Les agrumes

[1] - Jiménez A, Campo P, Rico P, Feliu A, Somoza ML, Crespo JF, et al. Randomized, double-blind, crossover challenge study in adult subjects reporting adverse reactions to orange (Citrus sinensis). EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°133

Background: Orange is by far the most important commercial citrus fruit. World production of oranges represents over 82% of the world‚s total production of citrus fruit with Spain as one of the export leaders. However, at present, only anecdotal cases of orange allergy have been reported, based mainly on case histories and allergy testing. The purpose of this investigation was to assess the clinical features of acute allergic reactions to orange objectively confirmed by the double-blind placebo- controlled food challenge (DBPCFC) procedure. Methods: Fifteen patients (range = 13 - 67 yr.; median = 25 yr.) complaining of adverse reactions to orange were included in the study protocol of the Food Allergy Unit from 'Hospital Universitario 12 de Octubre'. Diagnostic procedures included a clinical questionnaire, skin testing using the prick-prick technique and detection of specific IgE (CAP- FEIA), which were performed with several members of Rutaceae family and other foods reported as offenders. Patients underwent an open food challenge (OFC), unless they had a convincing history of severe anaphylaxis. Positive OFC reactions were subsequently evaluated by DBPCFC‚s, up to an cumulative dose of 200 g of fresh fruit. All negative results of DBPCFCs were followed by an open feeding. Results: Ten and 11 patients had positive results of skin prick test and CAP-FEIA with orange, respectively. The most frequent symptom was the oral allergy syndrome (80%), followed by acute urticaria (13%) and gastrointestinal anaphylaxis (7%). All patients underwent an OFC with orange. Five of them were positive, but only 4 of the 5 performed DBPCFC‚s elicited positive results. Overall, 8 reactions to other foods were diagnosed in the four patients with confirmed orange allergy. The foods associated were melon (4 patients), watermelon (2 patients) and banana (2 patients). All orange allergic patients tolerated grapefruit and lemon. All patients had seasonal rhinitis and/or asthma, and positive results of skin test to several pollens. Conclusions: Less than one third of reported reactions to orange are confirmed by DBPCFC. Isolated orange allergy seems to be infrequent, with all patients having clinical allergy to other foods, particularly to melon, and pollinosis.

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[2] - Raby P, Alonso R, Basagana M, Cistero-Bahima A. Orange allergy in a latex-allergic patient. Allergy 2007;62(suppl. 83):515

Background: Almost 45% of latex˜allergic patients present allergy to at least one fruit: banana (24%), avocado (24%), chestnut (22%) and kiwi (20%). The most important allergens responsible of the latex˜fruit syndrome are class I chitinases but they are not the only ones. An LTP of latex has also been described (Hev b 12). Citrus are clinically important allergic fruits but its incidence seems to be low. To our knowledge, citrus are not involved in the latex˜fruit syndrome. Orange major allergens are a germine˜like protein (Cit s 1) and a profiline (Cit s 2) but members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30˜50% of the allergic patients. Both orange, lemon and latex allergens show cross˜reactivity with the major peach allergen Pru p 3. Recently, a study of 15 patients with orange allergy (66% had specific IgE to germine˜like protein, 60% to profiline and 46% to LTPs) has demonstrated in vitro sensitization to latex in 9 of them. Case report: A 39˜year˜old woman with latex allergy since the age of 25 years attended our Allergy Department. She works as a podologist since 1995, three years before the onset of the symptoms. Five years after the onset of her latex allergy she presented cough after eating a kiwi and recently she suffered 2 anaphylactic reactions after ingestion of tangerine and orange juice. Results: Skin prick tests to usual airborne allergens and food extracts (ALK˜Abelló, Madrid, Spain) were positive to mites, latex and kiwi. All pollens were negative. Prick˜by˜prick with orange, tangerine, lemon and grapefruit juice, yielded an immediate positive wheal. Common blood parameters were within normal limits, including total serum IgE which was 41 KU/L. Serum specific˜IgE levels (Pharmacia, CAP System) were positive to Latex (1.36 KU/L), Kiwi (1.04 KU/L), and negative to Dermatophagoides pteronyssinus, Orange, Tangerine, Grapefruit and Lemon. An oral challenge with orange juice was positive with a systemic reaction after few minutes of the last dose. Conclusions: We present a case of latex allergy with cross˜reactivity with kiwi that presented in the evolution an anaphylactic reaction to tangerine and orange, demonstrated with an oral provocation test. We believe that this is due to a possible cross˜reactivity between citrus fruits and latex.

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[3] - Morimoto K, Tanaka T, Sugita Y, Hide M. Food-dependent exercise-induced anaphylaxis due to ingestion of orange. Acta Derm Venereol 2004;84:152-153

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[4] - Kim Y, Seo Y, Park H, Shim H, Lee K, Son D, et al. A case of anaphylaxis to orange juice products. ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° P139

Objectives : Anaphylaxis is one of urgent, life-threatening conditions associated with allergic reactions, and food is the leading cause of anaphylaxis in children. Recently we experienced a 10 year-old boy who showed anaphylactic reaction to orange, which is uncommon cause of food allergy. We attempted to investigate the allergenicity of orange. Methods : Blood was obtained from the patient with anaphylaxis to orange juice products. The diagnosis was confirmed by open food challenge test, in which urticaria, wheezing and hypotension developed within 15minutes after ingestion of a total of 400 mL of orange juice, although serum specific Ig E to orange was negative by CAP test. Proteins were extracted from crushed juice of fresh orange and from orange juice of 4 different manufacturers. The allergen in orange juice products was detected by SDS-PAGE and Western blot using protein extract from orange juice and the patient‚s serum specific Ig E. Results : The protein concentration of orange juice was 1.71 mg/mL from crushed juice from fresh orange and those from 4 different manufacturers ranged from 0.98 mg/mL to 1.33 mg/mL. More than 10 protein bands were observed from 10 kDa to 100 kDa in size in SDS-PAGE. Western blot showed 70 kDa-sized protein band, which was found in all the extracts regardless of whether it is from fresh orange or from commercially available orange juice. Our result suggested that allergen in orange juice products was derived from orange itself, not from the food additives. Conclusions : We presented a case of anaphylaxis to orange juice products, which might be caused by 70 kDa-sized protein in orange. However, we did not confirm whether this allergen was contained in peel, pulp, or seed of orange. Attention to orange should be paid for the risk of anaphylaxis, although it is not common.

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[5] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[6] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

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[7] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[8] - Lopez-Torrejon G, Ibañez MD, Ahrazem O, Sanchez-Monge R, Sastre J, Lombardero M, et al. Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. Allergy 2005;60:1424-1429

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables . METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays . RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract . CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

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[9] - Ebo DG, Ahrazem O, Lopez-Torrejon G, Bridts CH, Salcedo G, Stevens WJ. Anaphylaxis from Mandarin (Citrus reticulata) : Identification of Potential Responsible Allergens. Int Arch Allergy Immunol 2007;144:39-43

We report on a patient with anaphylaxis from mandarin. Temporal relationship between consumption of the fruit, the presence of positive specific IgE, the positive skin test and the basophil activation test for mandarin strongly supported the diagnosis of an IgE-mediated allergy from mandarin. The lipid transfer protein allergen from mandarin fruit was isolated and characterized. Specific IgE levels and IgE immunodetection data indicated the patient's sensitization to orange (Cit s 3) and mandarin (Cit r 3) lipid transfer protein allergens, as well as to germin-like (Cit s 1) allergen. These results were fully confirmed by skin prick test and basophil activation test (BAT) for lipid transfer proteins, and a BAT for Cit s 1. This case report has several particularities. First, in Central and Northern Europe, it is not widely appreciated that citrus fruits, particularly mandarin, can elicit anaphylaxis. Second, this case report re-emphasizes sensitization from lipid transfer proteins to predispose for severe allergic reactions. Finally, it provides an opportunity to summarize the applications of flow cytometry-assisted analysis and quantification of in vitro activated basophils in the diagnostic approach of anaphylaxis from food.

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[10] - Sierra EM, Pascual C, Thomas E, Bernal C, Martín Esteban M. Allergy to citrus in children. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°987

Background Citrus are a group of fruits from Rutaceae family. Despite its frequent consumption, there are not many documented cases of allergy to citrus fruits. Methods We evaluated a sample of patients with suggestive symptoms of allergy to citrus fruits, selected between all new studied patients by food allergy, under fifteen years old, from year 1990 to the present time. The patients were evaluated by means of clinical history, physical examination, skin prick test with fresh material (prick by prick), commercial extracts (prick) and specific IgE (CAP). Information was obtained about onset age of symptoms with citrus fruits, implicated citrus fruits, other allergic diseases and sensititazion to other food. Results The studied children with food allergy were 3870 and 59 children, 43 boys and 16 girls, aged of 4 months to 14 years (median age 2 years), were selected with suggestive clinical history of allergy to citrus fruits. Out of 59 patients, 56,9% had atopic familiar history, 50% atopic eczema dermatitis syndrome and 55,9% pollinosis. Out of 59 children, 39,7% presented oral allergy syndrome, 24,1% urticaria and/or angioedema, 19% atopic eczema dermatitis syndrome and 10,3% anafilactic reaction. The type of implicated citrus fruits in allergic reactions was: orange (81,4%), tangerine (13,6%) and lemon (5,1%). IgE mediated hipersensitivity to citrus fruits was confirmed in 37 children (group A), that represent the 0,9% of the 3870 selected children, and not confirmed in group B. The sensititazion to other food was: Group A: milk (30%), egg (40%), fish (30%), shellfish (13%), fresh fruit (84%), nuts (65%), legumes (35%), vegetables (46%) and, Group B: milk (32%), egg (54%), fish (14%), shellfish (9%), fresh fruit (36%), nuts (32%), legumes (4%), vegetables (18%); comparing the results of both groups, it is observed that the presence of sensitivity to other fresh fruits, nuts, vegetables and legumes showed statistically significative differences, being more frequent in group A. The association with pollinosis was of 67,6% in group A and of 27,3% in group B (p= 0.003). Conclusion 1. Despite of its wide consumption, the incidence of allergy to citrus fruits is very low in Spain. 2. The allergy to citrus fruits is associated with sensititazion to other vegetable food. 3. The presence of pollinosis is more frequent in group of patients with confirmed allergy to citrus fruits than in the group with not confirmed allergy.

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[11] - Sierra EM, Pascual C, Thomas E, Bernal C, Martín Esteban M. Allergy to citrus in children. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°987

Background Citrus are a group of fruits from Rutaceae family. Despite its frequent consumption, there are not many documented cases of allergy to citrus fruits. Methods We evaluated a sample of patients with suggestive symptoms of allergy to citrus fruits, selected between all new studied patients by food allergy, under fifteen years old, from year 1990 to the present time. The patients were evaluated by means of clinical history, physical examination, skin prick test with fresh material (prick by prick), commercial extracts (prick) and specific IgE (CAP). Information was obtained about onset age of symptoms with citrus fruits, implicated citrus fruits, other allergic diseases and sensititazion to other food. Results The studied children with food allergy were 3870 and 59 children, 43 boys and 16 girls, aged of 4 months to 14 years (median age 2 years), were selected with suggestive clinical history of allergy to citrus fruits. Out of 59 patients, 56,9% had atopic familiar history, 50% atopic eczema dermatitis syndrome and 55,9% pollinosis. Out of 59 children, 39,7% presented oral allergy syndrome, 24,1% urticaria and/or angioedema, 19% atopic eczema dermatitis syndrome and 10,3% anafilactic reaction. The type of implicated citrus fruits in allergic reactions was: orange (81,4%), tangerine (13,6%) and lemon (5,1%). IgE mediated hipersensitivity to citrus fruits was confirmed in 37 children (group A), that represent the 0,9% of the 3870 selected children, and not confirmed in group B. The sensititazion to other food was: Group A: milk (30%), egg (40%), fish (30%), shellfish (13%), fresh fruit (84%), nuts (65%), legumes (35%), vegetables (46%) and, Group B: milk (32%), egg (54%), fish (14%), shellfish (9%), fresh fruit (36%), nuts (32%), legumes (4%), vegetables (18%); comparing the results of both groups, it is observed that the presence of sensitivity to other fresh fruits, nuts, vegetables and legumes showed statistically significative differences, being more frequent in group A. The association with pollinosis was of 67,6% in group A and of 27,3% in group B (p= 0.003). Conclusion 1. Despite of its wide consumption, the incidence of allergy to citrus fruits is very low in Spain. 2. The allergy to citrus fruits is associated with sensititazion to other vegetable food. 3. The presence of pollinosis is more frequent in group of patients with confirmed allergy to citrus fruits than in the group with not confirmed allergy.

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[12] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[13] - Raby P, Alonso R, Basagana M, Cistero-Bahima A. Orange allergy in a latex-allergic patient. Allergy 2007;62(suppl. 83):515

Background: Almost 45% of latex˜allergic patients present allergy to at least one fruit: banana (24%), avocado (24%), chestnut (22%) and kiwi (20%). The most important allergens responsible of the latex˜fruit syndrome are class I chitinases but they are not the only ones. An LTP of latex has also been described (Hev b 12). Citrus are clinically important allergic fruits but its incidence seems to be low. To our knowledge, citrus are not involved in the latex˜fruit syndrome. Orange major allergens are a germine˜like protein (Cit s 1) and a profiline (Cit s 2) but members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30˜50% of the allergic patients. Both orange, lemon and latex allergens show cross˜reactivity with the major peach allergen Pru p 3. Recently, a study of 15 patients with orange allergy (66% had specific IgE to germine˜like protein, 60% to profiline and 46% to LTPs) has demonstrated in vitro sensitization to latex in 9 of them. Case report: A 39˜year˜old woman with latex allergy since the age of 25 years attended our Allergy Department. She works as a podologist since 1995, three years before the onset of the symptoms. Five years after the onset of her latex allergy she presented cough after eating a kiwi and recently she suffered 2 anaphylactic reactions after ingestion of tangerine and orange juice. Results: Skin prick tests to usual airborne allergens and food extracts (ALK˜Abelló, Madrid, Spain) were positive to mites, latex and kiwi. All pollens were negative. Prick˜by˜prick with orange, tangerine, lemon and grapefruit juice, yielded an immediate positive wheal. Common blood parameters were within normal limits, including total serum IgE which was 41 KU/L. Serum specific˜IgE levels (Pharmacia, CAP System) were positive to Latex (1.36 KU/L), Kiwi (1.04 KU/L), and negative to Dermatophagoides pteronyssinus, Orange, Tangerine, Grapefruit and Lemon. An oral challenge with orange juice was positive with a systemic reaction after few minutes of the last dose. Conclusions: We present a case of latex allergy with cross˜reactivity with kiwi that presented in the evolution an anaphylactic reaction to tangerine and orange, demonstrated with an oral provocation test. We believe that this is due to a possible cross˜reactivity between citrus fruits and latex.

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[14] - Sierra EM, Pascual C, Thomas E, Bernal C, Martín Esteban M. Allergy to citrus in children. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°987

Background Citrus are a group of fruits from Rutaceae family. Despite its frequent consumption, there are not many documented cases of allergy to citrus fruits. Methods We evaluated a sample of patients with suggestive symptoms of allergy to citrus fruits, selected between all new studied patients by food allergy, under fifteen years old, from year 1990 to the present time. The patients were evaluated by means of clinical history, physical examination, skin prick test with fresh material (prick by prick), commercial extracts (prick) and specific IgE (CAP). Information was obtained about onset age of symptoms with citrus fruits, implicated citrus fruits, other allergic diseases and sensititazion to other food. Results The studied children with food allergy were 3870 and 59 children, 43 boys and 16 girls, aged of 4 months to 14 years (median age 2 years), were selected with suggestive clinical history of allergy to citrus fruits. Out of 59 patients, 56,9% had atopic familiar history, 50% atopic eczema dermatitis syndrome and 55,9% pollinosis. Out of 59 children, 39,7% presented oral allergy syndrome, 24,1% urticaria and/or angioedema, 19% atopic eczema dermatitis syndrome and 10,3% anafilactic reaction. The type of implicated citrus fruits in allergic reactions was: orange (81,4%), tangerine (13,6%) and lemon (5,1%). IgE mediated hipersensitivity to citrus fruits was confirmed in 37 children (group A), that represent the 0,9% of the 3870 selected children, and not confirmed in group B. The sensititazion to other food was: Group A: milk (30%), egg (40%), fish (30%), shellfish (13%), fresh fruit (84%), nuts (65%), legumes (35%), vegetables (46%) and, Group B: milk (32%), egg (54%), fish (14%), shellfish (9%), fresh fruit (36%), nuts (32%), legumes (4%), vegetables (18%); comparing the results of both groups, it is observed that the presence of sensitivity to other fresh fruits, nuts, vegetables and legumes showed statistically significative differences, being more frequent in group A. The association with pollinosis was of 67,6% in group A and of 27,3% in group B (p= 0.003). Conclusion 1. Despite of its wide consumption, the incidence of allergy to citrus fruits is very low in Spain. 2. The allergy to citrus fruits is associated with sensititazion to other vegetable food. 3. The presence of pollinosis is more frequent in group of patients with confirmed allergy to citrus fruits than in the group with not confirmed allergy.

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[15] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[16] - Ebo DG, Ahrazem O, Lopez-Torrejon G, Bridts CH, Salcedo G, Stevens WJ. Anaphylaxis from Mandarin (Citrus reticulata) : Identification of Potential Responsible Allergens. Int Arch Allergy Immunol 2007;144:39-43

We report on a patient with anaphylaxis from mandarin. Temporal relationship between consumption of the fruit, the presence of positive specific IgE, the positive skin test and the basophil activation test for mandarin strongly supported the diagnosis of an IgE-mediated allergy from mandarin. The lipid transfer protein allergen from mandarin fruit was isolated and characterized. Specific IgE levels and IgE immunodetection data indicated the patient's sensitization to orange (Cit s 3) and mandarin (Cit r 3) lipid transfer protein allergens, as well as to germin-like (Cit s 1) allergen. These results were fully confirmed by skin prick test and basophil activation test (BAT) for lipid transfer proteins, and a BAT for Cit s 1. This case report has several particularities. First, in Central and Northern Europe, it is not widely appreciated that citrus fruits, particularly mandarin, can elicit anaphylaxis. Second, this case report re-emphasizes sensitization from lipid transfer proteins to predispose for severe allergic reactions. Finally, it provides an opportunity to summarize the applications of flow cytometry-assisted analysis and quantification of in vitro activated basophils in the diagnostic approach of anaphylaxis from food.

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[17] - Raby P, Alonso R, Basagana M, Cistero-Bahima A. Orange allergy in a latex-allergic patient. Allergy 2007;62(suppl. 83):515

Background: Almost 45% of latex˜allergic patients present allergy to at least one fruit: banana (24%), avocado (24%), chestnut (22%) and kiwi (20%). The most important allergens responsible of the latex˜fruit syndrome are class I chitinases but they are not the only ones. An LTP of latex has also been described (Hev b 12). Citrus are clinically important allergic fruits but its incidence seems to be low. To our knowledge, citrus are not involved in the latex˜fruit syndrome. Orange major allergens are a germine˜like protein (Cit s 1) and a profiline (Cit s 2) but members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30˜50% of the allergic patients. Both orange, lemon and latex allergens show cross˜reactivity with the major peach allergen Pru p 3. Recently, a study of 15 patients with orange allergy (66% had specific IgE to germine˜like protein, 60% to profiline and 46% to LTPs) has demonstrated in vitro sensitization to latex in 9 of them. Case report: A 39˜year˜old woman with latex allergy since the age of 25 years attended our Allergy Department. She works as a podologist since 1995, three years before the onset of the symptoms. Five years after the onset of her latex allergy she presented cough after eating a kiwi and recently she suffered 2 anaphylactic reactions after ingestion of tangerine and orange juice. Results: Skin prick tests to usual airborne allergens and food extracts (ALK˜Abelló, Madrid, Spain) were positive to mites, latex and kiwi. All pollens were negative. Prick˜by˜prick with orange, tangerine, lemon and grapefruit juice, yielded an immediate positive wheal. Common blood parameters were within normal limits, including total serum IgE which was 41 KU/L. Serum specific˜IgE levels (Pharmacia, CAP System) were positive to Latex (1.36 KU/L), Kiwi (1.04 KU/L), and negative to Dermatophagoides pteronyssinus, Orange, Tangerine, Grapefruit and Lemon. An oral challenge with orange juice was positive with a systemic reaction after few minutes of the last dose. Conclusions: We present a case of latex allergy with cross˜reactivity with kiwi that presented in the evolution an anaphylactic reaction to tangerine and orange, demonstrated with an oral provocation test. We believe that this is due to a possible cross˜reactivity between citrus fruits and latex.

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[18] - Kim Y, Seo Y, Park H, Shim H, Lee K, Son D, et al. A case of anaphylaxis to orange juice products. ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° P139

Objectives : Anaphylaxis is one of urgent, life-threatening conditions associated with allergic reactions, and food is the leading cause of anaphylaxis in children. Recently we experienced a 10 year-old boy who showed anaphylactic reaction to orange, which is uncommon cause of food allergy. We attempted to investigate the allergenicity of orange. Methods : Blood was obtained from the patient with anaphylaxis to orange juice products. The diagnosis was confirmed by open food challenge test, in which urticaria, wheezing and hypotension developed within 15minutes after ingestion of a total of 400 mL of orange juice, although serum specific Ig E to orange was negative by CAP test. Proteins were extracted from crushed juice of fresh orange and from orange juice of 4 different manufacturers. The allergen in orange juice products was detected by SDS-PAGE and Western blot using protein extract from orange juice and the patient‚s serum specific Ig E. Results : The protein concentration of orange juice was 1.71 mg/mL from crushed juice from fresh orange and those from 4 different manufacturers ranged from 0.98 mg/mL to 1.33 mg/mL. More than 10 protein bands were observed from 10 kDa to 100 kDa in size in SDS-PAGE. Western blot showed 70 kDa-sized protein band, which was found in all the extracts regardless of whether it is from fresh orange or from commercially available orange juice. Our result suggested that allergen in orange juice products was derived from orange itself, not from the food additives. Conclusions : We presented a case of anaphylaxis to orange juice products, which might be caused by 70 kDa-sized protein in orange. However, we did not confirm whether this allergen was contained in peel, pulp, or seed of orange. Attention to orange should be paid for the risk of anaphylaxis, although it is not common.

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[19] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[20] - 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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[21] - Raby P, Alonso R, Basagana M, Cistero-Bahima A. Orange allergy in a latex-allergic patient. Allergy 2007;62(suppl. 83):515

Background: Almost 45% of latex˜allergic patients present allergy to at least one fruit: banana (24%), avocado (24%), chestnut (22%) and kiwi (20%). The most important allergens responsible of the latex˜fruit syndrome are class I chitinases but they are not the only ones. An LTP of latex has also been described (Hev b 12). Citrus are clinically important allergic fruits but its incidence seems to be low. To our knowledge, citrus are not involved in the latex˜fruit syndrome. Orange major allergens are a germine˜like protein (Cit s 1) and a profiline (Cit s 2) but members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30˜50% of the allergic patients. Both orange, lemon and latex allergens show cross˜reactivity with the major peach allergen Pru p 3. Recently, a study of 15 patients with orange allergy (66% had specific IgE to germine˜like protein, 60% to profiline and 46% to LTPs) has demonstrated in vitro sensitization to latex in 9 of them. Case report: A 39˜year˜old woman with latex allergy since the age of 25 years attended our Allergy Department. She works as a podologist since 1995, three years before the onset of the symptoms. Five years after the onset of her latex allergy she presented cough after eating a kiwi and recently she suffered 2 anaphylactic reactions after ingestion of tangerine and orange juice. Results: Skin prick tests to usual airborne allergens and food extracts (ALK˜Abelló, Madrid, Spain) were positive to mites, latex and kiwi. All pollens were negative. Prick˜by˜prick with orange, tangerine, lemon and grapefruit juice, yielded an immediate positive wheal. Common blood parameters were within normal limits, including total serum IgE which was 41 KU/L. Serum specific˜IgE levels (Pharmacia, CAP System) were positive to Latex (1.36 KU/L), Kiwi (1.04 KU/L), and negative to Dermatophagoides pteronyssinus, Orange, Tangerine, Grapefruit and Lemon. An oral challenge with orange juice was positive with a systemic reaction after few minutes of the last dose. Conclusions: We present a case of latex allergy with cross˜reactivity with kiwi that presented in the evolution an anaphylactic reaction to tangerine and orange, demonstrated with an oral provocation test. We believe that this is due to a possible cross˜reactivity between citrus fruits and latex.

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[22] - Kim Y, Seo Y, Park H, Shim H, Lee K, Son D, et al. A case of anaphylaxis to orange juice products. ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° P139

Objectives : Anaphylaxis is one of urgent, life-threatening conditions associated with allergic reactions, and food is the leading cause of anaphylaxis in children. Recently we experienced a 10 year-old boy who showed anaphylactic reaction to orange, which is uncommon cause of food allergy. We attempted to investigate the allergenicity of orange. Methods : Blood was obtained from the patient with anaphylaxis to orange juice products. The diagnosis was confirmed by open food challenge test, in which urticaria, wheezing and hypotension developed within 15minutes after ingestion of a total of 400 mL of orange juice, although serum specific Ig E to orange was negative by CAP test. Proteins were extracted from crushed juice of fresh orange and from orange juice of 4 different manufacturers. The allergen in orange juice products was detected by SDS-PAGE and Western blot using protein extract from orange juice and the patient‚s serum specific Ig E. Results : The protein concentration of orange juice was 1.71 mg/mL from crushed juice from fresh orange and those from 4 different manufacturers ranged from 0.98 mg/mL to 1.33 mg/mL. More than 10 protein bands were observed from 10 kDa to 100 kDa in size in SDS-PAGE. Western blot showed 70 kDa-sized protein band, which was found in all the extracts regardless of whether it is from fresh orange or from commercially available orange juice. Our result suggested that allergen in orange juice products was derived from orange itself, not from the food additives. Conclusions : We presented a case of anaphylaxis to orange juice products, which might be caused by 70 kDa-sized protein in orange. However, we did not confirm whether this allergen was contained in peel, pulp, or seed of orange. Attention to orange should be paid for the risk of anaphylaxis, although it is not common.

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[23] - Ebo DG, Ahrazem O, Lopez-Torrejon G, Bridts CH, Salcedo G, Stevens WJ. Anaphylaxis from Mandarin (Citrus reticulata) : Identification of Potential Responsible Allergens. Int Arch Allergy Immunol 2007;144:39-43

We report on a patient with anaphylaxis from mandarin. Temporal relationship between consumption of the fruit, the presence of positive specific IgE, the positive skin test and the basophil activation test for mandarin strongly supported the diagnosis of an IgE-mediated allergy from mandarin. The lipid transfer protein allergen from mandarin fruit was isolated and characterized. Specific IgE levels and IgE immunodetection data indicated the patient's sensitization to orange (Cit s 3) and mandarin (Cit r 3) lipid transfer protein allergens, as well as to germin-like (Cit s 1) allergen. These results were fully confirmed by skin prick test and basophil activation test (BAT) for lipid transfer proteins, and a BAT for Cit s 1. This case report has several particularities. First, in Central and Northern Europe, it is not widely appreciated that citrus fruits, particularly mandarin, can elicit anaphylaxis. Second, this case report re-emphasizes sensitization from lipid transfer proteins to predispose for severe allergic reactions. Finally, it provides an opportunity to summarize the applications of flow cytometry-assisted analysis and quantification of in vitro activated basophils in the diagnostic approach of anaphylaxis from food.

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[24] - Morimoto K, Tanaka T, Sugita Y, Hide M. Food-dependent exercise-induced anaphylaxis due to ingestion of orange. Acta Derm Venereol 2004;84:152-153

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[25] - Kim Y, Seo Y, Park H, Shim H, Lee K, Son D, et al. A case of anaphylaxis to orange juice products. ACAAI Annual Meeting, New Orleans, 7-12 Nov. 2003, Poster n° P139

Objectives : Anaphylaxis is one of urgent, life-threatening conditions associated with allergic reactions, and food is the leading cause of anaphylaxis in children. Recently we experienced a 10 year-old boy who showed anaphylactic reaction to orange, which is uncommon cause of food allergy. We attempted to investigate the allergenicity of orange. Methods : Blood was obtained from the patient with anaphylaxis to orange juice products. The diagnosis was confirmed by open food challenge test, in which urticaria, wheezing and hypotension developed within 15minutes after ingestion of a total of 400 mL of orange juice, although serum specific Ig E to orange was negative by CAP test. Proteins were extracted from crushed juice of fresh orange and from orange juice of 4 different manufacturers. The allergen in orange juice products was detected by SDS-PAGE and Western blot using protein extract from orange juice and the patient‚s serum specific Ig E. Results : The protein concentration of orange juice was 1.71 mg/mL from crushed juice from fresh orange and those from 4 different manufacturers ranged from 0.98 mg/mL to 1.33 mg/mL. More than 10 protein bands were observed from 10 kDa to 100 kDa in size in SDS-PAGE. Western blot showed 70 kDa-sized protein band, which was found in all the extracts regardless of whether it is from fresh orange or from commercially available orange juice. Our result suggested that allergen in orange juice products was derived from orange itself, not from the food additives. Conclusions : We presented a case of anaphylaxis to orange juice products, which might be caused by 70 kDa-sized protein in orange. However, we did not confirm whether this allergen was contained in peel, pulp, or seed of orange. Attention to orange should be paid for the risk of anaphylaxis, although it is not common.

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[26] - Sierra EM, Pascual C, Thomas E, Bernal C, Martín Esteban M. Allergy to citrus in children. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°987

Background Citrus are a group of fruits from Rutaceae family. Despite its frequent consumption, there are not many documented cases of allergy to citrus fruits. Methods We evaluated a sample of patients with suggestive symptoms of allergy to citrus fruits, selected between all new studied patients by food allergy, under fifteen years old, from year 1990 to the present time. The patients were evaluated by means of clinical history, physical examination, skin prick test with fresh material (prick by prick), commercial extracts (prick) and specific IgE (CAP). Information was obtained about onset age of symptoms with citrus fruits, implicated citrus fruits, other allergic diseases and sensititazion to other food. Results The studied children with food allergy were 3870 and 59 children, 43 boys and 16 girls, aged of 4 months to 14 years (median age 2 years), were selected with suggestive clinical history of allergy to citrus fruits. Out of 59 patients, 56,9% had atopic familiar history, 50% atopic eczema dermatitis syndrome and 55,9% pollinosis. Out of 59 children, 39,7% presented oral allergy syndrome, 24,1% urticaria and/or angioedema, 19% atopic eczema dermatitis syndrome and 10,3% anafilactic reaction. The type of implicated citrus fruits in allergic reactions was: orange (81,4%), tangerine (13,6%) and lemon (5,1%). IgE mediated hipersensitivity to citrus fruits was confirmed in 37 children (group A), that represent the 0,9% of the 3870 selected children, and not confirmed in group B. The sensititazion to other food was: Group A: milk (30%), egg (40%), fish (30%), shellfish (13%), fresh fruit (84%), nuts (65%), legumes (35%), vegetables (46%) and, Group B: milk (32%), egg (54%), fish (14%), shellfish (9%), fresh fruit (36%), nuts (32%), legumes (4%), vegetables (18%); comparing the results of both groups, it is observed that the presence of sensitivity to other fresh fruits, nuts, vegetables and legumes showed statistically significative differences, being more frequent in group A. The association with pollinosis was of 67,6% in group A and of 27,3% in group B (p= 0.003). Conclusion 1. Despite of its wide consumption, the incidence of allergy to citrus fruits is very low in Spain. 2. The allergy to citrus fruits is associated with sensititazion to other vegetable food. 3. The presence of pollinosis is more frequent in group of patients with confirmed allergy to citrus fruits than in the group with not confirmed allergy.

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[27] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[28] - Lopez-Torrejon G, Ibañez MD, Ahrazem O, Sanchez-Monge R, Sastre J, Lombardero M, et al. Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. Allergy 2005;60:1424-1429

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables . METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays . RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract . CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

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[29] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[30] - Glaspole IN, de Leon MP, Rolland JM, O'Hehir RE. Anaphylaxis to lemon soap: citrus seed and peanut allergen cross-reactivity. Ann Allergy Asthma Immunol 2007;98:286-289

BACKGROUND: Many individuals allergic to peanuts have multiple allergen sensitivity. OBJECTIVE: To report the first case, to our knowledge, of a peanut allergic patient who exhibited cosensitivity to citrus seeds and who had experienced anaphylaxis to lemon soap. METHODS: Extracts of peanut and seeds from different varieties of citrus fruit (orange, lemon, and mandarin) were prepared and resolved with 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Direct and inhibition immunoblotting of the patient's serum on the extracts was used to examine the pattern of IgE reactivity and the presence of cross-reactive allergens. RESULTS: Numerous IgE reactive proteins were demonstrated in each citrus seed extract and the peanut extract. Complete IgE cross-reactivity was demonstrated among the different citrus seed extracts. Partial cross-reactivity was demonstrated between the peanut and orange seed extracts. CONCLUSIONS: Citrus seeds contain numerous IgE reactive proteins that are completely cross-reactive among orange, lemon, and mandarin. When peanut allergy coexists with citrus seed allergy, IgE cross-reactivity between peanut and citrus seed proteins can be demonstrated, suggesting a basis to this cosensitivity.

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[31] - Wang ET. Anaphylaxis caused by tangerine seeds but not tangerine fruit. Ann Allergy Asthma Immunol 2008;101:553-554

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[32] - Irañeta SG, Seoane MA, Laucella SA, Apicella C, Alonso A, Duschak VG. Antigenicity and Immunocrossreactivity of Orange Tree Pollen and Orange Fruit Allergenic Extracts. Int Arch Allergy Immunol 2005;137:265-272

BACKGROUND: It is important to study the crossreactivity between orange tree pollen (OTPE) and orange fruit (OFE) due to the high incidence of pollen/food-related allergies worldwide. The aim of the present study was to determine the antigenic relationship between OTPE and OFE . METHODS: OTPE and OFErabbit antisera as well as sera from patients allergic to OTPE and OFE were comparatively applied in IgG- and/or IgE-specific ELISA inhibition, crossover or inhibition immunoblotting assays using OTPE and OFE allergenic extracts as solid phase. Periodate and proteinase K treatments were used for carbohydrate and protein depletion, respectively . RESULTS: The antigenicity of OTPE and the presence of common structures between OTPE and OFE extracts were demonstrated by rabbit IgG-specific ELISA inhibition and crossover immunoblotting assays. A 30-kDa protein, common target of the IgE response on OTPE, OFE and mandarin extract, but absent in lemon extract, was identified by ELISA and immunoblot inhibition assays in patients suffering from primary sensitization to OTPE in the context of occupational exposure. Moreover, biochemical treatments showed that antigenic epitopes on the 30-kDa protein contain polypeptidic but no carbohydrate moieties . CONCLUSIONS: The antigenicity of OTPE, the presence of common antigenic determinants between pollen and citrus fruits and an IgE-specific crossreactive protein band of 30 kDa sharing carbohydrate-free epitopes were described. After isolation and purification, this common antigen might be useful for allergen immunotherapy in pollen/fruit-related allergic patients.

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[33] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[34] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

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[35] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[36] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[37] - Lopez-Torrejon G, Ibañez MD, Ahrazem O, Sanchez-Monge R, Sastre J, Lombardero M, et al. Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. Allergy 2005;60:1424-1429

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables . METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays . RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract . CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

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[38] - Lopez-Torrejon G, Ibañez MD, Ahrazem O, Sanchez-Monge R, Sastre J, Lombardero M, et al. Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. Allergy 2005;60:1424-1429

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables . METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays . RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract . CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

↩

[39] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

↩

[40] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[41] - Ebo DG, Ahrazem O, Lopez-Torrejon G, Bridts CH, Salcedo G, Stevens WJ. Anaphylaxis from Mandarin (Citrus reticulata) : Identification of Potential Responsible Allergens. Int Arch Allergy Immunol 2007;144:39-43

We report on a patient with anaphylaxis from mandarin. Temporal relationship between consumption of the fruit, the presence of positive specific IgE, the positive skin test and the basophil activation test for mandarin strongly supported the diagnosis of an IgE-mediated allergy from mandarin. The lipid transfer protein allergen from mandarin fruit was isolated and characterized. Specific IgE levels and IgE immunodetection data indicated the patient's sensitization to orange (Cit s 3) and mandarin (Cit r 3) lipid transfer protein allergens, as well as to germin-like (Cit s 1) allergen. These results were fully confirmed by skin prick test and basophil activation test (BAT) for lipid transfer proteins, and a BAT for Cit s 1. This case report has several particularities. First, in Central and Northern Europe, it is not widely appreciated that citrus fruits, particularly mandarin, can elicit anaphylaxis. Second, this case report re-emphasizes sensitization from lipid transfer proteins to predispose for severe allergic reactions. Finally, it provides an opportunity to summarize the applications of flow cytometry-assisted analysis and quantification of in vitro activated basophils in the diagnostic approach of anaphylaxis from food.

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[42] - Pöltl G, Ahrazem O, Paschinger K, Ibanez MD, Salcedo G, Wilson IB. Molecular and immunological characterization of the glycosylated orange allergen Cit s 1. Glycobiology 2007;17:220-230

The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) bind a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analysed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognise a range of glycoproteins and neoglycoconjugates containing beta1,2-xylose and core alpha1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients' sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients' IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1 using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein as compared to the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Due to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein, therefore, explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically-relevant allergen.

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[43] - Ahrazem O, Ibáñez MD, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Orange Germin-Like Glycoprotein Cit s 1: An Equivocal Allergen. Int Arch Allergy Immunol 2006;139:96-103

BACKGROUND: Orange allergens are virtually unknown, in spite of the large consumption of this fruit. Germin-like proteins, together with vicilins and legumins, form the cupin superfamily of plant proteins, which includes many seed allergens . METHODS: Twenty-nine patients with allergy to oranges were studied. A major IgE-binding protein from orange extracts was isolated by means of a two-step cation-exchange chromatographic protocol. The allergen was characterized by N-terminal amino acid sequencing and MALDI analysis, and its reactivity explored by specific IgE determination in individual sera, ELISA inhibition assays and in vivo skin prick tests (SPT). Chemical deglycosylation of the purified allergen was achieved by trifluoromethylsulfonate acid treatment . RESULTS: The 24-kDa purified allergen, designated Cit s 1, was identified as a germin-like glycoprotein, based on its N-terminal amino acid sequence, molecular size and recognition by rabbit anti-complex N-linked glycan antibodies. Specific IgE to Cit s 1 was detected in 62% of 29 individual sera from orange-allergic patients, whereas positive SPT responses to the purified allergen were obtained in only 10% of such patients. Deglycosylation of Cit s 1 resulted in a loss of its IgE-binding capacity. Moreover, the unrelated plant glycoprotein horseradish peroxidase inhibited over 70% the IgE-binding to Cit s 1 . CONCLUSIONS: Over 60% of patients with allergy to oranges show specific IgE to Cit s 1. However, the purified allergen exerts a low in vivo reactivity. Complex N-linked glycans seem to play a prominent role in the IgE-binding capacity of Cit s 1. This characteristic of Cit s 1, as well as of other orange glycoproteins, could lead to false positives if the diagnosis of allergy to oranges is mainly based on in vitro specific IgE determination.

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[44] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

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[45] - Pöltl G, Ahrazem O, Paschinger K, Ibanez MD, Salcedo G, Wilson IB. Molecular and immunological characterization of the glycosylated orange allergen Cit s 1. Glycobiology 2007;17:220-230

The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) bind a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analysed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognise a range of glycoproteins and neoglycoconjugates containing beta1,2-xylose and core alpha1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients' sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients' IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1 using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein as compared to the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Due to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein, therefore, explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically-relevant allergen.

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[46] - Karamloo F, Wangorsch A, Kasahara H, Davin LB, Haustein D, Lewis NG, et al. Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables. Eur J Biochem 2001;268:5310-5320

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.

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[47] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

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[48] - Smole U, Bublin M, Radauer C, Ebner C, Scheiner O, Breiteneder H. Identification of novel allergenic proteins from orange. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°515

Background and Aims: Orange (Citrus sinensis) is among the most widely consumed fruits. Previous studies carried out in Southern Europe showed the allergenic relevance of orange and identified three orange allergens. We aimed to characterise orange allergens involved in allergic reactions in the Central European population. Methods: Orange proteins were separated by 2D electrophoresis and chromatographic methods. Sera from 11 Austrian orange allergic patients were tested for their IgE binding to orange protein extract by IgE immunoblotting. Three IgE binding proteins were characterised by N-terminal sequencing. The relevance of N-glycosylation for IgE binding to orange allergens was tested by an ELISA inhibition assay with horseradish peroxidase that is carries N-linked glycans containing b-1,2-xylosyl and a-1,3-fucosyl residues. Results: IgE immunoblotting with individual sera revealed several IgE binding proteins of 12-33 kDa in the orange protein extract. Three of eleven patients' sera contained IgE reactive to a 17 kDa protein. One protein band with a molecular size of approximately 33 kDa reacted with 4 patients' sera and showed 100% N-terminal sequence identity to a glyoxalase from grapefruit (Citrus paradisi). A double-band of approximately 25 kDa was detected very strongly by 8 and with a lower intensity by another 2 patients' sera. Based on its N-terminal amino acid sequence, one protein could be identified as Cit s 1, a germin-like protein. The N-terminal sequence obtained for the second 25 kDa protein yielded 73% sequence identitiy to a chitinase from banana (Musa acuminata). IgE ELISA inhibition analysis of the identified IgE binding proteins confirmed that N-glycans carrying fucosyl and xylosyl residues contributed to the IgE binding. Conclusion: In this study, we present the identification of two novel orange allergens involved in the allergic reaction to oranges an Austrian population. The newly identified allergens were recognised by IgE of the majority of the tested sera from orange allergic patients.

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[49] - Raby P, Alonso R, Basagana M, Cistero-Bahima A. Orange allergy in a latex-allergic patient. Allergy 2007;62(suppl. 83):515

Background: Almost 45% of latex˜allergic patients present allergy to at least one fruit: banana (24%), avocado (24%), chestnut (22%) and kiwi (20%). The most important allergens responsible of the latex˜fruit syndrome are class I chitinases but they are not the only ones. An LTP of latex has also been described (Hev b 12). Citrus are clinically important allergic fruits but its incidence seems to be low. To our knowledge, citrus are not involved in the latex˜fruit syndrome. Orange major allergens are a germine˜like protein (Cit s 1) and a profiline (Cit s 2) but members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30˜50% of the allergic patients. Both orange, lemon and latex allergens show cross˜reactivity with the major peach allergen Pru p 3. Recently, a study of 15 patients with orange allergy (66% had specific IgE to germine˜like protein, 60% to profiline and 46% to LTPs) has demonstrated in vitro sensitization to latex in 9 of them. Case report: A 39˜year˜old woman with latex allergy since the age of 25 years attended our Allergy Department. She works as a podologist since 1995, three years before the onset of the symptoms. Five years after the onset of her latex allergy she presented cough after eating a kiwi and recently she suffered 2 anaphylactic reactions after ingestion of tangerine and orange juice. Results: Skin prick tests to usual airborne allergens and food extracts (ALK˜Abelló, Madrid, Spain) were positive to mites, latex and kiwi. All pollens were negative. Prick˜by˜prick with orange, tangerine, lemon and grapefruit juice, yielded an immediate positive wheal. Common blood parameters were within normal limits, including total serum IgE which was 41 KU/L. Serum specific˜IgE levels (Pharmacia, CAP System) were positive to Latex (1.36 KU/L), Kiwi (1.04 KU/L), and negative to Dermatophagoides pteronyssinus, Orange, Tangerine, Grapefruit and Lemon. An oral challenge with orange juice was positive with a systemic reaction after few minutes of the last dose. Conclusions: We present a case of latex allergy with cross˜reactivity with kiwi that presented in the evolution an anaphylactic reaction to tangerine and orange, demonstrated with an oral provocation test. We believe that this is due to a possible cross˜reactivity between citrus fruits and latex.

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[50] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

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[51] - Sierra EM, Pascual C, Thomas E, Bernal C, Martín Esteban M. Allergy to citrus in children. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°987

Background Citrus are a group of fruits from Rutaceae family. Despite its frequent consumption, there are not many documented cases of allergy to citrus fruits. Methods We evaluated a sample of patients with suggestive symptoms of allergy to citrus fruits, selected between all new studied patients by food allergy, under fifteen years old, from year 1990 to the present time. The patients were evaluated by means of clinical history, physical examination, skin prick test with fresh material (prick by prick), commercial extracts (prick) and specific IgE (CAP). Information was obtained about onset age of symptoms with citrus fruits, implicated citrus fruits, other allergic diseases and sensititazion to other food. Results The studied children with food allergy were 3870 and 59 children, 43 boys and 16 girls, aged of 4 months to 14 years (median age 2 years), were selected with suggestive clinical history of allergy to citrus fruits. Out of 59 patients, 56,9% had atopic familiar history, 50% atopic eczema dermatitis syndrome and 55,9% pollinosis. Out of 59 children, 39,7% presented oral allergy syndrome, 24,1% urticaria and/or angioedema, 19% atopic eczema dermatitis syndrome and 10,3% anafilactic reaction. The type of implicated citrus fruits in allergic reactions was: orange (81,4%), tangerine (13,6%) and lemon (5,1%). IgE mediated hipersensitivity to citrus fruits was confirmed in 37 children (group A), that represent the 0,9% of the 3870 selected children, and not confirmed in group B. The sensititazion to other food was: Group A: milk (30%), egg (40%), fish (30%), shellfish (13%), fresh fruit (84%), nuts (65%), legumes (35%), vegetables (46%) and, Group B: milk (32%), egg (54%), fish (14%), shellfish (9%), fresh fruit (36%), nuts (32%), legumes (4%), vegetables (18%); comparing the results of both groups, it is observed that the presence of sensitivity to other fresh fruits, nuts, vegetables and legumes showed statistically significative differences, being more frequent in group A. The association with pollinosis was of 67,6% in group A and of 27,3% in group B (p= 0.003). Conclusion 1. Despite of its wide consumption, the incidence of allergy to citrus fruits is very low in Spain. 2. The allergy to citrus fruits is associated with sensititazion to other vegetable food. 3. The presence of pollinosis is more frequent in group of patients with confirmed allergy to citrus fruits than in the group with not confirmed allergy.

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[52] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[53] - Lopez-Torrejon G, Ibañez MD, Ahrazem O, Sanchez-Monge R, Sastre J, Lombardero M, et al. Isolation, cloning and allergenic reactivity of natural profilin Cit s 2, a major orange allergen. Allergy 2005;60:1424-1429

BACKGROUND: Orange is among the most widely consumed fruits, and among the plant food sources causing allergic reactions according to popular perception. However, its relevant allergenic components are virtually unknown. Profilin is a well-defined minor plant panallergen, showing prevalences around 30% in fruits and vegetables . METHODS: Twenty-three orange-allergic patients were studied. Natural orange profilin, named Cit s 2, was purified by affinity chromatography and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization mass spectrometry analysis and isolation of its coding cDNA. Reactivity to Cit s 2 was analyzed in vivo by skin prick tests (SPT) and in vitro by IgE immunodetection, specific IgE determination in individual sera and enzyme-linked immunosorbent assay-inhibition assays . RESULTS: The N-terminal amino acid sequence and molecular mass of natural Cit s 2, both fully in agreement with the complete amino acid sequence deduced from its coding cDNA, demonstrated its profilin nature. An unexpectedly high reactivity to Cit s 2 was found in vivo (78% of positive SPT responses) and in vitro (87% of sera from orange allergic patients had specific IgE to Cit s 2). The purified allergen inhibited around 50% of the IgE binding to an orange pulp extract . CONCLUSION: Orange profilin Cit s 2, unlike other plant food profilins, is a major and highly prevalent allergen.

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[54] - Jiménez A, Campo P, Rico P, Feliu A, Somoza ML, Crespo JF, et al. Randomized, double-blind, crossover challenge study in adult subjects reporting adverse reactions to orange (Citrus sinensis). EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°133

Background: Orange is by far the most important commercial citrus fruit. World production of oranges represents over 82% of the world‚s total production of citrus fruit with Spain as one of the export leaders. However, at present, only anecdotal cases of orange allergy have been reported, based mainly on case histories and allergy testing. The purpose of this investigation was to assess the clinical features of acute allergic reactions to orange objectively confirmed by the double-blind placebo- controlled food challenge (DBPCFC) procedure. Methods: Fifteen patients (range = 13 - 67 yr.; median = 25 yr.) complaining of adverse reactions to orange were included in the study protocol of the Food Allergy Unit from 'Hospital Universitario 12 de Octubre'. Diagnostic procedures included a clinical questionnaire, skin testing using the prick-prick technique and detection of specific IgE (CAP- FEIA), which were performed with several members of Rutaceae family and other foods reported as offenders. Patients underwent an open food challenge (OFC), unless they had a convincing history of severe anaphylaxis. Positive OFC reactions were subsequently evaluated by DBPCFC‚s, up to an cumulative dose of 200 g of fresh fruit. All negative results of DBPCFCs were followed by an open feeding. Results: Ten and 11 patients had positive results of skin prick test and CAP-FEIA with orange, respectively. The most frequent symptom was the oral allergy syndrome (80%), followed by acute urticaria (13%) and gastrointestinal anaphylaxis (7%). All patients underwent an OFC with orange. Five of them were positive, but only 4 of the 5 performed DBPCFC‚s elicited positive results. Overall, 8 reactions to other foods were diagnosed in the four patients with confirmed orange allergy. The foods associated were melon (4 patients), watermelon (2 patients) and banana (2 patients). All orange allergic patients tolerated grapefruit and lemon. All patients had seasonal rhinitis and/or asthma, and positive results of skin test to several pollens. Conclusions: Less than one third of reported reactions to orange are confirmed by DBPCFC. Isolated orange allergy seems to be infrequent, with all patients having clinical allergy to other foods, particularly to melon, and pollinosis.

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[55] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[56] - Pauli G, Bessot JC, Braun PA, Dietemann-Molard A, Kopferschmitt-Kubler MC, Thierry R. Celery allergy: clinical and biological study of 20 cases. Ann Allergy 1988;60:243-246

In 20 patients, the ingestion of celery was responsible for mucocutaneous symptoms (generalized urticaria and angioedema) (18/20) and respiratory disorders (7/20). Four cases of systemic anaphylaxis were observed. The main associated allergic disorder was pollinosis (16/20). Food allergy to other vegetable products, mainly other Umbelliferae and apples, coexisted with celery allergy in 12 cases. It was found that celery allergy is mediated by IgE antibodies: it is easily diagnosed by skin tests (fresh extracts of celery may be used) and by adequate RAST (17 positive results). Cosensitization with mugwort pollen (14 cases) and birch pollen (9 cases) was found. Celery allergens responsible for clinical sensitization originate chiefly in the tuber and are at least partly thermally labile. The frequent association with pollen sensitization suggests the existence of common antigenic epitopes in celery extracts and mugwort and birch pollens. The immunologic investigations carried out so far (RAST inhibition and immunoprint) seem to support this hypothesis.

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[57] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[58] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

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[59] - Ahrazem O, Ibáñez D, López-Torrejón G, Sánchez-Monge R, Sastre J, Lombardero M, et al. Lipid Transfer Proteins and Allergy to Oranges. Int Arch Allergy Immunol 2005;137:201-210

BACKGROUND: Lipid transfer proteins (LTPs) are relevant fruit allergens, recently proposed as model plant food allergens. No citrus fruit allergen has been characterized to date. We sought to identify and isolate citrus fruit LTPs and to explore their relevance in orange allergy . METHODS: Twenty-seven patients, showing mainly oral allergy syndrome after orange ingestion, as well as positive prick responses and serum-specific IgE levels to orange, were selected. Natural orange and lemon LTPs, as well as a recombinant orange LTP isoform expressed in Pichia pastoris, were isolated by chromatographic methods and characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionizaion mass spectrometry, and DNA sequencing of the corresponding cDNA in the case of the recombinant allergen. Specific IgE determination, immunodetection, ELISA-inhibition assays and in vivo skin prick tests (SPTs) were performed with all three purified allergens and with the major peach LTP allergen, Pru p 3 . RESULTS: The natural allergens purified from orange (nCit s 3) and lemon (nCit l 3) showed very similar N-terminal amino acid sequences (18 out of 20 identical residues), typical of LTPs, and molecular masses of 9,610 and 9,618 Da, respectively. The recombinant orange isoform (rCit s 3) expressed in P. pastoris (16 out of 20 residues identical to its natural counterpart in the N-terminal region) presented 92 amino acid residues and 9,463 Da, and 67% sequence identity with rPru p 3. Of the 27 sera analyzed, specific IgE to the purified allergens was found in 54% for nCit l 3, 48% for nCit s 3, 46% for rCit s 3 and 37% for rPru p 3. Positive SPT responses were obtained in 7 out of 26 patients tested for nCit s 3, 3 out of 8 for nCit l 3 and 10 out of 26 for nPru p 3. ELISA-inhibition assays showed an equivalent IgE-binding pattern for the natural and recombinant orange LTPs, and IgE cross-reactivity among the purified orange, lemon and peach LTP allergens . CONCLUSIONS: Members of the LTP allergen family are involved in allergy to oranges, displaying positive in vitro and in vivo reactions in 30-50% of the patients studied. Both orange and lemon allergens show cross-reactivity with the major peach allergen Pru p 3.

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[60] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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[61] - Asero R, Mistrello G, Roncarolo D, Amato S, Zanoni D, Barocci F, et al. Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods. J Allergy Clin Immunol 2003;112:427-432

BACKGROUND: A proper classification of patients allergic to plant-derived foods is of pivotal importance because the clinical features of allergic reactions to fruits and vegetables depend on the nature and characteristics of proteins responsible for sensitization. However, in normal clinical settings this is presently impossible . OBJECTIVE: We sought to detect clinical markers of sensitization to profilin . METHODS: Seventy-one patients allergic to fruits and vegetables but not sensitized to lipid transfer protein or natural rubber latex were studied. Food allergy was ascertained on the basis of clinical history and positive skin prick test responses with fresh foods, commercial extracts, or both. Allergies to foods that had caused less than 2 adverse reactions were confirmed by means of open oral challenge. IgE reactivity to rBet v 1/rBet v 2 and to natural Phleum species profilin were detected. Moreover, IgE to the 30- to 40-kd and 60- to 90-kd birch pollen-enriched fractions, which also can be involved in cross-reactivity phenomena, were measured in sera from 52 patients by means of ELISA . RESULTS: On the basis of in vitro tests, 24, 18, and 25 patients turned out to be sensitized to Bet v 1, Bet v 2, or both, respectively. Four patients had negative test results for both allergens. Hypersensitivity to Bet v 2 was strongly associated with clinical allergy to citrus fruits (39% in patients monosensitized to Bet v 2 vs 4% in patients monosensitized to Bet v 1, P <.025), melon or watermelon (67% vs 0%, P <.001), banana (66% vs 8%, P <.001), and tomato (33% vs 0%, P <.05), whereas Bet v 1 sensitivity was associated with clinical allergy to apple (100% vs 39%, P <.001) and hazelnut (56% vs 0%, P <.001). The sensitivity of a history of allergy to gourd fruits, citrus fruits, tomato, banana, or a combination thereof as a means to detect profilin-hypersensitive patients was 85% (41/48). The specificity of an allergy to any of these fruits exceeded 85%, with positive predictive values ranging between 68% and 91% . CONCLUSION: In clinical settings in which laboratory investigations are not easily accessible, allergy to melon, watermelon, citrus fruits, tomato, and banana can be used as a marker of profilin hypersensitivity once a sensitization to natural rubber latex and lipid transfer protein is ruled out.

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[62] - Asero R, Jimeno L, Barber D. Component-resolved diagnosis of plant food allergy by SPT. Eur Ann Allergy Clin Immunol 2008;40:115-121

BACKGROUND: Fruits and vegetables may contain both labile and stable allergens. The former induce only OAS, whereas stable allergens may induce systemic reactions. Component-resolved diagnosis (CRD) of allergy to plant foods is therefore essential for the clinical management of allergic patients. METHODS: 80 adults allergic to plant foods underwent SPT with purified natural date palm profilin (Pho d 2), purified Mal d 1, a peach extract containing uniquely LTP, and with a kiwi extract containing uniquely stable allergens. RESULTS: 58 (72%) patients were monosensitized: 24 to Mal d 1, 24 to profilin, 7 to LTP, and 3 to kiwi. 22 patients were multi-sensitised: 14 to Mal d 1 and profilin, 2 to Mal d 1 and kiwi, 1 to LTP and profilin, 3 to LTP and Mal d 1, and 2 to LTP, Mal d 1 and profilin. Mal d 1 and LTP sensitisation were associated with apple and peach allergy, respectively, whereas profilin sensitisation was associated with allergy to melon, watermelon, banana, tomato and citrus fruits. 18/21 kiwi-allergic patients were sensitised to one of the cross-reacting allergens, but 2/18 reacted to kiwi-specific allergens as well. CONCLUSIONS: In patients with allergy to plant-derived foods CRD can be performed by SPT with purified allergen proteins. In the future, the availability of a larger number of purified natural or recombinant allergens for SPT will represent a simple means to classify food-allergic patients properly on the first visit.

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[63] - Asero R, Mistrello G, Roncarolo D, de Vries SC, Gautier MF, Ciurana CL, et al. Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion. Int Arch Allergy Immunol 2000;122:20-32

Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

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[64] - 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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[65] - Asero R, Mistrello G, Roncarolo D, Amato S. Relationship between peach lipid transfer protein specific IgE levels and hypersensitivity to non-Rosaceae vegetable foods in patients allergic to lipid transfer protein. Ann Allergy Asthma Immunol 2004;92:268-272

BACKGROUND: Lipid transfer protein (LTP), the major allergen in Rosaceae in geographic areas where the prevalence of birch pollen allergy is low, is a widely cross-reacting pan-allergen, but the pattern of cross-reactivity to plant-derived foods botanically unrelated to Rosaceae shows much variability. OBJECTIVE: To examine the relationship between peach LTP specific IgE levels and cross-reactivity to several non-Rosaceae, plant-derived foods. METHODS: IgE specific for peach LTP was measured by enzyme-linked immunosorbent assay in serum samples from 40 patients with Rosaceae allergy monosensitized to LTP. Patients were considered monosensitized to this protein in the absence of sensitization to other cross-reacting, plant-derived foods as shown by negative skin prick test (SPT) results with both birch and mugwort pollen. SPTs with commercial extracts of walnut, hazelnut, peanut, celery, maize, rice, tomato, orange, and onion were performed to detect possible immunologic cross-reactivity to these foods. RESULTS: Patients with negative SPT results with non-Rosaceae foods showed significantly lower levels of IgE to peach LTP than patients showing skin reactivity to one or more non-Rosaceae foods (P < .001). A significant difference in specific IgE to peach LTP between patients with positive or negative SPT results was observed with each individual food (P < .001 in all cases). The level of IgE to peach LTP was strongly related to the number of positive SPT results with non-Rosaceae foods (r = 0.78; P < .001). Increasing levels of IgE to peach LTP were associated with skin reactivity to nuts (29/40 [72%]), peanut (27/40 [67%]), maize (16/39 [41%]), rice (14/39 [36%]), onion (13/37 [35%]), orange (9/32 [28%]), celery (11/40 [27%]), and tomato (8/39 [20%]). CONCLUSIONS: This study suggests that all allergenic determinants in LTP from vegetable foods other than peach cross-react with peach LTP determinants, whereas only some peach LTP epitopes cross-react with allergenic determinants on botanically unrelated, plant-derived foods. The high levels of IgE to peach LTP seem to reflect the presence of IgE targeting common allergenic determinants of LTP, causing cross-reactivity to botanically unrelated, vegetable foods. In LTP-allergic patients, increasing levels of IgE to peach LTP are paralleled by an increasing number of foods other than Rosaceae positive on SPT that cause clinical symptoms.

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[66] - Asero R, Mistrello G, Roncarolo D, Amato S. Detection of Some Safe Plant-Derived Foods for LTP-Allergic Patients. Int Arch Allergy Immunol 2007;144:57-63

BACKGROUND: Lipid transfer protein (LTP) is a widely cross-reacting plant pan-allergen. Adverse reactions to Rosaceae, tree nuts, peanut, beer, maize, mustard, asparagus, grapes, mulberry, cabbage, dates, orange, fig, kiwi, lupine, fennel, celery, tomato, eggplant, lettuce, chestnut and pineapple have been recorded . OBJECTIVE: To detect vegetable foods to be regarded as safe for LTP-allergic patients . METHODS: Tolerance/intolerance to a large spectrum of vegetable foods other than Rosaceae, tree nuts and peanut was assessed by interview in 49 subjects monosensitized to LTP and in three distinct groups of controls monosensitized to Bet v 1 (n = 24) or Bet v 2 (n = 18), or sensitized to both LTP and birch pollen (n = 16), all with a history of vegetable food allergy. Patients and controls underwent skin prick test (SPT) with a large spectrum of vegetable foods. The absence of IgE reactivity to foods that were negative in both clinical history and SPT was confirmed by immunoblot analysis and their clinical tolerance was finally assessed by open oral challenge (50 g per food) . RESULTS: All patients reported tolerance and showed negative SPT to carrot, potato, banana and melon; these foods scored positive in SPT and elicited clinical symptoms in a significant proportion of patients from all three control groups. All patients tolerated these four foods on oral challenge. Immunoblot analysis confirmed the lack of IgE reactivity to these foods by LTP-allergic patients . CONCLUSION: Carrot, potato, banana and melon seem safe for LTP-allergic patients. This finding may be helpful for a better management of allergy to LTP.

↩

[67] - Asero R, Jimeno L, Barber D. Component-resolved diagnosis of plant food allergy by SPT. Eur Ann Allergy Clin Immunol 2008;40:115-121

BACKGROUND: Fruits and vegetables may contain both labile and stable allergens. The former induce only OAS, whereas stable allergens may induce systemic reactions. Component-resolved diagnosis (CRD) of allergy to plant foods is therefore essential for the clinical management of allergic patients. METHODS: 80 adults allergic to plant foods underwent SPT with purified natural date palm profilin (Pho d 2), purified Mal d 1, a peach extract containing uniquely LTP, and with a kiwi extract containing uniquely stable allergens. RESULTS: 58 (72%) patients were monosensitized: 24 to Mal d 1, 24 to profilin, 7 to LTP, and 3 to kiwi. 22 patients were multi-sensitised: 14 to Mal d 1 and profilin, 2 to Mal d 1 and kiwi, 1 to LTP and profilin, 3 to LTP and Mal d 1, and 2 to LTP, Mal d 1 and profilin. Mal d 1 and LTP sensitisation were associated with apple and peach allergy, respectively, whereas profilin sensitisation was associated with allergy to melon, watermelon, banana, tomato and citrus fruits. 18/21 kiwi-allergic patients were sensitised to one of the cross-reacting allergens, but 2/18 reacted to kiwi-specific allergens as well. CONCLUSIONS: In patients with allergy to plant-derived foods CRD can be performed by SPT with purified allergen proteins. In the future, the availability of a larger number of purified natural or recombinant allergens for SPT will represent a simple means to classify food-allergic patients properly on the first visit.

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[68] - Cuesta J, Blanco C, Fernández-Rivas M, Villalba M. Vegetalia. J Investig Allergol Clin Immunol 2006;16(Suppl. 2):40-44

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[69] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

↩

[70] - 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.

↩

[71] - Asero R, Mistrello G, Roncarolo D, Amato S. Relationship between peach lipid transfer protein specific IgE levels and hypersensitivity to non-Rosaceae vegetable foods in patients allergic to lipid transfer protein. Ann Allergy Asthma Immunol 2004;92:268-272

BACKGROUND: Lipid transfer protein (LTP), the major allergen in Rosaceae in geographic areas where the prevalence of birch pollen allergy is low, is a widely cross-reacting pan-allergen, but the pattern of cross-reactivity to plant-derived foods botanically unrelated to Rosaceae shows much variability. OBJECTIVE: To examine the relationship between peach LTP specific IgE levels and cross-reactivity to several non-Rosaceae, plant-derived foods. METHODS: IgE specific for peach LTP was measured by enzyme-linked immunosorbent assay in serum samples from 40 patients with Rosaceae allergy monosensitized to LTP. Patients were considered monosensitized to this protein in the absence of sensitization to other cross-reacting, plant-derived foods as shown by negative skin prick test (SPT) results with both birch and mugwort pollen. SPTs with commercial extracts of walnut, hazelnut, peanut, celery, maize, rice, tomato, orange, and onion were performed to detect possible immunologic cross-reactivity to these foods. RESULTS: Patients with negative SPT results with non-Rosaceae foods showed significantly lower levels of IgE to peach LTP than patients showing skin reactivity to one or more non-Rosaceae foods (P < .001). A significant difference in specific IgE to peach LTP between patients with positive or negative SPT results was observed with each individual food (P < .001 in all cases). The level of IgE to peach LTP was strongly related to the number of positive SPT results with non-Rosaceae foods (r = 0.78; P < .001). Increasing levels of IgE to peach LTP were associated with skin reactivity to nuts (29/40 [72%]), peanut (27/40 [67%]), maize (16/39 [41%]), rice (14/39 [36%]), onion (13/37 [35%]), orange (9/32 [28%]), celery (11/40 [27%]), and tomato (8/39 [20%]). CONCLUSIONS: This study suggests that all allergenic determinants in LTP from vegetable foods other than peach cross-react with peach LTP determinants, whereas only some peach LTP epitopes cross-react with allergenic determinants on botanically unrelated, plant-derived foods. The high levels of IgE to peach LTP seem to reflect the presence of IgE targeting common allergenic determinants of LTP, causing cross-reactivity to botanically unrelated, vegetable foods. In LTP-allergic patients, increasing levels of IgE to peach LTP are paralleled by an increasing number of foods other than Rosaceae positive on SPT that cause clinical symptoms.

↩

[72] - Asero R, Mistrello G, Roncarolo D, Amato S. Detection of Some Safe Plant-Derived Foods for LTP-Allergic Patients. Int Arch Allergy Immunol 2007;144:57-63

BACKGROUND: Lipid transfer protein (LTP) is a widely cross-reacting plant pan-allergen. Adverse reactions to Rosaceae, tree nuts, peanut, beer, maize, mustard, asparagus, grapes, mulberry, cabbage, dates, orange, fig, kiwi, lupine, fennel, celery, tomato, eggplant, lettuce, chestnut and pineapple have been recorded . OBJECTIVE: To detect vegetable foods to be regarded as safe for LTP-allergic patients . METHODS: Tolerance/intolerance to a large spectrum of vegetable foods other than Rosaceae, tree nuts and peanut was assessed by interview in 49 subjects monosensitized to LTP and in three distinct groups of controls monosensitized to Bet v 1 (n = 24) or Bet v 2 (n = 18), or sensitized to both LTP and birch pollen (n = 16), all with a history of vegetable food allergy. Patients and controls underwent skin prick test (SPT) with a large spectrum of vegetable foods. The absence of IgE reactivity to foods that were negative in both clinical history and SPT was confirmed by immunoblot analysis and their clinical tolerance was finally assessed by open oral challenge (50 g per food) . RESULTS: All patients reported tolerance and showed negative SPT to carrot, potato, banana and melon; these foods scored positive in SPT and elicited clinical symptoms in a significant proportion of patients from all three control groups. All patients tolerated these four foods on oral challenge. Immunoblot analysis confirmed the lack of IgE reactivity to these foods by LTP-allergic patients . CONCLUSION: Carrot, potato, banana and melon seem safe for LTP-allergic patients. This finding may be helpful for a better management of allergy to LTP.

↩

[73] - Crespo JF, Retzek M, Fötisch K, Sierra-Maestro E, Cid-Sanchez AB, Pascual CY, et al. Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits. Mol Nutr Food Res 2006;50:282-290

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

↩

[74] - Ibáñez MD, Sastre J, Martinez san Ireneo M, Laso MT, Barber D, Lombardero M. Different patterns of allergen recognition in children allergic to orange. J Allergy Clin Immunol 2004;113:175-176

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Les allergènes des fruits des Rutacées

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Les "fruits rouges"

Le raisin (et ses dérivés)

Les Cucurbitacées

Les Liliacées

Les Astéracées (Lactucées, artichaut...)

Divers aliments végétaux

Les épices, herbes aromatiques et autres condiments