Lipid transfer protein and vicilin are important walnut allergens in patients not allergic to pollen

Lipid transfer protein and vicilin are important walnut allergens in patients not allergic to pollen Elide A. Pastorello, MD,a Laura Farioli, BSc,b Valerio Pravettoni, MD,a Anna M. Robino, MD,a Joseph Scibilia, MD,c Donatella Fortunato, BSc,d Amedeo Conti, PhD,d Linda Borgonovo, MD,a Anders Bengtsson, BSc,e and Claudio Ortolani, MDf Milan and Turin, Italy, and Uppsala, Sweden

Background: Walnut is the most common cause of allergic reactions to tree nuts, as reported by large population studies. Two major allergens of walnut have been identified up until now: a 2S albumin and a vicilin-like protein. Objective: This study was designed to identify the walnut major allergens in the Italian population and to compare the walnut IgE-binding profile in patients with or without pollen allergy. Methods: We selected 46 patients either with oral allergy syndrome confirmed by open oral challenge or with systemic symptoms after ingestion of walnut. These patients’ sera were used for the immunoblotting of walnut extract; the identified allergens were purified by HPLC and sequenced. A peach-walnut cross-inhibition study was then performed. Results: The only major allergen recognized by our study population was a 9-kd lipid transfer protein (LTP), recognized by 37 patients. Two other minor allergens of approximately 9-kd molecular weight, both belonging to the vicilin family, were recognized by 10 patients. IgE binding to walnut LTP was completely inhibited by peach LTP. Conclusion: In Italian patients with walnut allergy confirmed by documented history of severe systemic reactions or by open oral food challenge, the major allergen is an LTP. The sensitization to this protein seems to be secondary to the sensitization to peach LTP, which acts as the primary sensitizer. LTP and vicilins were able to sensitize patients not allergic to pollen. (J Allergy Clin Immunol 2004;114:908-14.) Food allergy, dermatologic diseases, and anaphylaxis

Key words: Walnut, food allergy, nuts allergy, anaphylaxis, vicilin, lipid transfer protein

Walnut together with other tree nuts and after peanut represents the food most frequently involved in anaphylaxis and anaphylaxis deaths. From athe Allergy Center, Department of Internal Medicine and Dermatology, Ospedale Maggiore Istituto di Ricovero e Cura a Carattere Scientifico, Milan; bIstituti Clinici di Perfezionamento, Milan; cBizzozero Division, Niguarda Ca’ Granda Hospital, Milan; dthe National Research Council, Istituto di Scienze delle Produzioni Alimentari, Turin; ePharmacia Diagnostics, Uppsala; and fIstituto Allergologico Lombardo, Cesano Boscone, Milan. Received for publication May 4, 2004; revised June 11, 2004; accepted for publication June 11, 2004. Reprint requests: Elide Anna Pastorello, MD, Allergy Center, Division of Internal Medicine and Dermatology, Ospedale Maggiore IRCCS, Via Pace 9, 20122 Milan, Italy. E-mail: [email protected]. 0091-6749/$30.00 Ó 2004 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2004.06.020

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Abbreviations used LTP: Lipid transfer protein OAS: Oral allergy syndrome

The register of food anaphylaxis-related fatalities in the United States reported that 3 of 32 cases between 1994 and 1999 were caused by walnut.1 Similarly, in the United Kingdom register of anaphylaxis deaths between 1992 and 1998, 37 food-induced fatalities were recorded, 5 caused by walnut.2,3 Severe anaphylaxis to walnut is reported in 2 studies in Great Britain. The first described 55 severe or fatal food allergic reactions in a pediatric population: 1 near fatal reaction was caused by walnut.4 The second study observed, in 1000 patients allergic to nut, 30 cases of severe allergic reactions to walnut.5 The importance of allergy to walnut and other nuts is related not only to the severity of the allergic reactions but also to the high prevalence in the general population, around 0.2% to 0.7%.6,7 The US Peanut and Tree Nut Allergy Registry,8 collecting 5149 patients (mainly children), reports walnut as the first cause of allergic reactions to tree nuts (34% of reactions). For all of these reasons, walnut is included by European laws in the list of allergens that must be always declared in food labeling, whatever the quantity.9 Despite such precautions, nuts still represent a very dangerous food allergen, because they are often hidden in prepackaged foods and very small amounts can cause severe reactions. For example, in the United States, 20% of the food product recalls for undeclared allergens related to nut-containing products, causing 20 of 34 allergic reactions.10 A better knowledge of the allergens involved in these reactions is thus necessary to set up sensitive detection systems. The walnut allergens so far identified are Jug r 111 and Jug r 2,12 corresponding to walnut 2S albumin and to vicilin-like proprotein, respectively. In the current study, we described 46 patients with severe reactions to walnut, with the aim of identifying the major allergens recognized by this Italian population and comparing them with already known allergens. Moreover,

we defined the relationship between allergy to walnut and allergy to pollens by using selected groups of patients with walnut allergy with or without pollen sensitization.

METHODS Patients Patients referred to the two Allergy Centres in Milan were selected on the basis of documented allergic reactions to walnut. All of the patients were recruited and studied according to a strict protocol: (1) a careful clinical history with reaction documentation, (2) positive skin prick tests with commercial walnut extract (Stallergenes Italia, Milan, Italy), (3) positive walnut-specific IgE antibodies by CAP System (Pharmacia & Upjohn, Uppsala, Sweden), and (4) positive oral provocation test in patients without history of severe systemic symptoms. Allergy to pollens was evaluated by clinical history of respiratory symptoms during the pollen season and positive skin prick tests (Stallergenes Italia) and CAP system results with commercial pollen extracts. Blood was taken from all of the patients, and sera were collected and stored at 280°C.

In vivo study Skin prick tests. Skin prick tests with commercial walnut extract were performed on the volar side of the forearm with a 1-mm–tip lancet. After 20 minutes, the average diameter of the wheal was measured and compared with that of the wheal induced by histamine, 10 mg/mL. Only wheals with diameter of 4 mm or greater were considered positive. Open oral provocation test. Patients without clinical history of life-threatening reactions to walnut underwent an oral provocation test with fresh walnut, following a previously described protocol.13 Briefly, the patients were asked to chew and swallow increasing amounts of walnut, from 1 to 15 g (the approximate mean weight of a single walnut kernel is 5 g). The challenge was stopped when symptoms arose.

Identification of walnut allergens Walnut extract. Walnuts were ground in a kitchen mill, defatted with acetone (1:5 wt/vol) on a stirrer for 2 hours, and then filtered through a Bu¨chner funnel. Defatting for 30 minutes and filtering were repeated twice before leaving the powder to dry overnight. The nut powder was diluted 1:10 wt/vol in 0.1 mol/L phosphate buffer (pH 7.4) and shaken for 2 hours at 4°C to 8°C; the suspension was then centrifuged for 45 minutes at 4°C at 18000 rpm. The protein concentration of this extract, by the Lowry method,14 was 7.46 mg/mL. The extract was then concentrated by membrane ultrafiltration with Amicon cell (Amicon Inc, Beverly Millipore Corp, Bedford, Mass) with a molecular weight cutoff at 3500 d to reach a protein concentration of approximately 12 mg/mL. SDS-PAGE/immunoblotting. The IgE-binding profile of walnut extract and the IgE reactivity of the walnut allergens purified by HPLC were determined by SDS-PAGE/immunoblotting. SDS/PAGE was performed as previously described.15 The sample was diluted 1:2 with buffer containing 3% 2-mercaptoethanol, 2% SDS, 7% glycerol, and 0.001% bromphenol blue as tracking dye. Electrophoresis was run at 6 mA/gel for 18 to 20 hours in a BioRad Protein IIxi vertical electrophoresis slab cell (Bio-Rad Labs, Richmond, Calif), yielding 0.3 mg proteins (for walnut extract) and 0.03 mg purified proteins (for allergens) in each centimeter of gel. The separated proteins were transferred onto a nitrocellulose membrane (0.4 mm; Amersham, Buckinghamshire, United Kingdom)

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by electrophoretic transfer (Bio-Rad Trans blot cell), as described elsewhere.15 Unoccupied protein binding sites were blocked by placing it in PBS pH 7.4 with 0.1% TWEEN 20 for 30 minutes at 37°C. The nitrocellulose was then cut into strips and incubated with the serum of each patient with walnut allergy; after washing, a second incubation was performed with 125I-labeled antihuman IgE (Pharmacia & Upjohn). The strips were washed, dried, and exposed on x-ray film at 270°C.

Purification of walnut allergens Gel filtration. By using an HPLC system (AKTA Purifier; Amersham Pharmacia Biotech, Uppsala), 1 mL concentrated walnut extract was injected on a HiLoad Superdex 75 16/60 column (Amersham Pharmacia Biotech), equilibrated, and eluted with 35 mmol/L sodium phosphate buffer pH 7.5, 0.3 mol/L NaCl, at a flow rate of 1 mL/min. The absorbance was monitored at 280 nm. The fraction corresponding to proteins of approximately 9 kd, analyzed by SDS-PAGE, was also found to contain proteins of higher molecular weight; further resolution was obtained by cationic exchange. Cationic exchange chromatography. The concentrated sample was desalted by HiTrap Desalting column (Amersham Biosciences AB, Uppsala) and then loaded onto a cationic exchange column Resource S (bed volume, 1 mL; Amersham Pharmacia Biotech), equilibrated with 30 mmol/L sodium citrate dihydrate buffer pH 2.2, and eluted with a salt gradient from 0 to 1 mol/L NaCl in 25 column volumes at a flow rate of 2 mL/min. The well-defined peaks were analyzed by SDS-PAGE and by immunoblotting with the sera of patients with walnut allergy. Anionic exchange chromatography. The concentrated walnut extract was also separated on an anionic exchange Resource Q column (bed volume, 6 mL; Amersham Pharmacia Biotech) equilibrated with 20 mmol/L Tris-HCl buffer pH 8 and eluted with a salt gradient from 0 to 1 mol/L NaCl in 15 column volumes at a flow rate of 5 mL/min. The eluted peaks were analyzed by SDS-PAGE; the peak containing several bands at low molecular weight was used in a further purification step by gel filtration. Gel filtration. The sample was concentrated and loaded onto a Hiload Superdex 75 10/30 column (Amersham Pharmacia Biotech) equilibrated and eluted with 20 mmol/L Tris-HCl buffer pH 8, 150 mmol/L NaCl, at a flow rate of 0.6 mL/min. The fraction containing a protein of approximately 9 kd was analyzed by SDS-PAGE and IgE immunoblotting with the sera of patients with walnut allergy.

Biochemical characterization of walnut allergens Amino acid sequencing. The N-terminal sequences of the allergenic molecules were determined analyzing the purified proteins with an apparent molecular weight of 9 kd on a Perkin-Elmer Applied Biosystems 492 Procise sequencer (Applied Biosystems, Foster City, Calif) on pulse-liquid mode. All chemicals were from Perkin-Elmer Applied Biosystems. Electrospray ionization–mass spectrometry. An aliquot of the purified proteins was dialyzed and concentrated by ZipTipC4 devices (Millipore, Mass), following the manufacturer’s instructions. The protein eluted with 60% acetonitrile/1% formic acid was applied into a gold-coated borosilicate capillary and analyzed by a LCQ Finnigan MAT ThermoQuest (San Jose, Calif) ion trap mass spectrometer fitted with a nano ESI source. The capillary voltage was set at a voltage of 46 V and spray voltage of 1.9 kV. The data were managed by the Xcalibur software (Finnigan MAT ThermoQuest). Isoelectrofocusing. The purified proteins were focused by a Pharmacia-LKB Phast System Ready using Phast Gel, according to the manufacturer’s instructions. The gel was fixed and stained with Coomassie brilliant blue R-250.

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FIG 1. IgE immunoblotting of walnut extract with single sera from 46 walnut-allergic patients. Patients no. 1-18 were not allergic to pollens; patients no. 19-46 had pollinosis.

Periodic acid-Schiff stain. Periodic acid-Schiff staining was performed to detect glycosylation of the purified proteins. The periodic acid-Schiff stain was performed as described elsewhere.16

Immunoblotting inhibition Once one of the 9-kd proteins had been identified as a lipid transfer protein (LTP), an immunoblotting inhibition experiment was performed to verify whether the purified LTP really did inhibit the 9-kd allergen in the total walnut extract and to evaluate the cross-reactivity with peach LTP by using a pool of sera from patients exclusively reacting to the LTP of walnut and peach (patients no. 2, 8, 9, 20, 38). Briefly, 500 mL of peach LTP and 500 mL walnut LTP at different protein content (400, 40, and 4 mg) were each incubated with 500 mL pooled sera. After 1 hour of incubation, immunoblotting of walnut and peach extracts was performed by using the inhibited sera, as previously described.

RESULTS

Food allergy, dermatologic diseases, and anaphylaxis

Patients Forty-six patients (14 male patients, 32 female patients; mean age, 30 years) with clinical symptoms after walnut ingestion were recruited for this study. Table I reports their demographic data, symptoms, CAP/RAST, and open oral challenge results to walnut. The patients submitted to the open food challenge had symptoms whose severity was assessed following the classification of oral allergy syndrome (OAS) already described.16 SDS-PAGE/immunoblotting The SDS-PAGE of walnut extract showed different proteins with apparent molecular weights ranging from 9 to 80 kd. Walnut IgE immunoblotting of each single patient is shown in Fig 1. Forty-two of 46 patients (91.3%) had specific IgE against the allergenic components at 9 kd. Other allergenic polypeptides were detected at 14 kd (9 patients; 19.5%), 17 kd (17 patients; 37%), 20 kd (9 patients; 19.5%), 23 kd (6 patients; 13%), 25 kd (7 patients; 15.2%), 30 kd (11 patients; 24%), 34, 37, 49, and 52 kd (9 patients; 19.5%), 55 kd (5 patients; 10.8%), 63 kd (6 patients; 13%), 84 kd (3 patients; 6.5%) and 95 kd (1 patient; 2%). Patients number 1 to 18, not sensitized to pollens, recognized only the 9 kd proteins. Patients number 19 to

46 had pollinosis: some of them (patients no. 21-34, 36, 37, 41-46) recognized higher molecular weight allergens, whereas the remaining (patients no. 19, 20, 35, 38-40) recognized only the 9-kd allergens. This latter group included patients allergic only to pollens other than birch.

Purification of walnut allergens LTP and vicilin A. The size-exclusion separation of the walnut extract revealed a peak corresponding to proteins of approximately 9 kd molecular weight, which was not pure by SDS-PAGE and was then separated by cationic exchange. The resulting peaks were analyzed by SDS-PAGE/immunoblotting, and 2 of them contained pure allergenic proteins, subsequently sequenced and identified as an LTP and a vicilin-like protein precursor (vicilin A), respectively. Vicilin B. The anionic exchange of the walnut extract showed several peaks, one of which contained several components approximately 9 kd by SDS-PAGE. These were separated by gel filtration chromatography and analyzed by SDS-PAGE/immunoblotting. One of them was subsequently sequenced and identified as a vicilin precursor (vicilin B). Biochemical characterization of walnut allergens N-terminal sequence. The N-terminal sequence of the 9-kd purified allergens was determined for the proteins in solution and compared with those of known proteins by a database search. The 2 pure proteins obtained by cationic exchange corresponded to an LTP (amino acid sequence Val-Ile-Thr-Cys-Gly-Gln-Val-Ala-Ser-Ser) and to a fragment of the vicilin-like protein precursor of walnut (amino acid sequence Pro-Arg-Asp-Pro-Arg-Glu/Gln-Gln-TyrArg-Gln). Table II shows the N-terminal amino acid alignment of the first 30 amino acids of walnut LTP with other known allergenic LTPs. The walnut LTP showed a high degree of homology with peach and apricot LTPs (80%). The N-terminal sequence of the other 9-kd allergen purified by anionic exchange/gel filtration was ProArg-Asp-Pro-Arg-Gln-Gln-Tyr-Glu-Glu. This sequence

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Patient no.

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 41 42 43 44 45 46

Age

Sex

Symptoms to walnut

Walnut CAP System

Symptoms to walnut open oral challenge (OAS severity grade)

32 30 27 38 34 44 39 35 31 20 28 34 19 21 49 31 30 28 32 40 20 24 32 26 18 29 30 20 38 28 21 31 12 32 35 28 60 35 41 18 33 30 27 32 19 31

F F M M M F F F F F F M F F F F M F M F F F F F F M F M F F M M F F F F F F M M M M F F F F

GI, GE Urticaria, shock Urticaria/AE, GE Shock Shock Urticaria, GE OAS Shock Urticaria/AE, shock GE OAS Shock OAS AE, GE OAS OAS OAS OAS OAS OAS, AE, GE OAS Shock, AE, GE OAS GE Urticaria/AE, GE OAS OAS AD AD, asthma Urticaria/AE, GE Shock OAS OAS OAS OAS OAS OAS Shock OAS OAS OAS GE AE, GE OAS, shock AE, GE OAS

11.6 3.25 1.46 11.3 7.27 4.17 1.16 14 18 11.9 8.27 12.2 0.70 9.88 1.88 2.89 19.4 0.78 31 8.25 1.32 1.54 1.17 18.8 0.53 1.55 20 13.6 1.41 74.1 3.24 11.3 11.4 4.53 3.9 6.28 6.57 7.52 1.71 17.8 27.9 18.7 24.7 2.64 14.1 5.6

/ / / / / / OI (I) / / / OPI (I) / OI (I) / OI (I) OI (I) OPI 1 N (II) OI (I) OPI 1 LAE (I) / OI (I) / OI (I) / / OI 1 N 1 GP (II) OPI 1 LAE (I) / / / / OPI (I) OPI 1 LAE 1 N 1 GP (II) OI (I) OI (I) OPI (I) OI (I) / OI 1 LAE (I) OPI 1 GB (II) OI 1 LAE 1 N 1 GP (II) / / / / OI 1 GP (II)

AD, Atopic dermatitis; AE, angioedema; GB, gastric burning; GE, glottis edema; GI, gastrointestinal symptoms; GP, gastric pain; LAE, lip angioedema; N, nausea; OAS, oral allergy syndrome; OI, oral itching; OPI, oral and pharyngeal itching.

corresponded to another vicilin showing 90% identity with the vicilin protein precursor of cocoa (Theobroma cacao). We decided to name the vicilin-like protein precursor vicilin A and this latter vicilin precursor vicilin B. Electrospray ionization–mass spectrometry. The molecular mass of LTP was 9177.6 6 0.6 d, whereas that of vicilin A was 6445.2 6 1.5 d.

Isoelectrofocusing. Walnut LTP had an isoelectric point of 9.45 after Coomassie brilliant blue R-250 staining. Periodic acid-Schiff stain. On the purified LTP, periodic acid-Schiff stain yielded a negative result, excluding any glycosylation. Immunoblotting of LTP and vicilins. The SDS-PAGE and IgE-immunoblotting of the three 9-kd proteins

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TABLE I. Characteristics of the 46 selected patients

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TABLE II. N-terminal amino acid alignment of the first 30 amino acids of walnut LTP with other known allergenic LTPs Amino acid 1 2

Walnut Mais Peach Apricot Apple Almond Hazelnut

V A — — — — A

I I I I I I I

3

4

5

6

7

8

T S T T T T ST

C C C C C C C

G G G G G G GP

Q Q Q Q Q Q SQ

V V V V V V VI

A A S S T S TK

9 10 11

S S S S S S G

S A A S S N N

V I L L L L L

12

G A A A A A VTA

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Identity %

S P P P P P

C C C C C C

I I I I I I

G S P G G P

Y Y Y Y Y Y

L A V V V V

R R R R R R

G G — — — —

— Q — — — —

T G G G S G

V S G G G G

P G G G G G

T P A A A A

V S V V V V

P — P P P P

P A P P P P

SA G A A A A

C C C C C C

43 53 60 60 53

obtained by HPLC showed that all of them were pure and IgE binding (data not shown). The sera of all of the patients showing an IgE reactivity to the 9-kd components of walnut (42 of 46) were used for the immunoblotting analysis of pure LTP and vicilin A. Thirty-six of them reacted to walnut LTP, whereas only 9 revealed specific IgE binding to vicilin A (patients no. 4, 22, 26, 29, 30, 33, 39, 41, 46). Because of the difficulties in vicilin B purification, few patients (no. 4, 12, 20, 22, 24, 30) were tested for IgE reactivity to this protein, and patient 12 was the only one who recognized this protein. Fig 2 shows the results of all of the patients tested also for vicilin B.

Food allergy, dermatologic diseases, and anaphylaxis

Immunoblotting inhibition The preincubation of the serum pool with 400, 40, and 4 mg walnut purified LTP totally prevented the IgE binding to the 9-kd band of walnut raw extract, thus confirming that the 9-kd protein corresponded to the LTP (Fig 3). Fig 3 also shows the results of immunoblotting crossinhibition between walnut and peach, using a serum pool of patients allergic to both of them: 400 and 40 mg walnut and peach LTP totally inhibited the IgE binding to the 9-kd component of walnut, and a faint band reappeared only at an inhibitor concentration of 4 mg (Fig 3, A). Although as little as 4 mg peach LTP completely inhibited the IgE binding to the peach 9-kd protein, 400 mg walnut LTP was not sufficient to give a total inhibition of IgE binding to peach (Fig 3, B). DISCUSSION The current study confirmed that walnut is able to provoke very severe allergic reactions, because many of the selected patients had a convincing history of lifethreatening reactions to walnut, and some of the patients who reported a history of oral itching (OAS grade I) had more severe symptoms at the oral provocation test (OAS grade II). In previous studies, Teuber et al11,12 identified 2 major walnut allergens: the 2S albumin Jug r 111 and the vicilinlike proprotein Jug r 2.12 In our population, we found that the major allergen of walnut was an LTP, which was submitted to the International Union of Immunological Societies’ Allergen Nomenclature Subcommittee and registered as Jug r 3. This 9-kd protein was recognized by serum IgE antibodies of 36 of 46 subjects (78.2%), thus

FIG 2. IgE immunoblotting of three 9-kd purified allergens with single sera of patients no. 20, 24, 22, 4, 30, 12.

proving the only major allergen. All of the other allergenic proteins were able to bind IgE from a minority of the patients: among these, we identified 2 other 9-kd allergens, corresponding to vicilins, recognized by 10 of 46 patients (21.7%). In our population, the patients without pollen sensitization showed IgE reactivity only to the 9-kd components. It is noteworthy that the pollen-sensitized patients who exclusively reacted to the 9-kd allergen were not sensitized to birch pollen; all of the birch-sensitized patients showed a more complex allergenic pattern with higher molecular weight allergens. This finding confirms our previous results, which showed that sensitization to LTP is not a birch pollen–related allergy.13,15 Walnut LTP showed IgE cross-reactivity with the peach major allergen LTP, as demonstrated by the crossinhibition experiment depicted in Fig 3. The inhibition was stronger when peach LTP was used as inhibitor: this means that all of the IgE binding epitopes of walnut LTP are present in peach LTP, which also contains some additional IgE binding sites. In fact, many of the patients allergic to walnut were first sensitized to peach, which acted as a primary sensitizer, and only secondarily to walnut. To date, very little is known about the B-epitopes of LTPs. An exception is the major peach allergen Pru p 3: after its direct amino acid sequence determination,17 its IgE-binding sequential epitopes have recently been published.18 Although only 1 of the reported sequential epitopes comprises amino acid reported in Table II, it is interesting to note that the 3 essential amino acids for IgE binding within this epitope, C13, Y16, and R18, are

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conserved in the walnut, as well as maize, apricot, apple, and almond LTP sequences. In our study, the patients allergic to walnut reacting to LTP often showed severe symptoms; this finding is consistent with findings of previous studies.19,20 Moreover, they were usually also allergic to other fruits containing LTPs, like Prunoideae (apricot, cherry, plum), apple, grape, maize and hazelnut; thus, allergy to walnut can be considered a clinical manifestation of the LTP syndrome. In our study, vicilins were minor allergens: 9 patients reacted to a vicilin-like protein precursor (vicilin A) and 1 patient to a vicilin precursor with 90% sequence homology to the vicilin precursor from cocoa (vicilin B). It is interesting to notice that the only 2 patients in our study who were exclusively allergic to walnut (patients no. 4 and 26) were sensitized to vicilin A. Most of the vicilinsensitized patients had severe reactions. In particular, patients 4, 12, and 22, who were IgE reactive only to vicilin, had several episodes of anaphylactic shock and/or glottis edema, even after ingestion of minute quantities of walnut (for example, when walnut was present as a hidden allergen). This finding confirms the observations by Teuber et al,12 whose patients had life-threatening reactions. Moreover, 2 of these patients were not allergic to pollens, thus demonstrating that sensitization to vicilin can occur through the gastrointestinal route. Vicilins are seed storage proteins belonging to the 7S globulins and typically exist as homo-trimers with molecular weight of 150 to 190 kd, whose subunits have a molecular weight of around 40 to 80 kd.21 Even

though the molecular weight of the entire walnut vicilin is approximately 48 kd, IgE immunoblotting with sera from our patients showed a stained band at 9 kd and not at higher molecular weights as expected; a possible explanation is that the 9-kd band corresponds to a fragment of the entire vicilin, which is the only allergenic part of the protein. We are reasonably sure that this is the allergenic part of the molecule, because patient 4, who was clinically monosensible to walnut and IgE reactive only to vicilin, reacted only to the 9-kd band in the total walnut extract. The existence of IgE-reactive bands with different molecular weights from that expected for the entire vicilin has already been reported by other authors. Teuber et al12 found that preincubation of sera with Jug r 2 abolished the IgE binding to walnut proteins of 28, 44, 48, and 52 kd; whether these proteins are different subunits, isoforms, glycosylated forms, or fragments of the entire vicilin has yet to be clarified. Lopez-Torrejon et al22 found that the major allergen of lentil, a vicilin of 50-kd molecular weight, was able to inhibit the IgE binding to lentil proteins of 16 and 26 kd, which were probably 2 subunits derived from a posttranslational processing of the major allergen. In conclusion, our study underlined the importance of LTP as a major allergen of walnut and demonstrated that both LTP and vicilin are true food allergens, because they are able to sensitize patients not allergic to pollen. REFERENCES 1. Bock SA, Munoz-Furlong A, Sampson HA. Fatalities due to anaphylactic reactions to foods. J Allergy Clin Immunol 2001;107:191-3.

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FIG 3. A, Inhibition of IgE immunoblotting of walnut extract by walnut and peach LTP at different protein contents. B, Inhibition of IgE immunoblotting of peach extract by peach and walnut LTP at different protein contents.

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2. Pumphrey RSH. Lessons for management of anaphylaxis from a study of fatal reactions. Clin Exp Allergy 2000;30:1144-50. 3. Pumphrey RSH, Roberts ISD. Postmortem findings after fatal anaphylactic reactions. J Clin Pathol 2000;53:273-6. 4. Macdougall CF, Cant AJ, Colver AF. How dangerous is food allergy in childhood? the incidence of severe and fatal allergic reactions across the UK and Ireland. Arch Dis Child 2002;86:236-9. 5. Clark AT, Ewan PW. Interpretation of tests for nut allergy in one thousand patients, in relation to allergy or tolerance. Clin Exp Allergy 2003;33:1041-5. 6. Sicherer SH, Mun˜oz-Furlong A, Burks AW, Sampson HA. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J Allergy Clin Immunol 1999;103:559-62. 7. Emmett SE, Angus FJ, Fry JS, Lee PN. Perceived prevalence of peanut allergy in Great Britain and its association with other atopic conditions and with peanut allergy in other household members. Allergy 1999;54:380-5. 8. Sicherer SH, Furlong TJ, Mun˜oz-Furlong A, Burks AW, Sampson HA. A voluntary registry for peanut and tree nut allergy: characteristics of the first 5149 registrants. J Allergy Clin Immunol 2001;108:128-32. 9. Directive 2003/89/EC of the European Parliament and of the Council of 10 November 2003, amending Directive 2000/13/EC as regards indication of the ingredients present in foodstuffs. Published in the Official Journal of the European Unit. November 25, 2003. 10. Vierk K, Falci K, Wolyniak C, Klontz KC. Recalls of foods containing undeclared allergens reported to the US Food and Drug Administration, fiscal year 1999. J Allergy Clin Immunol 2002;109:1022-6. 11. Teuber SS, Dandekar AM, Peterson R, Sellers CL. Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia), a major food allergen. J Allergy Clin Immunol 1998;101:807-14. 12. Teuber SS, Jarvis KC, Dandekar AM, Peterson WR, Ansari AA. Identification and cloning of a complementary DNA encoding a vicilinlike proprotein, Jug r 2, from English walnut kernel (Juglans regia), a major food allergen. J Allergy Clin Immunol 1999;103:1311-20.

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Food allergy, dermatologic diseases, and anaphylaxis

Correction With regard to the July 2004 article entitled ‘‘New developments in food allergy: Old questions remain’’ (2004;114:127-30): The second paragraph on page 129 should have appeared as follows: Another problematic issue in managing adult patients with food allergies is whether patients with food allergy requiring Epipen should also be prescribed a b-blocker after a myocardial infarct or after the development of congestive cardiac failure. TenBrook et al18 describe a computer-simulated decisionanalytical model. Certain assumptions were made that incorporated a bias against b-blocker treatment as augmenting the risk of anaphylactic mortality 10-fold. Despite this bias the model predicted an overall improvement in life expectancy with b-blocker treatment in patients with peanut allergy and myocardial infarction (9.4 month increase) or congestive cardiac failure (17 months). This analysis makes a strong case for appropriate treatment of myocardial infarct or congestive heart failure with b-blockers (despite the existence of peanut allergy), although individual patient circumstances may influence the decision (eg, severity and frequency of anaphylactic reactions and coexistence of asthma).