Clinically relevant peach allergy is related to peach lipid transfer protein, Pru p 3, in the Spanish population

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1 Clinically relevant peach allergy is related to peach lipid transfer protein, Pru p 3, in the Spanish population Montserrat Fernández-Rivas, MD, PhD, a Eloína González-Mancebo, MD, a Rosa Rodríguez-Pérez, PhD, a Cristina Benito, BS, a Rosa Sánchez-Monge, PhD, b Gabriel Salcedo, PhD, b M. Dolores Alonso, MD, a Ana Rosado, MD, a Miguel Angel Tejedor, MD, PhD, a Concepción Vila, MD, a and M. Luisa Casas, MD c Madrid, Spain Background: Sensitization to peach and related Rosaceae fruits without clinical expression is commonly observed as the result of the extensive cross-reactivity of IgE antibodies directed toward lipid transfer proteins (LTPs), Bet v 1 homologues, profilins, and carbohydrate determinants. Objective: We aimed to study whether there are any clinical or immunologic differences between patients allergic to peach and those who have a current clinically irrelevant sensitization to this fruit. Methods: One hundred subjects with adverse reactions to peach were evaluated by medical history, skin prick tests with fresh peach and purified peach LTP (Pru p 3), and specific IgE determinations to peach, rbet v 1, and rbet v 2 (birch profilin). Clinical reactivity to peach was established by doubleblind, placebo-controlled food challenges. The clinical characteristics and the in vivo and in vitro tests were compared between allergic and nonallergic patients. Results: Peach allergy was confirmed in 76 patients and ruled out in 16; 2 patients dropped out, and the study was not conclusive in 6 individuals (placebo reactors). Pollen allergy was found in 76% of the allergic patients and in 100% of the nonallergic patients. Positive responses to Pru p 3, rbet v 1, and rbet v 2 were observed in 62%, 7%, and 34% of patients allergic to peach, respectively. The sensitization rate to Pru p 3 was significantly higher among subjects allergic than nonallergic to peach (62% vs 31%, P =.02). IgE responses to rbet v 2 were more frequent among subjects allergic to pollen, but no difference was observed in the presence or absence of peach allergy. Conclusions: Pru p 3 is the major allergen of peach in our population, and the IgE response to this allergen is related to the clinical expression of peach allergy. Sensitization to profilin is observed in those patients with an associated pollen allergy but does not appear to be related to the clinical reactivity to peach. (J Allergy Clin Immunol 2003;112: ) From a Unidad de Alergia, Fundacion Hospital Alcorcon, Alcorcon, Madrid, Spain, b Unidad de Bioquimica, Departamento de Biotecnologia, E.T.S. de Ingenieros Agronomos, Madrid, Spain, and c Area de Laboratorio, Fundacion Hospital Alcorcon, Alcorcon, Madrid, Spain. Received for publication March 12, 2003; revised July 17, 2003; accepted for publication July 18, Reprint requests: Dr Montserrat Fernandez-Rivas, Fundacion Hospital Alcorcon, Unidad de Alergia, C/ Budapest, 1, Alcorcon, Madrid, Spain. Supported by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Fondo de Investigacion Sanitaria (FIS grant 00/0179), and by the KPMG- FHA 2000 Research Award American Academy of Allergy, Asthma and Immunology /2003 $ doi: /mai Key words: Double-blind placebo-controlled food challenge, food allergy, lipid transfer protein, peach, Pru p 3, rbet v1, rbet v2, Rosaceae fruits, skin prick tests Peach is the fresh fruit most frequently involved in allergic reactions in the adult patients of the Mediterranean area, 1-3 and it is also the most frequent food allergy in our population in patients older than 3 years of age (unpublished). The clinical presentation includes both mild and severe symptomatology. Local reactions such as contact urticaria (CU) and oral allergy syndrome (OAS) are observed in >50% of patients. 2,4-7 Systemic reactions are frequently observed (18% to 44%), even in the absence of OAS. 2-7 The severity of this fruit allergy is probably related to the high stability (to heat treatment and pepsin digestion) 8,9 of its major allergen, Pru p 3, the peach lipid transfer protein (LTP). Sensitization to Pru p 3 has been detected in >90% of patients allergic to peach in the Mediterranean area, and it is the only allergen identified thus far in patients allergic to peach without an associated pollen allergy. 5,13,14 The majority of patients allergic to peach present an associated pollen allergy, and they also exhibit other food allergies, mainly to other Rosaceae species but also to taxonomically unrelated vegetable foods. 1,2,4-6,12-15 Cross-reactive IgE antibodies directed toward LTPs, Bet v 1 homologues (in patients allergic to birch pollen), profilins, and carbohydrate determinants are based on these associations. 4,9,10,12-14,16-23 Clinically relevant cross-reactivity among LTPs from botanically distant plants has been demonstrated. 12,23 As for any food, the diagnosis of peach allergy is based on the clinical history, supported by the demonstration of specific IgE, and confirmed by a positive oral challenge. Kivity et al 3 and Rodriguez et al 7 have reported positive responses in double-blind, placebo-controlled food challenges (DBPCFCs) to peach in 75% and 71% of patients, respectively. Similar results (83%) had been found by Pastorello et al 13 by means of open food challenges (OFC). There is, therefore, a group of patients who report adverse reactions to peach, who have specific IgE to this fruit but tolerate its ingestion when challenged (current clinically irrelevant sensitizations). Are there any clinical and/or immunologic differences that can account for this different clinical outcome upon 789

2 790 Fernández-Rivas et al J ALLERGY CLIN IMMUNOL OCTOBER 2003 Abbreviations used CU: Contact urticaria DBPCFC: Double-blind placebo-controlled food challenge LTP: Lipid transfer protein OAS: Oral allergy syndrome OFC: Open food challenge SPT: Skin prick test ingestion of this fruit? To investigate this point, we studied a group of 100 patients with adverse reactions to peach, in whom the actual clinical tolerance or reactivity was established by DBPCFCs. The aim of this study was to compare the clinical features and the reactivity to the allergens Pru p 3, rbet v 1, and rbet v 2 (birch profilin) of patients allergic to peach with those of subjects who tolerated the ingestion of this fruit. METHODS Patients One hundred subjects were consecutively selected among the patients 6 years old referred to the Allergy Unit of the Fundacion Hospital Alcorcon complaining of adverse reactions after the ingestion and/or contact with peach. The study was performed from May to October 2001, which is the peach season in Spain. The group comprised 40 male and 60 female subjects, 6 to 69 years old, with a mean age of years (SEM = 1.14). The study was performed with the approval of the ethics committee, and all the patients (or their representatives) gave written consent. Clinical evaluation The first step comprised a medical history, skin prick tests (SPTs), and blood sampling. A complete and careful medical history was obtained for each patient to fully characterize the reported reaction(s) to peach. On a second step, the actual clinical reactivity to peach was assessed by DBPCFCs, except in those patients (n = 13) who had specific IgE to peach and a convincing history of a severe anaphylaxis immediately related to peach ingestion in the 2 previous years. If the DBPCFC was negative, the patient was subsequently challenged in an open way (OFC) with fresh peach. Oral challenges were performed according to previously published procedures. 24,25 The amount of peach given was estimated on the basis of the weight of an average peach, which is approximately 150 to 200 g (with the stone). For the DBPCFC, a total of 150 g of fresh peach (peel and pulp) was masked in a vehicle (used alone as placebo food) that consisted of orange and pineapple juices (130 ml), coconut yogurt (125 g), soluble coffee (1 small spoonful), soluble cereals (20 g), and dried coconut (1 spoonful). Verum and placebo were randomly administered on separate days. Challenge foods were prepared immediately before the challenge, and 3 doses (one eighth, one fourth, and five eighths of total weight) were administered at 1-hour intervals. In the OFC, the patient received 30 g and 120 g of whole peach (peel and pulp) with a 1-hour interval. Rub tests with peach were performed in those patients who reported CU when handling peaches. The patient s forearm was rubbed with peach (peel) for 30 seconds and examined 15 to 20 minutes later. 6 Peach oral challenges and rub tests were performed out of the pollen season in those patients who had an associated pollen allergy. All the challenges were conducted in the hospital setting, with careful medical monitoring of the patients, and full emergency treatment was readily available. The reactivity or tolerance to other foods was carefully collected in the medical history, but it was not systematically assessed by oral challenges. Inhalant allergies were diagnosed by medical history and corresponding SPTs to the relevant aeroallergens of the area. Purified Pru p 3 Natural Pru p 3 was purified as previously described 10 and quantified by the bicinchoninic acid test. 26 It was tested at a concentration of 20 µg/ml in 0.9% NaCl, 50% glycerol, and 0.4% phenol, as previously published. 12 Skin prick tests All the patients underwent SPTs with fresh peach (peel and pulp separately) performed after the prick-prick method 27,28 and with Pru p 3 (in duplicate). SPTs were performed by the same investigator, on the volar surface of the forearm by using a standard 1-mmtip lancet, following the recommendations of the EAACI. 29 Histamine hydrochloride (10 mg/ml) and saline (ALK-Abello, S.A., Madrid, Spain) served as positive and negative controls, respectively. SPTs with pollens, mites, molds, cockroach, cat, and dog (ALK- Abello, S.A.) were performed in all subjects. The pollens tested were grass, olive, Cupressus arizonica and sempervirens, Platanus acerifolia, Artemisia vulgaris, Amaranthus retroflexus, Chenopodium album, Salsola kali, Parietaria judaica, and Plantago lanceolata. Any food different from peach involved in adverse reactions was also evaluated by SPTs. The wheal areas were measured in millimeters squared, using the Prick-Film System (Inmunotek, Madrid, Spain). An SPT reaction was considered positive if the wheal area was 7 mm 2 (diameter, 3 mm) greater than the negative control. 30 In vitro tests Total IgE and specific IgE to rbet v 1, rbet v 2, peach, and to any other food involved in adverse reactions were determined by the Pharmacia CAP System FEIA standard procedure (Pharmacia Diagnostics, Uppsala, Sweden). Statistical analysis Statistical analysis was performed with the use of Epi Info and SPSS software. Descriptive statistics included frequency of positive results (as percentage) with its 95% confidence interval for qualitative variables and mean ± SEM for quantitative variables. A χ 2 test was used for comparisons of frequencies. Differences in quantitative variables between allergic and nonallergic subgroups were compared by a Mann-Whitney U test. Paired correlations were calculated by Pearson test (r). Values were considered significant at a value of P <.05. RESULTS The results of the evaluation of the patients are presented in Fig 1. Allergy to peach was diagnosed in 76 patients (76%; 95% CI, 66% to 84%) (peach allergy group). These 76 subjects had specific IgE to peach (positive SPTs and/or CAP), and reactivity to the ingestion and/or contact with this fruit was demonstrated by oral challenges and/or rub tests in 68 of 76 subjects. Because of severe anaphylaxis, 13 subjects were not eligible for oral challenge tests, but CU was demonstrated in 5 of them. DBPCFCs were performed in 63 of the 76 patients allergic to peach. Positive results were obtained in 31 subjects in the blind challenges, whereas 32 did not react. These 32 subjects underwent OFCs that were positive in 21 and negative in 11. These 11 subjects tolerated the

3 J ALLERGY CLIN IMMUNOL VOLUME 112, NUMBER 4 Fernández-Rivas et al 791 FIG 1. Clinical evaluation of 100 patients reporting adverse reactions to peach contact and/or ingestion TABLE I. Patient characteristics Allergic to peach n = 76 Nonallergic to peach n = 16 P value Sex 31 male (41%) 6 male (38%) > female (59%) 10 female (62%) Age at study y (SEM 1.25) y (SEM 3.12) Age at first reaction to peach y (SEM 1.32) y (SEM 3.54) Time of peach avoidance 8.53 y (SEM 0.84) 8.44 y (SEM 1.60) Family history of atopy 45 (63%) 10 (62%) Pollen allergy 59 (78%) 16 (100%) Age at onset of pollen allergy (SEM 0.99) (SEM 2.29) Pollens involved in pollen allergy Grass 9/59 (15%) 1/16 (6%) Olive 2/59 (3.5%) 1/16 (6%) Grass + olive 30/59 (51%) 9/16 (56%) Grass + cupressus 16/59 (27%) 5/16 (31%) Platanus 2/59 (3.5%) 0 Other (potential) food allergies 61 (80%) 10 (62%) Data of family history of atopy were collected for 71 of the 76 patients allergic to peach. intake of peach but had CU, which was confirmed by positive rub tests. Actual clinical allergy to peach was ruled out in 16 patients (16%; 95% CI, 9% to 25%) who tolerated the contact and ingestion of the fruit (nonallergic group). Two patients dropped out (one moved to another city and the other one did not adhere to the study protocol), and the study was not conclusive in 6 individuals who reacted similarly with verum and placebo foods (placebo reactors). These 6 subjects reported oropharyngeal itching with no objective signs. The ingredients of the placebo food were well tolerated in their normal feeding, and sensitization to them was ruled out by SPTs. Even with a multiple cross-over in the DBPCFCs and the aid of visual scales for scoring the intensity of symptoms, no differences could be established between verum and placebo days (data not shown). CU was confirmed by positive rub tests in 42 patients allergic to peach (42 of 76: 55%; 95% CI, 43% to 67%). No systemic reactions were reported by patients in association with peach handling; neither were they observed in positive rub tests. In the 76 patients allergic to peach, 3 subgroups could be found: 34 subjects (45%; 95% CI, 33% to 57%) who reacted to peach ingestion but tolerated its contact, 31 individuals (41%; 95% CI, 30% to 53%) who reacted to both peach intake and handling, and 11 patients (14%; 95% CI, 7% to 24%) who had CU but tolerated peach intake (Fig 1). Because no differences were found among them in respect to the clinical and immunologic parameters presented in Tables I and II, all the allergic subjects were combined into one group. Patient characteristics are presented in Table I. No significant differences were found between the 2 groups in any of the clinical features. Female patients reported adverse reactions to peach with a higher frequency than did male subjects (60% female, 40% male, P <.05), and this proportion was maintained in the peach-allergic (59% female, 41% male) and nonallergic (62% female, 38% male) groups. Pollen allergy was present in 59 patients allergic to peach (78%; 95% CI, 67% to 86%) and in all the nonallergic subjects (100%; 95% CI, 79% to 100%). No differences were found in the reported symptomatology induced by peach intake between allergic and nonallergic subjects (Fig 2). For this analysis, we used the reported symptomatology of 59 of 76 subjects of the peach-allergy group and of 10 of 16 nonallergic patients (the remaining individuals reported exclusively CU). The

4 792 Fernández-Rivas et al J ALLERGY CLIN IMMUNOL OCTOBER 2003 FIG 2. Reported symptomatology after peach intake. TABLE II. SPTs and total and specific IgE results Allergic to peach n = 76 Nonallergic to peach n = 16 n Freq (%) 95% CI, % Mean SEM P value n Freq (%) 95% CI, % Mean SEM SPT peach peel > SPT peach pulp > SPT Pru p Total IgE > CAP peach > CAP rbet v > CAP rbet v > Mean values for SPTs and CAP are given in mm 2 and ku/l, respectively. CAP to peach, rbetv 1, and rbet v 2 were performed in 75, 74, and 73 peach allergic patients, respectively. The P value given is for the comparison of frequencies. n, Number of patients in the group with a positive test result (SPT wheal area, 7 mm 2 ; CAP, 0.35 ku/l; Freq, frequency of positive test results in the group. symptomatology observed in the oral challenges was not used to establish the clinical presentation of peach allergy because it does not reflect the true severity of this food allergy. Oral challenges were performed to confirm the patient s actual reactivity to peach but not to confirm the severity of the original reported reaction. The symptomatology has been classified in isolated OAS, isolated systemic symptoms (not preceded by OAS), and OAS plus systemic symptoms (OAS immediately followed by a systemic reaction) (Fig 2, left bars). Systemic reactions were reported by 28 of 59 of the allergic subjects and by 4 of 10 of the nonallergic individuals and comprised generalized urticaria, anaphylaxis (generalized systemic reaction involving the skin plus other target organ/s), asthma, and rhinoconjunctivitis (Fig 2, right bars). The latter 2 manifestations were not associated with OAS or skin involvement, appeared 30 to 60 minutes after peach ingestion, and were confirmed by DBPCFCs. SPTs and total and specific IgE results are presented in Table II. In the group of subjects allergic to peach, positive SPT reactions to peach peel and pulp were observed in 91% and 80% of patients tested (P =.06), whereas only 68% had a positive CAP to peach (SPT peel versus CAP, P <.001; SPT pulp versus CAP, P =.08). A trend to higher CAP values was observed in the group of allergic patients (4.38 ku/l vs 1.85 ku/l, P =.06). Positive responses to Pru p 3, rbet v 1, and rbet v 2 were observed in 62%, 7%, and 34% of allergic patients, respectively. The combination of positive results to Pru p 3 and rbet v 2 identified 63 of 76 patients allergic to peach (83%), and the combination of the 3 allergens covered 64 of 76 subjects (84%). The rate of positive SPT results to Pru p 3 was higher in the group of subjects allergic to peach (62% vs 31%, P =.02). No difference was found in the rates of sensitization to Pru p 3 among patients allergic to peach in relation to the severity of the clinical presentation (52% in OAS patients vs 71% in subjects with isolated systemic symptoms, P =.13). In a second step, the IgE responses to Pru p 3, rbet v 1, and rbet v 2 were analyzed, considering peach and pollen allergies (Fig 3). Sensitization to Pru p 3 was found with a significantly higher frequency among patients allergic to peach, and it was not related to the pollen allergy (61% in patients with peach and pollen allergies vs 59% in subjects allergic to peach without pollinosis, P >.05). In contrast, IgE responses to rbet v 2 were observed with a significantly higher frequency among patients allergic to pollen, but no difference (P >.05) was observed in the

5 J ALLERGY CLIN IMMUNOL VOLUME 112, NUMBER 4 Fernández-Rivas et al 793 FIG 3. Patterns of sensitization to the allergens Pru p 3, rbet v 1, and rbet v 2. presence (43%) or absence (50%) of peach allergy. Only 1 of 25 of the patients with a positive CAP to rbet v 2 was not allergic to pollen. This patient had OAS to peach, melon, and banana and had positive SPT results to the majority of pollens tested but without clinical expression (perhaps sensitization to profilin was enough to induce OAS to food ingestion but not to trigger rhinoconjunctivitis and/or asthma upon pollen exposure). Other (potential) food allergies were present in 61 subjects allergic to peach (80%) and in 10 of the peach-tolerant patients (62%) (P >.05). All the foods were of plant origin (not shown). Because the diagnosis was made on the basis of convincing histories and positive SPT results and/or CAP to the foods involved, the term potential food allergies is used. Patients allergic to peach reacted to a higher number of Rosaceae species (2.22 vs 0.75, P <.001) and to a higher number of different foods (4.22 vs 2.19, P =.005) that belonged to more different botanical families (2.74 vs 1.62, P =.01). The number of foods and of botanical families were correlated to the peach CAP (r = 0.37 and r = 0.38, respectively, P <.01) and to the rbet v 2 CAP (r = 0.37 and r = 0.38, respectively, P <.01). The number of Rosaceae fruits was also correlated to the peach CAP (r = 0.31, P =.008). DISCUSSION In this study, the prevalence of actual peach allergy established by DBPCFCs was 76%. This result is in agreement with the only 2 studies that have also assessed peach reactivity by DBPCFCs, 3,7 and it is significantly higher than the reported figures (generally <50%) for other foods evaluated by DBPCFCs. 24 By combining the series of Kivity et al, 3 Pastorello et al, 13 Rodriguez et al, 7 and ours, the prevalence of peach allergy confirmed by oral challenges among patients reporting adverse reactions to it is 76% (166 positive challenges in 219 patients), with a 95% CI of 70% to 81%. All the patients included in this work reported convincing histories of peach allergy, and followed the same study protocol, but 76% reacted in the peach challenges, whereas 16% did not. The outcome of the DBPCFC was not related to any clinical parameter. No differences were found in the reported symptomatology, with a similar distribution of mild local reactions such as CU or isolated OAS, and systemic symptoms (Fig 2). Both groups had a similar age at study and at first reported reaction to peach, and the time of avoidance was also similar (Table I). Peach allergy is a persistent food allergy in our population, since 76% of patients reacted to peach intake after a mean time of avoidance longer than 8 years. Mild local and severe systemic presentations of peach allergy are similarly persistent. In the patients allergic to peach, positive SPT results to peach peel (91%) and pulp (80%) were observed more frequently than positive CAP results to peach (68%). This reflects the already known higher sensitivity of SPTs with fresh fruits. 2,7,31 No differences were observed between allergic and nonallergic patients in the SPTs to peach due to the low specificity of these tests (<25% in our study). However, patients allergic to peach had higher specific IgE to peach and a wider pattern of reactivity to Rosaceae fruits and to other plant foods, which was correlated to the peach CAP. Additionally, the reactivity to any plant food was also correlated to the rbet v 2 CAP, which is in agreement with the ubiquitous presence of profilin within the plant kingdom 32,33 and has already been reported by other investigators. 34 This suggests that higher IgE titer antipeach and anti-profilin could broaden the reactivity (although not confirmed by oral challenges) to other Rosaceae fruits and to other plant foods.

6 794 Fernández-Rivas et al J ALLERGY CLIN IMMUNOL OCTOBER 2003 LTPs, Bet v 1 homologues, and profilins are the 3 most important allergens involved in Rosaceae fruit allergy. 4,9,10,12-14,16-23 In this work, we have evaluated reactivity to Pru p 3 by SPTs, whereas IgE responses to the peach Bet v 1 homologue and peach profilin were indirectly studied by means of CAP to rbet v 1 and rbet v 2 (birch profilin), respectively. Profilins and Bet v 1-related allergens from Rosaceae fruits have a high homology and a high degree of IgE cross-reactivity with their homologous allergens in birch ,22,35-37 However, the existence of epitope differences may decrease the sensitivity of the birch-related diagnostics to detect specific IgE to the homologous allergens in fruits. 37 Despite this, we have recently cloned peach profilin (rpru p 4) and found a complete correlation between IgE reactivity to rpru p 4 and rbet v In the group of patients allergic to peach, the rates of sensitization to Pru p 3, rbet v 2, and rbet v 1 were 62%, 34%, and 7%, respectively. These results confirm the role of Pru p 3 as the major allergen of peach in our population. Profilin is a minor allergen, and sensitization to rbet v 1 is exceptionally observed, as already reported 4 and expected in an area almost free of birch (and related Fagales) pollen. 39 From a diagnostic perspective, SPTs with Pru p 3 have a lower sensitivity (62%) than prick-prick tests with fresh peach peel (91%). The combination of SPTs with Pru p 3 and CAP to rbet v 2 increases sensitivity to 83%. The further addition of rbet v 1 only produced a 1% increase in sensitivity (up to 84%). However, 16% of patients allergic to peach were not detected by the combination of these allergens. These subjects had SPTs >7 mm 2 but below the histamine control and/or had low levels of specific IgE to peach (class 1 CAP). Factors such as antibody affinity, epitope differences between peach profilin and rbet v 2, or the involvement of allergens not tested could explain these negative results. SPTs to Pru p 3 had been previously performed by our group. 12 In that study, the rate of sensitization to Pru p 3 in 24 patients selected in our center (with positive case histories and specific IgE to peach but no oral challenges) was higher than the one found in the present study (83% vs 62%, P =.05), probably related to the different selection criteria. When the reactivity to Pru p 3, rbet v 1, and rbet v 2 was compared between allergic and nonallergic patients, a significantly higher frequency of sensitization to Pru p 3 was found, whereas the distribution of the other two allergens did not differ (Table II). The subsequent analysis of reactivity to Pru p 3 and rbet v 2 according to the presence or absence of peach and pollen allergies (Fig 3) shows a clearly cut, different scenario for these two allergens. Pru p 3 is a true food allergen, and the IgE response to it is linked to the clinical expression of peach allergy. In contrast, sensitization to profilin (rbet v 2) is related to the pollen allergy but not to the clinical reactivity to peach. A lack of clinical relevance of profilin has already been suggested in Dutch patients allergic to pollens and plant foods. 34 In summary, the analysis of the IgE responses to the allergens Pru p 3, rbet v 1, and rbet v 2 in relation to the outcome (positive or negative) of DBPCFCs with peach has allowed us to establish the crucial role of Pru p 3 in the clinical reactivity to peach. Further studies are needed to elucidate the clinical relevance of profilin, which should be performed in patients not sensitized to the major allergens such as LTPs or Bet v 1 homologues. REFERENCES 1. Hernandez J, Garcia Selles FJ, Pagan JA, Negro JM. Hipersensibilidad inmediata a frutas y verduras y polinosis. Allergol Immunopathol 1985;13: Ortolani C, Ispano M, Pastorello E, Bigi A, Ansaloni R. The oral allergy syndrome. Ann Allergy 1988;61: Kivity S, Dunner K, Marian Y. 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