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Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina Introdução: As reações alérgicas a frutas, anteriormente consideradas raras, são cada vez mais frequentes, principalmente em crianças mais velhas e adultos. Estas podem ser classificadas em primárias (classe I) ou secundárias (classe II) consoante a presença de reatividade cruzada com pólenes, sendo a clínica variável. As non-specific Lipid Transfer Proteins (nsLTPs) são os principais alergénios das alergias primárias (sem reatividade cruzada). As nsLTPs são mais resistentes ao calor e à digestão enzimática, o que resulta em reações mais heterogénias e de maior gravidade. As alergias secundárias incluem principalmente as Pathogenesis-Related Proteins 10 (PR-10) e as profilinas, que são sensíveis ao calor e à digestão enzimática, resultando em manifestações mais leves, como o Síndrome de Alergia Oral (SAO). O objetivo do presente estudo é caracterizar clínica e epidemiologicamente um grupo de crianças e adolescentes com alergia a frutas.Métodos: Incluíram-se os doentes observados numa consulta de Alergologia Pediátrica, nos últimos 5 anos, que cumpriram os critérios de diagnóstico de alergia a frutas frescas. O princípio da confidencialidade foi preservado. Na análise estatística descritiva foram utilizados diversos parâmetros para a distribuição das variáveis, nomeadamente a frequência, percentagem, média e desvio-padrão. Utilizaram-se os testes: Binominal e Q de Cochran.Resultados: Em 77 doentes registaram-se 134 alergias a 22 frutas diferentes, sendo as mais frequentes a kiwi (n=41; 30,6%), pêssego (n=29; 21,6%) e maçã (n=11; 8,2%). A mediana da idade da primeira reação foi de 4 anos. Observou-se rinite em 46 (59,74%), alergia/sensibilização a pólenes em 45 (58,44%), eczema atópico em 38 (49,35%), asma em 35 (45,45%). A manifestação mais frequente foi o SAO em 56 (41,79%), seguido de sintomas sistémicos em 47 (35,07%) e anafilaxia em 31 (23,13%). As 3 frutas mais frequentes, totalizaram 81/134 alergias (60,44%). Não se observaram diferenças estatisticamente significativas entre manifestações para cada uma destas 3 frutas. Os alergénios mais frequentes foram o Pru p 3, Act d 1 e o Mal d 1, para pêssego, kiwi e maçã, respetivamente. Nos doentes alérgicos a pêssego, 22/24 foram positivos para Pru p 3 (p

Background Natural rubber latex and chlorhexidine have previously been identified as causative substances in perioperative anaphylaxis. A pelvic examinations is generally considered noninvasive, however, this procedure is rarely associated with severe allergic reactions. We reported a rare case of dual latex and chlorhexidine allergies which caused anaphylaxis after pelvic examination in a woman with a history of latex-related fruits allergy. Case presentation A 54-year-old woman had severe anaphylaxis after a pelvic examination due to dual latex and chlorhexidine (CHX) allergies. The gynecologist used CHX for the vaginal preparation and wore latex-containing gloves with lubricating gel during the examination. In vivo and in vitro tests revealed CHX sensitization by a positive skin prick test to chlorhexidine at a very low concentration (0.002 mg/mL), and a positive basophil activation test to CHX. Latex allergy was confirmed by a positive specific IgE to latex and a positive glove-use test at 20 min. An analysis of specific IgE to latex component revealed positive results for Hev b 1, 5, 6.02, and 11. As she also had a past history of fruit allergy, prick-to-prick testing with latex-related fruits was performed. The results were positive for avocado, banana, jackfruit, kiwi, and longan. Conclusions Concomitant mucosal exposure of both natural rubber latex and CHX in highly sensitized patients during pelvic examinations can lead to severe anaphylaxis. Pre-procedural screening for an allergy to latex or CHX, or to any other allergen, should be performed in patients where there is suspicion of a specific allergy due to a previous allergic reaction. Increased awareness of these two allergens in all healthcare settings may improve patient safety.

Background: Peanut storage proteins (Ara h 1, Ara h 2, and Ara h 3) have been described as the major peanut allergens in children, although not all peanut-sensitized individuals have clinical reactivity after exposure. Objectives: We studied the sensitization profile of peanut-allergic and peanut-tolerant children in a pediatric cohort. Methods: The clinical features and sensitization profile to the peanut storage proteins Ara h 9 and Pru p 3 were compared between peanut-allergic and peanut-tolerant children using component-resolved diagnostics. Results: Thirty-three peanut-sensitized children were included: 22 allergic and 11 tolerant patients. Seventy-two percent of the peanut-allergic children were sensitized to at least one peanut storage protein. The rates of sensitization to Ara h 1, Ara h 2, and Ara h 3 were 63.6, 68.1, and 68.1%, respectively, among the peanut-allergic children and 27.2, 18.1, and 45.4% among the peanut-tolerant children. IgE from the sera of 18% of the peanut-allergic patients recognized Ara h 9, whereas no sensitization to Ara h 9 was detected in the peanut-tolerant children. A total of 59% of the peanut-allergic and 27% of the peanut-tolerant children were sensitized to Pru p 3. Sensitization to Ara h 1 and Ara h 2 was more frequent among the peanut-allergic children (p < 0.05). Although the levels of specific IgE against peanut storage proteins were higher in peanut allergy, there were not statistically significantly different from the levels in peanut tolerance, probably due to the small number of patients included. Conclusions: In our population, the peanut-allergic children were mainly sensitized to peanut storage proteins, and Ara h 2 sensitization allows a more accurate diagnosis of clinical reactivity to peanuts. More than half of the peanut-allergic patients were sensitized to peach Pru p 3, and 50% of them had fruit allergy at the time of the study.

Background Severe allergy to fruits mediated by a 7 kDa allergen belonging to the gibberellin-regulated protein (GRP) family is known to be associated with Cupressaceae pollinosis. Objective To identify and characterize Cupressaceae pollen allergens involved in GRP-related fruit allergy. Methods Pru p 7-related proteins from pollen of Cupressus sempervirens, Juniperus ashei and Cryptomeria japonica were identified using a rabbit anti-Pru p 7 antiserum, purified chromatographically and sequenced by mass spectrometry and bioinformatic comparisons. The C sempervirens protein was produced as a recombinant allergen in Pichia pastoris. IgE antibody binding to pollen GRP proteins was analysed in a peach allergic (n = 54) and a cypress pollen allergic (n = 88) patient population from southern France using ImmunoCAP. Results In each of the three Cupressaceae species studied, a 7 kDa pollen protein related to Pru p 7 was identified and found to comprise an amino acid sequence of 63 residues in length, 92%-98% identical to each other and 67%-68% identical to Pru p 7. The C sempervirens, J ashei and C japonica GRP allergens have been officially recognized by the WHO/IUIS Allergen Nomenclature Sub-Committee and named Cup s 7, Jun a 7 and Cry j 7, respectively. Recombinant Cup s 7 showed IgE antibody binding capacity comparable to that of the purified natural allergen. Among 51 peach allergic subjects sensitized to Pru p 7, substantially higher levels of IgE to Cup s 7 than to Pru p 7 were found. Further, the pollen protein was able to completely outcompete IgE binding to Pru p 7, while the reverse competition effect was modest, consistent with primary sensitization by the pollen allergen. Conclusion and clinical relevance Pru p 7-related pollen allergens from three Cupressaceae species have been characterized and may become useful for the identification of pollinosis patients at risk of developing severe fruit allergy.

Antecedentes: La prevalencia de la alergia al látex se desconoce en los estudiantes de medicina.Objetivo: Determinar prevalencia y factores asociados con autorreporte de alergia al látex en estudiantes de medicina.Métodos: Estudio transversal de estudiantes con o sin autorreporte de alergia al látex. Mediante cuestionario estructurado se indagó historia personal y familiar de enfermedad alérgica; tiempo y frecuencia de exposición a guantes de látex y síntomas tras la exposición a productos o alimentos relacionados con alergia al látex. Se realizaron modelos de regresión logística.Resultados: De 854 sujetos, 431 (50.5 %) fueron mujeres. La mediana de edad fue de 21 años. La prevalencia global de alergia al látex fue 4.3 % (IC 95 % = 3.1 a 5.9). Los factores de riesgo relacionados fueron edad (RM = 1.37; IC 95 % = 1.05 a 1.79), historia personal de dermatitis atópica (RM = 7.32; IC 95 % = 3.14 a 17.08), uso de guantes ≥ 15/semana (RM = 2.59; IC 95 % = 1.17 a 5.76), uso de productos con látex (RM = 5.76; IC 95 % = 2.15 a 15.49) y alergia a frutas (RM = 3.24; IC 95 % = 1.27 a 8.27).Conclusión: Cuatro de cada 100 estudiantes reportaron alergia al látex. La edad, la historia personal de dermatitis atópica, la mayor frecuencia de exposición a guantes de látex y antecedente de alergia a frutos fueron factores de riesgo.

Introduction: Component-resolved diagnostics is used to diagnose food allergies. However, few reports have evaluated the severity of peach fruit allergy using peach allergen components, including Pru p 7. Objective: This study aimed to predict peach fruit allergy severity based on the presence of specific IgE (sIgE) antibodies (Abs) to peach allergenic components. Methods: Twenty-seven patients with peach fruit allergy were enrolled and classified into two groups: the local reaction (LR) group, including 12 patients with only oral or throat mucosal symptoms, and the systemic reaction (SR) group, including 15 patients, 10 of whom experienced anaphylaxis. Serum sIgE Abs against crude peach extract – Pru p 1, 2, 3, 4, and 7 – and tree pollen were measured. Results: sIgE Ab titers of Pru p 1 and 4 and alder pollen in the LR group were significantly higher than those in the SR group. sIgE against Pru p 7 was significantly higher in the SR group than in the LR group. The frequencies of sIgE Abs against Pru p 1, 4, and 7 in the LR group were 91.7, 66.7, and 16.7%, respectively, while in the SR group these were 80, 20, and 60%. Sensitization to Pru p 2 and 3 was detected but limited in all patients. Conclusions: These findings suggest that sensitization to Pru p 1 and Pru p 4 is associated with local symptoms, and sensitization to Pru p 7 is associated with SR and anaphylaxis. To predict the severity of peach fruit allergy, it is useful to assess sIgE Ab reactions combining Pru p 1, 4, and 7.

Fruits are an important source of vitamins and antioxidants that can effectively delay aging and contribute to health and well-being of the human kind. However, they are growing to be one of the primary elicitors of food allergies around the world. Fruit allergens can induce an IgE-mediated (Immunoglobulin E) reaction, presenting with a symptom like localized oral allergy syndrome (OAS). Numerous studies showed that varying environmental and cultivation conditions can influence the fruit allergen content during flowering and ripening stages. Further, the variety, harvesting maturity, and storage conditions can also significantly influence the allergenicity potential. For example, unripe apples and tomatoes have lower levels of allergens compared to ripened fruits. Researchers have also reported that modified atmosphere packaging (MAP) can help reduce Mal d 3 content present in apples during storage. Post-harvest processing like peeling is also considered a good method to help reduce the overall allergenicity in few fruits whose peel might contain majority of the allergens. This review will discuss the overall influence of both pre-harvest and post-harvest factors on the fruit allergens. We will also discuss the progress regarding the cause, symptoms and diagnostic methods of fruit based allergies.

Lipid transfer protein (LTP) is a major fruit allergen. It has, however, recently been revealed that the systemic reaction in peach-allergic patients is related not only to LTP (Pru p 3) but also to gibberellin-regulated protein (Pru p 7). We investigated recombinant Pru p 7 (rPru p 7) for its potential use in worldwide standardization for the diagnosis of peach allergy.Natural Pru p 7 (nPru p 7) was purified from peach crude extract using a monoclonal antibody affinity column. Complementary DNA for Pru p 7 was cloned and expressed in Escherichia coli and Pichia pastoris. Serum immunoglobulin (Ig) E in peach-allergic patients was examined by enzyme-linked immunosorbent assay (ELISA) using nPru p 7 and rPru p 7 (E. coli product: erPru p 7 and P. pastoris product: prPru p 7).Peach-allergic patients (n=27) were diagnosed and categorized into oral reaction (n=10) or systemic reaction (n=17). The nPru p 7 positivity based on serum IgE levels was 52% in the systemic-reaction group and 0% in the oral-reaction group (P0.05). In the systemic-reaction group, there was no significant difference in reactivity between nPru p 7 and prPru p 7, but the reactivity of erPru p 7 was significantly lower than those of nPru p 7 and prPru p 7 (P0.05).We found that prPru p 7 exhibited reactivity in ELISA comparable to that of nPru p 7 for the diagnosis of peach allergy with systemic reaction.

Scope The aim was to investigate the potential contribution of a major birch pollen Bet v 1-homologous allergen to birch pollen-associated tomato fruit allergy. Methods and results Two isoforms of a Bet v 1-homologous protein (designated Sola l 4) from tomato fruit were identified by cDNA-cloning and produced as recombinant proteins. Allergen-specific IgE levels to tomato, birch pollen, Bet v 1, and Sola l 4 were determined in birch pollen allergic patients with allergy or tolerance to tomato. Sola l 4 was recognized in 76% of birch/tomato allergic patients, while tomato- and Bet v 1-specific IgE was detectable in 64% and 81% of sera. Almost all patients sensitized to Bet v 1 reacted with Sola l 4. Both Sola l 4 isoforms displayed allergenic potency and IgE-cross-reactivity with Bet v 1 as investigated by competitive ELISA and in vitro mediator release assay. Nevertheless, the reactivity pattern of patients' sera was diverse. Conclusion Sola l 4, a novel pathogenesis related-10 protein, qualifies as major allergen in tomato fruits. Data suggest Sola l 4 as class II allergen. IgE-testing using Sola l 4 showed low clinical specificity, but high sensitivity in tomato allergic patients and will further improve component-resolved allergy diagnosis.

Gibberellin-regulated protein (GRP) is a new allergen in peach allergy, with an amino acid sequence very well conserved through several botanical species. We investigated the allergenicity of GRP in fruit allergies other than peaches and identified the clinical characteristics of fruit allergy patients with GRP sensitization. One hundred consecutive Japanese patients with fruit allergies were enrolled in the present study. To identify the features of GRP sensitization, we selected patients with negative ImmunoCAP results for Bet v 1 homologs and profilin, which are marker allergens for pollen-food allergy syndrome (PFAS), or lipid transfer protein. These patients underwent specific immunoglobulin E measurements by enzyme-linked immunosorbent assay (ELISA) and skin prick tests (SPT) using purified nPru p 7. Twenty of 100 consecutive patients with fruit allergies had negative ImmunoCAP results for Bet v 1 homologs and profilin. Thirteen (65.0%) of the 20 patients had positive ELISA and/or SPT results using nPru p 7, whereas one of the 20 patients had positive ImmunoCAP results for Pru p 3. In 13 nPru p 7-sensitized patients, the causative foods were peaches (92.3%), apricots (61.5%), oranges (46.2%) and apples (30.8%). Ten patients (76.9%) had multiple causative fruits. Frequent symptoms included facial edema (92.3%) and laryngeal tightness (66.7%). In eight patients (61.5%), exercise or aspirin intake enhanced the allergic reaction onset as cofactors. The prevalence of GRP sensitization was high in Japanese fruit allergy patients except for PFAS patients. In conclusion, GRP-sensitized patients may have allergies to multiple fruits and may show peculiar characteristics such as facial swelling and cofactor dependence.

BACKGROUND: Up to 6.6% of adults and 11.5% of children aged between 0 and 6 years are reporting a perceived fruit allergy. In the last group 8.5% perceived reactions against apple. To keep allergic reactions as low as possible and to make apple consumption more tolerable for patients allergic to apple, theMal d 1 content of four apple cultivars (‘Boskoop’, ‘Elise’, ‘Pinova’, ‘Santana’ and ‘Topaz’) were analyzed, to identify apple cultivars with only a low Mal d 1 content. Further, the development of Mal d 1 content of low-allergenic cultivars should be investigated during long-term storage. RESULTS: The lowest Mal d 1 content was measured for fruits of the cultivar ‘Santana’ at harvest, followed by ‘Pinova’ and ‘Elise’ after storage without 1-MCP. Nearly all fruits treated with 1-MCP had a significantly lower Mal d 1 content than fruits without 1-MCP, except for ‘Boskoop’ fruits after 44 weeks of storage. Apple cultivars with a low Mal d 1 content are not a cross of ‘Golden Delicious’ with another cultivar, with the exception of ‘Pinova’ which is a cultivar from the so-called ‘Pi-series’, which have a moderate resistance to scab and mildew. First experiments on tomatoes and peanuts have shown a reduction of allergic proteins in older and child generations after transfer of RNA. This reduction is probably based on an alteration of gene expression regulating the synthesis of allergic proteins, leading to changes in protein composition in the fruit. CONCLUSION: Individuals allergic to apple should choose the described cultivars with relative low Mal d 1 content. If breeders want to breed cultivars with low allergy, they should avoid crossings with ‘Golden Delicious’.

Chinese bayberry Myrica rubra is a very popular fruit in southeastern China. In spite of its wide consumption, no allergies to this fruit have been reported previously. Here we report on a 40-year-old woman suffering from anaphylaxis to Chinese bayberry fruit. Prick-prick skin tests revealed strong reactions to fresh Chinese bayberry fruits as well as to peach, and weaker reactions to some other fruits including apple, melon, and banana. ImmunoCAP analysis revealed identical titers of specific IgE (4.3 kU(A)/L) to peach extract and its lipid transfer protein (LTP, rPru p 3), which was confirmed by detection of a 9 kD band following immunoblotting. Immunoblot analysis with Chinese bayberry extract gave bands of 22, 45, and 90 kD, but no 9 kD band was recognized. There was also no evidence of LTP recognition for loquat (36 kD) or melon (24 kD). This first report of a severe allergic reaction to Chinese bayberry fruit in a patient with LTP-mediated peach allergy indicates that other as yet unidentified non-pollen related fruit allergens are involved in this new severe fruit allergy.

BACKGROUND: Peach allergy can be caused by the allergen Pru p 1. This occurs by cross-reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)-binding protein extracted from peach fruit has never been reported. RESULTS: Phosphate-buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach-allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two-dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. CONCLUSION: Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1-homologous peach allergen Pru p 1. Copyright © 2011 Society of Chemical Industry

Banana fruit allergy is well known, but neither immunoglobulin E recognition patterns to purified plant food allergens nor true prevalences of putative banana allergens have been established. This study aimed to characterize β-1,3-glucanase and thaumatin-like protein (TLP) as banana allergens, testing them, together with other plant food allergens, in 51 children with allergic reactions after banana ingestion and both positive specific IgE and skin prick test (SPT) to banana. Banana β-1,3-glucanase and TLP were isolated and characterized. Both banana allergens, together with kiwifruit TLP Act d 2, avocado class I chitinase Pers a 1, palm pollen profilin Pho d 2 and peach fruit lipid transfer protein (LTP) Pru p 3, were tested by in vitro and in vivo assays. Banana β-1,3-glucanase (Mus a 5) was glycosylated, whereas banana TLP (Mus a 4) was not, in contrast with its homologous kiwi allergen Act d 2. Specific IgE to both banana allergens, as well as to peach Pru p 3, was found in over 70% of sera from banana-allergic children, and Mus a 4 and Pru p 3 provoked positive SPT responses in 6 of the 12 tested patients, whereas Mus a 5 in only one of them. Both peptidic epitopes and cross-reactive carbohydrate determinants were involved in the IgE-binding to Mus a 5, whereas cross-reactivity between Mus a 4 and Act d 2 was only based on common IgE protein epitopes. Profilin Pho d 2 elicited a relevant proportion of positive responses on in vitro (41%) and in vivo (58%) tests. Therefore, Mus a 4 and LTP behave as major banana allergens in the study population, and profilin seems to be also a relevant allergen. Mus a 5 is an equivocal allergenic protein, showing high IgE-binding to its attached complex glycan, and low in vivo potency.

Background: Peanut (PN), tree nuts (TN) and fruits are frequent causes of food allergy (FA). Peanut and TN are believed to cause more severe reactions than fruits. However, there are no studies comparing the severity of PN, TN and fruit allergy within one patient group. Methods: Four-hundred and eleven adult patients referred to our tertiary allergy center with suspicion of FA completed a standardized questionnaire. Patients with a typical history of immunoglubulin E (IgE)-mediated allergy, e.g. oropharyngeal symptoms to PN, TN (hazelnut, walnut, cashew nut) or fruit (apple, kiwi, peach, pear and cherry) were recruited (218/411). The objective was to evaluate differences in clinical severity between PN, TN and fruit allergy and how this was reflected by prescription of emergency medication and impact on daily life. Results: Eighty-two percent of the included 218 patients were sensitized to the respective foods. The percentages of severe symptoms (i.e. respiratory or cardiovascular symptoms) in PN, TN and fruit allergic patients were respectively 47%, 39% and 31% (respiratory) and 11%, 5.0% and 3.4% (cardiovascular). Prescription and use of emergency medication (epinephrine, antihistamines and steroids) did not differ among the three groups. The majority of patients with a PN or TN allergy (72%) and fruit allergy (62%) reported that FA influences their daily life considerably. Conclusions: Fruit allergy causes less severe symptoms than TN and especially PN allergy. However, this is not reflected in the prescription or use of emergency medication. This may indicate that physicians are not fully acquainted with the guidelines for prescription of emergency medication. A high impact on daily life was found both in PN, TN and in fruit allergy.

In the last decades allergies are increasing in the western countries and different protein families were identified as determinants of allergenicity. In apple fruit, one of the most important fruit crop in the world, four allergen families (Mal d 1–4) have been identified so far, including pathogenesis-related proteins 10, thaumatin-like proteins, lipid transfer proteins and profilins. Moreover, it has been demonstrated that patient sensitivity differs in relation to apple variety, fruit tissue, cultivation and conservation conditions, increasing the difficulties to associate genetic, molecular and biochemical data with the results of clinical tests. Mapping studies and the availability of the complete apple genome sequence confirmed that allergens are organized in large families with many different isoforms, the contribution of which to allergenicity is still largely unknown. The unravelling of allergy determinants is crucial for different reasons: • to better understand environmental influences on allergenicity. • to produce genetically modified plants with a reduced allergen content through a targeted-gene silencing approach. • to drive future breeding programs towards hypoallergenic apples through the exploitation of natural variability. Finally, the above-mentioned knowledge will be essential to allow the consumption of this fruit by individuals who are allergic to apples.

In the last decades allergies are increasing in the western countries and different protein families were identified as determinants of allergenicity. In apple fruit, one of the most important fruit crop in the world, four allergen families (Mal d 1 to 4) have been identified so far, including pathogenesis-related proteins 10, thaumatin-like proteins, lipid-transfer proteins and profilins. Moreover, it has been demonstrated that patient’s sensitivity differs in relation to apple variety, fruit tissue, cultivation and conservation conditions, increasing the difficulties to associate genetic, molecular and biochemical data with the results of clinical tests. Mapping studies and the availability of the complete apple genome sequence confirmed that allergens are organized in large families with many different isoforms, which contribution to allergenicity is still largely unknown. The unravelling of allergy determinants is crucial for different reasons: i) to better understand environmental influences on allergenicity; ii) to produce genetically modified plants with a reduced allergen content through a targeted-gene silencing approach and iii) to drive future breeding programs toward hypoallergenic apple through the exploitation of natural variability. Finally, the above-mentioned knowledge will be essential to allow the consumption of this fruit by individuals who are allergic to apple.

Background Allergy to a plant food can either result from direct sensitization to that food or from primary sensitization to pollen, latex, or another food. Objective We sought to investigate the primary sensitizers in apple allergy across Europe, the individual allergens involved, and whether these differences determine the clinical presentation. Methods Patients (n = 389) with positive case histories and skin prick test responses to fresh apple were selected in the Netherlands, Austria, Italy, and Spain. Skin prick tests and RASTs to a panel of pollens and plant foods were performed, as well as RASTs to Bet v 1 and the apple allergens Mal d 1, 2, 3, and 4. Results In the Netherlands, Austria, and Italy apple allergy is mild (>90% isolated oral symptoms) and related to birch pollinosis and sensitization to Bet v 1 and its apple homologue, Mal d 1, which has an odds ratio of local reactions of 2.85 (95% CI, 1.47-5.55). In Spain apple allergy is severe (>35% systemic reactions) and related to peach allergy and sensitization to Mal d 3 (nonspecific lipid transfer protein), which has an odds ratio of systemic reactions of 7.76 (95% CI, 3.87-15.56). Conclusion The analysis of individual apple allergens in a clinical context has provided insight into the sensitization pathway and into the intrinsic risk an allergen bears to induce mild or severe food allergy. Clinical implications Information on the sensitization pathway is essential to develop preventive strategies in food allergy. The application of individual food allergens with a known intrinsic risk will improve the prognostic value of diagnostic tests.

The importance of apple allergens, in particular Mal d 1, a Bet v 1 homologue for the pollen-fruit syndrome in Northern Europe, and Mal d 3, responsible for true fruit allergy in Southern Europe, has been repeatedly emphasized. However, little is known about the distribution pattern of major allergens in fruits and whether differences exist among different cultivars. Transcript expression of Mal d 1 isoforms and Mal d 3 was examined by RealTime-PCR and Northern analysis, respectively. An immuno-tissue-print (ITP) assay was developed to localise major allergens in apple fruit tissue and a Mal d 1 specific, patient independent ELISA was established. ITP analyses show that Mal d 1 and Mal d 2 are distributed throughout the apple pulp and peel, while Mal d 3 is restricted to the peel. Data obtained by ELISA reveal a variation of Mal d 1 content ranging from 0.84 to 33.17 μg/g fresh weight in 39 selected cultivars. Different apple cultivars show a markedly different expression of major allergens; this finding will influence the development of diagnostic tools as well as the dietary management of allergic individuals.

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

Two cDNA clones (named Pp-LTP1 and Pp-LTP2 ) corresponding to different lipid transfer protein (LTP) genes have been isolated from peach ( Prunus persica L. Batsch) epicarp and ovary, respectively. Sequence analysis revealed that the two fragments share 54% identity at nucleotide level and show common features of plant LTP genes, such as conserved cysteine residues and lipid-binding motifs. Phylogenetic analysis grouped Pp-LTP1 and Pp-LTP2 in two distinct clusters, the former with most of LTP genes sequenced in the Rosaceae family, the latter only with one almond LTP. Genomic Southern data indicated that a small LTP gene family is present in peach. Pp-LTP1 and Pp-LTP2 have been used as gene-specific probes to describe expression in flowers and fruits throughout development. In petals, sepals and stamen only Pp-LTP1 was expressed whereas transcripts of Pp-LTP2 strongly accumulated in non-pollinated and pollinated ovary with a decreasing trend in the period of four weeks after pollination. In fruits, a dramatic accumulation of Pp-LTP1 mRNA was detected in epicarp at all stages of fruit development and, with the exception of the early growth stage, no Pp-LTP1 transcripts have been detected in mesocarp. When Pp-LTP2 was used as a probe in the same fruit tissues, a faint hybridisation signal was observed only in epicarp of fruitlets collected at an early growth stage. Infection with Monilia induced only a slight increase of Pp-LTP1 transcript in epicarp of pre-climacteric and climacteric fruits. These results support the hypothesis of multiple roles played by LTPs and, considering that LTPs have been recognised as the major allergen of peach, indicate that Pp-LTP1 could be related to the allergenicity of peach.

Background: Lipid transfer proteins (LTPs) are major allergens of Rosaceae fruits in the Mediterranean area. IgE-cross-reactivity has been demonstrated in vitro among LTPs from peach, apple, chestnut and Artemisia pollen. The aim of this study was to evaluate the reactivity to LTPs from peach, apple, chestnut and Artemisia pollen by means of skin prick tests (SPTs). Methods: Forty-seven patients allergic to peach (peach group), 20 patients sensitized to Artemisia pollen with no food allergies (Artemisia group), and 12 control subjects were skin tested with fresh peach, as well as with whole extracts and purified LTPs of peach, apple, chestnut and Artemisia pollen. Results: The rates of positive SPTs for peach, apple, chestnut and Artemisia LTPs were, respectively, 91, 77, 23, and 36% in the peach group, and 30, 5, 15 and 40% in the Artemisia group. No response was observed in the control subjects. SPTs with peach LTP strongly correlated with SPTs conducted with fresh peach. In the peach group, the most frequent pattern of reactivity to LTPs was the combination peach-apple (45%), followed by peach-apple-Artemisia-chestnut (21%). Significant correlations were found between peach and apple LTPs, and between Artemisia and chestnut LTPs. Positive SPTs to chestnut LTP were only observed in patients with positive SPTs to Artemisia LTP. All the patients with positive case histories to chestnut reacted to chestnut LTP. Conclusions: LTPs are plant panallergens with different patterns of cross-reactivity. They are major allergens of Rosaceae fruits and seem to be involved in allergic reactions to unrelated foodstuffs such as chestnut, probably through sensitization to the cross-reactive Artemisia LTP. Rosaceae LTPs could be useful tools for in vivo diagnosis of Rosaceae fruit allergy.

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.