Le kiwi

	Kiwi vert	Kiwi doré	“ kiwai ”
Fonction	A. deliciosa	A. chinensis	A. arguta	A. eriantha
cystéine protéase	Act d 1 (actinidine)	Act c 1	Act a 1	Act e 1
thaumatine-like	Act d 2	Act c 2
??	Act d 3
cystatine	Act d 4	Act c 4
??	Act d 5 (kiwelline)	Act c 5
inhib. pectinestérase	Act d 6
pectinestérase	Act d 7
PR-10 (Bet v 1-like)	Act d 8	Act c 8
profiline	Act d 9
LTP	Act d 10	Act c 10
« major latex protein »	Act d 11
chitinase classe 1	présence	présence

[1] - Henríquez Santana A, González Cervera J, Carnés J, Berges Gimeno P, Álvarez Cuesta R. Anaphylaxis associated with the ingestion of gold kiwifruit. Allergol Immunopathol (Madr) 2008;36:366-367

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[2] - Nishiyama I. Fruits of the actinidia genus. Adv Food Nutr Res 2007;52:293-324

Kiwifruit is the most well-known crop in the genus Actinidia. Although Actinidia fruit sales in the international market are dominated by a single kiwifruit cultivar Actinidia deliciosa "Hayward," there are a considerable number of cultivars and selections in the genus that have widely diverse shape, size, and hairiness. They also offer a wide variation in sensory attributes such as flesh color, flavor, and taste, and in nutritional attributes such as the vitamin C level and carotenoid content. The level of actinidin, which is a cysteine protease in kiwifruit, also varies greatly among cultivars. This chapter reviews available information related to several important components, allergenic properties, and health benefits of Actinidia fruits.

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[3] - Goodman RE, Chen L, Lucas J, Hourihane JO, Taylor SL. IgE from Some Green Kiwifruit Allergic Individuals Binds to Proteins in Hardy Kiwifruit, a Third Cultivated Species of the Genus Actinidia. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°175

RATIONALE: Since hardy kiwifruit (Acintinidia arguta) is now cultivated in western North America, we tested protein extracts of hardy, green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit for IgE binding using sera from individuals with clinically diagnosed food allergies to green kiwifruit, to evaluate potential cross-reactivity METHODS: Sera from twelve green kiwifruit-allergic subjects (eight were positive by DBPCFC, four severe reactors were not challenged but had positive ImmunoCAP) and control subjects were assayed for IgE binding to soluble proteins in green, gold and hardy kiwifruits using reducing and non-reducing SDS-PAGE immunoblots and direct enzyme linked immunosorbent assays (ELISA) RESULTS: IgE-ELISA results of all kiwi-allergic subjects were positive compared to controls for one or more kiwifruit extracts. Immunoblot results demonstrated individual patient and species variability. Two sera with strong ELISA positive results to both hardy and green, but not gold kiwifruit did not show marked IgE binding to kiwifruit proteins on immunoblots with reduced, heat denatured extracts, but clearly bound two or more proteins from non-reduced, unheated extracts of hardy, but not green kiwifruit. One kiwifruit-allergic individual had marked binding to non-reduced, unheated gold kiwifruit proteins. Only one kiwifruit-allergic individual had marked binding to proteins of all species on the reduced gel blot. Some control sera showed marked IgE binding to high molecular weight proteins on immunblots CONCLUSIONS: These results suggest some kiwifruit-allergic individuals may suffer allergic cross-reactions if they consume hardy kiwifruit They also demonstrate the difficulty in developing in vitro reagents for accurate diagnosis of kiwifruit allergy and for correctly identifying major allergens Funding: Efficas, Inc.

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[4] - Senna GE, Crivellaro MA, Dama AR, Lombardi C, Falagiani P. Oral allergy syndrome to Actinidia arguta: a case report. Allergy 1997;52(suppl. 37):116

Kiwi (Actinidia deliciosu) is a fruit of the Actinidiae family very popular in Europe. Arguta (Actinidia argufu) belonging to the same family is rarely commercialized in Italy. PG is a 32 old man suffering from allergic rhinitis due to birch and Parietaria pollens. He presented mild oral itching immediately after eating fruits of arguta, followed in half an hour by a generalized urticaridangioedema and difficulty in breathing. He was treated in emergency room with steroids e.v. and antihistamines i.m. Twenty days after a new assuption of arguta determined the same oral complaints, but not generalized urticaria, because the patient spitted the fruit. The prick by prick with fresh fruit was clearly positive (+++) and also RAST showed a clear positivity for arguta (4,9%, class 2). Prick test and RAST were negative in seven control subjects. Double blind challenge was not performed for ethical reasons. To our knowledge this is the first report in 'Europe of oral allergy syndrome due to arguta. We are scheduling future studies by immunoblotting in order to assess a cross reactivity between arguta and kiwi or other fruits.

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[5] - Rancé F, Dutau G. Asthme et allergies alimentaires: à propos de 163 observations pédiatriques. Arch Pediatr 2002;9(suppl. 3):402-407

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[6] - Lucas JSA, Lewis SA, Hourihane JOB. Kiwi fruit allergy: A review. Pediatr Allergy Immunol 2003;14:420-428

Allergy to kiwi fruit was first described in 1981, and there have since been reports of the allergy presenting with a wide range of symptoms from localized oral allergy syndrome (OAS) to life-threatening anaphylaxis. The article reviews the available information concerning the clinical features of kiwi fruit allergy and the role of clinical investigations for diagnosis. Work identifying the major allergens in kiwi fruit has resulted in conflicting results, the possible reasons for which are discussed. The clinical associations of kiwi fruit allergy with allergies to pollens or latex are reviewed.

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[7] - Lucas JSA, Collins K, Grimshaw K, Warner J, Hourihane J. The Clinical Investigation of Kiwi Fruit Allergy. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°477

Rationale Allergy to kiwi fruit appears increasingly common, but few studies have evaluated its clinical characteristics. Methods 273 subjects with a history suggestive of allergy to kiwi completed a questionnaire. 45 were investigated by double blind placebo controlled food challenge (DBPCFC), prick-to-prick skin testing with fresh kiwi pulp, and measuring CAP specific IgE. 19 subjects were also skin tested using a commercially available solution. Results The most frequently reported symptom was oral pruritus (65%), but severe symptoms (wheeze, cyanosis or collapse) were reported by 18% of subjects. Young children were significantly more likely than adults to react on their first known exposure (p<0.001), and to report severe symptoms (p=0.008). 23 of 45 subjects (50%) had allergy confirmed by DBPCFC. Prick to prick skin test with fresh kiwi was positive in 95% of subjects who had allergy confirmed by DBPCFC, but also in 69% of subjects with a negative food challenge. The commercial extract was significantly less sensitive, but with fewer false positive reactions. CAP sIgE was only positive in 60% of subjects who had a positive challenge. Conclusions DBPCFC confirmed allergy to kiwi fruit in 50% of the subjects tested, who had a previous history suggestive of kiwi allergy. Skin testing with fresh fruit has good sensitivity (95%), but poor specificity (31%) in this population. CAP sIgE and a commercially available skin test solution were both much less sensitive (60%; 72%) but had better specificity (83%; 67%). Kiwi fruit should be considered a significant food allergen particularly for young children.

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[8] - Fritsche P, Pfister M, Theler B, Helbling A, Ballmer-Weber B. Kiwi: a food allergy with different clinical faces !. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°527

Background: We observe an increasing prevalence of kiwi allergy in Switzerland. Objectives: The aim of the present study was to investigate the clinical characteristics of kiwi allergic patients and to validate the currently used routine diagnostic tools. Methods: Patients with a positive case history of a kiwi allergy underwent a double-blind placebo-controlled food challenge (DBPCFC) with fresh kiwi, skin prick test (SPT) with extracts from kiwi, latex, different pollen and foods, prick-to-prick-test with native kiwi and serum-IgE analysis to birch pollen allergens, latex and kiwi. Personal history of allergy and the family history were recorded with a standardized questionnaire. Results: In 30 out of 38 patients (32.1 +/- 13.0 years) the case history of a kiwi allergy was confirmed by a positive DBPCFC. The most frequently reported symptoms were localized to the oral mucosa (100%), but severe symptoms (emesis, dyspnea or collapse) were reported by 23% of subjects. According to the case histories and the pattern of sensitisation in SPT and serum analysis we could recognise three different groups of patients: Eight patients were monosensitized to kiwi (group A), 17 patients were sensitized to kiwi and different types of pollen (group B), in particular to birch (71%) and grass pollen (77%), and 5 patients suffered from a combined kiwi and latex allergy (group C). Sensitivity of the SPT with a commercial kiwi extract was 71% and 80% for group A and C, respectively, but just 18% for group B. The sensitivity of the kiwi-CAP and the prick-prick-test with native kiwi was 17% and 83%, respectively, for all kiwi allergic patients. 30% of the patients with combined kiwi-pollen allergy were sensitized to latex without suffering from a relevant latex allergy. Four of the 5 kiwi-latex allergic patients suffered first from a pollen allergy, whereas the latex and the kiwi allergy developed after an average interval of more than 10 years. In conclusion: Kiwi may induce severe and even anaphylactic reactions. Kiwi allergy may manifest as a ''monoallergy'' or in association with a pollen and/or latex-allergy. Identification of cross-reactive allergens and inhibition assays will identify the primary sensitizer in latex-pollen-kiwi allergic patients. In patients with combined kiwi-pollen hypersensitivity kiwi allergy is most likely mediated by a labile protein, underrepresented in commercial extracts.

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[9] - Alemán A, Sastre J, Quirce S, de las Heras M, Carnés J, Fernández-Caldas E, et al. Allergy to kiwi: A double-blind, placebo-controlled food challenge study in patients from a birch-free area. J Allergy Clin Immunol 2004;113:543-550

Background Allergy to kiwi fruit is being increasingly reported, but it has never been evaluated by means of a double-blind, placebo-controlled food challenge (DBPCFC) study. Objective : We sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitized patients from a birch-free area. Method s : Forty-three patients with allergy symptoms who were sensitized to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The pattern of allergen recognition was assessed by means of IgE immunoblotting. Sequence analysis of IgE-binding bands was performed by using Edman degradation. Result s : DBPCFCs were performed in 33 patients; 4 patients had experienced severe anaphylaxis, and 6 patients declined informed consent. DBPCFC results were positive in 23 patients and negative in 10 patients. The most frequent clinical manifestation was oral allergy syndrome. Twenty-one percent of the patients were not allergic to pollen. Forty-six percent of patients experienced systemic symptoms, and this happened with higher frequency in patients not allergic to pollen (100%). Twenty-eight percent of the patients were sensitized to latex. The IgE-binding bands in kiwi extract more frequently recognized by patient sera were those of 30, 24, 66, and 12 kd, and they could not be associated with any pattern of kiwi-induced allergic reactions. Conclusion : The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi. One of 5 patients with kiwi allergy was not allergic to pollen, and these patients had the highest risk of systemic reactions to kiwi.

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[10] - Pumphrey RS. Fatal anaphylaxis in the UK, 1992-2001. Novartis Found Symp 2004;257:116-128

Each year in the UK, around nine deaths are attributed to anaphylaxis to pharmaceuticals, six to food and four to stings. I have identified 214 deaths associated with anaphylaxis, and have sufficient information for 196 to determine that 88 deaths were due to shock, 96 to asphyxia. Five deaths followed epinephrine overdose, seven were complicated by disseminated intravascular coagulation. There will have been other unrecognized fatal antibiotic and asthmatic food reactions. For foods, peak age was 17-27 with a female and atopic predominance; the first arrest was commonly from asthma 25-35 minutes after the implicated food. For stings, peak age was 45-70 with male and non-atopic predominance; death was commonly from shock 10-15 minutes after the sting. A majority of deaths from pharmaceuticals in hospital took 5 minutes or less from dose to arrest; peak age was 60-75. Maximum time for any cause from trigger to first arrest was 6 hours. The danger of epinephrine overdose and its limitations in reversing anaphylaxis must be recognized. The patient should remain supine with legs raised throughout sting and other shock reactions. Prevention of fatal food reactions will depend on avoidance and optimal daily control of asthma.

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[11] - Beezhold DH, Hickey VL, Slater JE, Sussman GL. Human IgE-binding epitopes of the latex allergen Hev b 5. J Allergy Clin Immunol 1999;103:1166-1172

BACKGROUND: Hev b 5 is an acidic protein (isoelectric point, 3.5) rich in glutamic acid with 9 repeated amino acid (AA) sequences of XEEX or XEEEX. Although its function in Hevea brasiliensis is unknown, Hev b 5 has been identified as a major latex allergen. Immunoblot inhibition studies suggest Hev b 5 exists as multiple isoforms or contains a common epitope found in several other proteins. OBJECTIVE: The purpose of this study was to further characterize Hev b 5 and to identify linear IgE-binding epitopes. METHODS: Octapeptides spanning the entire Hev b 5 protein were synthesized on a derivatized cellulose membrane. The membrane was reacted with sera pooled from health care workers allergic to latex or rabbits immunized with latex proteins. B-cell epitopes were identified by subsequent incubations with the appropriate secondary antibodies and detected by using chemifluorescence. RESULTS: Sera from patients allergic to latex recognized 6 IgE-binding regions located throughout the molecule. Two epitopes (2 and 4) had the common AA sequence of KTEEP. Epitopes 3 and 5 had a similar AA sequence of EEXXA, where X was P, T, or K. Epitopes 1 and 6 appeared to be unrelated to the other epitopes. Database analysis could not identify other proteins with similar sequences. Neither of the XEEEX sequences bound IgE. Control sera failed to react to any peptides. CONCLUSIONS: Hev b 5 exists as multiple isoforms, but only small amounts are present in the nonammoniated latex preparations, such as those used for diagnostic tests, and this may help to explain the relatively poor sensitivity of some in vitro tests.

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[12] - Ledger SE, Gardner RC. Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa). Plant Mol Biol 1994;25:877-886

Five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward) were isolated from a library made from young fruit, 8-10 days after anthesis. One gene (pKIWI503) has low levels of expression in young fruit but is induced late in fruit development and during fruit ripening, and has some homology to plant metallothionein-like proteins. The other four genes are highly expressed in young fruit with reduced expression in the later stages of fruit development. pKIWI504 has strong homology to plant metallothionein-like proteins and pKIWI505 exhibits homology to the beta-subunit of the mitochondrial ATP synthase gene. The two other genes (pKIWI501 and 502) encode proteins with no significant homology to other known sequences.

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[13] - Ibarrola I, Arilla MC, Martinez A, Asturias JA. Identification of a polygalacturonase as a major allergen (Pla a 2) from Platanus acerifolia pollen. J Allergy Clin Immunol 2004;113:1185-1191

Background Planetree pollen allergy is a clinical disorder affecting human populations in cities of the United States and Western Europe, but little is known about its relevant allergens. Objective : We sought to purify, characterize, and clone the 43-kd allergen from Platanus acerifolia. Method s : P acerifolia pollen extract was fractionated by using ion-exchange and gel-permeation chromatography. Analyses were carried out by using ELISA, SDS-PAGE, isoelectrofocusing, and immunoblotting. Partial amino acid sequence was obtained by means of Edman sequencing of cyanogen bromideˆdigested peptides. Specific cDNA was cloned by using reverse transcription, followed by PCR, with amino acid sequences from peptides of the allergen. Result s : The allergen isolated from P acerifolia pollen, Pla a 2, is a glycoprotein with an observed molecular mass of 43 kd and an isoelectric point value of 9.3. It is involved in the allergic responses of 84% of patients with planetree-induced pollinosis and represented 52% of the total IgE-binding capacity of the P acerifolia extract. Pla a 2 displays polygalacturonase (PG) activity, being the first PG with functional enzyme activity from an angiosperm plant pollen described as an allergen. The cDNA allergen sequence codified for a 372-residue protein with 56% and 42% sequence identity to PGs from pollen and fruits, respectively. Western blot analysis showed that Pla a 2 is present in pollen and stems and has IgG cross-reactivity with a PG from tomato and pectate lyases from Cupressaceae pollen. Conclusion : Pla a 2, a major allergen of P acerifolia pollen with PG activity has been purified, characterized, and cloned.

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[14] - Swoboda I, Grote M, Verdino P, Keller W, Singh MB, De Weerd N, et al. Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles. J Immunol 2004;172:6490-6500

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

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[15] - Stumvoll S, Lidholm J, Thunberg R, DeWitt AM, Eibensteiner P, Swoboda I, et al. Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential. Biol Chem 2002;383:1383-1396

Almost 500 million people worldwide suffer from Type I allergy, a genetically determined immunodisorder which is based on the production of IgE antibodies against per se harmless antigens (allergens). Due to their worldwide distribution and heavy pollen production, grasses represent a major allergen source for approximately 40% of allergic patients. We purified Phl p 4, a major timothy grass (Phleum pratense) pollen allergen with a molecular mass of 61.3 kDa and a pl of 9.6 to homogeneity. Circular dichroism spectroscopical analysis indicates that Phl p 4 contains a mixed alpha-helical/beta-pleated secondary structure and, unlike many other allergens, showed no reversible unfolding after thermal denaturation. We show that Phl p 4 is a major allergen which reacts with IgE antibodies of 75% of grass pollen allergic patients (n=150) and induces basophil histamine release as well as immediate type skin reactions in sensitized individuals. Phl p 4-specific IgE from three patients as well as two rabbit-anti Phl p 4 antisera cross-reacted with allergens present in pollen of trees, grasses, weeds as well as plant-derived food. Rabbit antibodies raised against Phl p 4 also inhibited the binding of allergic patients IgE to Phl p 4. Phl p 4 may thus be used for diagnosis and treatment of sensitized allergic patients.

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[16] - Asturias JA, Ibarrola I, Eraso E, Arilla MC, Martinez A. The major Platanus acerifolia pollen allergen Pla a 1 has sequence homology to invertase inhibitors. Clin Exp Allergy 2003;33:978-985

BACKGROUND: Sycamores or plane trees are an important source of airborne allergens in many cities of the United States and Western Europe. Pla a 1 has been described as a major allergen from Platanus acerifolia (London plane tree) . OBJECTIVE: To clone and characterize the cDNA for Pla a 1 and to express the recombinant protein . METHODS: Pla a 1 was isolated by cationic exchange, gel filtration, and reverse-phase chromato-graphies. Pla a 1 cDNA was cloned by reverse transcription followed by polymerase chain reaction, using amino acid sequences from tryptic peptides of the allergen. The Pla a 1 encoding sequence has been subcloned into the pKN172 expression vector and expressed in Escherichia coli as a non-fusion protein. Purified recombinant protein has been tested for its IgE-binding capacity in immunoblot, immunoblot inhibition, and ELISA . RESULTS: Pla a 1 reacted with serum IgE from 35 of the 42 (83.3%) Platanus-allergic patients studied and represented 60% of the total IgE-binding capacity of the P. acerifolia pollen extract. The allergen displayed 43% sequence identity to a grape invertase inhibitor and showed a predicted secondary structure characteristic of all-alpha proteins. Serological analysis revealed that both natural and recombinant forms of Pla a 1 displayed similar IgE-binding capacity . CONCLUSIONS: Pla a 1 belongs to a new class of allergens related to proteinaceous invertase inhibitors. Recombinant Pla a 1 binds IgE in vitro like its natural counterpart and, therefore, it can be useful for specific diagnosis and structural studies.

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[17] - Polovic N, Cirkovic Velickovic T, Burazer L, Atanaskovic-Markovic M, Vuckovic O, Jankov R, et al. Partial biochemical characterization of Art v 1 cross-reactive IgE-binding protein isolated from kiwi fruit. Allergy Clin Immunol Int 2005;17(Suppl. 1):347

Background: The kiwi fruit allergy is frequently associated to pollen allergies, especially birch, timothy and mugwort pollen allergies. Among the mugwort pollen allergens so far, only a 60 kD protein was identified to be involved in OAS to kiwi. The aim of this study was to investigate the significance of another highly glycosylated mugwort pollen allergen, a defensin-like protein (Art v 1) in relation to kiwi fruit allergy and to biochemically characterize the crossreacting kiwi fruit protein. Methods: The rabbit polyclonal antibodies to Art v 1 were purified by affinity chromatography on the column made by coupling of Art v 1 to CNBr activated matrix. They were used to recognize the cross-reacting proteins in kiwi fruit extract. The kiwi protein fraction was isolated by a combination of anion and cation exchange HPLC and characterized by reverse phase HPLC and 2D PAGE. Sugars percentage was determinated by phenol-sulfuric acid method. Pooled sera from five kiwi allergic patients were used to investigate IgE binding. For the cross-reactivity examination immunoblot and immunoblot inhibition experiments were performed using anti-Art v 1 rabbit antibodies and a pool of sera from mugwort pollen allergic patients, previously inhibited with protein purified from the kiwi fruit extract. Results: The cross-reacting protein was identified as a mixture of different isoforms, with molecular weights of 20, 26, and 27 kD in SDS PAGE and pI values of 7.4, 7.1, and 6.5, respectively. The mobilities of isoforms in reverse phase HPLC suggest that the microheterogeneity could arise from the glycocomponent of the protein (percentage of total sugar was 35) as seen for Art v 1. All isoforms bind IgE from the pool of kiwi allergic patients' sera. Furthermore, proteins of 26 and 27 kD have been recognized by anti-Art v 1 antibodies. When the pool of mugwort pollen allergic patients' sera was used the kiwi protein fraction showed inhibition of IgE binding to Art v 1 in a dose dependant manner. Similar results were obtained using anti-Art v 1 rabbit polyclonal antibodies. Conclusion: We isolated the highly glycosylated kiwi protein fraction that shares epitopes with the major mugwort pollen allergen Art v 1. According to our results, the Art v 1 could be involved in OAS to kiwi fruit.

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[18] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[19] - Möller M, Paschke A, Vieluf D, Kayma M, Vieths S, Steinhart H. Characterization of allergens in kiwi fruit and detection of cross-reactivities with allergens of birch pollen and related fruit allergens. Food Agric Immunol 1997;9:107-121

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[20] - Giangrieco I, Tuppo L, Palazzo P, Camardella L, Bernardi ML, Scala E, et al. Analysis of green and gold kiwi fruit protein pattern as a function of the ripening stage. Allergy 2007;62(suppl. 83):341

Background: Allergy to kiwi fruit has been the object of extensive investigation in the last years. Probably due to the experimental procedures used and/or to the tissue characteristics, the extracts can be variable both in the number and the amount of their components. In order to verify if the ripening process of the fruit affects the allergen pattern, the analysis of the protein components in the edible part of green (GrK) and gold kiwi (GoK) fruit, at different ripening stages, has been undertaken. Methods: Samples of GrK and GoK fruits were collected every two weeks from July to November/December 2006. Kiwi fruits were from Italian plantations, coming from nearby areas. For each fruit sample a soluble protein fraction (SF) and a saline extract of the cell wall (CW) were obtained. The total protein content of the extracts was determined by means Biorad Protein Assay (Bio-Rad, CA). The protein pattern of the SF and CW fractions was analyzed by SDS-PAGE and the identification by N-terminal amino acid sequencing of the electroblotted components has been undertaken by means Applied Biosystems Procise 492 Automatic Sequencer (Applied Biosystems, CA). Results: Ten samples have been collected in the frame period for each species. The total protein content of both GrK and GoK fruit increased during the ripening process. The two kiwi species showed a different protein pattern upon SDS-PAGE. In GrK fruit, the allergens actinidin, thaumatin-like protein (TLP) and kiwellin were already observed in the fruit samples collected in July and their amount progressively increased. During the ripening process two new protein bands were observed at 20 and 17 kDa, respectively, becoming progressively more abundant. Samples of GoK fruit showed less variations than GrK. The TLP was observed in significant amount in all the GoK kiwi samples. High amount of a double protein band at 16 kDa, absent in earlier stages, was also observed. Preliminary comparative evaluation of SF and CW fractions showed differences in the localization of allergenic proteins in fruit tissues. Conclusion: Data so far obtained show that the protein pattern of GrK and GoK fruit is affected by both the ripening process and extraction methods. Several differences in whole proteins and allergenic contents have been recorded comparing the two kiwi species. Immunochemical studies are needed in order to verify differences in the allergenicity pattern.

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[21] - Aleman A, Sastre J, Quirce S, Cuesta J, De Las Heras M, Carnés J, et al. Heterogeneity of kiwi allergy: recognition of allergens in kiwi-allergic patients. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°707

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[22] - Merima B, Radauer C, Lebens A, Knulst A, Scheiner O, Breiteneder H. Purification and partial characterization of a 40 kDa allergen from kiwifruit (Actinidia delicosa). EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°505

Introduction: In recent years, there has been an increasing number of reports of allergic reactions to kiwifruit. Two major allergens of kiwifruit have been described so far: Act c 1, the thiol protease actinidin, and Act c 2, the thaumatin like protein. According to our previous study, most of kiwi allergic patients presented specific IgE to 40 kDa protein. Our aim was to identify the 40 kDa allergen and to compare frequencies of IgE binding to Act c 1, Act c 2 and the 40 kDa allergen in a group of Dutch kiwi allergic patients. Methods: The 40 kDa allergen was purified from a kiwi protein extract by cation exchange chromatography. The protein was identified by sequencing of the N-terminus and an internal fragment obtained after cleavage by cyanogen bromide. Fifteen patients were selected based on the presence of reported allergy to kiwifruit. All patients had positive skin prick tests (> 1) and positive radioallergosorbent tests (> 0.35) to kiwifruit. The patients showed moderate to severe symptoms such as OAS, rhinitis, urticaria, eczema, asthma, angioedema or abdominal pain. Sera were tested for IgE binding to purified actinidin (Act c 1), the thaumatin-like protein (Act c 2) and to the 40 kDa allergen by ELISA. Additionally, ELISA inhibitions and immunoblots were performed with selected sera. Results: We obtained 1.1 mg of purified 40 kDa protein from 2 kg of kiwifruit. The protein was identified as a glycoprotein with a Pfam profile PF04862 but with unknown function. In IgE ELISA 88% of the patients gave positive results to Act c 1, 15% to Act c 2, and 66% to the 40 kDa allergen. Carbohydrate analysis of the 40 kDa allergen showed the existence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. However, the overall range of inhibtion capacity by HRP was only 15-60%. Conclusions: The study shows that 40 kDa allergen in kiwifruit is a new food allergen.

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[23] - Lewis SA, Pearce A, Lucas J, Hourihane J. An in vitro study of the dominant kiwi fruit allergens in a UK population and comparison of the allergenicity of two varieties of kiwi fruit. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°660

Background: To date, different studies have reported different dominant kiwi fruit allergens, possibly due to inter-population variations. The aim of this study was to identify the dominant kiwi fruit allergens in a UK population. We also aimed to compare the in vitro allergenicity of two varieties of kiwi fruit currently available in the UK: the Hayward (Zespri ˙Green) and Zespri˙Gold varieties. Methods: Serum was collected from 30 patients with raised serum kiwi-specific IgE (ten of which also had a positive DBPCFC). IgE binding patterns to a protein extract prepared from Hayward kiwi fruits were determined using western blotting. Protein extracts were prepared from both Hayward (Zespri˙Green) and Zespri˙Gold varieties and analysed using 1 and 2D SDS-PAGE and western blotting with pooled sera. The effects of reducing and non-reducing gel conditions were explored and mass spectrometry was used to identify proteins of interest. Results: 1D IgE binding patterns to the Hayward kiwi fruit extract, separated under reducing conditions, identified two major allergens at ~30 and ~38kDa respectively. These major bands did not change under non-reducing conditions. These allergens, one of which appears to be Act c 1, were not seen in a Zespri˙Gold extract. Using 2D SDS-PAGE, a highly expressed protein in the Hayward variety, with weight and pI features of Act c 1, was missing in the Zespri˙ Gold variety when gels were stained using Coomassie. Identification of the protein as Act c 1 was confirmed by mass spectrometry. Conclusions: There appear to be 2 dominant kiwi fruit (Hayward variety) allergens for the UK population (one of which may be Act c1), with linear IgE binding epitopes. There are differences in the in vitro allergenicity of the Hayward (Zespri˙ Green) and Zespri˙ Gold varieties of kiwi fruit.

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[24] - Lucas JSA. Relationship of Kiwi Fruit Allergens to Clinical Characteristics. J Allergy Clin Immunol 2005;115(2 suppl.):S93

RATIONALE: Kiwi allergy appears to be increasing, but information regarding the allergy remains limited. This is the first study to investigate a relationship between IgE binding to kiwi allergens and the clinical characteristics of the allergy METHODS: Kiwi proteins were separated by 1D- and 2D-SDS-PAGE. 5 proteins were investigated by MALDI-TOF and ESI-NanoLC tandem MS to obtain de novo sequence data. IgE binding to proteins by Western blotting was assessed using sera from 57 individuals with reported kiwi allergy, 40 of whom had undergone DBPCFC, (age range 4-72 years; symptom severity range anaphylaxis-OAS). Blot images were analyzed to estimate molecular weight and peak intensity of each band. Associations between the presence or absence of a band with the following variables were sought: symptom severity, age of subjects, outcome of DBPCFC, and associated allergies. Correlations between these variables and the maximum band intensity recorded for a patient blot, or the sum of band intensities for an individual blot were sought RESULTS: 1D gels revealed 6 major protein bands, and 2D analysis showed 15 discrete protein spots. De novo sequencing of 3 proteins showed sequence homology with pathogenesis related proteins. Subject sera identified 17 IgE binding bands. Only the 36kDa band was bound by >50% of patients. No associations were found between the binding characteristics and subjects‚ clinical characteristics, including symptom severity CONCLUSIONS: This UK population recognizes different kiwi allergens to those described in predominantly birch pollen areas, or in Southern Europe. IgE recognition of individual kiwi allergens does not predict severity of clinical symptoms

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[25] - Lucas JSA, Lewis SA, Trewin JB, Grimshaw KEC, Warner JO, Hourihane JO’B. Comparison of the allergenicity of Actinidia deliciosa (kiwi fruit) and Actinidia chinensis (gold kiwi). Pediatr Allergy Immunol 2005;16:647-654

Actinidia chinensis (gold kiwi) is a newly available fruit which has been shown to have in vitro immunoglobulin E (IgE) cross-reactivity with green kiwi. This is the first study to investigate clinical reactivity of gold kiwi. Five patients clinically allergic to green kiwi were investigated by skin test and double-blind placebo controlled food challenge (DBPCFC) with gold kiwi fruit. IgE-binding patterns of individual sera from the five challenged patients and a pool of sera from a further nine patients with kiwi allergy were compared in the two fruits by Western blotting. Cross reactivity of proteins in the two fruits was assessed by inhibition of immunoblots and by IgE enzyme-linked immunosorbent assay (ELISA) inhibition. Four of the five patients had a positive DBPCFC to gold kiwi. Western blotting showed marked differences in the allergen patterns of green and gold kiwi. However, inhibition of the immunoblots and ELISA assay reveals extensive inhibition of IgE binding to proteins in each fruit by the alternative species. Gold kiwi fruit is allergenic and patients allergic to green kiwi are at risk of reacting to the gold kiwi fruit. Despite having different protein profiles and IgE-binding patterns, the two species have proteins that extensively cross-inhibit the binding to IgE.

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[26] - Lucas JSA, Nieuwenhuizen NJ, Atkinson RG, MacRae EA, Cochrane SA, Warner JO, et al. Kiwifruit allergy: actinidin is not a major allergen in the United Kingdom. Clin Exp Allergy 2007;37:1340-1348

BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy . METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera . RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA . CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

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[27] - Lucas JSA, Lewis SA, Hourihane JOB. Kiwi fruit allergy: A review. Pediatr Allergy Immunol 2003;14:420-428

Allergy to kiwi fruit was first described in 1981, and there have since been reports of the allergy presenting with a wide range of symptoms from localized oral allergy syndrome (OAS) to life-threatening anaphylaxis. The article reviews the available information concerning the clinical features of kiwi fruit allergy and the role of clinical investigations for diagnosis. Work identifying the major allergens in kiwi fruit has resulted in conflicting results, the possible reasons for which are discussed. The clinical associations of kiwi fruit allergy with allergies to pollens or latex are reviewed.

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[28] - Voitenko V, Poulsen LK, Nielsen L, Norgaard A, Bindslev-Jensen C, Skov PS. Allergenic properties of kiwi-fruit extract: cross-reactivity between kiwi-fruit and birch-pollen allergens. Allergy 1997;52:136-143

Our investigation aimed to produce and characterize a kiwi extract and to use this extract to investigate a possible cross-reactivity with birch pollen. Kiwi was extracted in two buffers: phosphate-buffered saline (PBS) and borate-buffered saline (BBS). Extraction in BBS produced a double amount of protein, and a more stabile extract. Tandem crossed-immunoelectrophoresis showed that the BBS and PBS extracts had several common, but also a few individual, proteins. The mixture of both extracts was assumed to represent the most complete allergen extract. The allergenic properties of the kiwi extract were investigated by immunoblotting (IB), RAST, and histamine-release (HR) test in 15 birch-pollen-allergic patients (eight of them with clinical kiwi allergy) and one with clinical monoallergy to kiwi. All eight birch-pollen-allergic patients with kiwi allergy and the kiwi-monoallergic patient were positive in kiwi IB binding most frequently to proteins of 10-12 and 20-25 kDa. With our extract, RAST was positive in four kiwi-allergic and one non-kiwi-allergic patient, whereas the HR test was positive in five kiwi-allergic patients and negative in all non-kiwi-allergic patients. RAST and IB inhibition demonstrated cross-reactivity between birch-pollen and kiwi allergens due to a 10-12 kDa protein. In conclusion, a kiwi extract with allergenic properties was produced, and, by the methods used, cross-reactivity was demonstrated between birch-pollen and kiwi allergens.

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[29] - Goodman RE, Chen L, Lucas J, Hourihane JO, Taylor SL. IgE from Some Green Kiwifruit Allergic Individuals Binds to Proteins in Hardy Kiwifruit, a Third Cultivated Species of the Genus Actinidia. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°175

RATIONALE: Since hardy kiwifruit (Acintinidia arguta) is now cultivated in western North America, we tested protein extracts of hardy, green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit for IgE binding using sera from individuals with clinically diagnosed food allergies to green kiwifruit, to evaluate potential cross-reactivity METHODS: Sera from twelve green kiwifruit-allergic subjects (eight were positive by DBPCFC, four severe reactors were not challenged but had positive ImmunoCAP) and control subjects were assayed for IgE binding to soluble proteins in green, gold and hardy kiwifruits using reducing and non-reducing SDS-PAGE immunoblots and direct enzyme linked immunosorbent assays (ELISA) RESULTS: IgE-ELISA results of all kiwi-allergic subjects were positive compared to controls for one or more kiwifruit extracts. Immunoblot results demonstrated individual patient and species variability. Two sera with strong ELISA positive results to both hardy and green, but not gold kiwifruit did not show marked IgE binding to kiwifruit proteins on immunoblots with reduced, heat denatured extracts, but clearly bound two or more proteins from non-reduced, unheated extracts of hardy, but not green kiwifruit. One kiwifruit-allergic individual had marked binding to non-reduced, unheated gold kiwifruit proteins. Only one kiwifruit-allergic individual had marked binding to proteins of all species on the reduced gel blot. Some control sera showed marked IgE binding to high molecular weight proteins on immunblots CONCLUSIONS: These results suggest some kiwifruit-allergic individuals may suffer allergic cross-reactions if they consume hardy kiwifruit They also demonstrate the difficulty in developing in vitro reagents for accurate diagnosis of kiwifruit allergy and for correctly identifying major allergens Funding: Efficas, Inc.

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[30] - Lucas JSA, Lewis SA, Trewin JB, Grimshaw KEC, Warner JO, Hourihane JO’B. Comparison of the allergenicity of Actinidia deliciosa (kiwi fruit) and Actinidia chinensis (gold kiwi). Pediatr Allergy Immunol 2005;16:647-654

Actinidia chinensis (gold kiwi) is a newly available fruit which has been shown to have in vitro immunoglobulin E (IgE) cross-reactivity with green kiwi. This is the first study to investigate clinical reactivity of gold kiwi. Five patients clinically allergic to green kiwi were investigated by skin test and double-blind placebo controlled food challenge (DBPCFC) with gold kiwi fruit. IgE-binding patterns of individual sera from the five challenged patients and a pool of sera from a further nine patients with kiwi allergy were compared in the two fruits by Western blotting. Cross reactivity of proteins in the two fruits was assessed by inhibition of immunoblots and by IgE enzyme-linked immunosorbent assay (ELISA) inhibition. Four of the five patients had a positive DBPCFC to gold kiwi. Western blotting showed marked differences in the allergen patterns of green and gold kiwi. However, inhibition of the immunoblots and ELISA assay reveals extensive inhibition of IgE binding to proteins in each fruit by the alternative species. Gold kiwi fruit is allergenic and patients allergic to green kiwi are at risk of reacting to the gold kiwi fruit. Despite having different protein profiles and IgE-binding patterns, the two species have proteins that extensively cross-inhibit the binding to IgE.

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[31] - Rudeschko O, Fahlbusch B, Steurich F, Schlenvoigt G, Jäger L. Kiwi allergens and their cross-reactivity with birch, rye, timothy, and mugwort pollen. J Investig Allergol Clin Immunol 1998;8:78-84

In order to study kiwi allergens and examine their cross-reactivity to birch, rye, timothy, and mugwort pollen, immunoblot and enzyme immunoassay (EIA) inhibition tests were performed with self-prepared kiwi extract. For the investigations, the sera of 22 kiwi-allergic patients were used, which were characterized by radioallergosorbent (RAST) measurements for kiwi, birch pollen, and apple with commercial allergen disks. The RAST values for kiwi were compared with those obtained by self-prepared kiwi extract disks. In the RAST, the allergen potency of this extract was found to be very similar to that of the commercial extracts. This extract was able to bind immunoglobulin E from kiwi-allergic patients in the immunoblots and EIA. Immunoblot results revealed a broad spectrum of IgE specificities

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[32] - Lucas JSA, Nieuwenhuizen NJ, Atkinson RG, MacRae EA, Cochrane SA, Warner JO, et al. Kiwifruit allergy: actinidin is not a major allergen in the United Kingdom. Clin Exp Allergy 2007;37:1340-1348

BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy . METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera . RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA . CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

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[33] - Lucas JSA, Cochrane SA, Warner JO, Hourihane JOB. The effect of digestion and pH on the allergenicity of kiwifruit proteins. Pediatr Allergy Immunol 2008;19:392-398

It is suggested that patients with oral allergy syndrome (OAS) respond to pepsin-sensitive allergens, and systemic reactors identify pepsin-resistant allergens. We sought to assess the digestibility of kiwifruit proteins in simulated gastric fluid (SGF), and to compare the immunogenicity of the digests in patients with isolated oral and systemic reactions to kiwifruit. In addition, the effect of pH on digestibility of kiwifruit proteins was investigated. The in vitro resistance of kiwifruit proteins to digestion was determined using SGF. G-immunoglobulin (IgE) binding to digested proteins was investigated by Western blotting using sera from children and adults (aged 5-72 yr) with systemic reactions and patients with isolated oral symptoms. To determine whether pH conditions influence digestion of kiwifruit extracts, digestion at pHs 1.5-7 were compared by SDS-PAGE. Patients with systemic reactions showed IgE binding to digestion-resistant allergens, but patients with oral symptoms reacted only to digestion-labile allergens. An increase in pH from 1.5 to 2.5 significantly reduced pepsin breakdown of kiwifruit allergens. Immunoreactive digested protein fragments were detectable by immunoblot but not Coomassie stain. This study confirms a difference in the lability of food allergens recognized by patients with systemic reactions and those with OAS. Pepsin digestion of kiwifruit proteins was impaired by hypoacidic conditions suggesting that patients with hypoacidic gastric conditions are at increased risk of systemic absorption of allergens. The data indicate that commonly used methods for predicting allergenicity of novel proteins using Coomassie stains may be flawed.

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[34] - Möller M, Paschke A, Vieluf D, Kayma M, Vieths S, Steinhart H. Characterization of allergens in kiwi fruit and detection of cross-reactivities with allergens of birch pollen and related fruit allergens. Food Agric Immunol 1997;9:107-121

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[35] - Gavrovic-Jankulovic M, Circovic T, Vuckovic O, Atanaskovic-Markovic M, Petersen A, Gojgic G, et al. Isolation and biochemical characterization of a thaumatin-like kiwi allergen. J Allergy Clin Immunol 2002;110:805-810

BACKGROUND: Kiwi fruit allergy, as well as its association with hypersensitivity to other foods and to pollen, has been extensively reported in the last few years. Several IgE-binding components have been detected in kiwi extract, but only one 30- kd allergen has been isolated; it was identified as actinidin (Act c 1). Recently, we have reported a 24-kd kiwi protein to be a potential major allergen in a group of patients with oral allergy syndrome (OAS) . OBJECTIVE: The aim of this study was to purify and characterize the 24-kd kiwi allergen biochemically . METHODS: Seven polysensitized patients with OAS to kiwi were used in this study. The kiwi allergen was isolated by using a combination of gel permeation, ion exchange, and immobilized metal ion affinity chromatography. Its biochemical characterization included determination of its isoelectric point, molecular weight, N-terminal sequencing, concanavalin A -binding ability, digestibility in simulated gastric fluid, and antifungal activity. Western blotting, 2-dimensional PAGE immunoblotting, and skin prick tests were performed to characterize the isolated protein immunochemically . RESULTS: All 7 patients recognized the isolated 24-kd kiwi protein as an allergen. The isolated protein consisted of 2 isoforms with isoelectric points of 9.4 and 9.5 migrated as one protein band of 20 kd after SDS-PAGE under nonreducing conditions or at 24 kd under reducing conditions. The partial N-terminal sequence revealed that it is a thaumatin-like protein (TLP) with concanavalin A -binding ability. The protein showed antifungal activity toward Saccharomyces carlsbergensis, and Candida albicans. The protein was degraded by the simulated gastric fluid within 1 minute. Both isoforms bound IgE from a pool of sera in a 2-dimensional PAGE immunoblot. The TLP elicited positive skin prick test responses in 4 (80 %) of 5 patients with OAS . CONCLUSION: This study reported isolation and full characterization of a new kiwi allergen, TLP (isoelectric points of 9.4 and 9.5 and molecular weight of 24 kd), which belongs to the family of pathogenesis-related proteins. The isolated protein expressed antifungal activity toward S carlsbergensis and C albicans.

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[36] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[37] - Ciardiello MA, Giangrieco I, Tuppo L, Tamburrini M, Buccheri M, Palazzo P et al. Influence of the Natural Ripening Stage, Cold Storage, and Ethylene Treatment on the Protein and IgE-Binding Profiles of Green and Gold Kiwi Fruit Extracts. J Agric Food Chem 2009;57:1565-1571

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

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[38] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[39] - Palacin A, Rodriguez J, Blanco C, Lopez-Torrejon G, Sanchez-Monge R, Varela J, et al. Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms. Clin Exp Allergy 2008;38:1220-1228

BACKGROUND: Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients . OBJECTIVE: To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population . METHODS: A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs . RESULTS: The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity . CONCLUSION: Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

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[40] - Pastorello EA, Conti A, Pravettoni V, Farioli L, Rivolta F, Ansaloni R, et al. Identification of actinidin as the major allergen of kiwi fruit. J Allergy Clin Immunol 1998;101:531-537

Allergic reactions to fruits and vegetables are among the most frequent food allergies in adults. Kiwi fruit (Actinidia chinensis) is commonly involved, causing local mucosal, systemic, or both types of symptoms by an IgE-mediated mechanism. In a previous study on 30 patients allergic to kiwi, we identified a major allergen of 30 kd against which all sera tested clearly reacted. Other allergens were detected at 12, 24, and 28 kd. OBJECTIVE: The aim of this study was to fully characterize the major kiwi fruit allergen of 30 kd. METHODS: Allergens were separated and purified by high-performance liquid chromatography with anion-exchange columns. The purity of the single proteins was checked by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and their allergenicity was checked by immunoblotting with a pool of sera from patients allergic to kiwi. The allergens were characterized by isoelectrofocusing and amino acid sequencing, and periodic acid-Schiff stain was used to detect glycoproteins. RESULTS: Proteins of 30, 28, 24, and 17 kd were purified by high-performance liquid chromatography. IgE binding indicated the 30 kd protein, which showed an isoelectric point of 3.5, as the major allergen of kiwi. Determination of its partial amino acid sequence and comparison with the Swiss Protein Bank showed that this was actinidin, the main protein component of kiwi. The 24 and 28 kd proteins had the same N-terminal sequence, which did not correspond to any known protein. The 17 kd protein had a blocked N-terminal sequence. CONCLUSIONS: These results demonstrate that the major allergen of kiwi fruit, Act c 1, is actinidin, a proteolytic enzyme belonging to the class of thiol-proteases. Two other allergens of 24 and 28 kd appear identical on amino acid sequencing.

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[41] - Palacin A, Quirce S, Sánchez-Monge R, Fernández-Nieto M, Varela J, Sastre J, et al. Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens. Ann Allergy Asthma Immunol 2008;101:200-205

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

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[42] - Merima B, Radauer C, Ebner C, Allwardth D, Thomas WR, Mari A, et al. IgE Cross-reactivity between the Cysteine Proteases Der p 1 and Act c 1, the Major Allergens from House Dust Mites and Kiwifruit. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°194

RATIONALE: The cysteine proteases Act c 1 and Der p 1 are welldescribed major allergens in mites and kiwifruit, respectively. However, there are no data about IgE cross-reactivity between these allergens that are taxonomically not related but belong to the same protein family METHODS: Crystal structures and sequences of Der p 1 and Act c 1 were used to locate identical residues on the molecules' surfaces. Characterization of IgE binding and inhibition experiments were performed using Act c 1 and Der p 1. Sera of 16 patients were selected who had allergy to kiwifruit and/or sensitization to mites RESULTS: Sequence of Der p 1 and Act c 1 showed 30.6% identity and 41.8% similarity. Structural analysis of exterior amino acid side chains showed 18.7% identity and 29.1% similarity. The highest structural similarity was found within and in the vicinity of the active site of the two molecules. All sera tested contained IgE reactive to Der p 1 and 10 sera to Act c 1. The overall range of inhibitory capacity of Act c 1 upon IgE binding to Der p 1 was 20% to 61%. Der p 1 inhibited IgE binding to Act c 1 from 25% to 92% CONCLUSIONS: Here we demonstrate that homologous cysteine proteases of diverse sources share common IgE epitopes despite espite low sequence and structural similarity This work was supported by the Austrian Science Fund grant SFBF01802 Funding: Austrian Science Fund SFB-F01802.

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[43] - Nieuwenhuizen NJ, Beuning LL, Sutherland PW, Sharma NN, Cooney JM, Bieleski LRF, et al. Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit. Funct Plant Biol 2007;34:946-961

Abstract Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

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[44] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[45] - Ciardiello MA, Giangrieco I, Tuppo L, Tamburrini M, Buccheri M, Palazzo P et al. Influence of the Natural Ripening Stage, Cold Storage, and Ethylene Treatment on the Protein and IgE-Binding Profiles of Green and Gold Kiwi Fruit Extracts. J Agric Food Chem 2009;57:1565-1571

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

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[46] - Giangrieco I, Tuppo L, Palazzo P, Camardella L, Bernardi ML, Scala E, et al. Analysis of green and gold kiwi fruit protein pattern as a function of the ripening stage. Allergy 2007;62(suppl. 83):341

Background: Allergy to kiwi fruit has been the object of extensive investigation in the last years. Probably due to the experimental procedures used and/or to the tissue characteristics, the extracts can be variable both in the number and the amount of their components. In order to verify if the ripening process of the fruit affects the allergen pattern, the analysis of the protein components in the edible part of green (GrK) and gold kiwi (GoK) fruit, at different ripening stages, has been undertaken. Methods: Samples of GrK and GoK fruits were collected every two weeks from July to November/December 2006. Kiwi fruits were from Italian plantations, coming from nearby areas. For each fruit sample a soluble protein fraction (SF) and a saline extract of the cell wall (CW) were obtained. The total protein content of the extracts was determined by means Biorad Protein Assay (Bio-Rad, CA). The protein pattern of the SF and CW fractions was analyzed by SDS-PAGE and the identification by N-terminal amino acid sequencing of the electroblotted components has been undertaken by means Applied Biosystems Procise 492 Automatic Sequencer (Applied Biosystems, CA). Results: Ten samples have been collected in the frame period for each species. The total protein content of both GrK and GoK fruit increased during the ripening process. The two kiwi species showed a different protein pattern upon SDS-PAGE. In GrK fruit, the allergens actinidin, thaumatin-like protein (TLP) and kiwellin were already observed in the fruit samples collected in July and their amount progressively increased. During the ripening process two new protein bands were observed at 20 and 17 kDa, respectively, becoming progressively more abundant. Samples of GoK fruit showed less variations than GrK. The TLP was observed in significant amount in all the GoK kiwi samples. High amount of a double protein band at 16 kDa, absent in earlier stages, was also observed. Preliminary comparative evaluation of SF and CW fractions showed differences in the localization of allergenic proteins in fruit tissues. Conclusion: Data so far obtained show that the protein pattern of GrK and GoK fruit is affected by both the ripening process and extraction methods. Several differences in whole proteins and allergenic contents have been recorded comparing the two kiwi species. Immunochemical studies are needed in order to verify differences in the allergenicity pattern.

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[47] - Lewis SA, Warner JO, Hourihane JO, Lucas JSA. Gold Kiwi Fruit - A New Food Allergen. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°514

Rationale The allergic potential of all novel foods needs to be assessed, but to date there have been no studies investigating the allergic potential of gold kiwi in subjects allergic to green kiwi fruit (Actinidia deliciosa). It has been suggested the gold kiwi fruit (Actinidia chiensis) is less allergenic than green kiwi fruit because it contains extremely low levels (=10 fold less) of actinidin, the major allergen in kiwi fruit. Method s : Five subjects with allergy to green kiwi fruit confirmed by DBPCFC were investigated by skin test and DBPCFC with gold kiwi fruit. Result s : Four of the five subjects had a positive DBPCFC to gold kiwi. One subject with systemic symptoms to green kiwi fruit had a moderately severe reaction to the gold fruit. Two subjects with OAS to green kiwi fruit had similar symptoms with gold kiwi, but noted that the itching was less intense than that experienced during DBPCFC with green kiwi fruit. A subject who reacts systemically to green kiwi had oral pruritus with gold kiwi fruit. Conclusions : Gold kiwi fruit is allergenic despite extremely low levels of the major allergen in green kiwi, actinidin. Subjects allergic to green kiwi are at risk of reacting to the gold kiwi fruit.

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[48] - González G, Jiménez G, Moneo I, Caballero ML, García-Menaya JM. Actinidin (Act c 1), a probably PRP from kiwi's peel as a cause of anaphylaxis. Allergy Clin Immunol Int 2005;17(Suppl. 1):348

Background: In the last few years, it has been reported 12 relevant allergens from kiwi fruit, most of them represent commom allergenic structures with pollen, latex and other fruits. Actinidin (Act c 1) has been identified as a major allergen of kiwi, probably suposed to be a specific allergen that belongs to pathogenesis-related protein. We report a 15-year-old female, with personal and familiar history of atopy, who, in two occasions inmediately after eating kiwi (previously tolerated), suffered from nauseas, diarrhoea, vomiting, sneezing, rhinorrea, pharynx pruritus and difficulty to swallow saliva. She tolerates contact to latex and comsumption of other foods. Material and Methods: We performed skin prick tests (SPT) to commom inhalants, commercial extracts to latex and foods. Serum total IgE and specific IgE (UniCap Pharmacia method) to milk, egg and their proteinic fractions, kiwi and latex; hemogram and biochemical parameters were carried out. SDS PAGE and immunoblotting from crude self-prepared extracts of pulp, peel, central column and brushing of kiwi were performed. Results: SPT were positive to commercial extracts of kiwi, mites, grass and tree pollens, cat and dog danders. Total serum IgE was 358 KU/l. Specific serum IgE to kiwi was 1,35 KU/l, casein 0,71 KU/l, whole milk 0,92 KU/l, white egg 0,43 KU/l and alfa-lactoglobulin 0,39 KU/l. Several protein bands ranging from 10 kD to 100 kD were detected by means of SDS PAGE. Sera patient recognized a prominent IgE-binding band of 30 kD that belongs to kiwi's peel extract. Rest of complementary tests were negative. Conclusions: We report a patient with anaphylaxis to kiwi confirmed by means of in vivo and in vitro tests, without cross-reactivity to latex or other fruits. 30 kD-IgE-binding band detected could correspond to Act c 1, major allergen of kiwi. It is important to note that we could only obtained this band from the extract of kiwi's peel, so, it might act as a pathogenesis-related protein.

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[49] - Nieuwenhuizen NJ, Beuning LL, Sutherland PW, Sharma NN, Cooney JM, Bieleski LRF, et al. Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit. Funct Plant Biol 2007;34:946-961

Abstract Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

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[50] - Radauer C, Bublin M, Scheiner O, Breiteneder H. Molecular characterization of common and cultivar-specific kiwifruit allergens in the varieties Hayward and Hort16A. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°986

Background: Kiwifruits (Actinidia spp.) are commonly consumed in Europe since the 1980s. Since the first report of kiwifruit allergy in 1981, the prevalence of this new food allergy has apparently increased. The world market for kiwifruits is dominated by a single variety, A. deliciosa cv. Hayward. In 1998, a novel, yellow-fleshed variety, A. chinensis cv. Hort16A, was introduced. To date, there are only limited data on kiwifruit allergens. In particular, no studies dealing with Hort16A and other novel varieties have been published. The aim of this study was the identification of allergens in Hayward and Hort16A kiwifruit extracts by protein sequencing. Methods: Protein extracts of Hayward and Hort16A kiwifruits were separated by anion exchange chromatography and proteins in the chromatographic fractions were probed by IgE immunoblotting using kiwi-allergic patients' sera. IgE binding bands were excised from the blots and sequenced. Results: Total extracts as well as chromatographic fractions of the two kiwifruit varieties showed marked differences in their SDS-PAGE band patterns and in their compositions of IgE binding proteins. Allergens with molecular masses of 12, 22, 32 and 43 kDa were detected in both extracts. The 12 kDa allergens had identical N-terminal sequences similar to plant cystatins, cysteine protease inhibitors being parts of the plant defense system. The 22 kDa allergens were identified as thaumatin-like proteins; the 32 kDa proteins contained an N-terminal hevein-like domain. Members of both families have previously been described as allergens. The 43 kDa protein, which was found in much higher amounts in the Hort16A extract, was N-terminally blocked. The major kiwifruit allergen actinidin was detected exclusively in the Hayward extract. The Hort16A extract contained a different allergen of similar molecular mass whose N-terminus was blocked. Conclusions: The kiwifruit varieties Hayward and Hort16A differed in their allergen compositions qualitatively as well as quantitatively. This may have consequences for their risk potentials for kiwi-allergic patients and for their cross-reactivity to pollen and latex.

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[51] - Alemán A, Sastre J, Quirce S, de las Heras M, Carnés J, Fernández-Caldas E, et al. Allergy to kiwi: A double-blind, placebo-controlled food challenge study in patients from a birch-free area. J Allergy Clin Immunol 2004;113:543-550

Background Allergy to kiwi fruit is being increasingly reported, but it has never been evaluated by means of a double-blind, placebo-controlled food challenge (DBPCFC) study. Objective : We sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitized patients from a birch-free area. Method s : Forty-three patients with allergy symptoms who were sensitized to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The pattern of allergen recognition was assessed by means of IgE immunoblotting. Sequence analysis of IgE-binding bands was performed by using Edman degradation. Result s : DBPCFCs were performed in 33 patients; 4 patients had experienced severe anaphylaxis, and 6 patients declined informed consent. DBPCFC results were positive in 23 patients and negative in 10 patients. The most frequent clinical manifestation was oral allergy syndrome. Twenty-one percent of the patients were not allergic to pollen. Forty-six percent of patients experienced systemic symptoms, and this happened with higher frequency in patients not allergic to pollen (100%). Twenty-eight percent of the patients were sensitized to latex. The IgE-binding bands in kiwi extract more frequently recognized by patient sera were those of 30, 24, 66, and 12 kd, and they could not be associated with any pattern of kiwi-induced allergic reactions. Conclusion : The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi. One of 5 patients with kiwi allergy was not allergic to pollen, and these patients had the highest risk of systemic reactions to kiwi.

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[52] - Palazzo P, Giangrieco I, Bernardi ML, Tamburrini M, Giani M, Ciardiello MA, et al. IgE reactivity to kiwellin (Act d 28kD), actinidin (Act d 1) and thaumatin-like protein (Act d 2) from green kiwi fruit in kiwi allergic patients tested by SDS-PAGE arrayed molecules. Allergy 2007;62(suppl. 83):341

Background Several allergens have been identified and characterized in green kiwi fruit, but little is know about patients' profiles in terms of IgE reactivity to single kiwi fruit extract components. We sough to describe kiwi sensitization profiles by testing three kiwi allergens and three more plant-derived food allergens. Methods Kiwellin (Act d 28kD), Actinidin (Act d 1) and thaumatin-like protein (Act d 2) were purified from green kiwi fruit by ionic exchange chromatography on DE-52 and SP-Sepharose. The last purification step was carried out by FPLC (Mono S HR 10/10 or Q HR 10/10, Amersham-Pharmacia, Sweden). Proteins were pure upon SDS-PAGE and characterized by N-terminal amino acid sequencing. The three purified proteins were mixed at 1:1:1 ratio for immunoblotting analysis. Molecules underwent to a reducing SDS-PAGE, were electroblotted onto PVDF membranes, and probed with sera from kiwi allergic subjects. Sera were selected on the basis of clinical reactivity. IgE testing was also performed on ISAC system microarrayed allergens (VBC, Austria) and reactivity to profilin, LTP and Bet v 1-like allergens was recorded. Results 30 patients have been selected. Sera of 14 (47%) patients showed IgE reactivity to Act d 1, 10 (33%) to Act d 28kD, and 9 (30%) recognized Act d 2. The combined reactivity to the three components was recorded in 2 subjects (6%). Reactivity to Act d 1 and Act d 2 was detected in 4 subjects (13%), whereas Act d 1 plus Act d 28kD, and Act d 2 plus Act d 28kD were positive in 4 and 3 subjects (13% and 10%), respectively. A single allergen was positive in 5 (Act d 1, 16%), 4 (Act d 28kD, 13%), and 2 (Act d 2, 6%) subjects. 8 subjects were negative to all three green kiwi molecules (26%). 22 serum of 30 patients were tested on ISAC system microarrayed allergens. 14 sera were positive to at least one kiwi allergen. 4 subjects were positive to Bet v 1-like molecules, 7 to LTP and 4 to Profilin. 2 serum showed co-reactivity to LTP and Profilins, one to Bet v 1-like and Profilins and one to Bet v 1-like and LTP. 3 of 8 kiwi allergen negative sera were scored positive to at least one of the three additional food allergens. Conclusion IgE are detected for all kiwi allergens and differ in their prevalence. Patients with mono-sensitization are rare compared to those who show co-reactivity to 2 or more green kiwi allergens. Kiwi allergic subjects may show an IgE reactivity to additional plant-related allergens.

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[53] - Aleman A, Sastre J, Quirce S, Cuesta J, De Las Heras M, Carnés J, et al. Heterogeneity of kiwi allergy: recognition of allergens in kiwi-allergic patients. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°707

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[54] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[55] - Fahlbusch B, Rudeschko O, Schumann C, Steurich F, Henzgen M, Schlenvoigt G, et al. Further characterization of IgE-binding antigens in kiwi, with particular emphasis on glycoprotein allergens. J Investig Allergol Clin Immunol 1998;8:325-332

Fruit allergy is frequently associated with birch pollinosis. The aim of this study was to investigate which kiwi allergens were involved in subjects allergic to fruit alone and in patients allergic to both fruit and birch pollen. Sera of nine patients (five with both kiwi and birch pollen allergy and four with isolated kiwi allergy) were studied by immunoblot of kiwi extract. Eight of the nine sera reacted with the 30 kDa protein. Furthermore, IgE-binding proteins were seen at approximately 23 kDa (detected by five sera), 43 kDa and 80 kDa (four sera), and > 80 kDa (two sera). One serum showed no IgE binding to any kiwi allergen. The 30 kDa is the major allergen in kiwi and was purified by anion-exchange chromatography and characterized by isoelectrofocusing and amino acid sequencing. The comparison of its partial amino acid sequence with data from the Swiss Protein Bank revealed that this protein is actinidine. The carbohydrate structures in kiwi and birch pollen extracts were investigated with seven lectins. On kiwi blot, Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues) to the components of 35 to 92 kDa, while concanavalin A (indicating mannose, glucose or N-acetylglucosamine residues) showed weak binding at 67 kDa. In contrast, strong binding of Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A was found on birch pollen blots. The presence of IgE against carbohydrate structures was determined by means of enzyme-linked immunosorbent assay (ELISA) after periodate treatment of kiwi extract. The IgE binding was reduced by periodate treatment of kiwi coated microtiter plates, but not by sera reacting exclusively with the 30 kDa protein. Furthermore, selected sera were treated with proteinase K-digested kiwi and birch pollen extracts as the sources of crossreactive carbohydrate determinants. In accordance with the results of sodium periodate treatment, significant levels of anti-cross-reactive carbohydrate determinant IgE were found in sera from patients allergic to both kiwi and birch pollen. Our results show that the major allergen for kiwi allergy is the 30 kDa protein and additionally that the cross-reaction between kiwi and birch pollen allergy is mainly due to carbohydrate moieties

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[56] - Möller M, Paschke A, Vieluf D, Kayma M, Vieths S, Steinhart H. Characterization of allergens in kiwi fruit and detection of cross-reactivities with allergens of birch pollen and related fruit allergens. Food Agric Immunol 1997;9:107-121

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[57] - Möller M, Kayma M, Steinhart H, Paschke A. Isolation and characterization of a major allergen in kiwi fruit. Eur Food Res Technol 1997;205:364-369

The isolation of an important allergen in kiwi fruit ( Actinidia chinensis) by ion-exchange chromatography (IEC) and micropreparative SDS-PAGE followed by electroelution is reported. The purity of the allergen was analysed by SDS-PAGE and immunoblotting with sera from patients who have an allergy to kiwi. The allergen was shown to have a molecular weight of 43 kDa by SDS-PAGE/immunoblotting and an isoelectric point of approximately 6.9 as estimated by IEC. In accordance with World Health Organization nomenclature, this allergen is called Act c 2. By immunoblot inhibition it was shown that epitopes from different allergens in kiwi fruit are also located on Act c 2. N-terminal amino acid sequencing of 17 amino acid residues did not reveal homology with the major allergens in birch pollen (Bet v 1), apple (Mal d 1) or with other proteins of allergenic plant foods. In addition, the isoelectric point of a 67-kDa allergen in kiwi fruit was estimated to be 7.4 by IEC, but micropreparative isolation of this allergen failed because of its very low content in the fruit.

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[58] - Pastorello EA, Pravettoni V, Ispano M, Farioli L, Ansaloni R, Rotondo F, et al. Identification of the allergenic components of kiwi fruit and evaluation of their cross-reactivity with timothy and birch pollens. J Allergy Clin Immunol 1996;98:601-610

Only a few food allergens have as yet been identified, mainly because of the difficulty of obtaining a sufficient number of patients who are clinically sensitized to a given food. This is more feasible in the case of the oral allergy syndrome (OAS), a common form of food allergy, which is especially prevalent in patients with pollinosis. OBJECTIVE: We designed a study to identify the allergens of kiwi fruit (Actinidia chinensis) by analyzing the sera of patients with OAS for kiwi and to examine the cross-reactivity of these allergens with timothy and birch pollen allergens. METHODS: Twenty-seven patients with OAS for kiwi, a positive skin prick test response and serum IgE antibody to kiwi, and a positive open kiwi challenge test result and three patients who had OAS with severe systemic symptoms, which excluded a challenge test, were included in this study. The different polypeptide components of an extract of fresh kiwi were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and analyzed by IgE immunoblotting with sera from these patients. Cross-reactivity with the two pollen extracts was assessed by inhibition of the immunoblots with pooled and individual patients' sera. RESULTS: Twelve IgE-binding components with molecular weights ranging from 12 to 64 kd were identified in the kiwi extract, but only a 30 kd component acted as major allergen, being recognized by sera of 100% of these patients. Inhibition of kiwi immunoblots with timothy and birch pollen extracts demonstrated strong cross-reactivity with some of the kiwi allergens, suggesting complete identity between certain food and pollen allergens; whereas others, particularly the 30 kd allergen, were only partially inhibited, suggesting much weaker cross-reactivity. CONCLUSIONS: Kiwi fruit contains a large number of allergens widely cross-reacting with allergens in grass and birch pollen extracts. Nevertheless, the major allergen at 30 kd appears to be specific for kiwi.

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[59] - Gavrovic-Jankulovic M, Circovic T, Vuckovic O, Atanaskovic-Markovic M, Petersen A, Gojgic G, et al. Isolation and biochemical characterization of a thaumatin-like kiwi allergen. J Allergy Clin Immunol 2002;110:805-810

BACKGROUND: Kiwi fruit allergy, as well as its association with hypersensitivity to other foods and to pollen, has been extensively reported in the last few years. Several IgE-binding components have been detected in kiwi extract, but only one 30- kd allergen has been isolated; it was identified as actinidin (Act c 1). Recently, we have reported a 24-kd kiwi protein to be a potential major allergen in a group of patients with oral allergy syndrome (OAS) . OBJECTIVE: The aim of this study was to purify and characterize the 24-kd kiwi allergen biochemically . METHODS: Seven polysensitized patients with OAS to kiwi were used in this study. The kiwi allergen was isolated by using a combination of gel permeation, ion exchange, and immobilized metal ion affinity chromatography. Its biochemical characterization included determination of its isoelectric point, molecular weight, N-terminal sequencing, concanavalin A -binding ability, digestibility in simulated gastric fluid, and antifungal activity. Western blotting, 2-dimensional PAGE immunoblotting, and skin prick tests were performed to characterize the isolated protein immunochemically . RESULTS: All 7 patients recognized the isolated 24-kd kiwi protein as an allergen. The isolated protein consisted of 2 isoforms with isoelectric points of 9.4 and 9.5 migrated as one protein band of 20 kd after SDS-PAGE under nonreducing conditions or at 24 kd under reducing conditions. The partial N-terminal sequence revealed that it is a thaumatin-like protein (TLP) with concanavalin A -binding ability. The protein showed antifungal activity toward Saccharomyces carlsbergensis, and Candida albicans. The protein was degraded by the simulated gastric fluid within 1 minute. Both isoforms bound IgE from a pool of sera in a 2-dimensional PAGE immunoblot. The TLP elicited positive skin prick test responses in 4 (80 %) of 5 patients with OAS . CONCLUSION: This study reported isolation and full characterization of a new kiwi allergen, TLP (isoelectric points of 9.4 and 9.5 and molecular weight of 24 kd), which belongs to the family of pathogenesis-related proteins. The isolated protein expressed antifungal activity toward S carlsbergensis and C albicans.

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[60] - Lucas JSA, Nieuwenhuizen NJ, Atkinson RG, MacRae EA, Cochrane SA, Warner JO, et al. Kiwifruit allergy: actinidin is not a major allergen in the United Kingdom. Clin Exp Allergy 2007;37:1340-1348

BACKGROUND: Actinidin has previously been reported as the major allergen in kiwifruit. Objectives To investigate the relevance of actinidin in a well-characterized population of UK patients with kiwifruit allergy . METHODS: To identify the allergens in kiwifruit, using Western blots, we examined the IgE-binding patterns of 76 patients with a history of kiwifruit allergy, 23 of who had had a positive double-blind, placebo-controlled food challenge. In addition, IgE binding to purified native actinidin was studied in 30 patients, and to acidic and basic isoforms of recombinant actinidin in five patients. Inhibition of IgE binding to kiwifruit protein extract by purified native actinidin was investigated by both inhibition immunoblots and inhibition ELISAs using pooled sera . RESULTS: Twelve protein bands in kiwifruit protein extract were bound by IgE. A protein band with a molecular weight of 38 kDa was the major allergen recognized by 59% of the population. IgE did not bind to actinidin in the kiwifruit protein extract, or to purified native or recombinant forms of actinidin during Western blotting. Pooled sera bound to kiwifruit protein extract but not purified actinidin on ELISA, and pre-incubating sera with actinidin did not inhibit IgE binding to kiwifruit protein extract on immunoblot or ELISA . CONCLUSION: A novel 38 kDa protein, not actinidin, is the major allergen in this large study population. Identification of major allergens in one patient group is therefore not necessarily reproducible in another; therefore, major allergens should not be defined until there is a sufficient body of data from diverse geographical and cultural populations.

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[61] - Merima B, Radauer C, Lebens A, Knulst A, Scheiner O, Breiteneder H. Purification and partial characterization of a 40 kDa allergen from kiwifruit (Actinidia delicosa). EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°505

Introduction: In recent years, there has been an increasing number of reports of allergic reactions to kiwifruit. Two major allergens of kiwifruit have been described so far: Act c 1, the thiol protease actinidin, and Act c 2, the thaumatin like protein. According to our previous study, most of kiwi allergic patients presented specific IgE to 40 kDa protein. Our aim was to identify the 40 kDa allergen and to compare frequencies of IgE binding to Act c 1, Act c 2 and the 40 kDa allergen in a group of Dutch kiwi allergic patients. Methods: The 40 kDa allergen was purified from a kiwi protein extract by cation exchange chromatography. The protein was identified by sequencing of the N-terminus and an internal fragment obtained after cleavage by cyanogen bromide. Fifteen patients were selected based on the presence of reported allergy to kiwifruit. All patients had positive skin prick tests (> 1) and positive radioallergosorbent tests (> 0.35) to kiwifruit. The patients showed moderate to severe symptoms such as OAS, rhinitis, urticaria, eczema, asthma, angioedema or abdominal pain. Sera were tested for IgE binding to purified actinidin (Act c 1), the thaumatin-like protein (Act c 2) and to the 40 kDa allergen by ELISA. Additionally, ELISA inhibitions and immunoblots were performed with selected sera. Results: We obtained 1.1 mg of purified 40 kDa protein from 2 kg of kiwifruit. The protein was identified as a glycoprotein with a Pfam profile PF04862 but with unknown function. In IgE ELISA 88% of the patients gave positive results to Act c 1, 15% to Act c 2, and 66% to the 40 kDa allergen. Carbohydrate analysis of the 40 kDa allergen showed the existence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. However, the overall range of inhibtion capacity by HRP was only 15-60%. Conclusions: The study shows that 40 kDa allergen in kiwifruit is a new food allergen.

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[62] - Palacin A, Quirce S, Sánchez-Monge R, Fernández-Nieto M, Varela J, Sastre J, et al. Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens. Ann Allergy Asthma Immunol 2008;101:200-205

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

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[63] - Palacin A, Rodriguez J, Blanco C, Lopez-Torrejon G, Sanchez-Monge R, Varela J, et al. Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms. Clin Exp Allergy 2008;38:1220-1228

BACKGROUND: Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients . OBJECTIVE: To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population . METHODS: A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs . RESULTS: The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity . CONCLUSION: Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

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[64] - Lidholm J. Allergens and allergy diagnosis in Europrevall : component resolved diagnosis of food allergy. Communication, 28th Congress of EAACI, Warszaw, June 6-10 2009

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[65] - Gavrovic-Jankulovic M, Circovic T, Vuckovic O, Atanaskovic-Markovic M, Petersen A, Gojgic G, et al. Isolation and biochemical characterization of a thaumatin-like kiwi allergen. J Allergy Clin Immunol 2002;110:805-810

BACKGROUND: Kiwi fruit allergy, as well as its association with hypersensitivity to other foods and to pollen, has been extensively reported in the last few years. Several IgE-binding components have been detected in kiwi extract, but only one 30- kd allergen has been isolated; it was identified as actinidin (Act c 1). Recently, we have reported a 24-kd kiwi protein to be a potential major allergen in a group of patients with oral allergy syndrome (OAS) . OBJECTIVE: The aim of this study was to purify and characterize the 24-kd kiwi allergen biochemically . METHODS: Seven polysensitized patients with OAS to kiwi were used in this study. The kiwi allergen was isolated by using a combination of gel permeation, ion exchange, and immobilized metal ion affinity chromatography. Its biochemical characterization included determination of its isoelectric point, molecular weight, N-terminal sequencing, concanavalin A -binding ability, digestibility in simulated gastric fluid, and antifungal activity. Western blotting, 2-dimensional PAGE immunoblotting, and skin prick tests were performed to characterize the isolated protein immunochemically . RESULTS: All 7 patients recognized the isolated 24-kd kiwi protein as an allergen. The isolated protein consisted of 2 isoforms with isoelectric points of 9.4 and 9.5 migrated as one protein band of 20 kd after SDS-PAGE under nonreducing conditions or at 24 kd under reducing conditions. The partial N-terminal sequence revealed that it is a thaumatin-like protein (TLP) with concanavalin A -binding ability. The protein showed antifungal activity toward Saccharomyces carlsbergensis, and Candida albicans. The protein was degraded by the simulated gastric fluid within 1 minute. Both isoforms bound IgE from a pool of sera in a 2-dimensional PAGE immunoblot. The TLP elicited positive skin prick test responses in 4 (80 %) of 5 patients with OAS . CONCLUSION: This study reported isolation and full characterization of a new kiwi allergen, TLP (isoelectric points of 9.4 and 9.5 and molecular weight of 24 kd), which belongs to the family of pathogenesis-related proteins. The isolated protein expressed antifungal activity toward S carlsbergensis and C albicans.

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[66] - Radauer C, Bublin M, Scheiner O, Breiteneder H. Molecular characterization of common and cultivar-specific kiwifruit allergens in the varieties Hayward and Hort16A. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°986

Background: Kiwifruits (Actinidia spp.) are commonly consumed in Europe since the 1980s. Since the first report of kiwifruit allergy in 1981, the prevalence of this new food allergy has apparently increased. The world market for kiwifruits is dominated by a single variety, A. deliciosa cv. Hayward. In 1998, a novel, yellow-fleshed variety, A. chinensis cv. Hort16A, was introduced. To date, there are only limited data on kiwifruit allergens. In particular, no studies dealing with Hort16A and other novel varieties have been published. The aim of this study was the identification of allergens in Hayward and Hort16A kiwifruit extracts by protein sequencing. Methods: Protein extracts of Hayward and Hort16A kiwifruits were separated by anion exchange chromatography and proteins in the chromatographic fractions were probed by IgE immunoblotting using kiwi-allergic patients' sera. IgE binding bands were excised from the blots and sequenced. Results: Total extracts as well as chromatographic fractions of the two kiwifruit varieties showed marked differences in their SDS-PAGE band patterns and in their compositions of IgE binding proteins. Allergens with molecular masses of 12, 22, 32 and 43 kDa were detected in both extracts. The 12 kDa allergens had identical N-terminal sequences similar to plant cystatins, cysteine protease inhibitors being parts of the plant defense system. The 22 kDa allergens were identified as thaumatin-like proteins; the 32 kDa proteins contained an N-terminal hevein-like domain. Members of both families have previously been described as allergens. The 43 kDa protein, which was found in much higher amounts in the Hort16A extract, was N-terminally blocked. The major kiwifruit allergen actinidin was detected exclusively in the Hayward extract. The Hort16A extract contained a different allergen of similar molecular mass whose N-terminus was blocked. Conclusions: The kiwifruit varieties Hayward and Hort16A differed in their allergen compositions qualitatively as well as quantitatively. This may have consequences for their risk potentials for kiwi-allergic patients and for their cross-reactivity to pollen and latex.

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[67] - Giangrieco I, Tuppo L, Palazzo P, Camardella L, Bernardi ML, Scala E, et al. Analysis of green and gold kiwi fruit protein pattern as a function of the ripening stage. Allergy 2007;62(suppl. 83):341

Background: Allergy to kiwi fruit has been the object of extensive investigation in the last years. Probably due to the experimental procedures used and/or to the tissue characteristics, the extracts can be variable both in the number and the amount of their components. In order to verify if the ripening process of the fruit affects the allergen pattern, the analysis of the protein components in the edible part of green (GrK) and gold kiwi (GoK) fruit, at different ripening stages, has been undertaken. Methods: Samples of GrK and GoK fruits were collected every two weeks from July to November/December 2006. Kiwi fruits were from Italian plantations, coming from nearby areas. For each fruit sample a soluble protein fraction (SF) and a saline extract of the cell wall (CW) were obtained. The total protein content of the extracts was determined by means Biorad Protein Assay (Bio-Rad, CA). The protein pattern of the SF and CW fractions was analyzed by SDS-PAGE and the identification by N-terminal amino acid sequencing of the electroblotted components has been undertaken by means Applied Biosystems Procise 492 Automatic Sequencer (Applied Biosystems, CA). Results: Ten samples have been collected in the frame period for each species. The total protein content of both GrK and GoK fruit increased during the ripening process. The two kiwi species showed a different protein pattern upon SDS-PAGE. In GrK fruit, the allergens actinidin, thaumatin-like protein (TLP) and kiwellin were already observed in the fruit samples collected in July and their amount progressively increased. During the ripening process two new protein bands were observed at 20 and 17 kDa, respectively, becoming progressively more abundant. Samples of GoK fruit showed less variations than GrK. The TLP was observed in significant amount in all the GoK kiwi samples. High amount of a double protein band at 16 kDa, absent in earlier stages, was also observed. Preliminary comparative evaluation of SF and CW fractions showed differences in the localization of allergenic proteins in fruit tissues. Conclusion: Data so far obtained show that the protein pattern of GrK and GoK fruit is affected by both the ripening process and extraction methods. Several differences in whole proteins and allergenic contents have been recorded comparing the two kiwi species. Immunochemical studies are needed in order to verify differences in the allergenicity pattern.

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[68] - Alemán A, Sastre J, Quirce S, de las Heras M, Carnés J, Fernández-Caldas E, et al. Allergy to kiwi: A double-blind, placebo-controlled food challenge study in patients from a birch-free area. J Allergy Clin Immunol 2004;113:543-550

Background Allergy to kiwi fruit is being increasingly reported, but it has never been evaluated by means of a double-blind, placebo-controlled food challenge (DBPCFC) study. Objective : We sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitized patients from a birch-free area. Method s : Forty-three patients with allergy symptoms who were sensitized to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The pattern of allergen recognition was assessed by means of IgE immunoblotting. Sequence analysis of IgE-binding bands was performed by using Edman degradation. Result s : DBPCFCs were performed in 33 patients; 4 patients had experienced severe anaphylaxis, and 6 patients declined informed consent. DBPCFC results were positive in 23 patients and negative in 10 patients. The most frequent clinical manifestation was oral allergy syndrome. Twenty-one percent of the patients were not allergic to pollen. Forty-six percent of patients experienced systemic symptoms, and this happened with higher frequency in patients not allergic to pollen (100%). Twenty-eight percent of the patients were sensitized to latex. The IgE-binding bands in kiwi extract more frequently recognized by patient sera were those of 30, 24, 66, and 12 kd, and they could not be associated with any pattern of kiwi-induced allergic reactions. Conclusion : The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi. One of 5 patients with kiwi allergy was not allergic to pollen, and these patients had the highest risk of systemic reactions to kiwi.

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[69] - Palazzo P, Giangrieco I, Bernardi ML, Tamburrini M, Giani M, Ciardiello MA, et al. IgE reactivity to kiwellin (Act d 28kD), actinidin (Act d 1) and thaumatin-like protein (Act d 2) from green kiwi fruit in kiwi allergic patients tested by SDS-PAGE arrayed molecules. Allergy 2007;62(suppl. 83):341

Background Several allergens have been identified and characterized in green kiwi fruit, but little is know about patients' profiles in terms of IgE reactivity to single kiwi fruit extract components. We sough to describe kiwi sensitization profiles by testing three kiwi allergens and three more plant-derived food allergens. Methods Kiwellin (Act d 28kD), Actinidin (Act d 1) and thaumatin-like protein (Act d 2) were purified from green kiwi fruit by ionic exchange chromatography on DE-52 and SP-Sepharose. The last purification step was carried out by FPLC (Mono S HR 10/10 or Q HR 10/10, Amersham-Pharmacia, Sweden). Proteins were pure upon SDS-PAGE and characterized by N-terminal amino acid sequencing. The three purified proteins were mixed at 1:1:1 ratio for immunoblotting analysis. Molecules underwent to a reducing SDS-PAGE, were electroblotted onto PVDF membranes, and probed with sera from kiwi allergic subjects. Sera were selected on the basis of clinical reactivity. IgE testing was also performed on ISAC system microarrayed allergens (VBC, Austria) and reactivity to profilin, LTP and Bet v 1-like allergens was recorded. Results 30 patients have been selected. Sera of 14 (47%) patients showed IgE reactivity to Act d 1, 10 (33%) to Act d 28kD, and 9 (30%) recognized Act d 2. The combined reactivity to the three components was recorded in 2 subjects (6%). Reactivity to Act d 1 and Act d 2 was detected in 4 subjects (13%), whereas Act d 1 plus Act d 28kD, and Act d 2 plus Act d 28kD were positive in 4 and 3 subjects (13% and 10%), respectively. A single allergen was positive in 5 (Act d 1, 16%), 4 (Act d 28kD, 13%), and 2 (Act d 2, 6%) subjects. 8 subjects were negative to all three green kiwi molecules (26%). 22 serum of 30 patients were tested on ISAC system microarrayed allergens. 14 sera were positive to at least one kiwi allergen. 4 subjects were positive to Bet v 1-like molecules, 7 to LTP and 4 to Profilin. 2 serum showed co-reactivity to LTP and Profilins, one to Bet v 1-like and Profilins and one to Bet v 1-like and LTP. 3 of 8 kiwi allergen negative sera were scored positive to at least one of the three additional food allergens. Conclusion IgE are detected for all kiwi allergens and differ in their prevalence. Patients with mono-sensitization are rare compared to those who show co-reactivity to 2 or more green kiwi allergens. Kiwi allergic subjects may show an IgE reactivity to additional plant-related allergens.

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[70] - Merima B, Radauer C, Lebens A, Knulst A, Scheiner O, Breiteneder H. Purification and partial characterization of a 40 kDa allergen from kiwifruit (Actinidia delicosa). EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°505

Introduction: In recent years, there has been an increasing number of reports of allergic reactions to kiwifruit. Two major allergens of kiwifruit have been described so far: Act c 1, the thiol protease actinidin, and Act c 2, the thaumatin like protein. According to our previous study, most of kiwi allergic patients presented specific IgE to 40 kDa protein. Our aim was to identify the 40 kDa allergen and to compare frequencies of IgE binding to Act c 1, Act c 2 and the 40 kDa allergen in a group of Dutch kiwi allergic patients. Methods: The 40 kDa allergen was purified from a kiwi protein extract by cation exchange chromatography. The protein was identified by sequencing of the N-terminus and an internal fragment obtained after cleavage by cyanogen bromide. Fifteen patients were selected based on the presence of reported allergy to kiwifruit. All patients had positive skin prick tests (> 1) and positive radioallergosorbent tests (> 0.35) to kiwifruit. The patients showed moderate to severe symptoms such as OAS, rhinitis, urticaria, eczema, asthma, angioedema or abdominal pain. Sera were tested for IgE binding to purified actinidin (Act c 1), the thaumatin-like protein (Act c 2) and to the 40 kDa allergen by ELISA. Additionally, ELISA inhibitions and immunoblots were performed with selected sera. Results: We obtained 1.1 mg of purified 40 kDa protein from 2 kg of kiwifruit. The protein was identified as a glycoprotein with a Pfam profile PF04862 but with unknown function. In IgE ELISA 88% of the patients gave positive results to Act c 1, 15% to Act c 2, and 66% to the 40 kDa allergen. Carbohydrate analysis of the 40 kDa allergen showed the existence of glycans carrying fucosyl and xylosyl residues, structures previously shown to bind IgE. However, the overall range of inhibtion capacity by HRP was only 15-60%. Conclusions: The study shows that 40 kDa allergen in kiwifruit is a new food allergen.

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[71] - Möller M, Paschke A, Vieluf D, Kayma M, Vieths S, Steinhart H. Characterization of allergens in kiwi fruit and detection of cross-reactivities with allergens of birch pollen and related fruit allergens. Food Agric Immunol 1997;9:107-121

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[72] - Lidholm J. Allergens and allergy diagnosis in Europrevall : component resolved diagnosis of food allergy. Communication, 28th Congress of EAACI, Warszaw, June 6-10 2009

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[73] - Aleman A, Sastre J, Quirce S, Cuesta J, De Las Heras M, Carnés J, et al. Heterogeneity of kiwi allergy: recognition of allergens in kiwi-allergic patients. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°707

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[74] - Gavrovic-Jankulovic M, Circovic T, Vuckovic O, Atanaskovic-Markovic M, Petersen A, Gojgic G, et al. Isolation and biochemical characterization of a thaumatin-like kiwi allergen. J Allergy Clin Immunol 2002;110:805-810

BACKGROUND: Kiwi fruit allergy, as well as its association with hypersensitivity to other foods and to pollen, has been extensively reported in the last few years. Several IgE-binding components have been detected in kiwi extract, but only one 30- kd allergen has been isolated; it was identified as actinidin (Act c 1). Recently, we have reported a 24-kd kiwi protein to be a potential major allergen in a group of patients with oral allergy syndrome (OAS) . OBJECTIVE: The aim of this study was to purify and characterize the 24-kd kiwi allergen biochemically . METHODS: Seven polysensitized patients with OAS to kiwi were used in this study. The kiwi allergen was isolated by using a combination of gel permeation, ion exchange, and immobilized metal ion affinity chromatography. Its biochemical characterization included determination of its isoelectric point, molecular weight, N-terminal sequencing, concanavalin A -binding ability, digestibility in simulated gastric fluid, and antifungal activity. Western blotting, 2-dimensional PAGE immunoblotting, and skin prick tests were performed to characterize the isolated protein immunochemically . RESULTS: All 7 patients recognized the isolated 24-kd kiwi protein as an allergen. The isolated protein consisted of 2 isoforms with isoelectric points of 9.4 and 9.5 migrated as one protein band of 20 kd after SDS-PAGE under nonreducing conditions or at 24 kd under reducing conditions. The partial N-terminal sequence revealed that it is a thaumatin-like protein (TLP) with concanavalin A -binding ability. The protein showed antifungal activity toward Saccharomyces carlsbergensis, and Candida albicans. The protein was degraded by the simulated gastric fluid within 1 minute. Both isoforms bound IgE from a pool of sera in a 2-dimensional PAGE immunoblot. The TLP elicited positive skin prick test responses in 4 (80 %) of 5 patients with OAS . CONCLUSION: This study reported isolation and full characterization of a new kiwi allergen, TLP (isoelectric points of 9.4 and 9.5 and molecular weight of 24 kd), which belongs to the family of pathogenesis-related proteins. The isolated protein expressed antifungal activity toward S carlsbergensis and C albicans.

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[75] - Gavrovic-Jankulovic M, Cirkovic T, Burazer L, Vuckovic O, Jankov RM. IgE cross-reactivity between meadow fescue pollen and kiwi fruit in patients' sera with sensitivity to both extracts. J Investig Allergol Clin Immunol 2002;12:279-286

BACKGROUND: The presence of IgE reactivity to kiwi fruit and grass pollen allergens which could be caused by cross-reactivity has been detected in many patients with allergy. Proper identification of allergens as well as cross-reactive components is essential for understanding fruit- and pollen-associated hypersensitivity. METHODS: Using the sera from the polysensitized patients with specific IgE to grass pollen and kiwi fruit we tested reactivity to both allergen sources. IgE reactivity was exhibited in 8 serum samples by immunoblot. A serum pool formed by 8 individual sera was used for the investigation of IgE crossreactivity. SDS-PAGE immunoblot-inhibition assay was performed by preincubation of the sera with meadow fescue pollen, kiwi fruit extract, and isolated 24 kDa kiwi protein. To determine the allergens of kiwi fruit extract, we performed 2D PAGE immunoblot. In order to detect the crossreactive components between two allergen sources, a specific IgE for the 24 kDa kiwi allergen was purified. RESULTS: SDS-PAGE immunoblot meadow fescue pollen showed allergens ranging from 94 to 16 kDa, and kiwi fruit had 12 allergens ranging from 94 to 17 kDa. 2D-PAGE analysis revealed at least 15 spots in the kiwi extract and about 10 allergens. The most prominent allergen in 2D PAGE immunoblot was protein with 24 kDa and pI 9.4-9.5. Using an affinity-purified specific IgE we found that the 24 kDa kiwi allergen shared IgE-reactive epitopes with the meadow fescue group 4 and allergen about 36 kDa. Crossreactivity between isolated 24 kDa kiwi allergen and Fes p 4 was confirmed by anti-grass group 4 moab 2D8. CONCLUSION: Our findings showed that fescue meadow pollen cross-sensitize to kiwi fruits. A 24 kDa kiwi glycoprotein represent potential major allergen, which share common epitopes with Fes p 4 and 36 kDa meadow fescue allergen.

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[76] - Smole U, Bublin M, Radauer C, Ebner C, Scheiner O, Breiteneder H. Allergenic Fruit TLPs Possess Different Degrees of IgE Crossreactivity. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°195

RATIONALE: Thaumatin-like proteins (TLPs) are important allergens of a number of plant foods. We aimed to examine the IgE cross-reactivity between the apple TLP Mal d 2 and TLPs from kiwi (Act c2), grape (VvTLP), and cherry (Pru av 2) METHODS: Amino acid sequences of apple, cherry, kiwi, and grape TLPs were aligned. The TLPs were purified. IgE ELISA and ELISA inhibitions were carried out using sera from 46 apple allergic patients. The relevance of N-glycosylation for IgE-binding was investigated by inhibition with the oligosaccharide BSA-MUXF RESULTS: Sequence alignments showed 33% identity of Mal d 2 to Act c 2, 35% to VvTLP, and 69 % to Pru av 2. Mal d 2 was IgE reactive with a prevalence of 39% (18/46). Nine of these 18 patients recognized N-glycan epitopes on Mal d 2, the 9 remaining showed binding to protein epitopes Two of the 9 protein-specific sera had IgE to all four TLPs, 6/9 to Mal d 2, Act c 2, and Pru av 2, and one to Mal d 2 and Pru av 2. In addition, Mal d 2 and Pru av 2 showed a high extent of cross-reactivity CONCLUSIONS: The glycoallergen Mal d 2 shares cross-reactive epitopes with other TLPs that encompass the Mal d 2 polypeptide and its N-glycan structure. Inhibition of IgE-Mal d 2 interactions with Pru av 2 demonstrated that many of the epitopes of Mal d 2 were cross-reactive with epitopes on Pru av 2 This work was supported by the Austrian Science Fund grant SFBF01802 Funding: Austrian Science Fund grant SFB-F01802

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[77] - Zuidmeer L, Bolhaar S, Knulst A, van Leeuwen WA, Aalbers M, Krebitz M, et al. Severe symptoms caused by thaumatin-like proteins in foods: a result of primary sensitization to birch pollen ? EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°196

Background: It has almost become a dogma that birch pollen-related food allergies are always mild, because Bet v 1 and its homologues from foods as well as profilins are extremely heat and protease-sensitive. Protease-resistant birch pollen allergens have so far not been reported. Methods: A panel of 477 sera from The Netherlands with >2.0 IU/ml of specific IgE against apple were analysed by RAST for specific IgE against 4 apple allergens: nMal d 1, rMal d 2 (thaumatin-like protein [TLP]), nMal d 3 (LTP) and nMal d 4 (profilin). rMal d 2-positive sera (>0.7 IU/ml) were further analysed by RAST inhibition with (protease-digested) birch pollen extract. Serum from a patient with severe food-related anaphylaxis was studied in more detail by RAST(-inhibition) and immunoblot(- inhibition). Results: As expected most sera contained significant titers of specific IgE against Mal d 1 (70% with > 1.0 IU/ml). Frequency of recognition of Mal d 2, 3 and 4 was much lower (23%, 9% and 19% respectively). For 55 sera with a clear positive RAST for rMal d 2 IgE-binding to this allergen was inhibited with birch pollen extract. On average the Mald 2 RAST was inhibited by 75%, indicating that birch pollen extract contains a crossreactive structure with food TLP. A similar inhibition was observed with protease-digested birch pollen extract, suggesting that the putative TLP-related allergen from birch pollen is extremely protease resistant. This observation fits with the high degree of stability observed for rMal d 2 in simulated gastric fluid. Considering this stability, it is not surprising that among the patients with TLP-specific IgE antibodies, severe food allergic symptoms were observed. One of these patients was further analysed in depth. This patient experienced anaphylactic shock upon consumption of hazelnut liquor, raspberry and several other fruits on different occasions. IgE binding to raspberry and several other foods could be inhibited by birch pollen and by rMal d 2 in both RAST- and immunoblot-inhibition, indicating that the severe food allergy of this patient is caused by TLP-like allergens and that sensitisation is most likely birch pollen driven. Conclusion: Birch pollen contain an allergen that is crossreactive with food TLPs and is most likely responsible for sensitisation. In contrast to most Bet v 1-related allergens, food TLPs can cause severe food allergy.

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[78] - Gavrovic-Jankulovic M, Petersen A, Milovanovic K, Cirkovic Velickovic T, Stojanovic M, Inic-Kanada A, et al. Characterisation of a novel acidic thaumatin-like allergen from green and gold kiwi fruit. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1503

Background: A considerable number of identified plant-derived allergens belong to pathogen-related (PR) proteins. The PR-5 family comprises of acidic and basic isoforms of thaumatin-like proteins (TLPs), localized in different cell compartments: the apoplast and the vacuole. Using monoclonal antibodies raised against the basic kiwi TLP (Act c 2), we identified a novel acidic thaumatin-like kiwi homologue in gold and green kiwi fruit. The aim of this work was to characterize this novel protein and to examine its allergenic properties. Methods: A three-step purification protocol was developed for the isolation of the novel kiwi protein from gold as well as green kiwi fruit extract. Purification to homogeneity was accomplished by fractionation of the proteins from green and gold kiwi by HPLC (AKTApurified) either by anionexchange/ reverse-phase (MiniQ/uRPC) or cation-exchange/reverse phase (MiniS/uRPC) chromatography, respectively. The isolated proteins were identified by two monoclonal antibodies raised against the Act c 2 in dot blot and Western blot analysis. The N-terminal amino acid sequences were determined by automated Edman degradation. Five patients with positive history of kiwi allergy entered the study for in vitro and in vivo analysis. IgE binding of novel kiwi protein was assessed by Western blot. Evaluation of biological allergenic potency of acidic kiwi TLPs was performed by skin prick testing. Results: The novel kiwi protein identified in gold as well as green kiwi fruit is an acidic TLP isoform according to 2D PAGE, with molecular mass of about 20 kD. The N-terminal region of acidic TLPs from both kiwi cultivars did not reveal any difference from the basic TLPs (ATFNIINNCPFTV). The presence of IgE-binding epitopes was confirmed in Western blot. Two tested patients showed a positive skin prick test to the acidic TLP. Conclusion: The novel kiwi allergen has been isolated and characterized as the acidic thaumatin-like protein isoform. Kiwi is the first fruit in which the acidic as well as the basic TLP homologue has been found. Due to the sequence identity in the N-terminal part, structural difference between two proteins should at least be confined to the C-terminal region, which is important for correct protein targeting in a particular plant cell compartment. Mapping of the IgE-binding epitopes should reveal similarities and/or differences reflecting the structural features contributing to the allergenicity of the kiwi TLP isoforms.

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[79] - Radauer C, Bublin M, Scheiner O, Breiteneder H. Molecular characterization of common and cultivar-specific kiwifruit allergens in the varieties Hayward and Hort16A. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°986

Background: Kiwifruits (Actinidia spp.) are commonly consumed in Europe since the 1980s. Since the first report of kiwifruit allergy in 1981, the prevalence of this new food allergy has apparently increased. The world market for kiwifruits is dominated by a single variety, A. deliciosa cv. Hayward. In 1998, a novel, yellow-fleshed variety, A. chinensis cv. Hort16A, was introduced. To date, there are only limited data on kiwifruit allergens. In particular, no studies dealing with Hort16A and other novel varieties have been published. The aim of this study was the identification of allergens in Hayward and Hort16A kiwifruit extracts by protein sequencing. Methods: Protein extracts of Hayward and Hort16A kiwifruits were separated by anion exchange chromatography and proteins in the chromatographic fractions were probed by IgE immunoblotting using kiwi-allergic patients' sera. IgE binding bands were excised from the blots and sequenced. Results: Total extracts as well as chromatographic fractions of the two kiwifruit varieties showed marked differences in their SDS-PAGE band patterns and in their compositions of IgE binding proteins. Allergens with molecular masses of 12, 22, 32 and 43 kDa were detected in both extracts. The 12 kDa allergens had identical N-terminal sequences similar to plant cystatins, cysteine protease inhibitors being parts of the plant defense system. The 22 kDa allergens were identified as thaumatin-like proteins; the 32 kDa proteins contained an N-terminal hevein-like domain. Members of both families have previously been described as allergens. The 43 kDa protein, which was found in much higher amounts in the Hort16A extract, was N-terminally blocked. The major kiwifruit allergen actinidin was detected exclusively in the Hayward extract. The Hort16A extract contained a different allergen of similar molecular mass whose N-terminus was blocked. Conclusions: The kiwifruit varieties Hayward and Hort16A differed in their allergen compositions qualitatively as well as quantitatively. This may have consequences for their risk potentials for kiwi-allergic patients and for their cross-reactivity to pollen and latex.

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[80] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[81] - Popovic M, Milovanovic M, Burazer L, Vuckovic O, Knulst A, Hoffmann-Sommergruber K et al. Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy. Allergy 2009;64(Suppl. 90):270-271

Background: Kiwifruit has become a frequent cause of fruit allergy in the recent years. The molecular basis of type I hypersensitivity to kiwifruit is attributed to ten IUIS allergens with Act d 1, Act d 2 and Act d 5 as the most relevant. However, other nominated allergens wait for evaluation of their clinical relevance. Act d 4 is one of three kiwi cystatin isoforms described as IgE binding component in green kiwifruit extract. Aim: The aim of this work was to biochemically characterize cystein protease inhibitor Act d 4 from green kiwifruit, and to explore its allergenic potential for in vivo and in vitro diagnosis of kiwifruit allergy. Methods: Act d 4 was purified by affinity and reversed phase chromatography. The primary structure was analyzed by Edman degradation and mass fingerprint. Immunological reactivity was analyzed by 2D PAGE immunoblot with rabbit antisera. IgE reactivity was tested in dot blot assay, and biological activity was assessed by basophil activation assay and skin prick test. Results: Fourteen amino acids determined as VAAGGXRPIESLNSA confirmed the N-terminal sequence of mature kiwifruit cystatin. Mass fingerprint confirmed the identity of isolated Act d 4 with more than 90% of its primary structure. Act d 4 reveals molecular masses of 10902.5 and 11055.2 Da and pI of 6.9. Purified Act d 4 preserved biological function with high inhibitory potential against papain (IC50 = 2.47 nM). IgE reactivity was detected in dot blot analysis with six kiwifruit patients sera. Positive skin prick reactivity with Act d 4 was induced in three kiwifruit allergic patients, as well as upregulation of CD63 and CD203c molecules on the basophil membrane. Conclusion: As activator of effectors cells in type I hypersensitivity Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.

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[82] - Rassam M, Laing WA. Purification and characterization of phytocystatins from kiwifruit cortex and seeds. Phytochemistry 2004;65:19-30

Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwifruit after inactivation of the abundant cortex cysteine proteinase actinidain. One major (KCPI1) and four minor cystatins were identified from Actinidia deliciosa ripe mature kiwifruit cortex as well as a seed KCPI from A. chinensis. The predominant cortex cystatin, KCPI1, inhibited clan CA, family C1 (papain family) cysteine proteinases (papain, chymopapain, bromelain, ficin, human cathepsins B, H and L, actinidain and the house dust mite endopeptidase 1), while cysteine proteinases belonging to other families, [clostripain (C11), streptopain (C10) and calpain (C2)] were not inhibited. Inhibition constants (K(I)) ranged between 0.001 nM for cathepsin L and 0.98 nM for endopeptidase 1. The K(I) (14 nM) for KCPI1 inhibiting actinidain is at least 2 orders of magnitude higher than for other plant proteinases measured. The cortex KCPI1 and a seed KCPI purified from seeds had the same N-terminal sequence (VAAGGWRPIESL NSAEVQDV). BLAST-matching the peptide sequence against an in-house generated Actinidia EST database, identified 81 cDNAs that exactly matched the measured KCPI1 peptide sequence. Peptide sequences of two other cortex KCPIs each exactly matched a predicted peptide sequence of a cDNA from kiwifruit. The predicted peptide sequence of KCPI1 of 116 amino acids encodes a signal peptide and does not contain cysteine. Without the signal peptide (mature protein), KCPI1 has a molecular mass of approximately 11 kDa, possesses the consensus sequence characteristic for the phytocystatins and shows the highest homology to a cystatin from Citrusxparadisi (52% identity). This is the first report of phytocystatins from the Ericales

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[83] - Bublin M, Radauer C, Hafner C, Ebner C, Mari A, Wagner S, et al. Act d 5 (kiwellin) and Act d 4 (cystatin), two new important kiwifruit allergens. Allergy 2007;62(suppl. 83):342

Introduction: Today, kiwifruit (Actinidia deliciosa cv. Hayward) is one of the prevailing sources of plant food allergy in Europe. Symptoms of kiwifruit allergy encompass the whole spectrum ranging from oral allergy syndrome to severe anaphylaxis.Two major allergens of kiwifruit have been described so far: actinidin (Act c 1) and the thaumatin-like protein (Act c 2). Kiwellin and cystatin have been identified as IgE binding proteins in kiwifruit protein extract. Kiwellin, a 26 kDa protein, is a member of a new class of proteins with unknown biological function. Cystatin, an 11 kDa protein, is a cysteine protease inhibitor assumed to be involved in plant defence against pathogen attack. However, no detailed studies about the relevance of the two proteins using a large population of patients have been reported so far. Methods: Kiwellin and cystatin were purified from protein extract of commercially available green kiwifruits by combinations of different chromatographic methods. The purity of proteins was checked by SDS-PAGE and IgE immunoblotting. Both proteins were subjected to N-terminal amino acid sequence analysis. For the investigations of allergenic properties of kiwellin and cystatin, the sera of 50 patients were selected based on the presence of reported allergy to kiwifruit. IgE binding to purified allergens was tested by ELISA. Results: We obtained 2.5 mg of purified kiwellin and 1.8 mg of cystatin from 0.5 kg of kiwifruit. Of 12 latex allergic patients with kiwifruit allergy, 9 were sensitized to kiwellin, and 4 to cystatin. Of 38 sera from patients with multiple pollen and kiwifruit allergy, 25 contained IgE specific to kiwellin and 23 to cystatin. Conclusion: The allergens have been assigned the official designations Act d 4 (cystatin) and Act d 5 (kiwellin) by the I.U.I.S. allergen nomenclature sub-committee. Taking into account that the frequencies of sensitization to kiwellin and cystatin were 68% and 54%, respectively, we conclude that both are major allergens in kiwifruit.

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[84] - Lidholm J. Allergens and allergy diagnosis in Europrevall : component resolved diagnosis of food allergy. Communication, 28th Congress of EAACI, Warszaw, June 6-10 2009

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[85] - Tamburrini M, Cerasuolo I, Carratore V, Stanziola AA, Zofra S, Romano L, et al. Kiwellin, a novel protein from kiwi fruit. Purification, biochemical characterization and identification as an allergen. Protein J 2005;24:423-429

Kiwellin is a novel protein of 28 kDa isolated from kiwi (Actinidia chinensis) fruit. It is one of the three most abundant proteins present in the edible part of this fruit. Kiwellin has been purified by ion exchange chromatography. Its N-terminal amino acid sequence revealed high identity with that previously reported for a 28 kDa protein described as one of the most important kiwi allergens. This observation prompted us to fully characterize this protein. The complete primary structure, elucidated by direct sequencing, indicated that kiwellin is a cysteine-rich protein. Serological tests and Western Blotting analysis showed that kiwellin is specifically recognized by IgE of patients allergic to kiwi fruit

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[86] - Giangrieco I, Tuppo L, Palazzo P, Camardella L, Bernardi ML, Scala E, et al. Analysis of green and gold kiwi fruit protein pattern as a function of the ripening stage. Allergy 2007;62(suppl. 83):341

Background: Allergy to kiwi fruit has been the object of extensive investigation in the last years. Probably due to the experimental procedures used and/or to the tissue characteristics, the extracts can be variable both in the number and the amount of their components. In order to verify if the ripening process of the fruit affects the allergen pattern, the analysis of the protein components in the edible part of green (GrK) and gold kiwi (GoK) fruit, at different ripening stages, has been undertaken. Methods: Samples of GrK and GoK fruits were collected every two weeks from July to November/December 2006. Kiwi fruits were from Italian plantations, coming from nearby areas. For each fruit sample a soluble protein fraction (SF) and a saline extract of the cell wall (CW) were obtained. The total protein content of the extracts was determined by means Biorad Protein Assay (Bio-Rad, CA). The protein pattern of the SF and CW fractions was analyzed by SDS-PAGE and the identification by N-terminal amino acid sequencing of the electroblotted components has been undertaken by means Applied Biosystems Procise 492 Automatic Sequencer (Applied Biosystems, CA). Results: Ten samples have been collected in the frame period for each species. The total protein content of both GrK and GoK fruit increased during the ripening process. The two kiwi species showed a different protein pattern upon SDS-PAGE. In GrK fruit, the allergens actinidin, thaumatin-like protein (TLP) and kiwellin were already observed in the fruit samples collected in July and their amount progressively increased. During the ripening process two new protein bands were observed at 20 and 17 kDa, respectively, becoming progressively more abundant. Samples of GoK fruit showed less variations than GrK. The TLP was observed in significant amount in all the GoK kiwi samples. High amount of a double protein band at 16 kDa, absent in earlier stages, was also observed. Preliminary comparative evaluation of SF and CW fractions showed differences in the localization of allergenic proteins in fruit tissues. Conclusion: Data so far obtained show that the protein pattern of GrK and GoK fruit is affected by both the ripening process and extraction methods. Several differences in whole proteins and allergenic contents have been recorded comparing the two kiwi species. Immunochemical studies are needed in order to verify differences in the allergenicity pattern.

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[87] - Tuppo L, Giangrieco I, Palazzo P, Bernardi ML, Scala E, Carratore V, et al. Kiwellin, a Modular Protein from Green and Gold Kiwi Fruits: Evidence of in Vivo and in Vitro Processing and IgE Binding. J Agric Food Chem 2008;56:3812-3817

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

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[88] - Tuppo L, Giangrieco I, Palazzo P, Bernardi ML, Scala E, Carratore V, et al. Kiwellin, a Modular Protein from Green and Gold Kiwi Fruits: Evidence of in Vivo and in Vitro Processing and IgE Binding. J Agric Food Chem 2008;56:3812-3817

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

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[89] - Ciardiello MA, Giangrieco I, Tuppo L, Tamburrini M, Buccheri M, Palazzo P et al. Influence of the Natural Ripening Stage, Cold Storage, and Ethylene Treatment on the Protein and IgE-Binding Profiles of Green and Gold Kiwi Fruit Extracts. J Agric Food Chem 2009;57:1565-1571

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

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[90] - Bublin M, Radauer C, Hafner C, Ebner C, Mari A, Wagner S, et al. Act d 5 (kiwellin) and Act d 4 (cystatin), two new important kiwifruit allergens. Allergy 2007;62(suppl. 83):342

Introduction: Today, kiwifruit (Actinidia deliciosa cv. Hayward) is one of the prevailing sources of plant food allergy in Europe. Symptoms of kiwifruit allergy encompass the whole spectrum ranging from oral allergy syndrome to severe anaphylaxis.Two major allergens of kiwifruit have been described so far: actinidin (Act c 1) and the thaumatin-like protein (Act c 2). Kiwellin and cystatin have been identified as IgE binding proteins in kiwifruit protein extract. Kiwellin, a 26 kDa protein, is a member of a new class of proteins with unknown biological function. Cystatin, an 11 kDa protein, is a cysteine protease inhibitor assumed to be involved in plant defence against pathogen attack. However, no detailed studies about the relevance of the two proteins using a large population of patients have been reported so far. Methods: Kiwellin and cystatin were purified from protein extract of commercially available green kiwifruits by combinations of different chromatographic methods. The purity of proteins was checked by SDS-PAGE and IgE immunoblotting. Both proteins were subjected to N-terminal amino acid sequence analysis. For the investigations of allergenic properties of kiwellin and cystatin, the sera of 50 patients were selected based on the presence of reported allergy to kiwifruit. IgE binding to purified allergens was tested by ELISA. Results: We obtained 2.5 mg of purified kiwellin and 1.8 mg of cystatin from 0.5 kg of kiwifruit. Of 12 latex allergic patients with kiwifruit allergy, 9 were sensitized to kiwellin, and 4 to cystatin. Of 38 sera from patients with multiple pollen and kiwifruit allergy, 25 contained IgE specific to kiwellin and 23 to cystatin. Conclusion: The allergens have been assigned the official designations Act d 4 (cystatin) and Act d 5 (kiwellin) by the I.U.I.S. allergen nomenclature sub-committee. Taking into account that the frequencies of sensitization to kiwellin and cystatin were 68% and 54%, respectively, we conclude that both are major allergens in kiwifruit.

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[91] - Palazzo P, Giangrieco I, Bernardi ML, Tamburrini M, Giani M, Ciardiello MA, et al. IgE reactivity to kiwellin (Act d 28kD), actinidin (Act d 1) and thaumatin-like protein (Act d 2) from green kiwi fruit in kiwi allergic patients tested by SDS-PAGE arrayed molecules. Allergy 2007;62(suppl. 83):341

Background Several allergens have been identified and characterized in green kiwi fruit, but little is know about patients' profiles in terms of IgE reactivity to single kiwi fruit extract components. We sough to describe kiwi sensitization profiles by testing three kiwi allergens and three more plant-derived food allergens. Methods Kiwellin (Act d 28kD), Actinidin (Act d 1) and thaumatin-like protein (Act d 2) were purified from green kiwi fruit by ionic exchange chromatography on DE-52 and SP-Sepharose. The last purification step was carried out by FPLC (Mono S HR 10/10 or Q HR 10/10, Amersham-Pharmacia, Sweden). Proteins were pure upon SDS-PAGE and characterized by N-terminal amino acid sequencing. The three purified proteins were mixed at 1:1:1 ratio for immunoblotting analysis. Molecules underwent to a reducing SDS-PAGE, were electroblotted onto PVDF membranes, and probed with sera from kiwi allergic subjects. Sera were selected on the basis of clinical reactivity. IgE testing was also performed on ISAC system microarrayed allergens (VBC, Austria) and reactivity to profilin, LTP and Bet v 1-like allergens was recorded. Results 30 patients have been selected. Sera of 14 (47%) patients showed IgE reactivity to Act d 1, 10 (33%) to Act d 28kD, and 9 (30%) recognized Act d 2. The combined reactivity to the three components was recorded in 2 subjects (6%). Reactivity to Act d 1 and Act d 2 was detected in 4 subjects (13%), whereas Act d 1 plus Act d 28kD, and Act d 2 plus Act d 28kD were positive in 4 and 3 subjects (13% and 10%), respectively. A single allergen was positive in 5 (Act d 1, 16%), 4 (Act d 28kD, 13%), and 2 (Act d 2, 6%) subjects. 8 subjects were negative to all three green kiwi molecules (26%). 22 serum of 30 patients were tested on ISAC system microarrayed allergens. 14 sera were positive to at least one kiwi allergen. 4 subjects were positive to Bet v 1-like molecules, 7 to LTP and 4 to Profilin. 2 serum showed co-reactivity to LTP and Profilins, one to Bet v 1-like and Profilins and one to Bet v 1-like and LTP. 3 of 8 kiwi allergen negative sera were scored positive to at least one of the three additional food allergens. Conclusion IgE are detected for all kiwi allergens and differ in their prevalence. Patients with mono-sensitization are rare compared to those who show co-reactivity to 2 or more green kiwi allergens. Kiwi allergic subjects may show an IgE reactivity to additional plant-related allergens.

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[92] - Lidholm J. Allergens and allergy diagnosis in Europrevall : component resolved diagnosis of food allergy. Communication, 28th Congress of EAACI, Warszaw, June 6-10 2009

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[93] - Ciardiello MA, Tamburrini M, Tuppo L, Carratore V, Giovane A, Mattei B, et al. Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor. J Agric Food Chem 2004;52:7700-7703

Pectin methylesterase was purified from kiwi (Actinidia chinensis) and kaki fruit (Diospyros kaki). The pH values of the fruit homogenates were 3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell wall and has a neutral-alkaline pI, whereas the kaki enzyme is localized in the soluble fraction and has a neutral-acidic pI. The molecular weights of the kiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymes showed a similar salt and pH dependence of activity, and a different pH dependence of the inhibition by the kiwi proteinaceous inhibitor.

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[94] - Bernardi M, Palazzo P, Tuppo L, Giangrieco I, Camardella L, Tamburrini M, et al. Pectin methylesterase (Act d 7) and pectin methylesterase inhibitor (Act d 6): two new kiwi fruit allergens. Allergy 2008;63(suppl. 88):585-586

Background: Several protein components of kiwi fruit extracts have been reported as IgE-binding bands. However, despite the remarkable number of published studies, few kiwi fruit allergens have been identified, isolated and characterized. Pectin methylesterase inhibitor (PMEI) is a 16 kDa protein isolated in green kiwi fruit. It defines a protein family including invertase inhibitors and the allergen Pla a 1. In kiwi fruit, the PMEI target is a glycosylated pectin methylesterase (PME) of 50 kDa. Its amino acid sequence has been elucidated by direct protein sequencing and the polysaccharide structure has been obtained by mass spectrometry experiments. In order to ascertain the possible allergenicity of kiwi fruit PME and PMEI, their IgE binding capacity has been tested. Methods: PME and PMEI were purified from green kiwi fruit (Actinidia deliciosa, GrK). Proteins were pure upon SDS-PAGE analysis and identified by N-terminal amino acid sequencing. They were stored at a concentration of 1 mg/mL, in 1 mM PBS buffer, pH 7.4. To define their IgE reactivity, molecules (1 mg of each protein) underwent SDS-PAGE under reducing conditions and electroblotting onto PVDF membranes. Transferred proteins were probed with sera from GrK allergic subjects. Sera were selected on the basis of clinical criteria of a reliable patient-reported allergic reaction after ingestion of GrK. The same allergen preparations were tested for detecting direct IgE binding using the proteomic microarray system (ISAC, VBC-Genomics, Vienna, Austria) and by skin prick testing (ST) at 500 mg/mL. Results: Fifty patients have been selected on the basis of established clinical criteria. Immunoblotting experiments showed that sera of 16 (32%) and 36 (72%) patients recognized PME and PMEI, respectively. The combined reactivity to the two proteins was recorded in 13 subjects. Following IgE testing on ISAC, sera of 9 patients showed IgE binding to PME, whereas all the 50 sera did not detect PMEI. ST was carried out on 13 patients. Only PME produced a positive reaction. Nevertheless, PMEI showed a positive ST in two subjects of a different study population. Conclusion: PME (Act d 7) and PMEI (Act d 6) are two new allergens identified in GrK both capable to bind IgE either on immunoblot or on a microarray system. Few subjects compared to in vitro testing reacted on ST. In the case of PME this could be ascribed to the glycan side chain, known to be incapable to exert any in vivo reactivity.

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[95] - Ciardiello MA, D'Avino R, Amoresano A, Tuppo L, Carpentieri A, Carratore V, et al. The peculiar structural features of kiwi fruit pectin methylesterase: Amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor. Proteins 2008;71:195-206

Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS-PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N-glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel-filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME.

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[96] - Oberhuber C, Bulley SM, Ballmer-Weber BK, Bublin M, Gaier S, DeWitt AM et al. Characterization of Bet v 1-related allergens from kiwifruit relevant for patients with combined kiwifruit and birch pollen allergy. Mol Nutr Food Res 2008;52(suppl. 2):S230-S240

Allergy to kiwifruit appears to have become more common in Europe and elsewhere during the past several years. Seven allergens have been identified from kiwifruit so far, with actinidin, kiwellin and the thaumatin-like protein as the most relevant ones. In contrast to other fruits, no Bet v 1 homologues were characterized from kiwifruit so far. We cloned, purified, and characterized recombinant Bet v 1-homologous allergens from green (Actinidia deliciosa, Act d 8) and gold (Actinidia chinensis, Act c 8) kiwifruit, and confirmed the presence of its natural counterpart by inhibition assays. Well-characterized recombinant Act d 8 and Act c 8 were recognized by birch pollen/kiwifruit (confirmed by double-blind placebo-controlled food challenge) allergic patients in IgE immunoblots and ELISA experiments. The present data point out that Bet v 1 homologues are allergens in kiwifruit and of relevance for patients sensitized to tree pollen and kiwifruit, and might have been neglected so far due to low abundance in the conventional extracts used for diagnosis.

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[97] - Oberhuber C, Bulley SM, Ballmer-Weber BK, Bublin M, Gaier S, DeWitt AM et al. Characterization of Bet v 1-related allergens from kiwifruit relevant for patients with combined kiwifruit and birch pollen allergy. Mol Nutr Food Res 2008;52(suppl. 2):S230-S240

Allergy to kiwifruit appears to have become more common in Europe and elsewhere during the past several years. Seven allergens have been identified from kiwifruit so far, with actinidin, kiwellin and the thaumatin-like protein as the most relevant ones. In contrast to other fruits, no Bet v 1 homologues were characterized from kiwifruit so far. We cloned, purified, and characterized recombinant Bet v 1-homologous allergens from green (Actinidia deliciosa, Act d 8) and gold (Actinidia chinensis, Act c 8) kiwifruit, and confirmed the presence of its natural counterpart by inhibition assays. Well-characterized recombinant Act d 8 and Act c 8 were recognized by birch pollen/kiwifruit (confirmed by double-blind placebo-controlled food challenge) allergic patients in IgE immunoblots and ELISA experiments. The present data point out that Bet v 1 homologues are allergens in kiwifruit and of relevance for patients sensitized to tree pollen and kiwifruit, and might have been neglected so far due to low abundance in the conventional extracts used for diagnosis.

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[98] - Wagner B, Buck D, Hafner C, Sowka S, Niggemann B, Scheiner O, et al. Hev b 7 is a Hevea brasiliensis protein associated with latex allergy in children with spina bifida. J Allergy Clin Immunol 2001;108:621-627

In addition to their disease-associated handicaps, patients with spina bifida (SB) are at high risk of developing latex allergy. Individuals with SB represent a special group of latex-allergic patients, inasmuch as their IgE-binding patterns differ from those of other populations of latex-allergic individuals. Two allergens strongly associated with latex allergy in patients with SB-Hev b 1 and Hev b 3-have already been identified. OBJECTIVE: We intended to identify a predominant IgE-binding band-in addition to Hev b 1 and 3-at 43 kDa in a study population of 38 latex-allergic (IgE antibodies to latex and symptoms on provocation with latex gloves) and 15 latex sensitized (IgE antibodies to latex but no symptoms on provocation) children with SB (mean age, 12.3 years) and to determine its frequency of recognition. METHODS: Sera of latex-sensitized or latex-allergic patients with SB were tested on latex C extract containing natural Hev b 1, Hev b 3, and Hev b 7 and with the recombinant 43-kDa Hev b 7 in immunoblot and inhibition studies. RESULTS: Natural Hev b 1 was recognized by 82% and natural Hev b 3 by 79% of the latex-allergic children with SB. In addition to some other proteins, 15 (39.5%) of 38 latex-allergic and 2 (13%) of 5 latex-sensitized children with SB revealed IgE binding to a 43-kDa band in the latex protein extract. We identified this 43-kDa IgE-binding band as natural Hev b 7 by immunoblotting and inhibition experiments using recombinant Hev b 7. CONCLUSION: From these data, we conclude that Hev b 7, the patatinlike Hevea latex protein, is the third SB-associated latex allergen. Future immunotherapy for latex-allergic individuals with SB will have to include Hev b 7 in addition to Hev b 1 and Hev b 3.

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[99] - Beezhold DH, Sussman GL, Liss GM, Chang NS. Latex allergy can induce clinical reactions to specific foods. Clin Exp Allergy 1996;26:416-422

The purpose of this study was to investigate crossreactivity between latex and foods, to identify crossreacting IgE binding proteins, and to assess the clinical significance. METHODS: Forty-seven latex allergic patients and 46 non-latex allergic patient controls were studied. Allergen sensitization was determined by skin-prick testing (SPT) and allergenic proteins were identified by immunoblot reactivity and amino acid sequence analysis. RESULTS: Immunological reactivity to foods was found to be common, occurring in 33 latex-allergic individuals but in only seven controls (P < 0.000001); 100 of 376 (27%) food skin-prick tests were positive in the latex-allergic subjects. Twenty-seven out of 100 positive food SPTs were associated with clinical symptoms. Seventeen patients manifested a clinical allergy to at least one food including 11 with anaphylaxis, and 14 with local sensitivity reactions. Positive food skin tests occurred most frequently with avocado (53%), potato (40%), banana (38%), tomato (28%), chestnut (28%), and kiwi (17%). Latex-allergic patients (23%) recognize a protein that had sequence homology to a broad class of plant proteins known as patatins. Crossreactivity between latex and several potato proteins was observed by immunoblot inhibition analysis. CONCLUSIONS: Sensitization to latex has extensive crossreactivity with certain foods and leads to clinical allergic reactions. Potatoes and tomatoes are newly reported cross-reacting foods. Plant proteins with structural homology to latex proteins may predispose to food allergy.

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[100] - Levy DA, Mounedji N, Noirot C, Leynadier F. Allergic sensitization and clinical reactions to latex, food and pollen in adult patients. Clin Exp Allergy 2000;30:270-275

Many latex-allergic patients are sensitized to one or more foods. Patients allergic to tree and/or grass pollens are also often sensitized to plant-derived foods. Atopy, defined in most studies as sensitivity to an aeroallergen, is a risk factor for latex allergy. The relative importance of pollen sensitivity, a sign of atopy, as a risk factor for food allergy in latex-allergic patients has not, however, been examined. OBJECTIVE: To investigate the relationship between pollen sensitivity and sensitivity to food in latex-allergic patients. METHODS: Forty-four latex-allergic patients (Groups 1 and 2), 24 of whom were also allergic to tree and/or grass pollen (Group 1) and 25 pollinosis patients who were not allergic to latex (Group 3) were studied. We obtained a history of reactions to food and skin tested them with 12 fresh-frozen fruits. RESULTS: All 12 foods induced a skin test reaction in at least one patient in each of the three Groups. There were, however, twice as many positive skin test reactions to food in patients with pollinosis, whether or not they were allergic to latex, as there were in patients allergic to latex but not to pollen. Latex-allergic patients were most likely to have a positive skin test and a history of a reaction to avocado or banana whereas patients with pollinosis only were most likely to have a positive skin test and a history of a reaction to apple, peach or celery. CONCLUSIONS: These results suggest that concomitant allergy to pollen is an important risk factor in determining which plant-derived foods sensitize latex-allergic patients.

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[101] - Isola S, Ricciardi L, Saitta S, Fedele R, Mazzeo L, Fogliani O, et al. Latex Allergy and Fruit Cross-Reaction in Subjects Who Are Nonatopic. Allergy Asthma Proc 2003;24:193-197

Abstract: Since the first case reported in 1927, latex allergy has attracted the attention of allergists including its capacity to cross-react with fruits. To evaluate the frequency of sensitivity to some fruit allergens shown to cross-react with latex, we evaluated 82 patients (43 men and 39 women, aged between 18 and 45 years) with latex allergy. All patients underwent skin tests with various fruit extracts that potentially cross-react with latex. Only patients with negative prick tests successively underwent prick-by-prick tests with fresh fruits. Thirty-nine of 82 patients (47.5%) were found to have positive skin tests. Prick tests with fruit extracts were positive in 28 patients (kiwi, 21 patients; banana, 17 patients; avocado, 8 patients; and papaya, 3 patients), and the prick-by-prick test had positive results in 11 patients (kiwi, 7 patients; banana, 4 patients; and avocado, 3 patients). In our experience patients with latex allergy are at a high risk of sensitization to some fruits and they often develop allergic reactions, even severe ones, after eating them; for this reason fruit sensitization should be taken into consideration when investigating patients allergic to natural rubber latex.

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[102] - Gaspar A, Raulf-Heimsoth M, Pires G, Rihs HP, Yeang HY, Matos V, et al. Latex allergen sensitization patterns in different risk groups and latex-fruit syndrome patients from Portugal. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°1188

Background: Latex allergy has been recognized as a clinically important health problem, mainly in some risk groups: spina bifida (SB), other congenital malformations submitted to multiple surgeries (MS) and health care workers (HCW). Purpose: To study IgE-binding reactivity to latex allergens, by using recombinant and natural allergens, in latex-allergic patients from different risk groups including patients with latex-fruit syndrome (LFS). Material and Methods: We selected sera of 51 latex-allergic patients within different risk groups: 20 with SB, 10 MS and 21 HCW; all these patients had positive skin prick tests with commercial latex extract (ALK-Abelló) and serum latex-specific IgE determined by UniCAP®(Pharmacia Diagnostics). Sixteen out of the 51 patients had LFS. A panel of single recombinant latex allergens was coupled to ImmunoCAPs (CAP system®) and the isolated natural allergen nHev b 2 was coupled on paper disks (EAST testing). This panel comprised rHev b 1, nHev b 2, rHev b 3, rHev b 5, rHev b 6.01, rHev b 7, rHev b 8, rHev b 9, rHev b 10 and rHev b 11. The recombinant allergens were produced as fusion protein with maltose-binding protein (MBP) in E. coli. MBP coupled on ImmunoCAPs served as control. Specific IgE values of >0.35kU/l were considered positive. Major allergen is defined if produces a positive IgE response in more than 50% of the tested group. Results: Recombinant Hev b 1 specific IgE antibodies were detected in 70% sera from SB, 30% from MS, 5% from HCW, and in 13% sera from LFS patients. For nHev b 2: SB-69%, MS-71%, HCW-71% and LFS-75%. For rHev b 3: SB-50%, MS-20%, HCW-10% and LFS-13%. For rHev b 5: SB-55%, MS-40%, HCW-62% and LFS-75%. For rHev b 6.01: SB-45%, MS-30%, HCW-76% and LFS-81%. For rHev b 7: SB-33%, MS-0%, HCW-16% and LFS-27%. For rHev b 8: SB-10%, MS-0%, HCW-5% and LFS-6%. For rHev b 9 and rHev b 10: SB-0%, MS-0%, HCW-5% and LFS-7%. For rHev b 11: SB-7%, MS-0%, HCW-5% and LFS-7%. Conclusions: The different routes of exposure influence the IgE antibody pattern. The major latex allergens identified in SB were Hev b 1, Hev b 2, Hev b 3 and Hev b 5, being Hev b 1 the most important one. The major allergen identified in MS was Hev b 2. For occupational exposure route, the major latex allergens identified in HCW were Hev b 2, Hev b 5 and Hev b 6.01, being prohevein the most important one. Regarding cross-reactivity with foods, the major allergens identified in LFS were Hev b 2, Hev b 5 and Hev b 6.01.

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[103] - Fernández-Nieto M, Quirce S, De las Heras M, Cuesta J, Aleman A, Sastre J. Latex-Fruit Syndrome: A Study on Health Care Workers Allergic to Natural Rubber Latex. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°866

We conducted a study on 30 health care workers with latex allergy diagnosed by skin prick test with a non-ammoniated latex extract 100 HEP/ml (ALK-Abelló, Madrid, Spain). Of these patients, 9 had asthma, 6 rhinoconjunctivitis and 15 contact urticaria from latex products, mainly latex gloves. The results of inhalation challenge and cutaneous provocation tests with latex gloves confirmed the diagnosis of latex allergy. Skin tests were performed using the „prick by prick‰ method with fresh kiwi, avocado, banana and chestnut. Specific IgE against latex and the aforementioned fruits or nuts was determined by the CAP method (Pharmacia, Uppsala, Sweden). A positive skin test to kiwi was observed in 66.6% of the patients and specific IgE to kiwi was confirmed in 13.3% of them. Nine out of the 30 patients (30%) presented with allergic symptoms after kiwi ingestion: 6 had oral allergy syndrome (OAS), 1 urticaria and angioedema, 1 anaphylaxis and 1 epigastralgia and diarrhea. Three patients had never eaten kiwi and the remaining patients tolerated kiwi ingestion without any reaction. A positive skin prick test with avocado was found in 50% of the patients and 20% of them had positive specific IgE to avocado. Seven patients (23.3%) presented with allergic reactions after eating avocado: 5 OAS and 2 urticaria and angioedema. Three patients did not eat avocado. The remaining patients could eat avocado without any ill effect. A positive skin test to banana was observed in 60% of the patients and 16.6% of them had positive specific IgE to this fruit. After banana ingestion, 9 patients (30%) suffered from allergic symptoms: 2 OAS, 4 urticaria and angioedema and 3 other symptoms. A positive skin test to chestnut was found in 53.3% of the patients and in 10% of them specific IgE was positive. Five patients (16.7%) showed allergic symptoms after eating chestnut: 1 anaphylaxis, 1 OAS, 2 urticaria and angioedema and 1 other symptoms. One patient had never eaten chestnut. The remaining patients tolerated chestnut ingestion. In our study, between 17% and 30% of the latex-allergic health care workers showed allergy symptoms after eating fruits. However, the prevalence of positive skin tests to these fruits ranged between 50 and 67%. The fruits most frequently implicated in the latex-fruit syndrome were kiwi and banana.

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[104] - Blanco C, Carillo T, Castillo R, Quiralte J, Cuevas M. Avocado hypersensivity. Allergy 1994;49:454-459

The avocado (Av) is a fruit that belongs to the Lauraceae family. We report 17 patients with immediate hypersensitivity to avocado. Clinical manifestations in relation to avocado ingestion were as follows: systemic anaphylaxis in seven patients, angioedema/urticaria in six, vomiting in two, bronchial asthma in one, and rhinoconjunctivitis in one. Skin prick test (SPT) with fresh avocado was positive in all patients with the Strong avocado variety (SAv) and in 14 patients with the Hass avocado variety (HAv). Our patient-associated sensitizations were as follows: 10 to latex, eight to chestnut, eight to banana, four to kiwi, and four to walnut. Avocado-sensitized patients with latex allergy were typically middle-aged women, professionally exposed to latex, who also exhibited frequent associated sensitizations to chestnut, banana, and other fruits. Specific IgE against avocado was demonstrated in 11 of our patients, by both commercial CAP and RAST with avocado extract coupled to nitrocellulose disks. Despite its lower protein content, SAv seems to be more allergenic than HAv, both in vivo and in vitro. On incubating a pool of sera from our patients with avocado, latex, chestnut, and banana extracts, a progressive RAST inhibition was obtained, with SAv- and chestnut-marked disks. This suggests the existence of common antigenic determinants among these allergens.

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[105] - Somoza ML, Rico P, Feliu A, Jiménez A, Rodriguez J, Crespo JF. Banana allergy confirmed by double-blind placebo-controlled food challenge (DBPCFC). EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°132

Background: Banana, a member of the Musaceae family, is widely consumed all over the world. It is available almost all year round in temperate climates due to heavy exporting from banana-growing countries. Banana allergy has been reported mainly associated to latex allergy. The aim of the study was to investigate clinical features of acute allergic reactions to banana confirmed by double-blind, placebo-controlled, food challenge (DBPCFC). Methods: Case series of 23 adult patients diagnosed with clinical banana allergy in the Food Allergy Unit from 'Hospital Universitario 12 de Octubre' (Madrid). Diagnostic procedure including a clinical questionnaire, skin testing by prick-prick with fresh fruit and detection of specific IgE (CAP FEIA) were performed in all patients reporting adverse reactions to foods. Patients first underwent an open food challenge (OFC), unless they had a convincing history of severe anaphylaxis. Positive OFC reactions were subsequently evaluated by DBPCFCs. All negative results of DBPCFCs were followed by an open feeding. Results: The age of patients ranged from 10 to 66 yr. (median= 24 yr.) with a female/male ratio of 2.8. Most patients (62%) experienced multiple allergic reactions after banana ingestion before being diagnosed with banana allergy. The most common clinical manifestation was the oral allergy syndrome (52%), followed by gastrointestinal anaphylaxis (8%) and acute respiratory symptoms (8%). The results of allergy testing were positive in 94% of the patients; however, an IgE-mediated mechanism could not be demostrated in two patients. In addition, 54 adverse reactions to other 20 different foods of vegetable origin were confirmed by DBPCFC‚s in the banana allergic patients; including melon, 12 patients; chestnut, 6 pt.; and avocado and kiwi, 5 pt. each. Latex allergy was diagnosed in 8 out of 23 (35%). Seventy-eight percent of the patients had pollen allergy. Conclusions: Banana can induce severe anaphylaxis, although most adverse reactions consist of oral allergy syndrome. Isolated banana allergy is uncommon, being associated frequently to melon allergy and latex allergy.

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[106] - Rico P, Somoza ML, Feliu A, Jimenez A, Crespo JF, Rodriguez J. Clinical features of chestnut allergy objectively confirmed by double-bind pacebo-controlled food challenges (DBPCFC's). EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°134

Background: Chestnuts (Castanea sativa) belong to the Fagaceae family, genus Castanea. Sensitization to chestnut has been reported in patients with other allergies, particularly in the 'atex-fruit syndrome' However, few studies have focused on chestnut allergy confirmed by DBPCFC‚s. We sought to analyze the clinical features of adult patients with objectively confirmed chestnut allergy. Methods: Fifteen adult patients diagnosed with chestnut allergy were included in this study. Case histories were evaluated following the diagnostic protocol of the Food Allergy Unit from ŒHospital Universitario 12 de Octubre'; including a detailed clinical history, skin testing by the prick-prick method with fresh foods, and detection of food specific serum IgE (CAP-FEIA, Pharmacia, Uppsala, Sweden). Clinical reactivity to foods was assessed first by open food challenges (OFCs), unless a convincing history of a recent severe anaphylaxis. Positive OFCs reactions were subsequently evaluated by DBPCFCs. All negative results of DBPCFC's were followed by a final open feeding. Results: The age of chestnut adverse reactions ranged from 15 to 70 years (median=28). The most common symptoms were systemic anaphylaxis (47%), isolated oral symptoms (27%), and generalized acute urticaria (14%). Interestingly, the results of allergy testing (both SPT and detection of specific IgE) were negative in 5 out of 15 patients with clinical reactivity to chestnut. Forty-nine reactions to other 13 different foods of vegetable origin were confirmed by DBPCFCs in patients with chestnut allergy, including banana (6 out of 15 patients), avocado (5/15 pt.) and kiwi (4/15 pt.). In addition, 7 out of 15 (47%) patients having clinical allergy to chestnut were found to be allergic to latex. Conclusions: Systemic anaphylaxis is a common clinical feature associated to adverse reactions after chestnut ingestion. Latex-associated allergy is found in nearly half of patients reacting to chestnut ingestion. Allergy testing is a poor predictor of clinical reactivity to chestnut.

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[107] - Guinnepain MT, Guilloux L, Laurent J. Buckwheat allergy: an additional cross-reactivity between latex and food. Rev Fr Allergol Immunol Clin 1997;37:1218

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[108] - Olsen E, Zhang L, Hill RD, Kisil FT, Sehon AH, Mohapatra SS. Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen. J Immunol 1991;147:205-211

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.

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[109] - Alemán A, Sastre J, Quirce S, de las Heras M, Carnés J, Fernández-Caldas E, et al. Allergy to kiwi: A double-blind, placebo-controlled food challenge study in patients from a birch-free area. J Allergy Clin Immunol 2004;113:543-550

Background Allergy to kiwi fruit is being increasingly reported, but it has never been evaluated by means of a double-blind, placebo-controlled food challenge (DBPCFC) study. Objective : We sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitized patients from a birch-free area. Method s : Forty-three patients with allergy symptoms who were sensitized to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The pattern of allergen recognition was assessed by means of IgE immunoblotting. Sequence analysis of IgE-binding bands was performed by using Edman degradation. Result s : DBPCFCs were performed in 33 patients; 4 patients had experienced severe anaphylaxis, and 6 patients declined informed consent. DBPCFC results were positive in 23 patients and negative in 10 patients. The most frequent clinical manifestation was oral allergy syndrome. Twenty-one percent of the patients were not allergic to pollen. Forty-six percent of patients experienced systemic symptoms, and this happened with higher frequency in patients not allergic to pollen (100%). Twenty-eight percent of the patients were sensitized to latex. The IgE-binding bands in kiwi extract more frequently recognized by patient sera were those of 30, 24, 66, and 12 kd, and they could not be associated with any pattern of kiwi-induced allergic reactions. Conclusion : The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi. One of 5 patients with kiwi allergy was not allergic to pollen, and these patients had the highest risk of systemic reactions to kiwi.

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[110] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[111] - Sabbah A, Bonneau JC, Hernandez C, Drouet M, Le Sellin J. Allergie croisée pomme, bouleau, kiwi: à propos de 2 observations. Allerg Immunol (Paris) 1985;17:524-525

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[112] - Jung P, Sesztak-Greinecker G, Wantke F, Goetz M, Jarisch R, Hemmer W. Prevalence of cross-sensitisation to soy allergens in patients with birch pollen allergy and allergenicity of different soy products. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1530

Background: Soybean Gly m 4, a member of the PR-10 protein family, has been recognized as a cross-reactive food allergen in birch pollen-related food hypersensitivity with the potential to elicit severe systemic reactions. We investigated the prevalence of cross-sensitisation to soybean and the allergenicity of various soy-based food items in patients with birch pollen allergy. Method: A commercial brand of soy milk was added to our routine skin prick test panels and tested in consecutive patients with suspect inhalant or food allergy. Consumption habits concerning soy products and eventual adverse reactions were recorded by questionnaire. Additional prick-to-prick testing with different soy products was done in selected patients. Results: Among 292 patients with a positive skin prick test to birch pollen, 72 (24.7%) reacted to soy milk but only 18 (6.2%) reacted to a commercial soy skin prick test. 34/97 (35%) of birch pollen-allergic patients reported to have knowingly consumed soy milk before with 11/34 (31%) of them having experienced side effects. Mild reactions to tofu and soybean sprouts were occasionally reported. Prick-to-prick testing with different soy products in 16 patients with a positive skin test to soy milk revealed positive reactions to raw and cooked soybean sprouts (94%/50%), raw and cooked tofu (87%/40%), soy dessert (86%), and soy joghurt (21%). No significant differences in skin test responses were seen between seven different brands of soy milk (protein content 3.0-3.7g/100ml). Skin tests remained positive even after boiling soy milk für 5, 10 and 30 minutes. Conclusions: Cross-sensitisation to soy is frequent among patients with birch pollen allergy and many soy-based foods retain considerable allergenicity. As soy products are becoming increasingly popular on the market, birch pollen-allergic patients may be at growing risk of experiencing allergic reactions to these products.

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[113] - Osterballe M, Hansen TK, Mortz CG, Bindslev-Jensen C. The clinical relevance of sensitization to pollen-related fruits and vegetables in unselected pollen-sensitized adults. Allergy 2005;60:218-225

BACKGROUND: Previous studies have described cross-reactivity between fresh fruits, vegetables and pollen. However, no data demonstrates the clinical relevance of sensitization to pollen-related fruits and vegetables in unselected pollen-sensitized adults with and without symptoms in the pollen season . OBJECTIVE: The aim of this study was to estimate the clinical relevance of sensitization to pollen-related fruits and vegetables in unselected pollen-sensitized adults and to examine the diagnostic value of skin-prick test (SPT), histamine release and specific IgE compared with the outcome of oral challenge . METHODS: In total, 936 unselected adults (female : male 479 : 457, median age 33.7 years) were examined for pollen sensitization and clinical cross-reactivity with pollen-related fruits and vegetables by questionnaire, SPT, histamine release, specific IgE and oral challenge . RESULTS: The prevalence of pollen sensitization was 23.8% (n = 223). The probability of a clinical reaction to pollen-related foods in the respective pollen-sensitized groups was: 24% (birch), 4% (grass), 10% (mugwort), 35% (birch + grass), 8% (grass + mugwort) and 52% (birch + grass + mugwort). The odds ratio of a clinical reaction to pollen-related fruits and vegetables in symptomatic pollen-sensitized adults was as high as four times (birch + grass) the odds ratio of a clinical reaction in asymptomatic pollen-sensitized adults . CONCLUSION: This study not only demonstrates a high prevalence of clinical reactions to fruits and vegetables in pollen-sensitized adults, but also a discrepancy between the prevalence of sensitization to fruits and vegetables and the clinical relevance in different pollen-sensitized groups with symptoms in the pollen season as a significant factor.

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[114] - Patriarca S, Voltolini S, Navone R, Martini S, Montanari C, Negrini A, et al. Biochemical and immunochemical characterization of hop-hornbeam (Ostrya Carpinifolia Scop.) pollen - A new allergenic pollen from the Corylaceae family. Aerobiologia 2000;16:255-260

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[115] - Mittag D, Vieths S, Vogel L, Becker WM, Rihs HP, Helbling A, et al. Soybean allergy in patients allergic to birch pollen: Clinical investigation and molecular characterization of allergens. J Allergy Clin Immunol 2004;113:148-154

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[116] - Karamloo F, Wangorsch A, Kasahara H, Davin LB, Haustein D, Lewis NG, et al. Phenylcoumaran benzylic ether and isoflavonoid reductases are a new class of cross-reactive allergens in birch pollen, fruits and vegetables. Eur J Biochem 2001;268:5310-5320

We investigated the biochemical function of the birch pollen allergen Bet v 6 and its role in the IgE-cross-reactivity between birch pollen and plant foods, and characterized Pyr c 5, a Bet v 6-related food allergen, from pear; the proteins were expressed as His-Tag fusion proteins in Eschershia coli and purified by Ni-chelate affinity chromatography under native conditions. Nonfusion proteins were obtained by factor Xa protease treatment. The highest degree of amino-acid sequence identity of Pyr c 5 and Bet v 6 was found with a plant protein related to a defense mechanism, which we have named phenylcoumaran benzylic ether reductase (PCBER) based on its ability to catalyze the NADPH-dependent reduction of 8-5' linked lignans such as dehydrodiconiferyl alcohol to give isodihydrodehydrodiconiferyl alcohol. Enzymatic assays with recombinant Pyr c 5 and Bet v 6 showed PCBER catalytic activity for both recombinant allergens. Both Pyr c 5 and Bet v 6 allergens had similar IgE binding characteristics in immunoblotting and enzyme allergosorbent tests (EAST), and bound IgE from 10 sera of birch-pollen-allergic patients including six pear-allergic subjects. EAST inhibition experiments with Pyr c 5 as the solid phase antigen suggested that homologous allergens may be present in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini (courgette), and carrot. In extracts of pear, apple, orange, and persimmon, the presence of proteins of approximately 30-35 kDa containing Bet v 6 cross-reactive epitopes was demonstrated with two Bet v 6-specific monoclonal antibodies. Recombinant Pyr c 5 triggered a strong, dose-dependent mediator release from basophils of a pear-allergic subject, suggesting that Pyr c 5 has the potential to elicit type I allergic reactions.

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[117] - Mittag D, Vieths S, Vogel L, Wagner-Loew D, Starke A, Hunziker P, et al. Birch pollen-related food allergy to legumes: identification and characterization of the Bet v 1 homologue in mungbean (Vigna radiata), Vig r 1. Clin Exp Allergy 2005;35:1049-1055

BACKGROUND: Recently allergic reactions to legumes mediated by Bet v 1-homologous food allergens were described for soy and peanut. In this study we assessed allergic reactions to another legume, to mungbean seedlings, and identified its Bet v 1-homologous allergen Vig r 1 . METHODS: Ten patients were selected who had a history of allergic reactions to mungbean seedlings and a respiratory allergy to birch pollen. The Bet v 1 homologue in mungbean seedlings, Vig r 1, was cloned by a PCR strategy, expressed in Escherichia coli, and purified by preparative SDS-PAGE. In all sera, specific IgE against birch pollen, Bet v 1, Bet v 2, Vig r 1, and the Bet v 1 homologues in soy (Gly m 4) and cherry (Pru av 1) was determined by CAP-FEIA. Cross-reactivity of specific IgE with Vig r 1, Bet v 1, Gly m 4, and Pru av 1 was assessed by immunoblot inhibition. Expression of Vig r 1 during development of mungbean seedlings and under wounding stress was analysed by immunoblotting. The Vig r 1 double band was analysed by matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography/tandem mass spectrometry (LC/MS/MS) . RESULTS: All patients were sensitized to birch pollen and Bet v 1, 20% to Bet v 2, and 90% to Gly m 4. Seventy percent of the patients showed IgE binding to a double band at 15 kDa in mungbean extract that was inhibited after pre-incubation of sera with rBet v 1. PCR cloning revealed that the mungbean homologue of Bet v 1 had a molecular weight of 16.2 kDa, a calculated pI of 4.6% and 42.8% amino acid sequence identity with Bet v 1. MS analysis confirmed similarity of the double band with the deduced Vig r 1 sequence, but also indicated the existence of other Vig r 1 isoforms. ImmunoCAP analysis detected IgE against Vig r 1 in 80% of the sera. IgE binding to Vig r 1 was inhibited with Gly m 4 in six of six and with rPru av 1 in four of six patients. Vig r 1 expression occurred during development of seedlings and was increased by wounding stress . CONCLUSIONS: Food allergy to mungbean seedlings can be caused by primary sensitization to birch pollen and is mediated by Vig r 1 in the majority of the patients with birch pollen-related allergy to mungbean seedlings.

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[118] - Kazemi-Shirazi L, Pauli G, Purohit A, Spitzauer S, Fröschl R, Hoffmann-Sommergruber K, et al. Quantitative IgE inhibition with purified recombinant allergens indicate pollen-derived allergens as the sensitizing agents responsible for many forms of plant food allergy. J Allergy Clin Immunol 2000;105:116-125

Type I allergic symptoms in the oropharyngeal mucosa upon contact with plant-derived food in patients with pollen allergies have been termed oral allergy syndrome (OAS). IgE cross-reactivity between pollen and food allergens represents the molecular basis for this phenomenon. The sensitizing allergen source (pollen or plant food) in OAS is a controversial issue. OBJECTIVE: We sought to determine the primary sensitizing molecules in patients with OAS. METHODS: We used recombinant birch pollen (rBet v 1 and rBet v 2) and plant food allergens (apple, rMal d 1; celery, rApi g 1; and carrot, rDau c 1), as well as natural pollen (birch and timothy grass) and plant food (apple, peach, kiwi, hazelnut, celery, and carrot) allergens, to identify cross-reactive allergens by using qualitative immunoblot inhibitions. In addition, we determined the percentage of plant food-specific IgE that can be preadsorbed with recombinant and natural pollen allergens by quantitative RAST inhibitions by using sera from 71 patients with OAS. RESULTS: Preincubation of sera with recombinant and natural pollen allergens led to an almost complete inhibition of IgE binding to plant food allergens in Western blots, as well as in RAST inhibition experiments. In contrast, recombinant plant food allergens poorly inhibited IgE binding to Bet v 1. CONCLUSION: Most IgE epitopes in plant food recognized by patients with OAS are resembled by pollen allergens. Thus pollen allergens may be responsible for the elicitation and maintenance of OAS.

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[119] - Rudeschko O, Fahlbusch B, Steurich F, Schlenvoigt G, Jäger L. Kiwi allergens and their cross-reactivity with birch, rye, timothy, and mugwort pollen. J Investig Allergol Clin Immunol 1998;8:78-84

In order to study kiwi allergens and examine their cross-reactivity to birch, rye, timothy, and mugwort pollen, immunoblot and enzyme immunoassay (EIA) inhibition tests were performed with self-prepared kiwi extract. For the investigations, the sera of 22 kiwi-allergic patients were used, which were characterized by radioallergosorbent (RAST) measurements for kiwi, birch pollen, and apple with commercial allergen disks. The RAST values for kiwi were compared with those obtained by self-prepared kiwi extract disks. In the RAST, the allergen potency of this extract was found to be very similar to that of the commercial extracts. This extract was able to bind immunoglobulin E from kiwi-allergic patients in the immunoblots and EIA. Immunoblot results revealed a broad spectrum of IgE specificities

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[120] - Voitenko V, Poulsen LK, Nielsen L, Norgaard A, Bindslev-Jensen C, Skov PS. Allergenic properties of kiwi-fruit extract: cross-reactivity between kiwi-fruit and birch-pollen allergens. Allergy 1997;52:136-143

Our investigation aimed to produce and characterize a kiwi extract and to use this extract to investigate a possible cross-reactivity with birch pollen. Kiwi was extracted in two buffers: phosphate-buffered saline (PBS) and borate-buffered saline (BBS). Extraction in BBS produced a double amount of protein, and a more stabile extract. Tandem crossed-immunoelectrophoresis showed that the BBS and PBS extracts had several common, but also a few individual, proteins. The mixture of both extracts was assumed to represent the most complete allergen extract. The allergenic properties of the kiwi extract were investigated by immunoblotting (IB), RAST, and histamine-release (HR) test in 15 birch-pollen-allergic patients (eight of them with clinical kiwi allergy) and one with clinical monoallergy to kiwi. All eight birch-pollen-allergic patients with kiwi allergy and the kiwi-monoallergic patient were positive in kiwi IB binding most frequently to proteins of 10-12 and 20-25 kDa. With our extract, RAST was positive in four kiwi-allergic and one non-kiwi-allergic patient, whereas the HR test was positive in five kiwi-allergic patients and negative in all non-kiwi-allergic patients. RAST and IB inhibition demonstrated cross-reactivity between birch-pollen and kiwi allergens due to a 10-12 kDa protein. In conclusion, a kiwi extract with allergenic properties was produced, and, by the methods used, cross-reactivity was demonstrated between birch-pollen and kiwi allergens.

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[121] - Voitenko V, Poulsen LK, Nielsen L, Norgaard A, Bindslev-Jensen C, Skov PS. Allergenic properties of kiwi-fruit extract: cross-reactivity between kiwi-fruit and birch-pollen allergens. Allergy 1997;52:136-143

Our investigation aimed to produce and characterize a kiwi extract and to use this extract to investigate a possible cross-reactivity with birch pollen. Kiwi was extracted in two buffers: phosphate-buffered saline (PBS) and borate-buffered saline (BBS). Extraction in BBS produced a double amount of protein, and a more stabile extract. Tandem crossed-immunoelectrophoresis showed that the BBS and PBS extracts had several common, but also a few individual, proteins. The mixture of both extracts was assumed to represent the most complete allergen extract. The allergenic properties of the kiwi extract were investigated by immunoblotting (IB), RAST, and histamine-release (HR) test in 15 birch-pollen-allergic patients (eight of them with clinical kiwi allergy) and one with clinical monoallergy to kiwi. All eight birch-pollen-allergic patients with kiwi allergy and the kiwi-monoallergic patient were positive in kiwi IB binding most frequently to proteins of 10-12 and 20-25 kDa. With our extract, RAST was positive in four kiwi-allergic and one non-kiwi-allergic patient, whereas the HR test was positive in five kiwi-allergic patients and negative in all non-kiwi-allergic patients. RAST and IB inhibition demonstrated cross-reactivity between birch-pollen and kiwi allergens due to a 10-12 kDa protein. In conclusion, a kiwi extract with allergenic properties was produced, and, by the methods used, cross-reactivity was demonstrated between birch-pollen and kiwi allergens.

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[122] - Möller M, Paschke A, Vieluf D, Kayma M, Vieths S, Steinhart H. Characterization of allergens in kiwi fruit and detection of cross-reactivities with allergens of birch pollen and related fruit allergens. Food Agric Immunol 1997;9:107-121

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[123] - Oberhuber C, Bulley S, Bublin M, Ballmer-Weber B, Vieths S, Hoffmann-Sommergruber K. Bet v 1 homologous proteins in kiwi – relevant allergens? Allergy 2007;62(suppl. 83):107

Background: In Central and Northern Europe birch pollen related food allergy is mainly based upon cross reactive IgE to Bet v 1 and homologues present in various plant derived foods. So far, actinidin, kiwellin and the thaumatin-like protein have been identified from kiwi as relevant allergens. But so far, nothing is known about Bet v 1 homologous proteins present in kiwi. Preliminary efforts to purify natural Bet v1 homologues in sufficient quantities from kiwi failed so far. Methods: A sequence homologous to Bet v 1 was identified from Actinidia chinensis (Gold kiwi), subcloned into the expression vector pMW 175 and expressed in E. coli. The resulting recombinant protein was purified and characterized according to established methods. The immune reactivity of the purified recombinant Bet v 1 homologous protein was tested by IgE-ELISA, immunoblots and immunoblot inhibitions with natural kiwi extracts. ELISA experiments were performed using birch pollen, kiwi and latex allergic patients‚ sera. Results: Bet v 1 homologues cross reactive to Bet v 1 were identified from green kiwi (A. deliciosa) and gold kiwi (A. chinensis) by inhibition assays. Alignment of recombinant kiwi Bet v 1 homologue and recombinant Bet v 1a revealed an amino acid sequence identity of 50%. Well characterised purified recombinant kiwi Bet v 1 homologue was used for IgE in vitro assays. The majority of birch pollen and kiwi sensitized patients (74%) displayed specific IgE directed to the Bet v 1 homologue from kiwi followed by latex and latex-kiwi allergic patients (40%). In kiwi monosensitized patients only 1/8 had Bet v 1 homologue specific IgE. Conclusion: This is the first evidence of cross reactive Bet v 1 homologous allergens present in gold and green kiwi fruits. The present data point out that Bet v 1 homologues are relevant allergens in kiwi especially for patients sensitized to tree pollen and kiwi fruits and might have been neglected so far due to low abundancy in the conventional extracts used for diagnosis.

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[124] - Oberhuber C, Bulley SM, Ballmer-Weber BK, Bublin M, Gaier S, DeWitt AM et al. Characterization of Bet v 1-related allergens from kiwifruit relevant for patients with combined kiwifruit and birch pollen allergy. Mol Nutr Food Res 2008;52(suppl. 2):S230-S240

Allergy to kiwifruit appears to have become more common in Europe and elsewhere during the past several years. Seven allergens have been identified from kiwifruit so far, with actinidin, kiwellin and the thaumatin-like protein as the most relevant ones. In contrast to other fruits, no Bet v 1 homologues were characterized from kiwifruit so far. We cloned, purified, and characterized recombinant Bet v 1-homologous allergens from green (Actinidia deliciosa, Act d 8) and gold (Actinidia chinensis, Act c 8) kiwifruit, and confirmed the presence of its natural counterpart by inhibition assays. Well-characterized recombinant Act d 8 and Act c 8 were recognized by birch pollen/kiwifruit (confirmed by double-blind placebo-controlled food challenge) allergic patients in IgE immunoblots and ELISA experiments. The present data point out that Bet v 1 homologues are allergens in kiwifruit and of relevance for patients sensitized to tree pollen and kiwifruit, and might have been neglected so far due to low abundance in the conventional extracts used for diagnosis.

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[125] - Patriarca S, Voltolini S, Navone R, Martini S, Montanari C, Negrini A, et al. Biochemical and immunochemical characterization of hop-hornbeam (Ostrya Carpinifolia Scop.) pollen - A new allergenic pollen from the Corylaceae family. Aerobiologia 2000;16:255-260

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[126] - Boehncke WH, Loeliger C, Kuehnl P, Kalbacher H, Bohm BO, Gall H. Identification of HLA-DR and -DQ alleles conferring susceptibility to pollen allergy and pollen associated food allergy. Clin Exp Allergy 1998;28:434-441

BACKGROUND: Allergenic crossreactivity of pollen and foods due to the antigeneic similarity of oligopeptides is a well established clinical phenomenon. OBJECTIVE: To determine the immunopathological relevance of antigen presentation, we analysed the HLA class-II genotype of patients with either pollen allergy or pollen associated food allergy. METHODS: One hundred and twenty patients with pollen allergy and 80 patients with pollen associated food allergy were evaluated by skin- prick tests, RAST, and HLA class-II genotyping. The control population comprised 4251 healthy blood and bone marrow donors. RESULTS: Monovalent pollen allergy was observed in 57% (n=68) of patients with pollinosis (57x grass pollen, 11x birch pollen), but only in 15% (n=12) of patients with food allergy (9x grass pollen, 3x birch pollen). Hazelnut (71%), almond (65%), walnut (44%) and apple (41%) were the most common food allergens and frequently associated with birch pollen allergy. Grass pollen allergy was associated with an increased frequency of HLA-DQB1*0301 (RR=2.3; EF=0.4; P=0.0016) when compared with the control population. HLA-DRB *08 conferred a sixfold higher risk for peanut allergy (EF=0.3; P=0.0013) and -DRB1*12 a 13-fold higher risk for carrot allergy (EF=0.3; P<0.000001). The differences on allele frequencies detected among patients with food allergies diminished or turned statistically insignificant when their genotypes were directly compared to those of patients with the corresponding pollen allergies. This was found in the case of birch pollen associated hazel nut allergy for the extended haplotype HLA-DRB1*01, -DQA1*0101, -DQB1*0501 as well as in grass pollen associated peanut allergy for HLA-DRB1*08 (from RR=6, P=0.0013 to insignificant) and in birch pollen associated carrot allergy for HLA-DRB1*12 (from RR=13, P < 0.000001 to insignificant). CONCLUSION: We were able to identify HLA class-II alleles associated with some allergies thus indicating that these alleles might confer susceptibility to the respective allergens. Similarities at the level of the HLA class-II genotype parallel the empirical finding of distinct cross-reactivity patterns thus complementing investigations of IgE specificities. Our observations provide evidence for the major importance of antigen presentation on the manifestation of distinct crossreactivity patterns.

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[127] - Lucas JSA, Grimshaw KEC, Collins WK, Warner JO, Hourihane JOB. Kiwi fruit is a significant allergen and is associated with differing patterns of reactivity in children and adults. Clin Exp Allergy 2004;34:1115-1121

BACKGROUND: Allergy to kiwi fruit appears increasingly common, but few studies have evaluated its clinical characteristics, or evaluated methods of investigating the allergy . OBJECTIVE: To characterize the clinical characteristics of kiwi fruit allergy and to study the role of double-blind placebo-controlled food challenge (DBPCFC), skin tests and specific IgE in the diagnosis of this food allergy . METHODS: Two-hundred and seventy-three subjects with a history suggestive of allergy to kiwi completed a questionnaire. Forty-five were investigated by DBPCFC, prick-to-prick skin testing with fresh kiwi pulp, and specific IgE measurement. Nineteen subjects were also skin tested using a commercially available solution . RESULTS: The most frequently reported symptoms were localized to the oral mucosa (65%), but severe symptoms (wheeze, cyanosis or collapse) were reported by 18% of subjects. Young children were significantly more likely than adults to react on their first known exposure (P<0.001), and to report severe symptoms (P=0.008). Twenty-four of 45 subjects (53%) had allergy confirmed by DBPCFC. Prick-to-prick skin test with fresh kiwi was positive in 93% of subjects who had allergy confirmed by DBPCFC, and also in 55% of subjects with a negative food challenge. The commercial extract was significantly less sensitive, but with fewer false-positive reactions. CAP sIgE was only positive in 54% of subjects who had a positive challenge . CONCLUSIONS: Kiwi fruit should be considered a significant food allergen, capable of causing severe reactions, particularly in young children. DBPCFC confirmed allergy to kiwi fruit in 53% of the subjects tested, who had a previous history suggestive of kiwi allergy. Skin testing with fresh fruit has good sensitivity (93%), but poor specificity (45%) in this population. CAP sIgE and a commercially available skin test solution were both much less sensitive (54%; 75%) but had better specificity (90%; 67%).

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[128] - Bublin M, Mari A, Ebner C, Knulst A, Scheiner O, Hoffmann-Sommergruber K, et al. IgE sensitization profiles toward green and gold kiwifruits differ among patients allergic to kiwifruit from 3 European countries. J Allergy Clin Immunol 2004;114:1169-1175

Background Kiwifruits have become a major elicitor of plant food allergy. Until recently, the only species of kiwifruit grown commercially was the common green-fleshed Actinidia deliciosa cv Hayward. In 1999, the yellow-fleshed cultivar Actinidia chinensis cv Hort16A was introduced into the international market. Objective : We compared the allergen compositions of green and gold kiwifruits and assessed the sensitization patterns of patients with kiwifruit allergy toward both varieties. Method s : Sera from 90 patients with kiwifruit allergy from Austria, central Italy, and the Netherlands were tested for IgE binding to green and gold kiwifruit protein extracts and to purified actinidin, the major kiwifruit allergen, by ELISA. In addition, ELISA inhibitions and immunoblots were performed with selected sera. Relevant allergens were identified by N-terminal sequencing and immunoblotting with allergen-specific antibodies. Result s : IgE immunoblotting showed marked differences in the allergen compositions of green and gold kiwifruit extracts. Phytocystatin, a novel plant food allergen, and a thaumatin-like protein were identified as allergens common for both cultivars. Two allergens with homologies to chitinases were found in gold kiwifruits, whereas actinidin was detected exclusively in green kiwifruits. Patients from Central Europe and central Italy showed distinct sensitization profiles toward green and gold kiwifruit extracts as well as actinidin. Whereas sera from Austrian and Dutch patients mainly recognized green kiwifruit extract and actinidin, almost all Italian sera showed IgE binding to both kiwifruit species, but only half of them contained actinidin-specific IgE. Green and gold kiwifruit extracts were shown to be highly cross-reactive as determined by IgE ELISA inhibition. Conclusion : The presence of common allergens and the IgE cross-reactivity to green kiwifruit qualifies gold kiwifruit as a potential new allergen source for patients allergic to green kiwifruits.

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[129] - de Amici M, Moratti R, Quaglini S, Vignini MA, Mosca M. IgE to Bet v 1/Bet v 2 in OAS. Allergy 2002;57:181-182

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[130] - Alemán A, Sastre J, Quirce S, de las Heras M, Carnés J, Fernández-Caldas E, et al. Allergy to kiwi: A double-blind, placebo-controlled food challenge study in patients from a birch-free area. J Allergy Clin Immunol 2004;113:543-550

Background Allergy to kiwi fruit is being increasingly reported, but it has never been evaluated by means of a double-blind, placebo-controlled food challenge (DBPCFC) study. Objective : We sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitized patients from a birch-free area. Method s : Forty-three patients with allergy symptoms who were sensitized to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The pattern of allergen recognition was assessed by means of IgE immunoblotting. Sequence analysis of IgE-binding bands was performed by using Edman degradation. Result s : DBPCFCs were performed in 33 patients; 4 patients had experienced severe anaphylaxis, and 6 patients declined informed consent. DBPCFC results were positive in 23 patients and negative in 10 patients. The most frequent clinical manifestation was oral allergy syndrome. Twenty-one percent of the patients were not allergic to pollen. Forty-six percent of patients experienced systemic symptoms, and this happened with higher frequency in patients not allergic to pollen (100%). Twenty-eight percent of the patients were sensitized to latex. The IgE-binding bands in kiwi extract more frequently recognized by patient sera were those of 30, 24, 66, and 12 kd, and they could not be associated with any pattern of kiwi-induced allergic reactions. Conclusion : The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi. One of 5 patients with kiwi allergy was not allergic to pollen, and these patients had the highest risk of systemic reactions to kiwi.

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[131] - Alemán A, Sastre J, Quirce S, de las Heras M, Carnés J, Fernández-Caldas E, et al. Allergy to kiwi: A double-blind, placebo-controlled food challenge study in patients from a birch-free area. J Allergy Clin Immunol 2004;113:543-550

Background Allergy to kiwi fruit is being increasingly reported, but it has never been evaluated by means of a double-blind, placebo-controlled food challenge (DBPCFC) study. Objective : We sought to assess kiwi allergy on the basis of a DBPCFC and identify the patterns of allergen recognition in sensitized patients from a birch-free area. Method s : Forty-three patients with allergy symptoms who were sensitized to kiwi were evaluated by means of clinical history, skin tests, IgE determinations, and DBPCFCs. The pattern of allergen recognition was assessed by means of IgE immunoblotting. Sequence analysis of IgE-binding bands was performed by using Edman degradation. Result s : DBPCFCs were performed in 33 patients; 4 patients had experienced severe anaphylaxis, and 6 patients declined informed consent. DBPCFC results were positive in 23 patients and negative in 10 patients. The most frequent clinical manifestation was oral allergy syndrome. Twenty-one percent of the patients were not allergic to pollen. Forty-six percent of patients experienced systemic symptoms, and this happened with higher frequency in patients not allergic to pollen (100%). Twenty-eight percent of the patients were sensitized to latex. The IgE-binding bands in kiwi extract more frequently recognized by patient sera were those of 30, 24, 66, and 12 kd, and they could not be associated with any pattern of kiwi-induced allergic reactions. Conclusion : The results provide evidence that kiwi allergy is not a homogeneous disorder because several clinical subgroups can be established. No definite allergen-recognition pattern was associated with the type of allergic reactions to kiwi. One of 5 patients with kiwi allergy was not allergic to pollen, and these patients had the highest risk of systemic reactions to kiwi.

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[132] - Palazzo P, Giangrieco I, Bernardi ML, Tamburrini M, Giani M, Ciardiello MA, et al. IgE reactivity to kiwellin (Act d 28kD), actinidin (Act d 1) and thaumatin-like protein (Act d 2) from green kiwi fruit in kiwi allergic patients tested by SDS-PAGE arrayed molecules. Allergy 2007;62(suppl. 83):341

Background Several allergens have been identified and characterized in green kiwi fruit, but little is know about patients' profiles in terms of IgE reactivity to single kiwi fruit extract components. We sough to describe kiwi sensitization profiles by testing three kiwi allergens and three more plant-derived food allergens. Methods Kiwellin (Act d 28kD), Actinidin (Act d 1) and thaumatin-like protein (Act d 2) were purified from green kiwi fruit by ionic exchange chromatography on DE-52 and SP-Sepharose. The last purification step was carried out by FPLC (Mono S HR 10/10 or Q HR 10/10, Amersham-Pharmacia, Sweden). Proteins were pure upon SDS-PAGE and characterized by N-terminal amino acid sequencing. The three purified proteins were mixed at 1:1:1 ratio for immunoblotting analysis. Molecules underwent to a reducing SDS-PAGE, were electroblotted onto PVDF membranes, and probed with sera from kiwi allergic subjects. Sera were selected on the basis of clinical reactivity. IgE testing was also performed on ISAC system microarrayed allergens (VBC, Austria) and reactivity to profilin, LTP and Bet v 1-like allergens was recorded. Results 30 patients have been selected. Sera of 14 (47%) patients showed IgE reactivity to Act d 1, 10 (33%) to Act d 28kD, and 9 (30%) recognized Act d 2. The combined reactivity to the three components was recorded in 2 subjects (6%). Reactivity to Act d 1 and Act d 2 was detected in 4 subjects (13%), whereas Act d 1 plus Act d 28kD, and Act d 2 plus Act d 28kD were positive in 4 and 3 subjects (13% and 10%), respectively. A single allergen was positive in 5 (Act d 1, 16%), 4 (Act d 28kD, 13%), and 2 (Act d 2, 6%) subjects. 8 subjects were negative to all three green kiwi molecules (26%). 22 serum of 30 patients were tested on ISAC system microarrayed allergens. 14 sera were positive to at least one kiwi allergen. 4 subjects were positive to Bet v 1-like molecules, 7 to LTP and 4 to Profilin. 2 serum showed co-reactivity to LTP and Profilins, one to Bet v 1-like and Profilins and one to Bet v 1-like and LTP. 3 of 8 kiwi allergen negative sera were scored positive to at least one of the three additional food allergens. Conclusion IgE are detected for all kiwi allergens and differ in their prevalence. Patients with mono-sensitization are rare compared to those who show co-reactivity to 2 or more green kiwi allergens. Kiwi allergic subjects may show an IgE reactivity to additional plant-related allergens.

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[133] - Asero R, Jimeno L, Barber D. Component-resolved diagnosis of plant food allergy by SPT. Eur Ann Allergy Clin Immunol 2008;40:115-121

BACKGROUND: Fruits and vegetables may contain both labile and stable allergens. The former induce only OAS, whereas stable allergens may induce systemic reactions. Component-resolved diagnosis (CRD) of allergy to plant foods is therefore essential for the clinical management of allergic patients. METHODS: 80 adults allergic to plant foods underwent SPT with purified natural date palm profilin (Pho d 2), purified Mal d 1, a peach extract containing uniquely LTP, and with a kiwi extract containing uniquely stable allergens. RESULTS: 58 (72%) patients were monosensitized: 24 to Mal d 1, 24 to profilin, 7 to LTP, and 3 to kiwi. 22 patients were multi-sensitised: 14 to Mal d 1 and profilin, 2 to Mal d 1 and kiwi, 1 to LTP and profilin, 3 to LTP and Mal d 1, and 2 to LTP, Mal d 1 and profilin. Mal d 1 and LTP sensitisation were associated with apple and peach allergy, respectively, whereas profilin sensitisation was associated with allergy to melon, watermelon, banana, tomato and citrus fruits. 18/21 kiwi-allergic patients were sensitised to one of the cross-reacting allergens, but 2/18 reacted to kiwi-specific allergens as well. CONCLUSIONS: In patients with allergy to plant-derived foods CRD can be performed by SPT with purified allergen proteins. In the future, the availability of a larger number of purified natural or recombinant allergens for SPT will represent a simple means to classify food-allergic patients properly on the first visit.

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[134] - van Ree R, Voitenko V, van Leeuwen WA, Aalberse RC. Profilin is a cross-reactive allergen in pollen and vegetable foods. Int Arch Allergy Immunol 1992;98:97-104

Sera with IgE antibodies against grass pollen often contain IgE against vegetable foods. We investigated the role of the ubiquitous protein profilin in this cross-reactivity. Profilin was purified from Lolium perenne grass pollen by means of affinity purification with Sepharose-coupled poly(L-proline). This solid phase was also used as capturing agent for profilin from pollen and food extracts for application in a radioallergosorbent test. It was shown that profilin is an allergen in grass pollen and in a wide range of vegetable foods, like potato and celery. Within a grass-pollen-sensitive population, patients with IgE to vegetable foods have a high incidence of antibodies against profilin. IgE antibodies against grass pollen profilin were shown to be cross-reactive with respect to vegetable foods

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[135] - Rudeschko O, Fahlbusch B, Steurich F, Schlenvoigt G, Jäger L. Kiwi allergens and their cross-reactivity with birch, rye, timothy, and mugwort pollen. J Investig Allergol Clin Immunol 1998;8:78-84

In order to study kiwi allergens and examine their cross-reactivity to birch, rye, timothy, and mugwort pollen, immunoblot and enzyme immunoassay (EIA) inhibition tests were performed with self-prepared kiwi extract. For the investigations, the sera of 22 kiwi-allergic patients were used, which were characterized by radioallergosorbent (RAST) measurements for kiwi, birch pollen, and apple with commercial allergen disks. The RAST values for kiwi were compared with those obtained by self-prepared kiwi extract disks. In the RAST, the allergen potency of this extract was found to be very similar to that of the commercial extracts. This extract was able to bind immunoglobulin E from kiwi-allergic patients in the immunoblots and EIA. Immunoblot results revealed a broad spectrum of IgE specificities

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[136] - Mempel M, Rakosi J, Ring J, Ollert M. Severe anaphylaxis to kiwi fruit: Immunologic changes related to successful sublingual allergen immunotherapy. J Allergy Clin Immunol 2003;111:1406-1409

ABSTRACT: BACKGROUND: CD4+ T-cell epitope immunodominance is not adequately explained by peptide selectivity in class II major histocompatibility proteins, but it has been correlated with adjacent segments of conformational flexibility in several antigens . METHODS: The published T-cell responses to two venom allergens and two aeroallergens were used to construct profiles of epitope dominance, which were correlated with the distribution of conformational flexibility, as measured by crystallographic B factors, solvent-accessible surface, COREX residue stability, and sequence entropy . RESULTS: Epitopes associated with allergy tended to be excluded from and lie adjacent to flexible segments of the allergen . CONCLUSION: During the initiation of allergy, the N- and/or C-terminal ends of proteolytic processing intermediates were preferentially loaded into antigen presenting proteins for the priming of CD4+ T cells.

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[137] - Lidholm J. Allergens and allergy diagnosis in Europrevall : component resolved diagnosis of food allergy. Communication, 28th Congress of EAACI, Warszaw, June 6-10 2009

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[138] - Asero R, Mistrello G, Roncarolo D, de Vries SC, Gautier MF, Ciurana CL, 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

Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

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[139] - Asero R, Mistrello G, Roncarolo D, Amato S. Detection of Some Safe Plant-Derived Foods for LTP-Allergic Patients. Int Arch Allergy Immunol 2007;144:57-63

BACKGROUND: Lipid transfer protein (LTP) is a widely cross-reacting plant pan-allergen. Adverse reactions to Rosaceae, tree nuts, peanut, beer, maize, mustard, asparagus, grapes, mulberry, cabbage, dates, orange, fig, kiwi, lupine, fennel, celery, tomato, eggplant, lettuce, chestnut and pineapple have been recorded . OBJECTIVE: To detect vegetable foods to be regarded as safe for LTP-allergic patients . METHODS: Tolerance/intolerance to a large spectrum of vegetable foods other than Rosaceae, tree nuts and peanut was assessed by interview in 49 subjects monosensitized to LTP and in three distinct groups of controls monosensitized to Bet v 1 (n = 24) or Bet v 2 (n = 18), or sensitized to both LTP and birch pollen (n = 16), all with a history of vegetable food allergy. Patients and controls underwent skin prick test (SPT) with a large spectrum of vegetable foods. The absence of IgE reactivity to foods that were negative in both clinical history and SPT was confirmed by immunoblot analysis and their clinical tolerance was finally assessed by open oral challenge (50 g per food) . RESULTS: All patients reported tolerance and showed negative SPT to carrot, potato, banana and melon; these foods scored positive in SPT and elicited clinical symptoms in a significant proportion of patients from all three control groups. All patients tolerated these four foods on oral challenge. Immunoblot analysis confirmed the lack of IgE reactivity to these foods by LTP-allergic patients . CONCLUSION: Carrot, potato, banana and melon seem safe for LTP-allergic patients. This finding may be helpful for a better management of allergy to LTP.

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[140] - Aleman A, Sastre J, Quirce S, Cuesta J, De Las Heras M, Carnés J, et al. Heterogeneity of kiwi allergy: recognition of allergens in kiwi-allergic patients. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°707

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[141] - Lewis SA, Pearce A, Lucas J, Hourihane J. An in vitro study of the dominant kiwi fruit allergens in a UK population and comparison of the allergenicity of two varieties of kiwi fruit. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°660

Background: To date, different studies have reported different dominant kiwi fruit allergens, possibly due to inter-population variations. The aim of this study was to identify the dominant kiwi fruit allergens in a UK population. We also aimed to compare the in vitro allergenicity of two varieties of kiwi fruit currently available in the UK: the Hayward (Zespri ˙Green) and Zespri˙Gold varieties. Methods: Serum was collected from 30 patients with raised serum kiwi-specific IgE (ten of which also had a positive DBPCFC). IgE binding patterns to a protein extract prepared from Hayward kiwi fruits were determined using western blotting. Protein extracts were prepared from both Hayward (Zespri˙Green) and Zespri˙Gold varieties and analysed using 1 and 2D SDS-PAGE and western blotting with pooled sera. The effects of reducing and non-reducing gel conditions were explored and mass spectrometry was used to identify proteins of interest. Results: 1D IgE binding patterns to the Hayward kiwi fruit extract, separated under reducing conditions, identified two major allergens at ~30 and ~38kDa respectively. These major bands did not change under non-reducing conditions. These allergens, one of which appears to be Act c 1, were not seen in a Zespri˙Gold extract. Using 2D SDS-PAGE, a highly expressed protein in the Hayward variety, with weight and pI features of Act c 1, was missing in the Zespri˙ Gold variety when gels were stained using Coomassie. Identification of the protein as Act c 1 was confirmed by mass spectrometry. Conclusions: There appear to be 2 dominant kiwi fruit (Hayward variety) allergens for the UK population (one of which may be Act c1), with linear IgE binding epitopes. There are differences in the in vitro allergenicity of the Hayward (Zespri˙ Green) and Zespri˙ Gold varieties of kiwi fruit.

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[142] - Asero R, Jimeno L, Barber D. Component-resolved diagnosis of plant food allergy by SPT. Eur Ann Allergy Clin Immunol 2008;40:115-121

BACKGROUND: Fruits and vegetables may contain both labile and stable allergens. The former induce only OAS, whereas stable allergens may induce systemic reactions. Component-resolved diagnosis (CRD) of allergy to plant foods is therefore essential for the clinical management of allergic patients. METHODS: 80 adults allergic to plant foods underwent SPT with purified natural date palm profilin (Pho d 2), purified Mal d 1, a peach extract containing uniquely LTP, and with a kiwi extract containing uniquely stable allergens. RESULTS: 58 (72%) patients were monosensitized: 24 to Mal d 1, 24 to profilin, 7 to LTP, and 3 to kiwi. 22 patients were multi-sensitised: 14 to Mal d 1 and profilin, 2 to Mal d 1 and kiwi, 1 to LTP and profilin, 3 to LTP and Mal d 1, and 2 to LTP, Mal d 1 and profilin. Mal d 1 and LTP sensitisation were associated with apple and peach allergy, respectively, whereas profilin sensitisation was associated with allergy to melon, watermelon, banana, tomato and citrus fruits. 18/21 kiwi-allergic patients were sensitised to one of the cross-reacting allergens, but 2/18 reacted to kiwi-specific allergens as well. CONCLUSIONS: In patients with allergy to plant-derived foods CRD can be performed by SPT with purified allergen proteins. In the future, the availability of a larger number of purified natural or recombinant allergens for SPT will represent a simple means to classify food-allergic patients properly on the first visit.

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[143] - Vocks E, Borga A, Szliska C, Seifert HU, Seifert B, Burow G, et al. Common allergenic structures in hazelnut, rye grain, sesame seeds, kiwi, and poppy seeds. Allergy 1993;48:168-172

Allergy to kiwi, poppy seeds, and/or sesame seeds often occurs in patients with a simultaneous sensitization to nuts and flour. Previously cross reactions have been verified by RAST inhibition. In this study the nature of this cross-reactivity is further characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting to nitrocellulose. The degree of cross-reactivity among kiwi, sesame seeds, poppy seeds, hazelnuts, and rye grain was found to be very high in the patients studied. The existence of both cross-reacting and unique components was observed; however, the cross-reacting and unique components could be different for different patients.

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[144] - Palacin A, Quirce S, Sánchez-Monge R, Fernández-Nieto M, Varela J, Sastre J, et al. Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens. Ann Allergy Asthma Immunol 2008;101:200-205

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

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[145] - Constantin C, Quirce S, Poorafshar M, Touraev A, Niggemann B, Mari A et al. Micro-arrayed wheat seed and grass pollen allergens for component-resolved diagnosis. Allergy 2009;64:1030-1037

BACKGROUND: Wheat is a potent allergen source and can cause baker's asthma, food and pollen allergy. The aim of the study was to develop an allergen micro-array for differential diagnosis of baker's asthma, wheat-induced food allergy and grass pollen allergy . METHODS: We analysed the immunoglobulin-E reactivity profiles of patients suffering from baker's asthma, wheat-induced food allergy and grass pollen allergy to micro-arrayed recombinant wheat flour allergens and grass pollen allergens and compared these results with clinical results and diagnostic tests based on crude wheat flour, wheat pollen and grass pollen allergen extracts . RESULTS: We identified recombinant wheat flour allergens, which are specifically recognized by patients suffering from baker's asthma, but not from patients with food allergy to wheat or pollen allergy. rPhl p 1 and rPhl p 5 were identified as marker allergens specific for grass pollen allergy. They can be used to replace grass pollen extracts for allergy diagnosis and to identify grass pollen allergic patients among patients suffering from baker's asthma and wheat-induced food allergy. Profilin was identified as a cross-reactive allergen recognized by patients suffering from baker's asthma, food and pollen allergy . CONCLUSIONS: Our results indicate that it will be possible to design serological tests based on micro-arrayed recombinant wheat seed and grass pollen allergens for the discrimination of baker's asthma, wheat-induced food allergy and grass pollen allergy.

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[146] - Palacin A, Quirce S, Sánchez-Monge R, Fernández-Nieto M, Varela J, Sastre J, et al. Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens. Ann Allergy Asthma Immunol 2008;101:200-205

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

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[147] - Yagami T, Haishima Y, Nakamura A, Osuna H, Ikezawa Z. Digestibility of allergens extracted from natural rubber latex and vegetable foods. J Allergy Clin Immunol 2000;106:752-762

BACKGROUND: Several cross-reactive allergens are now known to be involved in the defense responses of higher plants. Such proteins are drawing the attention of plant breeders because of their antimicrobial or stress-alleviating activities. Plants genetically modified to express defense-related proteins are being developed. The current concern is focused on the allergenicity of these intentionally expressed proteins. OBJECTIVE: It is believed that food allergens are proteins resistant to digestion. Digestibility tests have been accepted as an appropriate method for evaluating the allergenicity of newly introduced proteins. In this study we investigated the usefulness of this method for detecting allergens from natural rubber latex and vegetable foods. METHODS: Proteins were extracted from rubber latex, potato, and 5 kinds of fruits. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were used for the digestibility test. An aliquot of each digest was periodically withdrawn and analyzed. Allergens were detected with pooled sera from individuals with latex allergy or patients given a diagnosis of oral allergy syndrome. RESULTS: Most latex and vegetable food proteins were digested by the SGF within 4 minutes. Numerous allergens were also decomposed by the SGF within 8 minutes. Although vegetable food allergens were relatively stable in the SIF, kiwi allergens were substantially degraded by the SIF within 16 hours. CONCLUSION: The pronounced lability of the plant-derived allergens was thought to reflect the discrete sensitization and elicitation processes of patients with latex-fruit syndrome or oral allergy syndrome. These results indicate that the allergenicity of a newly expressed protein should be carefully evaluated according to not only its digestibility but also other important properties.

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[148] - Fu TJ, Abbott UR, Hatzos C. Digestibility of food allergens and nonallergenic proteins in simulated gastric fluid and simulated intestinal fluid-a comparative study. J Agric Food Chem 2002;50:7154-7160

Information on the comparative digestibility of food allergens and nonallergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. In this work, we compared the digestive stability of a number of food allergens and proteins of unproven allergenicity and examined whether allergens possess a higher stability than nonallergenic proteins of similar cellular functions, and whether there is a correlation between protein digestibility and allergenicity. The stability of groups of storage proteins, plant lectins, contractile proteins, and enzymes, both allergens and proteins with unproven allergenicity, in a standard simulated gastric fluid and a standard simulated intestinal fluid was measured. Food allergens were not necessarily more resistant to digestion than nonallergenic proteins. There was not a clear relationship between digestibility measured in vitro and protein allergenicity.

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[149] - Bublin M, Radauer C, Knulst A, Wagner S, Scheiner O, Mackie AR, et al. Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein. Mol Nutr Food Res 2008;52:1130-1139

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

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[150] - Bublin M, Radauer C, Knulst A, Wagner S, Scheiner O, Mackie AR, et al. Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein. Mol Nutr Food Res 2008;52:1130-1139

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

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[151] - Polovic N, Blanusa M, Gavrovic-Jankulovic M, Atanaskovic-Markovic M, Burazer L, Jankov R, et al. A matrix effect in pectin-rich fruits hampers digestion of allergen by pepsin in vivo and in vitro. Clin Exp Allergy 2007;37:764-771

Abstract Background It is a general belief that a food allergen should be stable to gastric digestion. Various acidic plant polysaccharides, including pectin, are ubiquitous in fruit matrixes and can form hydrogels under low-pH conditions. Objective The purpose of this study was to investigate the effect of hydrogel forming polysaccharide-rich fruit matrixes on in vivo gastric and in vitro pepsic digestion of fruit allergens. Methods Fruit extract proteins (kiwi, banana, apple and cherry) and a purified major kiwi allergen Act c 2 were digested with simulated gastric fluid in accordance with the US Pharmacopeia. In vivo experiments on kiwi fruit digestion were performed on four healthy non-atopic volunteers by examining the gastric content 1 h after ingestion of kiwi fruit. The Act c 2 and kiwi proteins were detected in immunoblots using monoclonal anti-Act c 2 antibodies and rabbit polyclonal antisera. Results Crude fruit extracts were resistant to digestion by pepsin when compared with commonly prepared extracts. In the gastric content of all volunteers, following kiwi fruit ingestion and immunoblotting, intact Act c 2 was detected with anti-Act c 2 monoclonal antibodies, while kiwi proteins of higher molecular weights were detected using rabbit polyclonal antisera. Addition of apple fruit pectin (1.5% and 3%) to the purified kiwi allergen was able to protect it from pepsin digestion in vitro. Conclusion The matrix effect in pectin-rich fruits can influence the digestibility of food proteins and thereby the process of allergic sensitization in atopic individuals.

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[152] - Bublin M, Radauer C, Knulst A, Wagner S, Scheiner O, Mackie AR, et al. Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein. Mol Nutr Food Res 2008;52:1130-1139

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

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[153] - Lucas JSA, Cochrane SA, Warner JO, Hourihane JOB. The effect of digestion and pH on the allergenicity of kiwifruit proteins. Pediatr Allergy Immunol 2008;19:392-398

It is suggested that patients with oral allergy syndrome (OAS) respond to pepsin-sensitive allergens, and systemic reactors identify pepsin-resistant allergens. We sought to assess the digestibility of kiwifruit proteins in simulated gastric fluid (SGF), and to compare the immunogenicity of the digests in patients with isolated oral and systemic reactions to kiwifruit. In addition, the effect of pH on digestibility of kiwifruit proteins was investigated. The in vitro resistance of kiwifruit proteins to digestion was determined using SGF. G-immunoglobulin (IgE) binding to digested proteins was investigated by Western blotting using sera from children and adults (aged 5-72 yr) with systemic reactions and patients with isolated oral symptoms. To determine whether pH conditions influence digestion of kiwifruit extracts, digestion at pHs 1.5-7 were compared by SDS-PAGE. Patients with systemic reactions showed IgE binding to digestion-resistant allergens, but patients with oral symptoms reacted only to digestion-labile allergens. An increase in pH from 1.5 to 2.5 significantly reduced pepsin breakdown of kiwifruit allergens. Immunoreactive digested protein fragments were detectable by immunoblot but not Coomassie stain. This study confirms a difference in the lability of food allergens recognized by patients with systemic reactions and those with OAS. Pepsin digestion of kiwifruit proteins was impaired by hypoacidic conditions suggesting that patients with hypoacidic gastric conditions are at increased risk of systemic absorption of allergens. The data indicate that commonly used methods for predicting allergenicity of novel proteins using Coomassie stains may be flawed.

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[154] - Gall H, Kalveram KJ, Forck G, Sterry W. Kiwi fruit allergy: a new birch pollen-associated food allergy. J Allergy Clin Immunol 1994;94:70-76

To determine the cross-reacting antigens of kiwi fruit and other foods and pollen, we investigated 22 patients allergic to kiwi fruit: 10 with severe systemic reactions and 12 with localized symptoms confined to oral and pharyngeal mucosa (oral allergy syndrome). Seven patients with birch pollen allergy who tolerated kiwi fruit were included as a control group. METHODS: All patients were evaluated by skin testing and RAST; three patients were evaluated by RAST inhibition assays. RESULTS: Prick tests showed positive reactions to kiwi fruit in all patients, whereas specific IgE to kiwi fruit could be demonstrated only in patients with generalized severe symptoms. Surprisingly, all 22 patients with clinical kiwi allergy showed positive prick test results and elevated IgE to birch pollen. Clinically, all complained of rhinitis during birch pollen season. Many patients showed sensitization to grass and mugwort pollen. Also, food allergy was found to be associated with kiwi allergy: we found strong reactions to apple and hazelnut; moderate reactions to carrot, potato, and avocado; and weak reactions to wheat and rye flour, pineapple and papaya, and their enzymes bromelain and papain. RAST inhibition studies revealed cross-reacting antigens between birch pollen and kiwi fruit. Interestingly, patients with birch pollen allergy without clinical signs of kiwi allergy had positive prick test reactions to kiwi. Patients with kiwi allergy showed higher concentrations to birch pollen IgE compared with patients with isolated birch pollen allergy. CONCLUSIONS: Our results indicate that kiwi allergy is a new manifestation of birch pollen-associated food allergy and is mediated by cross-reacting antigens in the kiwi fruit. Kiwi allergy can be expected in patients with birch pollen allergy exhibiting high levels of IgE to birch pollen.

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[155] - Besler M, Fahlbusch B, Quirce S. Allergen data collection: kiwi fruit (Actinidia chinensis). Internet Symp Food Allergens 2000;2(suppl. 2):1-11

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[156] - Fiocchi A, Restani P, Bernardo L, Martelli A, Ballabio C, D’Auria E, et al. Tolerance of heat-treated kiwi by children with kiwifruit allergy. Pediatr Allergy Immunol 2004;15:454-458

Kiwifruit allergy is increasing among children but whether heating affects clinical tolerance to kiwifruit is unknown. To assess tolerance to heated kiwifruit in children allergic to fresh kiwifruit. In this prospective trial, 20 children (median age 9.4 yr) with a history of immediate allergic reactions to fresh kiwifruit underwent double-blind placebo-controlled food challenges with steam-cooked (100 degrees C for 5') and industrially homogenised kiwifruit. Skin prick tests with a commercial kiwifruit allergen, raw kiwifruit and double-blind placebo-controlled food challenge with 25 g of fresh kiwifruit were used to confirm the history. Specific kiwifruit IgE to native and homogenized fruit were identified by immunoblotting. Fresh kiwifruit induced positive skin prick wheals in all children (confirmed during challenge in 19 patients). Commercial skin prick test elicited a positive response in five children, steam-cooked kiwifruit in five, and the homogenised kiwifruit preparation in none. UniCAP determinations were positive for kiwifruit in three patients. All children's sera showed specific IgE at immunoblotting with raw kiwifruit and one with the homogenised preparation (major allergens identified: Act c 1 and Act c 2). There was no clinical reactivity following challenge with homogenised kiwifruit but one child reacted to cooked kiwifruit challenge. Industrial heat treatment and homogenisation can make kiwifruit safe for children who are allergic to this increasingly popular fruit. This has dietary implications for children who are allergic to several fruit and vegetable proteins.

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[157] - Bublin M, Radauer C, Knulst A, Wagner S, Scheiner O, Mackie AR, et al. Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein. Mol Nutr Food Res 2008;52:1130-1139

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

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[158] - Lucas JSA, Cochrane SA, Warner JO, Hourihane JOB. The effect of digestion and pH on the allergenicity of kiwifruit proteins. Pediatr Allergy Immunol 2008;19:392-398

It is suggested that patients with oral allergy syndrome (OAS) respond to pepsin-sensitive allergens, and systemic reactors identify pepsin-resistant allergens. We sought to assess the digestibility of kiwifruit proteins in simulated gastric fluid (SGF), and to compare the immunogenicity of the digests in patients with isolated oral and systemic reactions to kiwifruit. In addition, the effect of pH on digestibility of kiwifruit proteins was investigated. The in vitro resistance of kiwifruit proteins to digestion was determined using SGF. G-immunoglobulin (IgE) binding to digested proteins was investigated by Western blotting using sera from children and adults (aged 5-72 yr) with systemic reactions and patients with isolated oral symptoms. To determine whether pH conditions influence digestion of kiwifruit extracts, digestion at pHs 1.5-7 were compared by SDS-PAGE. Patients with systemic reactions showed IgE binding to digestion-resistant allergens, but patients with oral symptoms reacted only to digestion-labile allergens. An increase in pH from 1.5 to 2.5 significantly reduced pepsin breakdown of kiwifruit allergens. Immunoreactive digested protein fragments were detectable by immunoblot but not Coomassie stain. This study confirms a difference in the lability of food allergens recognized by patients with systemic reactions and those with OAS. Pepsin digestion of kiwifruit proteins was impaired by hypoacidic conditions suggesting that patients with hypoacidic gastric conditions are at increased risk of systemic absorption of allergens. The data indicate that commonly used methods for predicting allergenicity of novel proteins using Coomassie stains may be flawed.

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[159] - Gavrovic-Jankulovic M, Polovic N, Prisic S, Jankov R, Atanaskovic-Markovic M, Vuckovic O, et al. Allergenic potency of kiwi fruit during fruit development. Food Agric Immunol 2005;16:117-128

Food allergies, including kiwi fruit allergy, have been the subject of extensive research in the last few years. The aim of this study was to examine a possible relationship between the developmental stage of kiwi fruit and its allergenic potency. The protein and allergen patterns of kiwi fruit extracts in September, October, November and December fruit in the period from 2000ˆ2002 were analysed. One of the factors that may contribute to the difficulties in proposing well-defined and standardized fruit extracts should also be the time of fruit harvesting. In this particular case, when the kiwi fruit was edible throughout November and December, we showed discrepancies in allergen content and potencies both in qualitative and quantitative terms. Two major allergens of kiwi fruit, Act c 1 and Act c 2, mainly accounted for the highest allergenic potential of November kiwi extract in vivo and in vitro . Not only the content of major allergens, but also the ratio of different proteins and even isoforms of the same allergen (Act c 2) change with fruit ripening. These findings should be taken into account during preparation of extracts for allergy diagnosis.

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[160] - Giangrieco I, Tuppo L, Palazzo P, Camardella L, Bernardi ML, Scala E, et al. Analysis of green and gold kiwi fruit protein pattern as a function of the ripening stage. Allergy 2007;62(suppl. 83):341

Background: Allergy to kiwi fruit has been the object of extensive investigation in the last years. Probably due to the experimental procedures used and/or to the tissue characteristics, the extracts can be variable both in the number and the amount of their components. In order to verify if the ripening process of the fruit affects the allergen pattern, the analysis of the protein components in the edible part of green (GrK) and gold kiwi (GoK) fruit, at different ripening stages, has been undertaken. Methods: Samples of GrK and GoK fruits were collected every two weeks from July to November/December 2006. Kiwi fruits were from Italian plantations, coming from nearby areas. For each fruit sample a soluble protein fraction (SF) and a saline extract of the cell wall (CW) were obtained. The total protein content of the extracts was determined by means Biorad Protein Assay (Bio-Rad, CA). The protein pattern of the SF and CW fractions was analyzed by SDS-PAGE and the identification by N-terminal amino acid sequencing of the electroblotted components has been undertaken by means Applied Biosystems Procise 492 Automatic Sequencer (Applied Biosystems, CA). Results: Ten samples have been collected in the frame period for each species. The total protein content of both GrK and GoK fruit increased during the ripening process. The two kiwi species showed a different protein pattern upon SDS-PAGE. In GrK fruit, the allergens actinidin, thaumatin-like protein (TLP) and kiwellin were already observed in the fruit samples collected in July and their amount progressively increased. During the ripening process two new protein bands were observed at 20 and 17 kDa, respectively, becoming progressively more abundant. Samples of GoK fruit showed less variations than GrK. The TLP was observed in significant amount in all the GoK kiwi samples. High amount of a double protein band at 16 kDa, absent in earlier stages, was also observed. Preliminary comparative evaluation of SF and CW fractions showed differences in the localization of allergenic proteins in fruit tissues. Conclusion: Data so far obtained show that the protein pattern of GrK and GoK fruit is affected by both the ripening process and extraction methods. Several differences in whole proteins and allergenic contents have been recorded comparing the two kiwi species. Immunochemical studies are needed in order to verify differences in the allergenicity pattern.

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[161] - Nieuwenhuizen NJ, Beuning LL, Sutherland PW, Sharma NN, Cooney JM, Bieleski LRF, et al. Identification and characterisation of acidic and novel basic forms of actinidin, the highly abundant cysteine protease from kiwifruit. Funct Plant Biol 2007;34:946-961

Abstract Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

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[162] - Ciardiello MA, Giangrieco I, Tuppo L, Tamburrini M, Buccheri M, Palazzo P et al. Influence of the Natural Ripening Stage, Cold Storage, and Ethylene Treatment on the Protein and IgE-Binding Profiles of Green and Gold Kiwi Fruit Extracts. J Agric Food Chem 2009;57:1565-1571

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

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[163] - Bublin M, Radauer C, Knulst A, Wagner S, Scheiner O, Mackie AR, et al. Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein. Mol Nutr Food Res 2008;52:1130-1139

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

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[164] - Lucas JSA, Collins K, Grimshaw K, Warner J, Hourihane J. The Clinical Investigation of Kiwi Fruit Allergy. AAAAI 60th Annual Meeting, San Francisco, 19-23 March 2004, Poster n°477

Rationale Allergy to kiwi fruit appears increasingly common, but few studies have evaluated its clinical characteristics. Methods 273 subjects with a history suggestive of allergy to kiwi completed a questionnaire. 45 were investigated by double blind placebo controlled food challenge (DBPCFC), prick-to-prick skin testing with fresh kiwi pulp, and measuring CAP specific IgE. 19 subjects were also skin tested using a commercially available solution. Results The most frequently reported symptom was oral pruritus (65%), but severe symptoms (wheeze, cyanosis or collapse) were reported by 18% of subjects. Young children were significantly more likely than adults to react on their first known exposure (p<0.001), and to report severe symptoms (p=0.008). 23 of 45 subjects (50%) had allergy confirmed by DBPCFC. Prick to prick skin test with fresh kiwi was positive in 95% of subjects who had allergy confirmed by DBPCFC, but also in 69% of subjects with a negative food challenge. The commercial extract was significantly less sensitive, but with fewer false positive reactions. CAP sIgE was only positive in 60% of subjects who had a positive challenge. Conclusions DBPCFC confirmed allergy to kiwi fruit in 50% of the subjects tested, who had a previous history suggestive of kiwi allergy. Skin testing with fresh fruit has good sensitivity (95%), but poor specificity (31%) in this population. CAP sIgE and a commercially available skin test solution were both much less sensitive (60%; 72%) but had better specificity (83%; 67%). Kiwi fruit should be considered a significant food allergen particularly for young children.

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[165] - Lucas JSA, Grimshaw KEC, Collins WK, Warner JO, Hourihane JOB. Kiwi fruit is a significant allergen and is associated with differing patterns of reactivity in children and adults. Clin Exp Allergy 2004;34:1115-1121

BACKGROUND: Allergy to kiwi fruit appears increasingly common, but few studies have evaluated its clinical characteristics, or evaluated methods of investigating the allergy . OBJECTIVE: To characterize the clinical characteristics of kiwi fruit allergy and to study the role of double-blind placebo-controlled food challenge (DBPCFC), skin tests and specific IgE in the diagnosis of this food allergy . METHODS: Two-hundred and seventy-three subjects with a history suggestive of allergy to kiwi completed a questionnaire. Forty-five were investigated by DBPCFC, prick-to-prick skin testing with fresh kiwi pulp, and specific IgE measurement. Nineteen subjects were also skin tested using a commercially available solution . RESULTS: The most frequently reported symptoms were localized to the oral mucosa (65%), but severe symptoms (wheeze, cyanosis or collapse) were reported by 18% of subjects. Young children were significantly more likely than adults to react on their first known exposure (P<0.001), and to report severe symptoms (P=0.008). Twenty-four of 45 subjects (53%) had allergy confirmed by DBPCFC. Prick-to-prick skin test with fresh kiwi was positive in 93% of subjects who had allergy confirmed by DBPCFC, and also in 55% of subjects with a negative food challenge. The commercial extract was significantly less sensitive, but with fewer false-positive reactions. CAP sIgE was only positive in 54% of subjects who had a positive challenge . CONCLUSIONS: Kiwi fruit should be considered a significant food allergen, capable of causing severe reactions, particularly in young children. DBPCFC confirmed allergy to kiwi fruit in 53% of the subjects tested, who had a previous history suggestive of kiwi allergy. Skin testing with fresh fruit has good sensitivity (93%), but poor specificity (45%) in this population. CAP sIgE and a commercially available skin test solution were both much less sensitive (54%; 75%) but had better specificity (90%; 67%).

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[166] - Florido Lopez JF, Quiralte Enriquez J, Arias de Saavedra Alías JM, Saenz de San Pedro B, Martin Casañez E. An allergen from Olea europaea pollen (Ole e 7) is associated with plant-derived food anaphylaxis. Allergy 2002;57(suppl. 71):53-59

Background: Several cross-reacting proteins have been identified as responsible of the co-occurrence of pollinosis and plant-derived food allergy. This association has been mainly described in the birch-apple syndrome but other pollens such as Olea europaea and other fruits may also contain homologous proteins. Objective: To evaluate the associations between sensitization to allergens of Olea europaea pollen and confirmed plant-derived food allergy, in addition to investigate if any pattern of clinical hypersensitivity of food allergy reaction (oral allergy syndrome (OAS) or anaphylaxis) and/or any fresh fruit or nut allergy, are associated to one or several Olea pollen allergen(s). Methods: 134 consecutive patients diagnosed with pollinosis by Olea were studied. Of these patients only 40, reported adverse reaction to plant-derived food. Twenty-one (group A) were classified as OAS and 19 (group B) as anaphylaxis. Skin-tests with six Olea pollen allergens and several groups of fruits, were performed. Double-blind placebo-controlled food challenge (DBPCFC), confirmed the diagnostics of food allergy with the exception of patients who suffered previous anaphylactic reaction. Results: All patients, showed a positive skin prick test (SPT), against one or more of Olea europaea allergens. Sensitization to Ole e 7, was more frequent (P = 0.02) in patients from group B. A total of 84 DBPCFC were performed with 44% positive results. Challenge confirmed at least the 50% of positive SPT in any case (peach: 68.42%; pear: 50%; melon: 71.42% and kiwi: 53.84%). In patients from group B, significant association with O. europaea pollen allergens were found between positive SPT to Rosaceae fruits and Ole e 3 (P = 0.045) and Ole e 7 (P = 0.03); Cucurbitaceae and Ole e 7 (P = 0.03) and Actinidiaceae with Ole e 3 (P = 0.04). Conclusions: The results of this study, establish a new spectrum of associations between pollens and plant-derived foods: sensitization to olive profilin (Ole e 2) is not more frequent in OAS patients. Patients with anaphylactic reaction after eating fruit are also sensitized to Ole e 7, a LTP present in Olea pollen, and suffer pollinic symptoms. Finally a polcalcin (Ole e 3) could be also associated to Olea pollen respiratory and food allergy.

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[167] - Palacin A, Rodriguez J, Blanco C, Lopez-Torrejon G, Sanchez-Monge R, Varela J, et al. Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms. Clin Exp Allergy 2008;38:1220-1228

BACKGROUND: Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients . OBJECTIVE: To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population . METHODS: A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs . RESULTS: The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity . CONCLUSION: Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

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[168] - Levy DA, Mounedji N, Noirot C, Leynadier F. Allergic sensitization and clinical reactions to latex, food and pollen in adult patients. Clin Exp Allergy 2000;30:270-275

Many latex-allergic patients are sensitized to one or more foods. Patients allergic to tree and/or grass pollens are also often sensitized to plant-derived foods. Atopy, defined in most studies as sensitivity to an aeroallergen, is a risk factor for latex allergy. The relative importance of pollen sensitivity, a sign of atopy, as a risk factor for food allergy in latex-allergic patients has not, however, been examined. OBJECTIVE: To investigate the relationship between pollen sensitivity and sensitivity to food in latex-allergic patients. METHODS: Forty-four latex-allergic patients (Groups 1 and 2), 24 of whom were also allergic to tree and/or grass pollen (Group 1) and 25 pollinosis patients who were not allergic to latex (Group 3) were studied. We obtained a history of reactions to food and skin tested them with 12 fresh-frozen fruits. RESULTS: All 12 foods induced a skin test reaction in at least one patient in each of the three Groups. There were, however, twice as many positive skin test reactions to food in patients with pollinosis, whether or not they were allergic to latex, as there were in patients allergic to latex but not to pollen. Latex-allergic patients were most likely to have a positive skin test and a history of a reaction to avocado or banana whereas patients with pollinosis only were most likely to have a positive skin test and a history of a reaction to apple, peach or celery. CONCLUSIONS: These results suggest that concomitant allergy to pollen is an important risk factor in determining which plant-derived foods sensitize latex-allergic patients.

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[169] - Casasnovas P, Oliver A, Sanchez-Jimenez M, Rodriguez J, Crespo JF. Receiver operating characteristics analyses of allergen skin test and specific IgE in the diagnosis of clinical allergy to kiwi. Allergy Clin Immunol Int 2005;17(Suppl. 1):67

Background: Few studies have evaluated IgE mediated hypersensitivity to kiwi with details of the diagnostic value of clinical investigation by skin tests and specific IgE antibody assay compared with a double-blind placebo-controlled food challenges (DBPCFC). Purpose: To evaluate the receiver operating characteristic (ROC) analysis in the diagnosis of clinical allergy to kiwi. Methods: Eighty six subjects (age range, 15-68 years) who reported a history of adverse reactions to kiwi underwent a systematic investigation of food allergy following the recommendations of the EAACI. Skin testing was performed by the prick-prick method using fresh kiwi. The quantifications of kiwispecific IgE antibody were performed by the Pharmacia UniCAP 100 System. Patients first underwent an open food challenge, unless a convincing history of severe anaphylaxis. Positive results of open food challenge were subsequently evaluated by double-blind placebo-controlled food challenges (DBPCFC). Negative results of DBPCFC were further evaluated by a final open feeding. The optimal threshold value for the average net orthogonal diameter of the skin wheal induced by the prick-prick testing and the level of kiwi-specific IgE were selected using the best operating point from the area under the resulting receiver-operating characteristic (ROC) curve predicting positive DBPCFC result. Results: Ten patients reported severe systemic reactions which precluded oral provocations. A total of 15 patients were considered as placebo reactors (10 patients reacted after ingestion of just placebo and 5 patients reacted after ingestion of both placebo and verum). In the remaining 61 patients, the result of the DBPCFC with kiwi was conclusive (n = 22 patients; positive). The estimated area under the curve for skin testing and specific IgE were 0.711 and 0.681, respectively. With results of skin testing and kiwi-specific IgE greater than 7 mm or 1.0 kUA/L cut-off points, the positive predictive values were respectively 80% and 75%. Conclusions: The quantitative analysis of the diameter of the skin wheal induced by the prick-prick testing and the level of specific IgE to kiwi could be useful predictors of clinical allergy and should be considered when selecting patients for oral challenge with this food.

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[170] - Lucas JSA. Relationship of Kiwi Fruit Allergens to Clinical Characteristics. J Allergy Clin Immunol 2005;115(2 suppl.):S93

RATIONALE: Kiwi allergy appears to be increasing, but information regarding the allergy remains limited. This is the first study to investigate a relationship between IgE binding to kiwi allergens and the clinical characteristics of the allergy METHODS: Kiwi proteins were separated by 1D- and 2D-SDS-PAGE. 5 proteins were investigated by MALDI-TOF and ESI-NanoLC tandem MS to obtain de novo sequence data. IgE binding to proteins by Western blotting was assessed using sera from 57 individuals with reported kiwi allergy, 40 of whom had undergone DBPCFC, (age range 4-72 years; symptom severity range anaphylaxis-OAS). Blot images were analyzed to estimate molecular weight and peak intensity of each band. Associations between the presence or absence of a band with the following variables were sought: symptom severity, age of subjects, outcome of DBPCFC, and associated allergies. Correlations between these variables and the maximum band intensity recorded for a patient blot, or the sum of band intensities for an individual blot were sought RESULTS: 1D gels revealed 6 major protein bands, and 2D analysis showed 15 discrete protein spots. De novo sequencing of 3 proteins showed sequence homology with pathogenesis related proteins. Subject sera identified 17 IgE binding bands. Only the 36kDa band was bound by >50% of patients. No associations were found between the binding characteristics and subjects‚ clinical characteristics, including symptom severity CONCLUSIONS: This UK population recognizes different kiwi allergens to those described in predominantly birch pollen areas, or in Southern Europe. IgE recognition of individual kiwi allergens does not predict severity of clinical symptoms

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[171] - Palacin A, Rodriguez J, Blanco C, Lopez-Torrejon G, Sanchez-Monge R, Varela J, et al. Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms. Clin Exp Allergy 2008;38:1220-1228

BACKGROUND: Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi-sensitized [skin prick test (SPT)-positive] and truly kiwi-allergic patients . OBJECTIVE: To isolate major kiwifruit allergens, and to explore their relevance by in vitro and in vivo methods in a large kiwi-sensitized and -allergic population . METHODS: A large group (n=92) of kiwi-sensitized patients with different clinical symptoms were selected, and double-blind, placebo-controlled, food challenges to kiwi were performed in 52 of them. The three major IgE-binding proteins from kiwifruit extracts were isolated and characterized by N-terminal amino acid sequencing and molecular size and glycosylation analysis. The allergenic potency of the three kiwi allergens, and of avocado Pers a 1 as a model allergen associated with the latex-fruit syndrome, was tested by specific IgE quantitation, immunodetection assays and SPTs . RESULTS: The isolated kiwifruit allergens were identified as actinidin Act d 1, glycosylated thaumatin-like Act d 2 and a novel 40 kDa glycoprotein designated as Act d 3.02. Specific IgE to each of the three allergens was found in over 60% of sera from kiwi-sensitized patients, and Act d 1 and Act d 2 induced positive SPT responses in over 50% of the tested patients. A significant link between IgE levels to Act d 1 and Act d 3 and anaphylaxis was uncovered. Avocado Pers a 1 showed an in vitro sensitization prevalence of around 45%, but a low in vivo reactivity . CONCLUSION: Act d 1, Act d 2 and Act d 3 are major allergens in the population studied. Severe symptoms after kiwi ingestion are associated with high IgE levels to Act d 1 and Act d 3.

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[172] - Lidholm J. Allergens and allergy diagnosis in Europrevall : component resolved diagnosis of food allergy. Communication, 28th Congress of EAACI, Warszaw, June 6-10 2009

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[173] - Malandain H, Giroux F, Cano Y. The influence of carbohydrate structures present in common allergen sources on specific IgE results. Eur Ann Allergy Clin Immunol 2007;39:216-220

BACKGROUND: Cross-reactive carbohydrate determinants (CCD) are well known interferants in specific IgE assays (sIgE). Glyco-epitopes are not restricted to CCD and extracts used to prepare in vitro tests contain many other glycoproteins able to bind glycan-specific IgE. The overall amounts of IgE-bindable glycan structures in allergen sources are unknown . OBJECTIVE: We aimed at quantifying the influence of N-glycan structures on IgE reactivity to commonly tested allergen sources . METHODS: IgE reactivity to 51 allergen extracts, one purified natural allergen and 10 recombinant allergens was measured on Phadia UniCAP system using 2 sera demonstrating significant levels of glycan-related IgE reactivity. Immobilized bromelain and horseradish peroxidase (HRP) were used to capture N-glycan-specific IgE from these sera. Residual IgE reactivity was measured for 42 allergen sources and 4 recombinant/purified allergens . RESULTS: An obviously excessive number of positive CAP-results were obtained with both sera, especially for plant-based allergen sources. Capture of glycan-specific IgE led to a decrease of serum IgE ractivity, variable among allergen sources and between sera. Among others, peanut results were proven largely interfered by the presence of glycan-specific IgE. Unexpectedly some allergen sources showed a slight influence of glycan-related reactivity, such as cockroach, mosquito, mussel, shrimp and domestic mites . CONCLUSION: In patients sensitized to pollens or to Hymenoptera venoms sIgE results should be interpreted with caution. One cannot substract the result of a glyco-reporter test (bromelain and/or HRP) in order to compute glycan-free slgE results for common allergen sources like peanuts. As long as the demonstration of a significant role for glycan structures in clinical allergic reactions is lacking, a simple pre-treatment able to discard glycan-specific IgE from serum would be useful to improve accuracy of in vitro diagnostic tests.

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[174] - Fahlbusch B, Rudeschko O, Schumann C, Steurich F, Henzgen M, Schlenvoigt G, et al. Further characterization of IgE-binding antigens in kiwi, with particular emphasis on glycoprotein allergens. J Investig Allergol Clin Immunol 1998;8:325-332

Fruit allergy is frequently associated with birch pollinosis. The aim of this study was to investigate which kiwi allergens were involved in subjects allergic to fruit alone and in patients allergic to both fruit and birch pollen. Sera of nine patients (five with both kiwi and birch pollen allergy and four with isolated kiwi allergy) were studied by immunoblot of kiwi extract. Eight of the nine sera reacted with the 30 kDa protein. Furthermore, IgE-binding proteins were seen at approximately 23 kDa (detected by five sera), 43 kDa and 80 kDa (four sera), and > 80 kDa (two sera). One serum showed no IgE binding to any kiwi allergen. The 30 kDa is the major allergen in kiwi and was purified by anion-exchange chromatography and characterized by isoelectrofocusing and amino acid sequencing. The comparison of its partial amino acid sequence with data from the Swiss Protein Bank revealed that this protein is actinidine. The carbohydrate structures in kiwi and birch pollen extracts were investigated with seven lectins. On kiwi blot, Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues) to the components of 35 to 92 kDa, while concanavalin A (indicating mannose, glucose or N-acetylglucosamine residues) showed weak binding at 67 kDa. In contrast, strong binding of Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A was found on birch pollen blots. The presence of IgE against carbohydrate structures was determined by means of enzyme-linked immunosorbent assay (ELISA) after periodate treatment of kiwi extract. The IgE binding was reduced by periodate treatment of kiwi coated microtiter plates, but not by sera reacting exclusively with the 30 kDa protein. Furthermore, selected sera were treated with proteinase K-digested kiwi and birch pollen extracts as the sources of crossreactive carbohydrate determinants. In accordance with the results of sodium periodate treatment, significant levels of anti-cross-reactive carbohydrate determinant IgE were found in sera from patients allergic to both kiwi and birch pollen. Our results show that the major allergen for kiwi allergy is the 30 kDa protein and additionally that the cross-reaction between kiwi and birch pollen allergy is mainly due to carbohydrate moieties

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Les allergènes du kiwi : les connus, les potentiels, les hypothétiques

Les différents cadres de réactivité au kiwi

Kiwi et stabilité à la chaleur et à la digestion

Kiwi et procédés techno-alimentaires

Kiwi et CCD

Rubriques

Dans la même rubrique

Autres "fruits exotiques"

La figue et autres Moracées

Le syndrome latex-aliments

L’avocat

La banane