medical recordings. SPT with a large series of plant-derived foods were carried out as well. The cross reactivity between LTPs from botanically unrelated plant-derived foods was assessed by ELISA inhibition tests using walnut and peanut extracts as substrate, and peach LTP as inhibitor . RESULTS: All patients reported allergic reactions after the ingestion of at least one from a large number of vegetable foods other than Rosaceae, and in several cases clinical reactions were very severe (anaphylaxis, asthma, urticaria/angioedema). Nuts and peanuts were the most frequently reported causes of allergic reactions (80% and 40% of patients, respectively). All patients showed positive SPT to several non-Rosaceae food extracts. SPT with nuts, peanut, legumes, celery, rice, and corn were positive in the majority of patients. In ELISA inhibition studies, absorption of sera with peach LTP caused complete inhibition of IgE reactivity to walnut and peanut in all cases . CONCLUSION: LTP is a clinically relevant pan-allergen. Most Rosaceae-allergic, LTP-hypersensitive patients experience adverse reactions after ingestion of botanically unrelated plant-derived foods as well. In view of the high prevalence and severity of the allergic reactions induced, hazelnut, walnut, and peanut should be regarded as potentially hazardous for these patients.

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{{}}	J. regia	J. nigra	J. cinerea	J. californica
2S albumines	Jug r 1	Jug n 1	Jug ci 1	Jug ca 1
Vicilines	Jug r 2	Jug n 2	Jug ci 2	Jug ca 2
Légumines	Jug r 4
Profilines	xx
Mn superoxyde dismutases		xx
Glycéraldéhyde-P deshydrogénases		xx

Amande

La noisette

Les noix

Noix de cajou

Noix de coco

Noix de macadamia

Noix de nangaille

Noix de pécan

Noix du Brésil

Pistache

Rubriques

Dans la même rubrique

Les fruits à coque : généralités

Les fruits à coque : réactivités croisées