Relationship between peach lipid transfer protein specific IgE levels and hypersensitivity to non-Rosaceae vegetable foods in patients allergic to lipid transfer protein

Relationship between peach lipid transfer protein specific IgE levels and hypersensitivity to non-Rosaceae vegetable foods in patients allergic to lipid transfer protein Riccardo Asero, MD*; Gianni Mistrello, BSc†; Daniela Roncarolo, BSc†; and Stefano Amato, BSc†

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. Ann Allergy Asthma Immunol. 2004;92:268–272.

INTRODUCTION Lipid transfer protein (LTP), the major allergen in Rosaceae in geographic areas where the prevalence of birch pollen allergy is low,1–5 is a potentially hazardous food allergen due to its extreme resistance to pepsin digestion.6 Because LTP is widely distributed throughout the plant kingdom and LTPs from distinct botanical sources show a moderate-to-high (35% to 95%) degree of sequence homology, it is not surprising that patients allergic to Rosaceae sensitized to this protein may show IgE reactivity, often associated with clinical allergy, to a number of botanically unrelated, plant-derived foods.6 –15 Since only a fraction of Rosaceae-allergic patients sensitized to LTP show cross-reactivity

to other vegetable foods, the spectrum of LTP allergy ranges from patients sensitized uniquely to peach or Rosaceae to patients hypersensitive to an array of fruits and vegetables; interestingly, no LTP-allergic patient hypersensitive to foods other than peach in the absence of sensitization to peach has been described so far.6 –15 These observations suggest that the peach probably represents the primary source of sensitization to LTP and that peach LTP contains all allergenic epitopes of LTPs from other vegetable foods. In the present study, we investigated whether the level of IgE to peach LTP is associated with immunologic cross-reactivity to botanically distinct, plant-derived foods in a cohort of LTP-hypersensitive patients.

* Ambulatorio di Allergologia, Clinica San Carlo, Paderno Dugnano, Milan, Italy. † Lofarma SpA, Milan, Italy. Received for publication July 18, 2003. Accepted for publication in revised form September 18, 2003.

METHODS Patients Forty adults with a history of oral allergy syndrome, urticaria, gastrointestinal symptoms, and/or anaphylaxis following the ingestion of Rosaceae (apple, pear, peach, cherry, plum,

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apricot, or almond) monosensitized to LTP were studied. Patients were screened from a population of approximately 700 patients with Rosaceae allergy on the basis of a positive skin prick test (SPT) result with a commercial plum extract (Dome-Hollister/Stier, Spokane, WA; 1:10 wt/vol); we previously demonstrated that this extract contains LTP but virtually lacks both Bet v 1–like proteins and profilin.16 On the basis of the well-known cross-reactivity between LTP from distinct Rosaceae,5 this commercial extract represents an excellent means to detect patients sensitized to LTP in the clinical practice. Patients were considered as monosensitized to LTP in the absence of cosensitization to other crossreacting, plant-derived food allergens as shown by negative SPT results with commercial extracts of both birch17–21 and mugwort22–24 pollen (Allergopharma, Reinbeck, Germany). All skin tests in this study were performed as a part of the routine evaluation of LTP-hypersensitive patients seen at this allergy unit. All the patients gave their consent to venipuncture for serologic investigations. Patients were thoroughly interviewed to detect all foods other than Rosaceae that had caused adverse reactions. Measurement of IgE to Peach LTP IgE reactivity to LTP was measured by enzyme-linked immunosorbent assay using the 10-kDa fraction of peach peel separated by gel chromatography6 as substrate. The LTP from peach was reconstituted in 50-mmol/L sodium bicarbonate, pH 9.6, and coated overnight onto polystyrene microtiter plates (100 ng per well). After washings, plates were saturated with 5% nonfat dry milk in 0.15-mmol/L Tris buffer, pH 7.2, for 1 hour. After further washing, serum samples were added (50 ␮L of serum plus 50 ␮L of Tris buffer) and incubated for 1 hour. Bound IgE was detected with peroxidase-conjugated anti-human IgE serum (Sigma, Milan, Italy) and tetramethylbenzidine as substrate. The enzyme reaction was stopped after 10 minutes by the addition of 1-mol/L sulfuric acid, and absorbance was read at 450 nm. IgE levels were expressed as absorbance (optical density [OD]); levels more than 2 times the average OD of a pool of normal sera (ie, levels ⬎66 OD) were considered positive. Skin Tests Patients underwent SPTs with commercial extracts (DomeHollister/Stier, 1:20 wt/vol) of representatives of several different botanical families: walnut, hazelnut, peanut, celery, maize, rice, tomato, orange, and onion. Other tree nuts possibly containing LTP (eg, chestnut, Brazil nut, pecan, pine nut, pistachio) were not available in the commercial panel used to perform this study. All skin tests were performed on the volar side of the forearm using sterile, 1-mm-tip lancets (Dome-Hollister/Stier). Readings were taken after 15 minutes. Reactions were expressed as mean wheal diameter (adding the longest diameter to the orthogonal diameter and dividing it by 2). A wheal diameter of 3 mm or more was considered positive.25 Histamine, 10 mg/mL, and saline were used as positive and negative controls, respectively.

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Statistical Methods Because of the skewed distribution of IgE levels to peach LTP, data were compared by nonparametric means using either the Mann-Whitney U test or Spearman rank correlation test where needed. P ⬍ .05 was considered statistically significant. RESULTS Levels of IgE to peach LTP and results of SPT with plantderived foods other than Rosaceae are shown in Table 1. IgE levels ranged from 79 OD (patient 1) to 3,576 OD (patient 40). Eight patients (20%) did not show any skin reactivity to non-Rosaceae foods; 7 of these ranked among the 10 patients who showed the lowest IgE levels. Patients who tested negative on SPT 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). Five patients had positive SPT results with all non-Rosaceae foods; in these patients, levels of IgE to peach LTP ranged from 1,688 to 3,576 OD, and 3 of them ranked among the 5 patients with the highest IgE levels (Table 1). 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%]) (Table 1). The number of positive SPT results and the reported offending foods other than Rosaceae are shown in Table 2. Although the spectrum of reported offending foods was much larger than the series of 8 foods considered for SPTs, growing levels of IgE to peach LTP were paralleled by an increasing number of foods both positive on SPTs and causing clinical symptoms (Table 2). DISCUSSION Although LTP from botanically unrelated, plant-derived foods share IgE-binding epitopes,6 –15 patients allergic to this protein show a high variability in their IgE reactivity to vegetable foods. In this study, we found that the likelihood of becoming sensitized to vegetable foods other than Rosaceae is directly related to peach LTP specific IgE levels and that specific patterns of cross-sensitization can be observed at different IgE levels. As previously noted,7,16 the foods most frequently involved in cross-reaction phenomena were nuts (walnut, hazelnut) and peanut; IgE reactivity to these foods was observed in approximately 70% of patients, from IgE levels as low as 262 and 135 OD, respectively. At the opposite end, skin reactivity to tomato was observed only in 20% of patients, all showing levels of IgE to peach LTP of more than 1,600 OD. We used commercial extracts to perform the skin tests. In view of the extreme resistance of LTP and of its

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Table 1. Levels of IgE to Peach LTP and Skin Reactivity to 8 Non-Rosaceae, Plant-Derived Foods Skin prick test result Rank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Peach LTP IgE, OD 79 98 127 130 135 148 229 262 306 446 551 615 962 973 980 991 1,084 1,097 1,249 1,294 1,560 1,688 1,720 1,816 1,888 2,046 2,070 2,152 2,313 2,341 2,441 2,558 2,711 2,940 3,069 3,258 3,346 3,405 3,568 3,576

Nuts*

Peanut

Maize

Rice

Onion

Orange

Celery

Tomato

Neg Neg Neg Neg Neg Neg Neg Pos Pos Neg Pos Pos Pos Pos Pos Pos Neg Pos Pos Neg Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Neg Neg Neg Neg Pos Neg Neg Neg Neg Neg Pos Neg Pos Pos Neg Neg Pos Pos Pos Pos Neg Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Pos Neg Pos Neg Pos Pos Pos Neg Neg Pos Pos Neg Pos Pos ND Pos Pos Pos Pos Pos Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Neg Pos Neg Pos Pos Pos Neg Neg Pos Pos Neg Pos Neg ND Pos Pos Pos Pos Pos Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos Neg ND Pos Neg ND Pos Neg Pos Pos Pos Pos Pos ND Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg ND Neg Neg Neg ND ND Neg Pos Neg ND Neg Pos Neg Pos ND Pos ND ND Pos Neg ND Pos Pos Pos ND Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Neg Pos Neg Pos Neg Pos Pos Pos Neg Neg Neg Neg Neg Neg Neg Neg Pos Pos Pos Pos Neg Pos

Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Pos Neg Neg Pos Pos Neg Neg Pos Neg Neg Pos Neg Neg Neg Pos Pos Neg ND Pos

Abbreviations: LTP, lipid transfer protein; Neg, negative; ND, not done; OD, optical density units; Pos, positive. * Walnut and/or hazelnut.

persistence in commercial extract of vegetable foods,16 it is unlikely that the use of fresh foods would have produced an increase in the number of positive test results. The possibility that these results may depend on the different concentrations of LTP in vegetable food extracts for SPTs seems very unlikely. If so, skin reactivity to all non-Rosaceae vegetable foods would show a linear increase that parallels levels of IgE to peach LTP in all patients; however, some patients who showed high IgE to peach LTP (eg, patients 33, 31, 27, and 21) had negative SPT results with non-Rosaceae foods. Another possible explanation for these findings might be that cross-reactivity reflects the degree of sequence homology

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between LTPs from peach and other plant-derived foods. However, if this were the case, all patients should react to foods that show a high degree of sequence homology to peach LTP; the different combinations of positive SPT results with botanically unrelated, plant-derived foods observed in LTPallergic patients do not seem to support this hypothesis. An alternative explanation might be that all allergenic determinants present in LTP from vegetable foods other than peach cross-react with peach determinants, whereas only some peach LTP epitopes cross-react with allergenic determinants in LTP from botanically unrelated fruits and vegetables. In such a model, high levels of IgE to peach LTP would reflect

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Table 2. Number of Positive SPT Results and Reported Offending Foods Other Than Rosaceae Rank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Peach LTP IgE, OD

No. of positive SPT results

79 98 127 130 135 148 229 262 306 446 551 615 962 973 980 991 1,084 1,097 1,249 1,294 1,560 1,688 1,720 1,816 1,888 2,046 2,070 2,152 2,313 2,341 2,441 2,558 2,711 2,940 3,069 3,258 3,346 3,405 3,568 3,576

0 0 0 0 1 0 0 1 1 0 2 1 2 2 1 1 1 2 3 5 0 8 2 6 6 8 2 2 6 5 2 6 4 3 6 8 8 7 4 8

Reported offending foods other than Rosaceae None None None None Nuts None None Nuts Nuts None Nuts, peanut Nuts Nuts, peanut, string bean Peanut Nuts None None Nuts, peanut, pineapple, mustard Maize Fennel, pesto sauce (pine nuts) None Nuts None Nuts, fennel Nuts, peanut, maize, celery, legumes, tomato Nuts, rice, maize, salad Nuts Nuts, peanut, lentil Nuts, tomato, kiwi, pineapple Nuts, peanut, grapes, eggplant Nuts Rice, onion Nuts Peanut, pistachio Nuts, peanut, beer, parsley, banana Nuts, grapes, watermelon Nuts, tomato, artichoke Nuts, peanut, maize, celery, fennel, onion, eggplant Nuts Beer

Abbreviations: LTP, lipid transfer protein; OD, optical density units; SPT, skin prick test.

the presence of IgE targeting common allergenic determinants of LTP (and, hence, causing cross-reactivity to botanically unrelated vegetable foods), whereas low levels of IgE to peach LTP would reflect the presence of IgE targeting non– cross-reactive, peach-specific determinants. Such a situation would be virtually identical to that of hypersensitivity to Apiaceae and Rosaceae in patients with birch pollen allergy.26 This view is supported by the findings of our previous studies that showed the immunologic cross-reactivity between LTP from botanically unrelated, plant-derived foods.6,7 In those studies, the inhibition experiments showed that preabsorption of sera from multireactive patients with peach peel extract totally abolishes IgE reactivity to other vegetable

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foods, whereas preabsorption of sera with concentrated extracts of vegetable foods other than peach induces only partial suppression of IgE reactivity to peach LTP. The present study investigated the immunologic crosssensitization to non-Rosaceae vegetable foods in LTP-allergic patients with a history of allergy to peach. Skin reactivity does not automatically mean clinical allergy, because this may depend on a number of variables, such as the dietary habits, the amount of food ingested on a single occasion, the frequency of intake of a certain food, and the association with cofactors such as exercise or drugs. The clinical aspects did not represent the primary scope of this study; nonetheless, the analysis showed that the number of non-Rosaceae offending

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foods roughly parallels the level of IgE to peach LTP. Altogether, this study clearly indicates which patients are at risk for adverse reactions to non-Rosaceae vegetable foods. In patients allergic to LTP, increasing levels of IgE to peach LTP are associated with immunologic cross-reactivity to different non-Rosaceae, plant-derived foods. Patients who show high levels of IgE to peach LTP are at risk for adverse reactions to vegetable foods other than Rosaceae. REFERENCES 1. Lleonart R, Cistero` A, Carreira J, Batista A, Moscoso del Prado J. Food allergy: identification of the major IgE-binding component of peach (Prunus persica). Ann Allergy. 1992;69:128 –130. 2. Pastorello EA, Farioli L, Pravettoni V, et al. The major allergen on peach (Prunus persica) is a lipid transfer protein. J Allergy Clin Immunol. 1999;103:520 –526. 3. Sanchez-Monge R, Lombardero M, Garcia-Selles FJ, Barber D, Salcedo G. Lipid transfer proteins are relevant allergens in fruit allergy. J Allergy Clin Immunol. 1999;103:514 –519. 4. Pastorello EA, Pravettoni V, Farioli L, et al. Clinical role of a lipid transfer protein that acts as a new apple-specific allergen. J Allergy Clin Immunol. 1999;104:1099 –1106. 5. Pastorello EA, Ortolani C, Farioli L, et al. Allergenic crossreactivity among peach, apricot, plum, and cherry in patients with oral allergy syndrome: an in-vivo and in-vitro study. J Allergy Clin Immunol. 1994;94:699 –707. 6. Asero R, Mistrello G, Roncarolo D, 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. 7. Asero R, Mistrello G, Roncarolo D, et al. Immunological crossreactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study. Allergy. 2002;57: 900 –906. 8. Asero R, Mistrello G, Roncarolo D, Amato S, van Ree R. A case of allergy to beer showing cross-reactivity between lipid transfer proteins. Ann Allergy Asthma Immunol. 2001;87: 63– 67. 9. Pastorello EA, Farioli L, Pravettoni V, et al. The maize major allergen, which is responsible for food-induced allergic reactions, is a lipid transfer protein. J Allergy Clin Immunol. 2000; 106:744 –751. 10. Pastorello EA, Farioli L, Prevettoni V, et al. Identification of grape and wine allergens as endochinase 4, a lipid transfer protein, and a thaumatin. J Allergy Clin Immunol. 2003;111: 350 –359. 11. Pastorello EA, Vieths S, Pravettoni V, et al. Identification of hazelnut major allergens in sensitive patients with positive double-blind, placebo-controlled food challenge results. J Allergy Clin Immunol. 2002;109:563–570. 12. Diaz-Perales A, Tabar AI, Sanchez-Monge R, et al. Characterization of asparagus allergens: a relevant role for lipid transfer proteins. J Allergy Clin Immunol. 2002;110:790 –796. 13. Navarro AM, Orta JC, Sanchez MC, et al. Primary sensitization to Morus alba. Allergy. 1997;52:1144 –1145.

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