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Fennel Allergy is an LTP (Lipid Tranfer Protein) Related Food Hypersensitivity Associated to Peach Allergy Elide A. Pastorello, Laura Farioli, Chrysi Stafylaraki, Joseph Scibilia, Maria G. Giuffrida, Ambra Mascheri, Marta Piantanida, Cristina Baro, Laura Primavesi, Michele Nichelatti, Jan W. Schroeder, and Valerio Pravettoni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf303291k • Publication Date (Web): 04 Dec 2012 Downloaded from http://pubs.acs.org on January 12, 2013
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Fennel Allergy is an LTP (Lipid Transfer Protein) Related Food Hypersensitivity
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Associated to Peach Allergy
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Elide A. Pastorello§*, Laura Farioli•, Chrysi Stafylaraki§, Joseph Scibilia§, Maria G. Giuffrida∫,
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Ambra Mascheri §, Marta Piantanida‡, Cristina Baro∫, Laura Primavesi‡, Michele Nichelatti₪, Jan
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W. Schroeder§, and Valerio Pravettoni‡.
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Additional short title: LTP is a relevant fennel allergen.
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§
Allergology and Immunology Unit, Niguarda Ca' Granda Hospital, Milan, Italy; •Department of
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Laboratory Medicine, Niguarda Ca' Granda Hospital, Milan, Italy; ∫ISPA-CNR, c/o Bioindustry
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Park Silvano Fumero, Colleretto Giacosa (Turin), Italy; ‡Clinical Allergy and Immunology Unit,
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Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy;
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Biostatistics, Niguarda Ca' Granda Hospital, Milan, Italy
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Service of
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*Corresponding author:
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Elide A. Pastorello, Piazza Ospedale Maggiore, 3 Milan 20162, Italy
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Tel: +39 02 64442751
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Fax number: +39 02 64442082
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e-mail address:
[email protected] 21
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Abstract
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Fennel allergy has been rarely reported and the association with peach allergy has never been described.
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Our aim was to: i)study the correlation between symptoms severity to peach and fennel, ii)identify fennel
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allergens and the role of rPru p3 antibodies in severe reactions to fennel.
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In 148 pts with peach allergy, we investigated 58 pts with symptoms and IgE antibodies positive to fennel.
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IgE to rPru p1-3-4, and rBet v1-2-4 were measured by immunoblotting and the N-terminal-amino-acid
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sequences and relevant allergens were determined.
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We found significant association between severe reactions to fennel and peach(p=0.0009). A major allergen
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was ~9 kDa, LTP cross-reactive with Pru p3, a 15 kDa protein identified as a Pathogenesis-Related-
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Protein-1 of the Bet v 1 family.
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In conclusion, peach and fennel severe allergic symptoms are significantly related, and LTP is a major
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fennel allergen. Fennel should be included n the LTP syndrome.
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Keywords: fennel allergy, Foeniculum vulgare, lipid transfer protein, peach allergy, Pru p 3.
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Introduction
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Fennel (Foeniculum vulgare) is a member of Apiaceae (formerly called Umbrelliferae) family, a large group
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of plants encompassing approximately 300 genera and more than 3000 species. These species include some
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important allergenic plants such as carrot and celery. Fennel is native to southern Europe where it has been
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used for centuries as a spice and for medicinal purposes1,2 and more recently consumed as a fresh vegetable.
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In contrast, in northern Europe fennel seeds are consumed in bread and sausages. Perhaps due to its infrequent
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consumption, fennel allergy has been rarely studied. The few publications which have addressed fennel
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allergy have focussed on the relationship to birch and mugwort pollen allergy in the so-called birch-weed or
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fruit-spice syndrome.3-5 In fact, fennel allergens are not included in the International Union of Immunological
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Societies (IUIS) database. The only previous manuscript regarding fennel allergy is that of Jensen-Jarolim et
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al.,6 who studied 6 fennel-allergic patients. These patients were suffering from mugwort and birch hay fever,
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and IgE from these patients reacted with 14 and 17 kDa allergens likely to be birch-related and with 50-70
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kDa mugwort-related allergens, thus demonstrating the immunologic basis of the clinical association between
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fennel and birch or mugwort pollen allergies. Similarly, in the other Apiaceae plant foods, represented by
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celery and carrot, the major allergens Api g 1 and Dau c 1 are Bet v 1 homologues and Api g 4 and Dau c 4
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are Bet v 2 homologues.7 We recently observed an unexpectedly high rate of fennel allergy in a group of
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severe peach-allergic patients,8 some of whom also suffered from birch and/or mugwort pollinosis. Fennel-
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induced symptoms ranged from mild oral allergy syndrome (OAS) to urticaria and anaphylaxis, suggesting a
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role of allergens unrelated to birch. In our study, peach-allergic patients were subdivided into those with mild
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symptoms and those with severe symptoms, thus defining a relationship between peach symptom severity
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and sensitisation to rPru p 3 allergen. Because we detected a high number of individuals with fennel allergic
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symptoms within the group of peach-allergic patients, we aimed to investigate the clinical relationship
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between peach and fennel symptom severity, the fennel allergens involved and the correlation between fennel
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symptom severity and positive responses to one or more fennel allergens.
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Materials and Methods
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Chemicals
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All reagents used for protein buffer extraction were by Carlo Erba (Milano, Italy) and those reagents used for
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SDS PAGE were by BIO-RAD Laboratories (Richmond, CA). For blotting we used a nitrocellulose
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membrane by Amersham Biosciences (GE Healthcare, UK) and 125I-labelled anti-human-IgE antibodies
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were provided by RADIM (Rome, Italy).
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Study population
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One hundred forty eight peach-allergic patients in whom peach, birch and specific recombinant (rPru p 1, 3, 4
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and rBet v 1, 2, 4) IgE levels were measured8 were also evaluated for hypersensitivity to other allergenic plant
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foods. The patients with a history of allergic reactions to fennel and a prick-prick positivity for fresh fennel9
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were recruited for further evaluations. Patients with a history of mild OAS were tested using an open food
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challenge (OFC). For the patients who reported severe reactions, the patients’ documentation was accurately
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reviewed.10 Fennel-induced symptoms were classified into four possible grades of severity11: grade I, only
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OAS symptoms (mild OAS); grade II, OAS and urticaria/angioedema; grade III, OAS with gastrointestinal
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symptoms and asthma; and grade IV, with life-threatening symptoms such as glottis oedema, hypotension
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and shock (severe symptoms included grades II, III, IV). The Ethics Committee approved the study (Clinical
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Trials.gov, protocol ID NCT00715156). Blood was drawn from all of the enrolled patients and serum was
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stored at -20°C.
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In vivo tests
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Fennel OFC. OFC was performed with fresh fennel in a graduated manner at a time when birch pollen levels
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were low. Patients were instructed to chew a small piece (0.5 g) of fennel, keep it in the oral cavity for 1 min,
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and then spit it out. Patients in whom no symptoms occurred then chewed and swallowed a second piece.
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Subsequent doses (also swallowed) were doubled until symptoms appeared or the entire dose of fennel was
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ingested (80 g). The additional doses were delivered at 15 minute intervals. The test was stopped and
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considered positive when symptoms appeared. Patients with a clear-cut history of anaphylaxis or documented
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severe systemic reaction to fennel were not challenged to avoid provoking severe reactions. The objective
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symptoms included urticaria, oedema, oral mucosal lesions, asthma, nasal secretion and sneezing; a
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complaint of itching of the oral mucosa without any further objective evidence was recorded as a subjective
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symptom.
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In vitro test
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Serum-specific IgE determination. All patients’ sera were tested for specific IgE towards fennel, peach, and to
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rPru p 1, 3, 4 and rBet v 1, 2, 4 by the ImmunoCAP System (Thermo Fisher Scientific, Milan, Italy),
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according to the manufacturer’s instructions. The results were expressed as kUA/L. IgE values were
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considered positive when a value greater than 0.10 kUA/L was obtained.
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Immunodetection of fennel allergens. Fresh fennel and peach extracts were prepared according to the
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Bjorksten method.12 After centrifugation, the supernatants were dialysed against phosphate buffered saline
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(PBS 0.1 M, pH 7.4) for 48 hour at 4°C. The protein contents, determined by colorimetric Lowry method,13
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for peach and fennel extracts were 5.47 mg/mL and 6.77 mg/mL respectively. Sodium dodecyl sulfate
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polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Neville.14 The proteins were
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separated in a discontinuous gel with a 6% stacking gel and a 7.5-20% separation gel at 6 mA for 16 hours in
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a BIO-RAD Protein II xi Vertical Electrophoresis Cell (BIO-RAD Laboratories, Richmond, CA), as
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previously reported.15 After electrophoresis, each fraction was electro-transferred to a nitrocellulose
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membrane (pore size 0.45 µm; Amersham Biosciences, GE Healthcare, UK) using a Trans-Blot Cell (Bio-
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Rad, Laboratories, Richmond, CA) at 0.45 A, 100 V for 4 hours at 4°C. After washing and blocking with
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PBS (pH 7.4 ± 0.2) and 0.5% Tween 20, the nitrocellulose membrane was cut into strips and incubated with
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the sera of each patient and control subject diluted 1:5 in PBS (pH 7.4 ± 0.2) and 0.1% Tween 20.16 In
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particular, sera from the first 25 enrolled patients were used to perform an IgE immunoblotting (in Table 2
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corresponding to # 1-25). The specific IgE-binding proteins were detected by incubation with 125I-labelled
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anti-human-IgE antibodies (RADIM, Rome, Italy) diluted 1:2 in the same buffer used for diluted sera and
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exposure to auto-radiographic film (Hyperfilm, Amersham) at -70°C for 4 -7 days.
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Protein identification: Bands corresponding to the IgE binding fennel proteins were excised from SDS-
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PAGE gel , passively eluted and microsequenced on a Procise 492 protein sequencer (Applied Biosystems,
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Foster City, CA, USA) as described by Pessione et al.17 The amino acids sequences were searched using the
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BLASTP software (http://www.expasy.ch/tools/blast) against both Uniprot KB and NCBInr.2011.01.09
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database.
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Band 2 from Figure 2 was also characterized by LC-MS/MS. For the LC-MS/MS the selected band was
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excised from the SDS-PAGE gel, destained, reduced, alkylated and digested with trypsin as described
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elsewhere.18 The peptide mixture was analysed by means of LC-MS/MS using Agilent-Technologies 1100
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series nano-HPLC, coupled to an Agilent XCT Plus ion trap fitted with a nano electrospray nebulizer. The
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chromatographic separations were run on a 150 x 0,075 mm C18 nanocolumn Zorbax 300SB (Agilent) using
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a linear 5-70% gradient of 0.1% formic acid in acetonitrile within 45 min, with a flow of 0.3 µL/min. The
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injection volume was 1 µL. The MS parameters were: capillary voltage 1600 V, fragmentation voltage 1.3V.
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The DataAnalysis for LC/MSD Trap Version 5.2 software (Agilent) was used to elaborate the LC and
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MS/MS
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(http://prospector.ucsf.edu/prospector/mshome.htm) was used to identify the protein against the
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NCBInr.2011.01.09 database and selecting the Apiaceae family. The parameters used for the search were: S-
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carbamidomethyl derivate on cysteine as fixed modification, oxidation on methionine as variable
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modification and two missed cleavage sites for trypsin digestion. The peptide mass tolerance was set to 0.6
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Da and the fragment mass tolerance to 0.8Da. The homology search was allowed as “single Base Change”.
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Protein hits were validated if the protein scores were above the MS Batch default and the Best Expected value
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(p
