La tomate

[1] - Westphal S, Kempf W, Foetisch K, Retzek M, Vieths S, Scheurer S. Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency. Allergy 2004;59:526-532

BACKGROUND: To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy . METHODS: The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed . RESULTS: Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils . CONCLUSION: The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.

↩

[2] - Westphal S, Kolarich D, Foetisch K, Lauer I, Altmann F, Conti A, et al. Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato. Eur J Biochem 2003;270:1327-1337

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Manalpha1-6(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Manalpha1-6(Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

↩

[3] - Lorenz Y, Fötisch K, LeQuynh L, Sonnewald U, Lauer I, Enrique E, et al. Frequency of specific IgE-sensitisation to tomato allergens in German and Spanish patients and identification of a new tomato allergen. Allergy Clin Immunol Int 2005;17(Suppl. 1):344

Background: The prevalence of tomato allergy ranges from about 1.5% to 16% among food-allergic patients. Profilin Lyc e 1 (~14 kDa) and invertase Lyc e 2 (52-60 kDa) were recently described as tomato allergens. Moreover, a tomato protein with an apparent molecular weight of 8 kDa showed IgE-reactivity with sera from a subgroup of tomato-allergic patients. Aim of this study was to identify the 8 kDa protein, and to compare frequencies of IgE binding to tomato allergens in a group of Spanish and German patients. METHODS: Patients (n=74) were recruited in Germany (n=39) and Spain (n=35). The most frequent symptom after ingestion of tomato was the oral allergy syndrome. Purified rLyc e 1 and nLyc e 2 were available from previous studies. The 8 kDa protein was N-terminal sequenced from tomato peel, two recombinant isoforms were cloned, and their secondary structure analysed by circular dichroism spectrometry. Their allergenic potency was tested by in vitro basophil histamine release assay. Prevalence of IgE-binding to crude tomato extract and the three purified tomato allergens was determined by immunoblotting and by EAST. RESULTS: Frequency of IgE binding (German/Spanish patients) to rLyc e 1, nLyc e 2 and the 8 kDa allergen were 29/48%, 38/58%, and 3/32%, respectively. Reactivity to at least one of the three evaluated allergens was 46% for German and 94% for Spanish patients (p<0.0001; Fisher´s exact test). The 8 kDa tomato allergen was identified as non-specific Lipid Transfer Protein (nsLTP) and designated Lyc e 3. Both rLyc e 3 isoforms (Lyc e 3.1 and Lyc e 3.2) exhibited mainly alpha helical secondary structure and appeared to be correctly folded. Sequence identity between the 2 isoforms is 73%. Prevalence of IgEbinding of sera from LTP-sensitised patients was more frequent to Lyc e 3.2 (8/12) than to Lyc e 3.1 (5/11) and specific IgE-titres were higher to Lyc e 3.2 compared to those to Lyc e 3.1. Both isoforms showed similar allergenic potency as indicated by histamine release assay. CONCLUSION: Lyc e 3 is a new minor allergen in tomato. Spanish patients showed a significant higher frequency of IgE-binding to the analysed tomato allergens compared to German patients. Therefore, other tomato allergens which are responsible for sensitisation of German patients should be identified. Also the question whether tomato LTP is the primary sensitising allergen in Spanish patients should be elucidated.

↩

[4] - Reche M, Pascual CY, Vicente J, Caballero T, Muñoz FM, Sanchez S, et al. Tomato allergy in children and young adults: cross-reactivity with latex and potato. Allergy 2001;56:1197-1201

Background: Several studies have shown that allergy to natural rubber latex is associated with cross-reactivity to certain foods such as tomato and potato. The objective was to investigate the clinical and immunologic differences between a group of patients with clinical allergy to tomato and latex and another which had only clinical allergy to tomato. We also aimed to assess, in vitro, the relationship of tomato and latex allergens, which could explain the cross-reactivity. Methods: Forty patients with histories of adverse reactions to tomato and IgE-mediated hypersensitivity were enrolled in the study. Tomato, latex, and potato components were analyzed by SDSˆPAGE immunoblotting. CAP and immunoblot inhibition were used to study allergen cross-reactivity. Results: Patients from group A had a mean age of 13.2 years, and in group B the mean age was 21.7 years. In group B, 9/10 patients belonged to the latex-fruits syndrome. All patients of both groups tolerated potato. Immunoblotting patterns obtained with patients' sera from pool A showed IgE-binding bands to tomato ranging from 44 to 46 kDa and a triple band at 67 kDa. For latex, there was a strong binding at 44 kDa, and potato showed a strong band of 44 kDa and a 67-kDa triple band. In pool B, the binding to the band of 44 kDa in latex and tomato was more intense than in pool A. In pool A, immunoblot inhibition with potato allergen showed an intense inhibition of the three allergens (potato, latex, and tomato); with latex, inhibition was partial and with tomato, a complete inhibition of tomato and latex was observed, and a partial inhibition of potato. In pool B, the inhibition pattern followed a similar tendency to pool A. The CAP inhibition confirmed the high rate of cross-reactivity between tomato, potato, and latex. Conclusions: In our study, tomato, potato, and latex showed a common band of 44ˆ46 kDa probably corresponding to patatin. This protein could be implicated in the high cross-reactivity between tomato, latex, and potato observed in the immunoblot and CAP inhibition.

↩

[5] - Castro L, Metz-Favre C, Villalba M, Purohit A, Rodrigez R, Pauli G et al. Incidence of profilin sensitisation in an ash allergic population with and without tomato sensitisation. Allergy 2009;64(Suppl. 90):106

Aim: To study the incidence of profilin sensitization in 74 rhinitic patients sensitized to ash, 26 of them being also sensitized to tomato. METHODS: 74 patients with Spring rhinitis were tested with ash pollen by prick tests. 30 of them had nasal challenge tests with the same extract (Stallergenes). Tomato allergy was detected by case history and cutaneous tests performed with tomato extract (ALK). IgE determination by ELISA was performed in all patients with tomato extract (in which the presence of profilin had been assessed by using polyclonal rabbit antibodies to profilin from chenopodium album) and recombinant profilin from chenopodium album (rChe a 2). Inhibition experiments with tomato extract and rChe a 2 were performed with pooled sera in immunoblot experiments and with individual sera in ELISA experiments. RESULTS: All patients had positive cutaneous tests to ash pollen and 25 out of 30 had positive nasal challenge tests (threshold <10 IR). Serological tomato sensitization was associated with clinical symptoms in half of the cases (8 OAS and 5 general reactions). The frequency of sensitization to profilin was 38 % in the patients sensitized to tomato and 4 % in the patients without sensitization to tomato. Inhibition experiments performed with the sera of patients with clinical symptoms to tomato showed partial inhibition of the IgE binding to rChe a 2 when tomato extract was used as inhibitor both in ELISA and immunoblot inhibition. CONCLUSIONS: This study confirms that prevalence of profilin sensitization is increased in patients sensitized to ash and tomato and that profilin may be a cross reacting allergen in these patients.

↩

[6] - Ferrer A, Andreu C, de Larramendi C, García-Abujeta J, Huertas J, Tella R, et al. Skin test evaluation of the allergenicity of seven tomato varieties, including peel, pulp, boiled and fried tomato extracts. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1541

Background: There is no evidence that different tomato varieties may express a different allergenicity in vivo. Therefore, we decided to analyse the allergenicity of several commonly consumed tomato varieties by skin testing in a group of tomato skin test positive individuals. Material & Methods: All consecutive individuals evaluated at the outpatient clinics of the participating Hospitals (located in different regions of the Mediterranean coast of Spain) for respiratory and/or cutaneous symptoms were skin tested with a battery of aeroallergens and with peel and pulp of the Canary variety tomato at 10 mg of freeze dried material/ml. Patients were also asked for any food related complaints, including oral allergy syndrome, contact urticaria, angioedema, anaphylaxis and/or gastrointestinal symptoms with the ingestion, or contact with tomato. All patients with a positive skin test and/or complaints related with tomatoes were asked to participate in the study and were skin tested with extracts of the following varieties: Canario, Pera, Kumato, Rambo, Raft, Cherry and Rama and canned fried tomato. All were purchased at a local market. The wheal sizes were measured and expressed in squared mm (± SD). Results: One hundred thirty six patients were recruited. The mean wheal sizes produced by the different extracts were: Canary peel: 33.6 ± 36.4; Canary pulp: 9.1 ± 13.2; Rama peel: 26.5 ± 36.7; Rama pulp: 9.8 ± 13.5; Cherry whole: 9.5 ± 14.7; Kumato peel: 26.9 ± 29.3; Kumato pulp: 17.7 ± 22.2; Rambo peel: 26.2 ± 26.9; Rambo pulp: 4.6 ± 8.6; Pera peel: 20.3 ± 26.7; Pera pulp: 13 ± 16.3; Raft peel: 25.3 ± 33; Raft pulp: 7.3 ± 13.5; Canned fried tomato: 11.4 ± 16.8; Rama boiled peel: 19.5 ± 29.5; Rama boiled pulp: 9.8 ± 16. Conclusions: The wheals skin test results obtained with all the peel extracts was, in all cases, greater than that obtained with the respective pulp extracts. The greatest mean skin test reaction induced by the peel extracts was obtained with the Canario and by the pulp extracts with the Kumato variety. Most of the Canario tomato positive patients were sensitised to all varieties studied. The Canario variety seems to be a good variety to diagnose in vivo sensitisation to tomatoes.

↩

[7] - Poza P, Somoza ML, Crespo JF, Izquierdo G, Campo P, Rodriguez J. Diagnostic evaluation of 27 patients reporting adverse reactions to tomato. EAACI 21th Congress, Naples, 1-5 June, 2002, Communication n°59

Background: Sensitization to tomato has been reported in patients with other allergies, particularly latex. IgE inhibition studies have shown cross-reactive components shared by latex, potato and tomato. Nevertheless, less information is available on the clinical relevance of tomato sensitization objectively confirmed by DBPCFCs. The purpose of this investigation was to assess clinical and immunological features of adverse reactions to tomato. Methods: Twenty-seven adult patients (median π28 years) complaining of adverse reactions to tomato were included in the study. Skin prick tests and detection of specific IgE (CAP) were performed in all patients with tomato, latex, pollen and other offending foods. Clinical reactivity to foods was evaluated first by open food challenges (OFCs), unless a convincing history of a recent severe anaphylaxis. Positive OFCs were subsequently evaluated by the DBPCFC method. Results: Twelve and 13 patients had positive skin prick tests and CAP FEIA with tomato, respectively. Two patients reported severe systemic anaphylaxis, which excluded any challenge. Twenty-five patients underwent OFC with tomato. Ten of 25 initial OFCs were positive, but only four of the nine performed DBPCFCs elicited positive results. An OFC induced a severe reaction that precluded further provocations. Overall, seven reactions were considered positive in 16 patients with positive results of skin tests and/or CAP FEIA. The positive predictive value of a skin prick test was 46 and 43% for specific IgE. The most frequent symptom was the oral allergy syndrome. Thirty-two reactions to other foods were confirmed in the seven patients with actual allergy to tomato. The most common foods associated were melon (four patients), watermelon (three), banana (three), kiwi (three), avocado (two), peach (two), chestnut (two), hazelnut (two), and peanut (two). Moreover, three out of seven patients allergic to tomato were found to be sensitive to latex (one patient had anaphylactic episodes upon latex exposure and two had positive results of the used tests with latex). All patients have seasonal rhinitis and/or asthma and positive results of skin tests to several pollens. Conclusions: Tomato is an allergenic source that can induce actual clinical allergy, as established by DBPCFCs. Although most patients experienced oral allergy syndrome, severe anaphylactic reactions could be elicited. Isolated allergy to tomato seems to be infrequent, most patients having allergy to a number of foods, pollen and/or latex.

↩

[8] - Willerroider M, Fuchs H, Ballmer-Weber BK, Focke M, Susani M, Thalhamer J, et al. Cloning and Molecular and Immunological Characterisation of Two New Food Allergens, Cap a 2 and Lyc e 1, Profilins from Bell Pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum). Int Arch Allergy Immunol 2003;131:245-255

BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food . OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins . METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA . RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin . CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.

↩

[9] - Fuchs HC, Willerroider M, Ballmer-Weber B, Susani M, Thalhamer J, Wüthrich B, et al. Molecular and immunological characterisation of two new food allergens, Lyc e 1 and Cap a 2, profilin from bell pepper (Capsicum annuum) and tomato (Lycopersicon esculentum). EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°219

Profilins are highly conserved ubiquitous proteins involved in the regulation of the actin cytoskeleton. In the past years a number of plant profilins have been identified as allergens in pollen from grasses, weeds and trees and in fruits and vegetables. Patients sensitised to profilins usually react to a broad range of inhalant and nutritive plant allergen sources. Here we present the characterisation of profilin from bell pepper (Capsicum annuum) and tomato (Lycopersicon esculentum) as food allergens. RNA was prepared from bell pepper and tomato fruits, respectively, according to standard procedures. The profilin encoding cDNAs sequences were obtained by RT-PCR using primers deduced from the sequence of a previously cloned tomato pollen profilin at the N-terminus and a poly-A primer at the C-terminus. Both profilins encompass 131 amino acid residues with a predicted molecular mass of 14,252 kDa and a pI of 4.44 for bell pepper profilin and 14,252 kDa and a pI of 4.46 for the corresponding protein from tomato. Both protein sequences display 91% identity whereas tomato pollen profilin and tomato fruit profilin share only 75% identity. Both profilin encoding sequences were ligated into a high level expression vector pMW 172 and expressed in E. coli upon induction with IPTG and were purified using their high binding capacity to poly-L-proline. The purified recombinant proteins were tested in IgE immunoblots and ELISAs for their immunological properties, displaying IgE-binding capacities comparable to the natural proteins. The predicted molecular mass was confirmed by size exclusion HPLC. We conclude that we have identified two new members of the protein family of profilins responsible for allergic reactions in a number of profilin sensitised patients.

↩

[10] - Willerroider M, Fuchs H, Ballmer-Weber BK, Focke M, Susani M, Thalhamer J, et al. Cloning and Molecular and Immunological Characterisation of Two New Food Allergens, Cap a 2 and Lyc e 1, Profilins from Bell Pepper (Capsicum annuum) and Tomato (Lycopersicon esculentum). Int Arch Allergy Immunol 2003;131:245-255

BACKGROUND: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food . OBJECTIVES: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins . METHODS: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA . RESULTS: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin . CONCLUSIONS: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food.

↩

[11] - Castro L, Metz-Favre C, Villalba M, Purohit A, Rodrigez R, Pauli G et al. Incidence of profilin sensitisation in an ash allergic population with and without tomato sensitisation. Allergy 2009;64(Suppl. 90):106

Aim: To study the incidence of profilin sensitization in 74 rhinitic patients sensitized to ash, 26 of them being also sensitized to tomato. METHODS: 74 patients with Spring rhinitis were tested with ash pollen by prick tests. 30 of them had nasal challenge tests with the same extract (Stallergenes). Tomato allergy was detected by case history and cutaneous tests performed with tomato extract (ALK). IgE determination by ELISA was performed in all patients with tomato extract (in which the presence of profilin had been assessed by using polyclonal rabbit antibodies to profilin from chenopodium album) and recombinant profilin from chenopodium album (rChe a 2). Inhibition experiments with tomato extract and rChe a 2 were performed with pooled sera in immunoblot experiments and with individual sera in ELISA experiments. RESULTS: All patients had positive cutaneous tests to ash pollen and 25 out of 30 had positive nasal challenge tests (threshold <10 IR). Serological tomato sensitization was associated with clinical symptoms in half of the cases (8 OAS and 5 general reactions). The frequency of sensitization to profilin was 38 % in the patients sensitized to tomato and 4 % in the patients without sensitization to tomato. Inhibition experiments performed with the sera of patients with clinical symptoms to tomato showed partial inhibition of the IgE binding to rChe a 2 when tomato extract was used as inhibitor both in ELISA and immunoblot inhibition. CONCLUSIONS: This study confirms that prevalence of profilin sensitization is increased in patients sensitized to ash and tomato and that profilin may be a cross reacting allergen in these patients.

↩

[12] - Westphal S, Kolarich D, Foetisch K, Lauer I, Altmann F, Conti A, et al. Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato. Eur J Biochem 2003;270:1327-1337

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Manalpha1-6(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Manalpha1-6(Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

↩

[13] - Lorenz Y, Fötisch K, LeQuynh L, Sonnewald U, Lauer I, Enrique E, et al. Frequency of specific IgE-sensitisation to tomato allergens in German and Spanish patients and identification of a new tomato allergen. Allergy Clin Immunol Int 2005;17(Suppl. 1):344

Background: The prevalence of tomato allergy ranges from about 1.5% to 16% among food-allergic patients. Profilin Lyc e 1 (~14 kDa) and invertase Lyc e 2 (52-60 kDa) were recently described as tomato allergens. Moreover, a tomato protein with an apparent molecular weight of 8 kDa showed IgE-reactivity with sera from a subgroup of tomato-allergic patients. Aim of this study was to identify the 8 kDa protein, and to compare frequencies of IgE binding to tomato allergens in a group of Spanish and German patients. METHODS: Patients (n=74) were recruited in Germany (n=39) and Spain (n=35). The most frequent symptom after ingestion of tomato was the oral allergy syndrome. Purified rLyc e 1 and nLyc e 2 were available from previous studies. The 8 kDa protein was N-terminal sequenced from tomato peel, two recombinant isoforms were cloned, and their secondary structure analysed by circular dichroism spectrometry. Their allergenic potency was tested by in vitro basophil histamine release assay. Prevalence of IgE-binding to crude tomato extract and the three purified tomato allergens was determined by immunoblotting and by EAST. RESULTS: Frequency of IgE binding (German/Spanish patients) to rLyc e 1, nLyc e 2 and the 8 kDa allergen were 29/48%, 38/58%, and 3/32%, respectively. Reactivity to at least one of the three evaluated allergens was 46% for German and 94% for Spanish patients (p<0.0001; Fisher´s exact test). The 8 kDa tomato allergen was identified as non-specific Lipid Transfer Protein (nsLTP) and designated Lyc e 3. Both rLyc e 3 isoforms (Lyc e 3.1 and Lyc e 3.2) exhibited mainly alpha helical secondary structure and appeared to be correctly folded. Sequence identity between the 2 isoforms is 73%. Prevalence of IgEbinding of sera from LTP-sensitised patients was more frequent to Lyc e 3.2 (8/12) than to Lyc e 3.1 (5/11) and specific IgE-titres were higher to Lyc e 3.2 compared to those to Lyc e 3.1. Both isoforms showed similar allergenic potency as indicated by histamine release assay. CONCLUSION: Lyc e 3 is a new minor allergen in tomato. Spanish patients showed a significant higher frequency of IgE-binding to the analysed tomato allergens compared to German patients. Therefore, other tomato allergens which are responsible for sensitisation of German patients should be identified. Also the question whether tomato LTP is the primary sensitising allergen in Spanish patients should be elucidated.

↩

[14] - Weangsripanaval T, Nomura N, Moriyama T, Ohta N, Ogawa T. Identification of suberization-associated anionic peroxidase as a possible allergenic protein from tomato. Biosci Biotechnol Biochem 2003;67:1289-1304

↩

[15] - Fötisch K, Son D, Altmann F, Aulepp H, Conti A, Haustein D, et al. Tomato (Lycopersicon esculentum) allergens in pollen-allergic patients. Eur Food Res Technol 2001;213:259-266

Abstract. This study was undertaken to investigate the relevance of tomato allergy in pollen-allergic patients, and to identify and characterize tomato allergens. Therefore, sera of a group of 32 pollen-allergic patients with a history of adverse reactions to tomato were investigated by means of immunoblotting and specific IgE measurements (CAP, EAST, EAST inhibition and CCD [cross-reactive carbohydrate determinants] - ELISA). IgE-reactive spots on 2D blots were analyzed by amino acid microsequencing. Tomato allergy was found to occur with a prevalence of about 9% in our group of birch pollen-allergic patients. Of the patients with adverse reactions to tomato, 44% presented IgE to tomato profilin and 35.5% specific IgE to CCD. Two patients were sensitized to a lipid transfer protein in tomato. Microsequencing of other IgE-reactive proteins, separated by 2D-gel electrophoresis and transferred to PVDF membranes, revealed beta-fructofuranosidase, polygalacturonase 2A, and pectinesterase as further tomato allergens. Our data show that tomato is a relevant allergenic food in pollen-allergic patients. It seems that the majority of pollen-associated allergies to tomato is caused by known ubiquitous allergenic structures such as profilin and cross-reactive IgE-binding N-glycans. Furthermore, lipid transfer protein, as well as polygalacturonase 2A, pectinesterase and beta-fructofuranosidase may represent birch pollen-independent tomato allergens for certain patients.

↩

[16] - Kondo Y, Urisu A, Tokuda R. Identification and Characterization of the Allergens in the Tomato Fruit by Immunoblotting. Int Arch Allergy Immunol 2001;126:294-299

Background: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Methods: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients' sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. Results: Four proteins binding with IgE from more than half of the OAS patients' sera were determined to be polygalacturonase 2A (PG2A), -fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, -fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. Conclusion: The concentrations of 3 of 4 tomato allergens increased during ripening.

↩

[17] - Palomares O, Villalba M, Quiralte J, Polo F, Rodriguez R. 1,3-beta-glucanases as candidates in latex-pollen-vegetable food cross-reactivity. Clin Exp Allergy 2005;35:345-351

Summary Background 1,3-beta-glucanases (group 2 of pathogenesis-related proteins) are enzymes widely distributed among higher plants and have been recently proven to be significant allergens. Objective The aim of this work was to study the potential implication of 1,3-beta-glucanases in cross-reactivities among latex, pollen and vegetable foods. Methods The cDNA encoding the N-terminal domain (NtD) of Ole e 9, a major allergenic 1,3-beta-glucanase from olive pollen, was amplified by polymerase chain reaction and produced as a recombinant protein in Pichia pastoris (recombinant N-terminal domain, rNtD). Circular dichroism, ELISA, immunoblotting and immunoblotting inhibition experiments were carried out. Sera from olive pollen allergic patients and a rNtD-specific polyclonal antiserum were used. Results The NtD of Ole e 9 has been produced at high yield in the yeast P. pastoris and possesses 1,3-beta-glucanase activity. The expressed polypeptide conserves IgE and IgG immunodominant epitopes of the whole Ole e 9. A rNtD-specific polyclonal antiserum and sera from olive pollen allergic patients allowed detection of IgG and IgE reactive peptidic epitopes common to 1,3-beta-glucanase Ole e 9 in extracts from ash and birch pollen, tomato, potato, bell-pepper, banana and latex. Conclusion rNtD and homologous glucanases are new molecules to be used in diagnostic protocols as they could help to identify allergic pollen patients who are at risk for developing allergic symptoms to fruits, vegetables and latex.

↩

[18] - Faurobert M, Mihr C, Bertin N, Pawlowski T, Negroni L, Sommerer N, et al. Major Proteome Variations Associated with Cherry Tomato Pericarp Development and Ripening. Plant Physiol 2007;143:1327-1346

Tomato (Solanum lycopersicum) is a model plant for studying fleshy fruit development. Several genetic and molecular approaches have been developed to increase our knowledge about the physiological basis of fruit growth but very few data are yet available at the proteomic level. Main stages of fruit development were first determined through the dynamics of fruit diameter and pericarp cell number. Then, total proteins were extracted from pericarp tissue, at 6 relevant developmental stages, and separated by two dimensional gel electrophoresis. The protein patterns were markedly different between stages. The proteins showing major variations were monitored. We identified ninety of the 1791 well-resolved spots either by MALDI-TOF peptide mass fingerprinting or LC-MS/MS sequencing and EST database searching. Clustered correlation analysis results pointed out groups of proteins with similar expression profiles during fruit development. In young fruit, spots linked to amino acid metabolism or protein synthesis were m ainly expressed during the cell division stage and down-regulated later. Some spots linked to cell division processes could be identified. During the cell expansion phase spots linked to photosynthesis and proteins linked to cell wall formation transiently increased. In contrast, the major part of the spots related to C-compounds and carbohydrate metabolism or oxidative processes were up-regulated during fruit development showing an increase in spot intensity during development and maximum abundance in mature fruit. This was also the case for the spots linked to stress responses and fruit senescence. We discuss the protein variations taking into account their potential role during fruit growth and comparing our results with already known variations at the mRNA and metabolite profiling levels

↩

[19] - Ferrer A, Huertas AJ, Larramendi CH, Pagán JA, Bartra J, García-Abujeta JL, et al. Antigenic and allergenic differences between green and mature tomatoes. J Investig Allergol Clin Immunol 2008;18:411-412

↩

[20] - Larramendi C, Ferrer A, Huertas A, Pagán J, Navarro L, Lavín J et al. Prevalence of skin test reactivity to tomato extracts of differing ripening state in the Mediterranean coast of Spain. Allergy 2009;64(Suppl. 90):488-489

Background: Tomato (Lycopersicum escolentum) belongs to the Solanaceae family and is a frequently ingested fruit worldwide. Previous studies have demonstrated that there are differences between the allergenicity of green and ripe tomatoes. The objective of this study was to investigate the prevalence of skin test reactivity to tomato at different ripening state in the Mediterranean coast of Spain. Methods: Canary variety tomatoes with different maturation stage were obtained from the same cultivar. Green, ripe and over-ripe tomatoes were peeled. Extracts from different peel tomatoes were prepared and skin prick tested in a double blind multicenter study (including 7 hospitals located in the Mediterranean Coast of Spain). A total of 611 consecutive patients residing in the Mediterranean area of Spain and referring respiratory and/or cutaneous symptoms, were skin prick tested with the 3 extracts and with a battery of common aeroallergens. Results: A total of 72 individuals (Mean age: 31.2 years +/- 14.5; 48.6% males and 51.4% females) residing were positive to at least one of the three extracts (11.5%). Sixty two patients (92.5 %) were positive to overripe, 50 (74.6%) to ripe and 58 (80.6%) to green tomato extract, respectively. Forty nine subjects (68%) were positive to all extracts, 6 to two extracts (8.3%) and 17 to only one extract (23.6%). Sixty one (84.7%) tomato positive skin prick tested patients were sensitised to inhalant aeroallergens: 60 (83.3%) to pollens, 38 (52.8%) to mites, 27 (37.5%) to epithelia and 5 (6.9%) to moulds. Only 10 (13.9%) reported symptoms after contact or ingestion with tomato. Conclusions: The prevalence of sensitisation to tomato is high. Pollen sensitization rate is very high among tomato sensitized individuals. Non-significant differences in the rate of sensitization were obtained between extracts at different ripening stage, but individual differences were seen.

↩

[21] - Ferrer A, Larramendi C, Huertas J, García-Abujeta J, Andreu C, Tella R, et al. Prevalence of Skin Test Reactivity to Tomato Peel and Pulp Extracts in the Mediterranean Coast of Spain. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°1164

RATIONALE: Tomatoes (Lycopersicon esculentum) are widely consumed The prevalence of sensitisation remains unknown. The objective of this study was to determine the prevalence of skin test reactivity to tomato allergens in the Mediterranean coast of Spain METHODS: One thousand and four hundred ninety eight consecutive individuals (675 males and 823 females; mean age, 31.4 ± 17.8 years-old) evaluated for respiratory or/and cutaneous symptoms at the Allergy Units of 4 hospitals located in the Mediterranean coast were included in the study. All patients were skin prick tested with a battery of common inhalant allergens (Laboratorios LETI, S.L.) and with extracts of peel and pulp of ripe Canary Island tomatoes at 10 mg/ml. The Fisher exact test was used to evaluate the results RESULTS: The prevalence of sensitization to tomato was 5.94 % (89/1498 patients; 51 males and 38 females; mean age, 30.2 ± 12.9 years) The tomato peel extract was positive in 87 patients (5.81 %) and the pulp extract in 33 patients (2.2 %); 2 patients were positive exclusively to pulp Eighty five (85.5%) tomato sensitised patients were sensitised to inhalant aeroallergens: 77 (86.5%) to pollens; 52 (58.4%) to mites, 33 (37.1%) to epithelia and 16 (18.0%) to moulds. Most of the sensitized subjects (91.1%) were asymptomatic and did not refer symptoms after tomato contact or ingestion CONCLUSIONS: The prevalence of sensitisation to tomato is high However, most sensitized subjects are asymptomatic. Sensitisation to tomato is more prevalent among pollen sensitised individuals, suggesting allergen cross-reactivity, or a genetic predisposing factors.

↩

[22] - Carnés J, López-Matas M, Ferrer M, Larramendi C, Huertas J, Casanovas M, et al. Immunochemical Characterization of Tomato Peel and Pulp Extracts. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°1165

RATIONALE: Tomatoes (Lycopersicum esculentum) are consumed worldwide and several allergens have been described, including Lyc e 1 (profilin), Lyc e 2 (b-fructofuranosidase) and Lyc e 3 (LTP). The aim of this study was to analyze the in vitro characteristics of peel and pulp tomato extracts in a Spanish population METHODS: Tomatoes of the Canary variety were purchased at a local market. Peel and pulp extracts were prepared, separately. Forty-three patients, residing in the Mediterranean Coast of Spain, with a positive skin prick test to peel extract (10 mg of freeze dried material/ml) were enrolled in the study. Specific IgE to peel and pulp extracts was measured by ELISA. The antigenic and allergenic profiles of the extracts were evaluated by SDS-PAGE, 2-D electrophoresis, IEF and immunoblots RESULTS: SDS-PAGE of the peel extract showed 7 prominent bands in a molecular weigh range of 8 to 50 kDa. The pulp extract had 2 main bands at 8 and 9 kDa. The protein content of the peel extract was 85 mcg/mg and of the pulp extract 37.5 mcg/mg of freeze-dried material Twenty two patients had detectable specific IgE against the peel extract and 4 against the pulp. Thirteen sera were used for immunoblots. Five patients (38%) recognized a 45 kDa band (probably polygalacturonase 2A, or Lyc c 2), 4 (30.8%) a 31 kDa band (probably pectinesterase) and 4 (30.8%) a band of 9 kDa (probably LTP) CONCLUSIONS: Several proteins seem to be implicated in tomato allergy. Most tomato allergic patients are sensitized to peel proteins Funding: Laboratorios LETI, S.L.

↩

[23] - Ferrer A, Andreu C, de Larramendi C, García-Abujeta J, Huertas J, Tella R, et al. Skin test evaluation of the allergenicity of seven tomato varieties, including peel, pulp, boiled and fried tomato extracts. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1541

Background: There is no evidence that different tomato varieties may express a different allergenicity in vivo. Therefore, we decided to analyse the allergenicity of several commonly consumed tomato varieties by skin testing in a group of tomato skin test positive individuals. Material & Methods: All consecutive individuals evaluated at the outpatient clinics of the participating Hospitals (located in different regions of the Mediterranean coast of Spain) for respiratory and/or cutaneous symptoms were skin tested with a battery of aeroallergens and with peel and pulp of the Canary variety tomato at 10 mg of freeze dried material/ml. Patients were also asked for any food related complaints, including oral allergy syndrome, contact urticaria, angioedema, anaphylaxis and/or gastrointestinal symptoms with the ingestion, or contact with tomato. All patients with a positive skin test and/or complaints related with tomatoes were asked to participate in the study and were skin tested with extracts of the following varieties: Canario, Pera, Kumato, Rambo, Raft, Cherry and Rama and canned fried tomato. All were purchased at a local market. The wheal sizes were measured and expressed in squared mm (± SD). Results: One hundred thirty six patients were recruited. The mean wheal sizes produced by the different extracts were: Canary peel: 33.6 ± 36.4; Canary pulp: 9.1 ± 13.2; Rama peel: 26.5 ± 36.7; Rama pulp: 9.8 ± 13.5; Cherry whole: 9.5 ± 14.7; Kumato peel: 26.9 ± 29.3; Kumato pulp: 17.7 ± 22.2; Rambo peel: 26.2 ± 26.9; Rambo pulp: 4.6 ± 8.6; Pera peel: 20.3 ± 26.7; Pera pulp: 13 ± 16.3; Raft peel: 25.3 ± 33; Raft pulp: 7.3 ± 13.5; Canned fried tomato: 11.4 ± 16.8; Rama boiled peel: 19.5 ± 29.5; Rama boiled pulp: 9.8 ± 16. Conclusions: The wheals skin test results obtained with all the peel extracts was, in all cases, greater than that obtained with the respective pulp extracts. The greatest mean skin test reaction induced by the peel extracts was obtained with the Canario and by the pulp extracts with the Kumato variety. Most of the Canario tomato positive patients were sensitised to all varieties studied. The Canario variety seems to be a good variety to diagnose in vivo sensitisation to tomatoes.

↩

[24] - Ferrer A, Larramendi CH, Huertas AJ, García-Abujeta JL, Andreu C, Lavin JR, et al. Prevalence of skin test reactivity to tomato peel and pulp extracts in the Mediterranean coast of Spain. Allergy Clin Immunol Int 2005;17(Suppl. 1):344

Background: Tomatoes are widely consumed in Mediterranean countries and other parts of the world. The prevalence and the relative importance of sensitization to tomato allergens in our area is unknown. OBJECTIVE: To determine the prevalence of skin test reactivity to tomato allergens in the Mediterranean coast of Spain. Material and Methods: Consecutive individuals attending for the first time the Allergy Unit of 3 hospitals in Alicante and Murcia (Spain), who consulted for respiratory or/and cutaneous symptoms in a two month period, were included. Peel and pulp extracts of the Canary tomato variety at 10 mg of freeze dried material/ml were prepared. The antigenic and allergenic profiles of both extracts were analyzed by SDS-PAGE and Immunoblots. The protein content was evaluated by the Lowry-Biuret method. Skin prick tests with the two tomato extracts and a battery of common inhalants (house dust mites, pollens, moulds and animal dander; Laboratorios LETI, S.L.) were performed. Fisher exact test was used for comparison between the non sensitized and the sensitized individuals. RESULTS: Two hundred thirty-six patients, 102 males (47%) and 125 females (53%), (mean age: 28.9; range 1-75 years) were included; 69% (167) were positive to at least one of the inhalants tested, 46% (109) of them to pollens and 43 % (102) to mites. Fourteen patients were sensitized to tomato: 4 to peel and pulp and 10 just to peel. Nine were males and 5 females with a mean age of 31.4 (range 13-75) years; 86% of them were sensitized to inhalant allergens, 4 (29%) to mites (p>0.05) and 11 (79%) to pollens (p<0.01). One patient was monosensitized to tomato and only one reported contact urticaria and oral allergy syndrome after contact with, or ingestion of tomato. Two tomato skin test-negative subjects reported mild symptoms (one, pruritus in hands and the other in mouth) upon contact with tomato. The prevalence of tomato sensitization in this population was 5.93% and that of clinical allergy to tomato was 0.4%. CONCLUSIONS: The prevalence of sensitization to tomato can be considered as high in the population studied, and comparable to that of some inhalant allergens. All positive patients were sensitized to the peel of the tomato variety analysed. Most of the sensitized subjects were asymptomatic and sensitized to pollens.

↩

[25] - Ferrer A, Larramendi C, Huertas J, García-Abujeta J, Andreu C, Tella R, et al. Prevalence of Skin Test Reactivity to Tomato Peel and Pulp Extracts in the Mediterranean Coast of Spain. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°1164

RATIONALE: Tomatoes (Lycopersicon esculentum) are widely consumed The prevalence of sensitisation remains unknown. The objective of this study was to determine the prevalence of skin test reactivity to tomato allergens in the Mediterranean coast of Spain METHODS: One thousand and four hundred ninety eight consecutive individuals (675 males and 823 females; mean age, 31.4 ± 17.8 years-old) evaluated for respiratory or/and cutaneous symptoms at the Allergy Units of 4 hospitals located in the Mediterranean coast were included in the study. All patients were skin prick tested with a battery of common inhalant allergens (Laboratorios LETI, S.L.) and with extracts of peel and pulp of ripe Canary Island tomatoes at 10 mg/ml. The Fisher exact test was used to evaluate the results RESULTS: The prevalence of sensitization to tomato was 5.94 % (89/1498 patients; 51 males and 38 females; mean age, 30.2 ± 12.9 years) The tomato peel extract was positive in 87 patients (5.81 %) and the pulp extract in 33 patients (2.2 %); 2 patients were positive exclusively to pulp Eighty five (85.5%) tomato sensitised patients were sensitised to inhalant aeroallergens: 77 (86.5%) to pollens; 52 (58.4%) to mites, 33 (37.1%) to epithelia and 16 (18.0%) to moulds. Most of the sensitized subjects (91.1%) were asymptomatic and did not refer symptoms after tomato contact or ingestion CONCLUSIONS: The prevalence of sensitisation to tomato is high However, most sensitized subjects are asymptomatic. Sensitisation to tomato is more prevalent among pollen sensitised individuals, suggesting allergen cross-reactivity, or a genetic predisposing factors.

↩

[26] - Ferrer A, Huertas AJ, Larramendi CH, García-Abujeta JL, Bartra J, Lavín JR et al. Usefulness of manufactured tomato extracts in the diagnosis of tomato sensitization: Comparison with the prick-prick method. Clin Mol Allergy 2008;6:1

ABSTRACT: BACKGROUND: Commercial available skin prick test with fruits can be negative in sensitized or allergic patients due to a reduction in biological activity during the manufacturing process. Prick-prick tests with fresh foods are often preferred, but they are a non-standardized procedure. The usefulness of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method in the diagnosis of tomato sensitization has been analyzed.The objective of the study was to assess the potential diagnostic of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method. METHODS: Two groups of patients were analyzed: Group I: 26 individuals reporting clinical symptoms induced by tomato contact or ingestion. Group II: 71 control individuals with no symptoms induced by tomato: 12 of them were previously skin prick test positive to a tomato extract, 39 were atopic and 20 were non-atopic. All individuals underwent prick-prick with fresh ripe peel Canary tomatoes and skin prick tested with freeze-dried peel and pulp extracts obtained from peel and pulp of Canary tomatoes at 10 mg/ml. Wheal sizes and prick test positivity (>/= 7 mm2) were compared between groups. RESULTS: In group I, 21 (81%) out of 26 patients were prick-prick positive. Twenty patients (77%) had positive skin prick test to peel extracts and 12 (46%) to pulp extracts. Prick-prick induced a mean wheal size of 43.81 +/- 40.19 mm2 compared with 44.25 +/- 36.68 mm2 induced by the peel extract (Not significant), and 17.79 +/- 9.39 mm2 induced by the pulp extract (p < 0.01).In group II, 13 (18%) out of 71 control patients were prick-prick positive. Twelve patients (all of them previously positive to peel extract) had positive skin prick test to peel and 3 to pulp. Prick-prick induced a mean wheal size of 28.88 +/- 13.12 mm2 compared with 33.17 +/- 17.55 mm2 induced by peel extract (Not significant), and 13.33 +/- 4.80 mm2 induced by pulp extract (p < 0.05 with peel extract and prick-prick). CONCLUSION: Canary peel tomato extract seems to be as efficient as prick-prick tests with ripe tomatoes to diagnose patients sensitized to tomato. The wheal sizes induced by prick-prick and peel extracts were very similar and showed a high correlation coefficient.

↩

[27] - Larramendi CH, Ferrer A, Huertas AJ, García-Abujeta JL, Andreu C, Tella R, et al. Sensitization to tomato peel and pulp extracts in the Mediterranean Coast of Spain: prevalence and co-sensitization with aeroallergens. Clin Exp Allergy 2008;38:169-177

BACKGROUND: Tomatoes (Lycopersicon esculentum) are consumed world-wide. The prevalence of sensitization to tomatoes remains unknown . OBJECTIVE: To determine the prevalence of skin test reactivity to tomato and to describe the characteristics of tomato-sensitized subjects . METHODS: Individuals attending for the first time during the period of the study to six Allergy centres, located along the Mediterranean coast of Spain, reporting respiratory and/or cutaneous symptoms, were included. All patients were skin prick tested with a battery of inhalant allergens and with peel and pulp of Canary tomato extracts . RESULTS: The study included 1734 individuals (757 males, 977 females; 31.9+/-17.8 years old). The prevalence of sensitization to tomato was 6.52% (113 patients; 65 males, 48 females; 29.5+/-13 years old). The peel extract was positive in 110 patients and the pulp extract in 47 patients; three patients were positive exclusively to pulp. Only 1.8% of individuals reported symptoms with tomato; 44% of them had skin test negative to both extracts. Among tomato-sensitized subjects, 16% reported symptoms with tomato, 97% were sensitized to inhalant aeroallergens, including 84% to pollens (mainly Artemisia vulgaris and Platanus hybrida), with differences between Northern and Southern centres . CONCLUSIONS: The prevalence found of skin test sensitivity to tomato is high. Peel extracts detected most of the sensitized subjects. Most of the sensitized subjects were asymptomatic and some patients reported symptoms without skin test sensitivity. Positive subjects were very frequently sensitized to pollens, suggesting allergen cross-reactivity. Regional differences may exist, possibly related to the pattern of sensitization to cross-reacting pollens.

↩

[28] - Ferrer A, Larramendi C, Huertas J, García-Abujeta J, Andreu C, Tella R, et al. Prevalence of Skin Test Reactivity to Tomato Peel and Pulp Extracts in the Mediterranean Coast of Spain. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°1164

RATIONALE: Tomatoes (Lycopersicon esculentum) are widely consumed The prevalence of sensitisation remains unknown. The objective of this study was to determine the prevalence of skin test reactivity to tomato allergens in the Mediterranean coast of Spain METHODS: One thousand and four hundred ninety eight consecutive individuals (675 males and 823 females; mean age, 31.4 ± 17.8 years-old) evaluated for respiratory or/and cutaneous symptoms at the Allergy Units of 4 hospitals located in the Mediterranean coast were included in the study. All patients were skin prick tested with a battery of common inhalant allergens (Laboratorios LETI, S.L.) and with extracts of peel and pulp of ripe Canary Island tomatoes at 10 mg/ml. The Fisher exact test was used to evaluate the results RESULTS: The prevalence of sensitization to tomato was 5.94 % (89/1498 patients; 51 males and 38 females; mean age, 30.2 ± 12.9 years) The tomato peel extract was positive in 87 patients (5.81 %) and the pulp extract in 33 patients (2.2 %); 2 patients were positive exclusively to pulp Eighty five (85.5%) tomato sensitised patients were sensitised to inhalant aeroallergens: 77 (86.5%) to pollens; 52 (58.4%) to mites, 33 (37.1%) to epithelia and 16 (18.0%) to moulds. Most of the sensitized subjects (91.1%) were asymptomatic and did not refer symptoms after tomato contact or ingestion CONCLUSIONS: The prevalence of sensitisation to tomato is high However, most sensitized subjects are asymptomatic. Sensitisation to tomato is more prevalent among pollen sensitised individuals, suggesting allergen cross-reactivity, or a genetic predisposing factors.

↩

[29] - Bassler OY, Weiss J, Wienkoop S, Lehmann K, Scheler C, Dolle S et al. Evidence for Novel Tomato Seed Allergens: IgE-Reactive Legumin and Vicilin Proteins Identified by Multidimensional Protein Fractionation-Mass Spectrometry and in Silico Epitope Modeling. J Proteome Res 2009;8:1111-1122

Tomato fruit and seed allergens were detected by IgE-immunoblotting using sera from 18 adult tomato-sensitized patients selected based on a positive history skin prick test (SPT) and specific Immunglobulin (Ig) E-levels. Isolated tomato seed total protein showed high SPT activity comparable or even higher than tomato fruit protein. For the molecular characterization of tomato seed allergens, a multidimensional protein fractionation strategy and LC-MS/MS was used. Two legumin- and vicilin-proteins were purified and showed strong IgE-reactivity in immunoblots. Individual patient sera exhibited varying IgE-sensitivity against the purified proteins. In silico structural modeling indicates high homology between epitopes of known walnut allergens and the detected IgE-crossreactive tomato proteins

↩

[30] - Pravettoni V, Primavesi L, Farioli L, Brenna OV, Pompei C, Conti A et al. Tomato Allergy: Detection of IgE-Binding Lipid Transfer Proteins in Tomato Derivatives and in Fresh Tomato Peel, Pulp, and Seeds. J Agric Food Chem 2009;57:10749-10754

There is an increasing consumption of tomatoes worldwide: fresh in salads, cooked in household sauces, or industrially processed. Although many tomato allergens have been identified, there is no information in the literature on the allergenic components found in commercial tomato products. The primary aim of the study was to evaluate the allergenic profile of commercial tomato products by skin prick tests (SPTs) and IgE/immunoblotting in tomato-allergic subjects. The secondary end point was the study of the IgE-binding profile of tomato peel, pulp, and seeds. Forty tomato-allergic patients, reporting oral allergy syndrome (OAS) at different grades of severity for fresh and, in some cases, also for cooked tomato, were selected on the basis of positive tomato allergy history or open food challenge (OFC). They were evaluated by SPTs with different experimental tomato extracts. SDS-PAGE/immunoblotting was performed to detect tomato allergens, which were then identified by Edman degradation. Twenty-three patients (57.5%) presented first-grade OAS at the OFC, whereas 17 (42.5%) reported severe symptoms. Ten of these 17 patients (25%) reported allergic reactions to cooked tomatoes; in immunoblotting tests, their sera reacted only to lipid transfer protein (LTP). In commercial products, LTP was the only detectable allergen. In contrast to other LTP-containing fruits, in tomato, an IgE-binding LTP was identified not only in the peel but also in the pulp and seeds. This study demonstrates that, in fresh tomato, different LTP isoforms are present and allergenic. Industrial tomato derivatives still contain LTP, thus presenting a problem for LTP-allergic patients.

↩

[31] - Hannuksela M, Lahti A. Immediate reactions to fruits and vegetables. Contact Dermatitis 1977;3:79-84

Common fruits and vegetables were tested as such with the "scratch-chamber" method in 388 patients with various atopic disorders. Of the patients with hypersensitivity to birch pollen, 36% showed immediate positive responses to these natural, fresh materials. Apple, carrot, parsnip and potato elicited reactions more often than, e.g. swede, tomato, onion and parsley. On the basis of clinical data, the relevancy of the results of the skin tests was 80-90%. Both "false positive" and "false negative" responses were seen. Itching and tingling with or without oedema of the lips, mouth and tongue were the most common complaints after eating raw fruits and vegetables. In addition, laryngeal and abdominal disturbances, rhinitis and hand dermatitis were recorded. Among patients without allergy to birch pollen, reactions to fruits and vegetables were rare. The nature of the allergens could not be determined.

↩

[32] - Kondo Y, Urisu A, Tokuda R. Identification and Characterization of the Allergens in the Tomato Fruit by Immunoblotting. Int Arch Allergy Immunol 2001;126:294-299

Background: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Methods: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients' sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. Results: Four proteins binding with IgE from more than half of the OAS patients' sera were determined to be polygalacturonase 2A (PG2A), -fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, -fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. Conclusion: The concentrations of 3 of 4 tomato allergens increased during ripening.

↩

[33] - Kawamoto H, Yamagata M, Nakashima H, Kambe M, Kuraoka T. [A case of tomato juice-induced oral allergy syndrome in which dyspnea onset occurred during the season of Japanese cedar pollen dispersion]. Nihon Kokyuki Gakkai Zasshi 2003;41:397-401

A 42-year-old-man with a history of Japanese cedar pollinosis repeatedly visited the emergency clinic due to dyspnea during the season of Japanese cedar pollen dispersion. Before each onset of this symptom, he had always drunk tomato juice. Swelling of the oral and nasal mucosa, and congestion of the bulbar conjuctiva was observed. No audible wheezing was present. His pulmonary function test results were normal (FEV 1.0 = 4.02 L, %FEV 1.0-124%, negative reversible test). The CAP RAST scores were 4 for tomatoes and 3 for Japanese cedar pollen. A result was obtained in a challenge test using tomato juice. Since tomato juice was involved in the development of the symptoms, a diagnosis of oral allergy syndrome induced by tomato juice was made. When tomato juice consumption was avoided, no symptoms developed. A common antigenicity was found between tomatoes and Japanese cedar pollen. This may be associated with the development of this allergy during the dispersion season of Japanese cedar pollen. The dyspnea may have reflected a feeling of pharyngeal narrowing which is a symptom of oral allergy syndrome. The possibility of oral allergy syndrome as the chief complaint should be considered also in patients with dyspnea. This is the first reported case of oral allergy syndrome induced by tomato juice.

↩

[34] - Kondo Y, Tokuda R, Urisu A, Matsuda T. Assessment of cross-reactivity between Japanese cedar (Cryptomeria japonica) pollen and tomato fruit extracts by RAST inhibition and immunoblot inhibition. Clin Exp Allergy 2002;32:590-594

Background An association between pollinosis and sensitivity to fruits and vegetables has been reported. Although Japanese cedar (Cryptomeria japonica) pollinosis is one of the most widespread diseases in Japan, there have been no reports demonstrating cross-reactivity between Japanese cedar pollen and other plant food. Objective : The aim of this study was to demonstrate cross-reactivity between Japanese cedar pollen and tomato fruit (Lycopersicon esculentum) using RAST inhibition and immunoblot inhibition. Method s : The RAST and immunoblot inhibition were performed using sera from patients with oral allergy syndrome (OAS) after ingesting fresh tomatoes. We identified some proteins that took part in cross-reactive IgE by the determination of N-terminal amino acid sequences and a homology search through the SWISS-PROT database. Result s : In the RAST inhibition, the bindings of IgE from the sera from four out of five (4/5) subjects to Japanese cedar pollen discs were inhibited by more than 50 by preincubation of the serum with tomato fruit extracts. Likewise, the IgE bindings to tomato fruit discs were inhibited more than 50 by Japanese cedar pollen extracts in 3/5 sera. In immunoblot inhibition, IgE binding activities of some protein bands on both membranes were decreased by heterologous inhibitors. However, the combinations of these protein bands involved in cross-reactivity were different between patients. Conclusion : We have demonstrated cross-reactivity between Japanese cedar pollen and tomato fruit using RAST inhibition and immunoblot inhibition

↩

[35] - Bonds RS, Tiwari R, Ning B, Czerwinski EW, Goldblum RG, Midoro-Horiuti T. Cross Reactivity in Mountain Cedar-Tomato Oral Allergy Syndrome. J Allergy Clin Immunol 2007;119(1 suppl):S113

RATIONALE: Oral allergy syndrome (OAS) is recognized in up to 50% of pollen-allergic adults. However, the mechanisms of cross-recognition are not well established. We describe here a mountain cedar-tomato OAS and propose a molecular basis for cross-reactivity. METHODS: Eleven patients with tomato OAS and cedar pollen hypersensitivity were identified by history and skin testing. A role for Jun a 1, the dominant allergen of mountain cedar, was evaluated by ImmunoCap inhibition. A panel of monoclonal antibodies (mAbs) to Jun a 1 was screened for reactivity with tomato crude extract (CE) by ELISA. Strongly reacting mAbs were tested by Western blotting and immunoprecipitation, followed by protein idenfification by mass spectrometry. RESULTS: Jun a 1 inhibited 38% of patient IgE binding to tomato ImmunoCaps. MAbs that reacted with partially heat sensitive (conformational) and heat resistant (linear) epitopes of Jun a 1 cross-reacted with tomato CE. Western blot analysis suggested that mAbs bound to several tomato proteins, including some known allergens. Immunoprecipitation and mass spectrometry indicated that the most striking interaction of several mAbs was with tomato pectin methylesterase. Computer overlays of the crystal structures of Jun a 1 and tomato pectin methylesterase demonstrated extensive structural homology, despite a very low degree of sequence identity. CONCLUSIONS: Our findings suggest extensive cross-reactivity between Jun a 1, the major allergen of mountain cedar, and tomato allergens, in particular pectin methylesterase. Careful analysis of the epitopes recognized by IgE in patients with cedar-tomato OAS may allow us to define patterns that predict local and systemic reactions to tomato.

↩

[36] - Fötisch K, Son D, Altmann F, Aulepp H, Conti A, Haustein D, et al. Tomato (Lycopersicon esculentum) allergens in pollen-allergic patients. Eur Food Res Technol 2001;213:259-266

Abstract. This study was undertaken to investigate the relevance of tomato allergy in pollen-allergic patients, and to identify and characterize tomato allergens. Therefore, sera of a group of 32 pollen-allergic patients with a history of adverse reactions to tomato were investigated by means of immunoblotting and specific IgE measurements (CAP, EAST, EAST inhibition and CCD [cross-reactive carbohydrate determinants] - ELISA). IgE-reactive spots on 2D blots were analyzed by amino acid microsequencing. Tomato allergy was found to occur with a prevalence of about 9% in our group of birch pollen-allergic patients. Of the patients with adverse reactions to tomato, 44% presented IgE to tomato profilin and 35.5% specific IgE to CCD. Two patients were sensitized to a lipid transfer protein in tomato. Microsequencing of other IgE-reactive proteins, separated by 2D-gel electrophoresis and transferred to PVDF membranes, revealed beta-fructofuranosidase, polygalacturonase 2A, and pectinesterase as further tomato allergens. Our data show that tomato is a relevant allergenic food in pollen-allergic patients. It seems that the majority of pollen-associated allergies to tomato is caused by known ubiquitous allergenic structures such as profilin and cross-reactive IgE-binding N-glycans. Furthermore, lipid transfer protein, as well as polygalacturonase 2A, pectinesterase and beta-fructofuranosidase may represent birch pollen-independent tomato allergens for certain patients.

↩

[37] - Reche M, Pascual CY, Vicente J, Caballero T, Muñoz FM, Sanchez S, et al. Tomato allergy in children and young adults: cross-reactivity with latex and potato. Allergy 2001;56:1197-1201

Background: Several studies have shown that allergy to natural rubber latex is associated with cross-reactivity to certain foods such as tomato and potato. The objective was to investigate the clinical and immunologic differences between a group of patients with clinical allergy to tomato and latex and another which had only clinical allergy to tomato. We also aimed to assess, in vitro, the relationship of tomato and latex allergens, which could explain the cross-reactivity. Methods: Forty patients with histories of adverse reactions to tomato and IgE-mediated hypersensitivity were enrolled in the study. Tomato, latex, and potato components were analyzed by SDSˆPAGE immunoblotting. CAP and immunoblot inhibition were used to study allergen cross-reactivity. Results: Patients from group A had a mean age of 13.2 years, and in group B the mean age was 21.7 years. In group B, 9/10 patients belonged to the latex-fruits syndrome. All patients of both groups tolerated potato. Immunoblotting patterns obtained with patients' sera from pool A showed IgE-binding bands to tomato ranging from 44 to 46 kDa and a triple band at 67 kDa. For latex, there was a strong binding at 44 kDa, and potato showed a strong band of 44 kDa and a 67-kDa triple band. In pool B, the binding to the band of 44 kDa in latex and tomato was more intense than in pool A. In pool A, immunoblot inhibition with potato allergen showed an intense inhibition of the three allergens (potato, latex, and tomato); with latex, inhibition was partial and with tomato, a complete inhibition of tomato and latex was observed, and a partial inhibition of potato. In pool B, the inhibition pattern followed a similar tendency to pool A. The CAP inhibition confirmed the high rate of cross-reactivity between tomato, potato, and latex. Conclusions: In our study, tomato, potato, and latex showed a common band of 44ˆ46 kDa probably corresponding to patatin. This protein could be implicated in the high cross-reactivity between tomato, latex, and potato observed in the immunoblot and CAP inhibition.

↩

[38] - Castro L, Metz-Favre C, Villalba M, Purohit A, Rodrigez R, Pauli G et al. Incidence of profilin sensitisation in an ash allergic population with and without tomato sensitisation. Allergy 2009;64(Suppl. 90):106

Aim: To study the incidence of profilin sensitization in 74 rhinitic patients sensitized to ash, 26 of them being also sensitized to tomato. METHODS: 74 patients with Spring rhinitis were tested with ash pollen by prick tests. 30 of them had nasal challenge tests with the same extract (Stallergenes). Tomato allergy was detected by case history and cutaneous tests performed with tomato extract (ALK). IgE determination by ELISA was performed in all patients with tomato extract (in which the presence of profilin had been assessed by using polyclonal rabbit antibodies to profilin from chenopodium album) and recombinant profilin from chenopodium album (rChe a 2). Inhibition experiments with tomato extract and rChe a 2 were performed with pooled sera in immunoblot experiments and with individual sera in ELISA experiments. RESULTS: All patients had positive cutaneous tests to ash pollen and 25 out of 30 had positive nasal challenge tests (threshold <10 IR). Serological tomato sensitization was associated with clinical symptoms in half of the cases (8 OAS and 5 general reactions). The frequency of sensitization to profilin was 38 % in the patients sensitized to tomato and 4 % in the patients without sensitization to tomato. Inhibition experiments performed with the sera of patients with clinical symptoms to tomato showed partial inhibition of the IgE binding to rChe a 2 when tomato extract was used as inhibitor both in ELISA and immunoblot inhibition. CONCLUSIONS: This study confirms that prevalence of profilin sensitization is increased in patients sensitized to ash and tomato and that profilin may be a cross reacting allergen in these patients.

↩

[39] - Kondo Y, Tokuda R, Urisu A, Matsuda T. Assessment of cross-reactivity between Japanese cedar (Cryptomeria japonica) pollen and tomato fruit extracts by RAST inhibition and immunoblot inhibition. Clin Exp Allergy 2002;32:590-594

Background An association between pollinosis and sensitivity to fruits and vegetables has been reported. Although Japanese cedar (Cryptomeria japonica) pollinosis is one of the most widespread diseases in Japan, there have been no reports demonstrating cross-reactivity between Japanese cedar pollen and other plant food. Objective : The aim of this study was to demonstrate cross-reactivity between Japanese cedar pollen and tomato fruit (Lycopersicon esculentum) using RAST inhibition and immunoblot inhibition. Method s : The RAST and immunoblot inhibition were performed using sera from patients with oral allergy syndrome (OAS) after ingesting fresh tomatoes. We identified some proteins that took part in cross-reactive IgE by the determination of N-terminal amino acid sequences and a homology search through the SWISS-PROT database. Result s : In the RAST inhibition, the bindings of IgE from the sera from four out of five (4/5) subjects to Japanese cedar pollen discs were inhibited by more than 50 by preincubation of the serum with tomato fruit extracts. Likewise, the IgE bindings to tomato fruit discs were inhibited more than 50 by Japanese cedar pollen extracts in 3/5 sera. In immunoblot inhibition, IgE binding activities of some protein bands on both membranes were decreased by heterologous inhibitors. However, the combinations of these protein bands involved in cross-reactivity were different between patients. Conclusion : We have demonstrated cross-reactivity between Japanese cedar pollen and tomato fruit using RAST inhibition and immunoblot inhibition

↩

[40] - Kondo Y, Urisu A, Tokuda R. Identification and Characterization of the Allergens in the Tomato Fruit by Immunoblotting. Int Arch Allergy Immunol 2001;126:294-299

Background: Although the tomato fruit (Lycopersicon esculentum) has been widely investigated for breeding purposes, there have been few studies on tomato allergenicity. We attempted to identify the tomato fruit allergens and to compare the concentrations of IgE-binding proteins among the different growth stages with sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Methods: An immunoblot experiment on tomato fruit extracts was performed using sera from 11 patients with oral allergy syndrome (OAS) to tomatoes. Bands reacting with IgE from more than half of the OAS patients' sera were excised and subjected to determination of N-terminal amino acid sequences using the automated Edman degradation method. Moreover, we compared the concentrations of these proteins at each growth stage of the tomato fruit with SDS-PAGE and immunoblotting. Results: Four proteins binding with IgE from more than half of the OAS patients' sera were determined to be polygalacturonase 2A (PG2A), -fructofuranosidase, superoxide dismutase (SOD) and pectinesterase (PE). The concentrations of PG2A, -fructofuranosidase and PE were highest in the red ripening stage with both SDS-PAGE and immunoblotting. Conclusion: The concentrations of 3 of 4 tomato allergens increased during ripening.

↩

[41] - Kondo Y, Tokuda R, Urisu A, Asano Y. Cross-reactivity between tomato fruit and Japanese cedar (Cryptomeria japonica) pollen in immunoblot inhibition. ICACI 2000, 15-20 Oct. 2000, Sydney, Australia, Abstract P-666

↩

[42] - Westphal S, Kempf W, Foetisch K, Retzek M, Vieths S, Scheurer S. Tomato profilin Lyc e 1: IgE cross-reactivity and allergenic potency. Allergy 2004;59:526-532

BACKGROUND: To date, very little data are available about the nature of tomato allergens. Immunoglobulin E (IgE) cross-reactive profilins have been suggested to account for allergic symptoms in patients suffering from tomato allergy . METHODS: The cDNA of tomato profilin was amplified by reversely transcribed polymerase chain reaction (RT-PCR) from total RNA extracted from ripe tomato fruit. The gene was cloned into the pET101D expression plasmid and the protein was produced in Escherichia coli BL21. Purification was performed via poly-l-proline (PLP) affinity chromatography. IgE reactivity of recombinant tomato profilin was investigated by immunoblot and enzyme-linked immunosorbent assay. IgE-inhibition studies were performed to analyse cross-reactivity with other profilins. To determine the allergenic activity of the recombinant protein, basophil histamine release assays using sera of patients with adverse reactions to tomato were performed . RESULTS: Profilin was identified as a new minor allergen in tomato fruits. The recombinant tomato profilin comprises 131 amino acids and high sequence identity to other allergenic food and pollen profilins. It was shown to be IgE-reactive with a prevalence of 22% (11/50) in tomato-allergic patients. In patients with tomato allergy and multiple sensitization to other foods and birch pollen, IgE directed against tomato profilin showed a strong cross-reactivity with profilins from plant food sources and birch pollen. The tomato profilin was able to induce mediator release from human basophils . CONCLUSION: The tomato profilin is a minor allergen in tomato fruit. Thus, it shows biological activity, as confirmed by in vitro histamine release assays with human basophils and thereby has the potential to account for clinical symptoms in tomato-allergic patients.

↩

[43] - Westphal S, Kolarich D, Foetisch K, Lauer I, Altmann F, Conti A, et al. Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato. Eur J Biochem 2003;270:1327-1337

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Manalpha1-6(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Manalpha1-6(Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

↩

[44] - Fötisch K, Son D, Altmann F, Aulepp H, Conti A, Haustein D, et al. Tomato (Lycopersicon esculentum) allergens in pollen-allergic patients. Eur Food Res Technol 2001;213:259-266

Abstract. This study was undertaken to investigate the relevance of tomato allergy in pollen-allergic patients, and to identify and characterize tomato allergens. Therefore, sera of a group of 32 pollen-allergic patients with a history of adverse reactions to tomato were investigated by means of immunoblotting and specific IgE measurements (CAP, EAST, EAST inhibition and CCD [cross-reactive carbohydrate determinants] - ELISA). IgE-reactive spots on 2D blots were analyzed by amino acid microsequencing. Tomato allergy was found to occur with a prevalence of about 9% in our group of birch pollen-allergic patients. Of the patients with adverse reactions to tomato, 44% presented IgE to tomato profilin and 35.5% specific IgE to CCD. Two patients were sensitized to a lipid transfer protein in tomato. Microsequencing of other IgE-reactive proteins, separated by 2D-gel electrophoresis and transferred to PVDF membranes, revealed beta-fructofuranosidase, polygalacturonase 2A, and pectinesterase as further tomato allergens. Our data show that tomato is a relevant allergenic food in pollen-allergic patients. It seems that the majority of pollen-associated allergies to tomato is caused by known ubiquitous allergenic structures such as profilin and cross-reactive IgE-binding N-glycans. Furthermore, lipid transfer protein, as well as polygalacturonase 2A, pectinesterase and beta-fructofuranosidase may represent birch pollen-independent tomato allergens for certain patients.

↩

[45] - Westphal S, Fötisch K, Lauer I, Conti A, Kolarich D, Altmann F, et al. Cloning and Characterization of beta-Fructofuranosidase, a Newly Identified Glycoprotein as a Minor Allergen of Tomato (Lycopersicon Esculentum). AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°947

Until now, only few attempts have been made to identify and characterize tomato allergens. In most reports, allergy to tomato is linked to other allergies. The prevalence of tomato allergy ranges from 1.5 % to 16 % among food-allergic populations and reaches up to 39 % among grass pollen-allergic subjects, showing that tomato seems to be a relevant allergenic food. Cross-reactive carbohydrate determinants (CCD) are found in many allergenic sources such as vegetables, pollen and insect venom, but the highest rate of serological reactions to CCD has been observed to plant food extracts. The analysis of free and linked N-glycans of tomato revealed the presence of a plant-characteristic glycan core with xylose and fucose known as IgE-binding epitope. Recently our group has identified 3 potential tomato allergens by two-dimensional immunoblotting using sera from tomato-allergic patients. These proteins, polygalacturonase 2A, pectinesterase and -fructofuranosidase seem to contain IgE-reactive N-glycans and may represent birch pollen-independent tomato allergens (Foetisch et al., Eur Food Res Technol 2001, published online). The aim of this study was to investigate the role of N-linked glycans in the IgE-response of tomato-allergic patients. We purified the -fructofuranosidase from tomato extract via hydrophobic interaction chromatography (HIC) followed by gel filtration with Superdex 75. The identity of the protein was confirmed by N-terminal sequencing. Out of 53 sera from patients with tomato allergy who had symptoms such as OAS, nausea, itching of the skin, urticaria, abdominal pain and spasm, 6 (11 %) reacted with the native protein in immunoblots. The enzyme allergosorbent test (EAST) with the purified protein covalently linked to CNBr-activated paper disks showed almost identical results, only one serum did not react in this assay. Blot inhibition studies with 3 different patients' sera showed specific inhibition of IgE binding to -fructofuranosidase by the glycopeptide MUXF purified from pineapple stem bromelain. Moreover, the cDNA of the -fructofuranosidase gene was obtained by RT-PCR and cloned into the expression vector pET100D. The sequence of the gene contains 4 putative N-glycosylation sites giving a hint that -fructofuranosidase may be a multivalent glycoprotein allergen. The recombinant protein was overexpressed in E. coli and purified via electroelution from SDS-PAGE gels. Immunoblot analysis showed no reaction of the patients' sera with the recombinant, non-glycosylated protein. These results strongly suggest an important role for N-glycans in IgE-response of tomato-allergic patients against the minor allergen -fructofuranosidase.

↩

[46] - Lorenz Y, Fötisch K, LeQuynh L, Sonnewald U, Lauer I, Enrique E, et al. Frequency of specific IgE-sensitisation to tomato allergens in German and Spanish patients and identification of a new tomato allergen. Allergy Clin Immunol Int 2005;17(Suppl. 1):344

Background: The prevalence of tomato allergy ranges from about 1.5% to 16% among food-allergic patients. Profilin Lyc e 1 (~14 kDa) and invertase Lyc e 2 (52-60 kDa) were recently described as tomato allergens. Moreover, a tomato protein with an apparent molecular weight of 8 kDa showed IgE-reactivity with sera from a subgroup of tomato-allergic patients. Aim of this study was to identify the 8 kDa protein, and to compare frequencies of IgE binding to tomato allergens in a group of Spanish and German patients. METHODS: Patients (n=74) were recruited in Germany (n=39) and Spain (n=35). The most frequent symptom after ingestion of tomato was the oral allergy syndrome. Purified rLyc e 1 and nLyc e 2 were available from previous studies. The 8 kDa protein was N-terminal sequenced from tomato peel, two recombinant isoforms were cloned, and their secondary structure analysed by circular dichroism spectrometry. Their allergenic potency was tested by in vitro basophil histamine release assay. Prevalence of IgE-binding to crude tomato extract and the three purified tomato allergens was determined by immunoblotting and by EAST. RESULTS: Frequency of IgE binding (German/Spanish patients) to rLyc e 1, nLyc e 2 and the 8 kDa allergen were 29/48%, 38/58%, and 3/32%, respectively. Reactivity to at least one of the three evaluated allergens was 46% for German and 94% for Spanish patients (p<0.0001; Fisher´s exact test). The 8 kDa tomato allergen was identified as non-specific Lipid Transfer Protein (nsLTP) and designated Lyc e 3. Both rLyc e 3 isoforms (Lyc e 3.1 and Lyc e 3.2) exhibited mainly alpha helical secondary structure and appeared to be correctly folded. Sequence identity between the 2 isoforms is 73%. Prevalence of IgEbinding of sera from LTP-sensitised patients was more frequent to Lyc e 3.2 (8/12) than to Lyc e 3.1 (5/11) and specific IgE-titres were higher to Lyc e 3.2 compared to those to Lyc e 3.1. Both isoforms showed similar allergenic potency as indicated by histamine release assay. CONCLUSION: Lyc e 3 is a new minor allergen in tomato. Spanish patients showed a significant higher frequency of IgE-binding to the analysed tomato allergens compared to German patients. Therefore, other tomato allergens which are responsible for sensitisation of German patients should be identified. Also the question whether tomato LTP is the primary sensitising allergen in Spanish patients should be elucidated.

↩

[47] - Lorenz Y, Fötisch K, LeQuynh L, Sonnewald U, Lauer I, Enrique E, et al. Frequency of specific IgE-sensitisation to tomato allergens in German and Spanish patients and identification of a new tomato allergen. Allergy Clin Immunol Int 2005;17(Suppl. 1):344

Background: The prevalence of tomato allergy ranges from about 1.5% to 16% among food-allergic patients. Profilin Lyc e 1 (~14 kDa) and invertase Lyc e 2 (52-60 kDa) were recently described as tomato allergens. Moreover, a tomato protein with an apparent molecular weight of 8 kDa showed IgE-reactivity with sera from a subgroup of tomato-allergic patients. Aim of this study was to identify the 8 kDa protein, and to compare frequencies of IgE binding to tomato allergens in a group of Spanish and German patients. METHODS: Patients (n=74) were recruited in Germany (n=39) and Spain (n=35). The most frequent symptom after ingestion of tomato was the oral allergy syndrome. Purified rLyc e 1 and nLyc e 2 were available from previous studies. The 8 kDa protein was N-terminal sequenced from tomato peel, two recombinant isoforms were cloned, and their secondary structure analysed by circular dichroism spectrometry. Their allergenic potency was tested by in vitro basophil histamine release assay. Prevalence of IgE-binding to crude tomato extract and the three purified tomato allergens was determined by immunoblotting and by EAST. RESULTS: Frequency of IgE binding (German/Spanish patients) to rLyc e 1, nLyc e 2 and the 8 kDa allergen were 29/48%, 38/58%, and 3/32%, respectively. Reactivity to at least one of the three evaluated allergens was 46% for German and 94% for Spanish patients (p<0.0001; Fisher´s exact test). The 8 kDa tomato allergen was identified as non-specific Lipid Transfer Protein (nsLTP) and designated Lyc e 3. Both rLyc e 3 isoforms (Lyc e 3.1 and Lyc e 3.2) exhibited mainly alpha helical secondary structure and appeared to be correctly folded. Sequence identity between the 2 isoforms is 73%. Prevalence of IgEbinding of sera from LTP-sensitised patients was more frequent to Lyc e 3.2 (8/12) than to Lyc e 3.1 (5/11) and specific IgE-titres were higher to Lyc e 3.2 compared to those to Lyc e 3.1. Both isoforms showed similar allergenic potency as indicated by histamine release assay. CONCLUSION: Lyc e 3 is a new minor allergen in tomato. Spanish patients showed a significant higher frequency of IgE-binding to the analysed tomato allergens compared to German patients. Therefore, other tomato allergens which are responsible for sensitisation of German patients should be identified. Also the question whether tomato LTP is the primary sensitising allergen in Spanish patients should be elucidated.

↩

[48] - Larramendi CH, Ferrer A, Huertas AJ, García-Abujeta JL, Andreu C, Tella R, et al. Sensitization to tomato peel and pulp extracts in the Mediterranean Coast of Spain: prevalence and co-sensitization with aeroallergens. Clin Exp Allergy 2008;38:169-177

BACKGROUND: Tomatoes (Lycopersicon esculentum) are consumed world-wide. The prevalence of sensitization to tomatoes remains unknown . OBJECTIVE: To determine the prevalence of skin test reactivity to tomato and to describe the characteristics of tomato-sensitized subjects . METHODS: Individuals attending for the first time during the period of the study to six Allergy centres, located along the Mediterranean coast of Spain, reporting respiratory and/or cutaneous symptoms, were included. All patients were skin prick tested with a battery of inhalant allergens and with peel and pulp of Canary tomato extracts . RESULTS: The study included 1734 individuals (757 males, 977 females; 31.9+/-17.8 years old). The prevalence of sensitization to tomato was 6.52% (113 patients; 65 males, 48 females; 29.5+/-13 years old). The peel extract was positive in 110 patients and the pulp extract in 47 patients; three patients were positive exclusively to pulp. Only 1.8% of individuals reported symptoms with tomato; 44% of them had skin test negative to both extracts. Among tomato-sensitized subjects, 16% reported symptoms with tomato, 97% were sensitized to inhalant aeroallergens, including 84% to pollens (mainly Artemisia vulgaris and Platanus hybrida), with differences between Northern and Southern centres . CONCLUSIONS: The prevalence found of skin test sensitivity to tomato is high. Peel extracts detected most of the sensitized subjects. Most of the sensitized subjects were asymptomatic and some patients reported symptoms without skin test sensitivity. Positive subjects were very frequently sensitized to pollens, suggesting allergen cross-reactivity. Regional differences may exist, possibly related to the pattern of sensitization to cross-reacting pollens.

↩

[49] - Ferrer A, Huertas J, de Larramendi CH, Pagan J, Garcia-Abujeta J, Lavin J et al. Cross-reactivity Between Tomato And Pollen Extracts. J Allergy Clin Immunol 2009;123:S30

RATIONALE: Cross reactivity among fruits and different pollen species have been extensively described. The objective of this study was to analyze cross-reactivity between tomato and other common pollen extracts. METHODS: A serum pool from 18 individuals (9 males and 9 females; age 25.4 ± 8.4) with positive skin prick test to peel tomato was prepared. Extracts from Olea europaea, Platanus acerifolia, Plantago lanceolata, Chenopodium album, Salsola kali, Betula alba, Artemisia vulgaris, Phoenix dactyliphera and grasses (Avena sativa and Poa pratensis) were prepared. Specific IgE was determined by direct ELISA. Allergenic profile of different extracts was evaluated by Immunoblot. Percentage of cross-reactivity between extracts was analyzed by CAP inhibition and the allergens implicated by immunoblot inhibition. RESULTS: Eleven individuals (61.1%) reported symptoms after ingestion and/or contact with tomato. Twelve (66.7%) individuals were sensitized to Chenopodiaceae, 11 (61.1%) to A. vulgaris, 10 (55.5%) to O. europaea, 3 (16.7%) to P. acerofila and 6 (33.3%) to grasses. The highest specific IgE levels were obtained to grasses, P. acerifolia, O. europaea and Chenopodiaceae. Most pollen extracts showed a similar capacity of inhibition (about 35%). A. sativa was capable to inhibit 53% and P. pratensis 47%. P. dactyliphera showed the less capacity of inhibition with 23.4%. The most strongly inhibited allergens corresponded to bands of 32 and 45 kDa. The band of 10 kDa was poorly inhibited by all extracts except by P. acerifolia. CONCLUSIONS: Cross-reactivity between tomato and pollen extracts have been demonstrated, especially in grasses. Allergens with high molecular weight range seem to be the responsible.

↩

[50] - de Martino M, Novembre E, Cozza G, de Marco A, Bonazza P, Vierruci A. Sensitivity to tomato and peanut allergens in children monosensititized to grass pollen. Allergy 1988;43:206-213

Possible associations between allergy to grass pollen and positive skin tests to food allergens were studied in 102 children monosensitized (as to inhalant allergens) to grass pollen, and in 117 children monosensitized (as to inhalant allergens) to Dermatophagoides. Thirty-two foods were tested by an epicutaneous method. Positive skin tests to food allergens were more frequent in children with allergy to grass pollen (59.8%) than in children with allergy to Dermatophagoides (9.4%). A considerably high frequency of positive reactions to tomato (39.2%), peanut (22,5%), green pea (13.7%), and wheat (11.7%) was observed in children with allergy to grass pollen. Positive skin tests to peanut closely correlated with positive RAST results and nasal provocation tests, whereas in children with skin test positivity to tomato a close correlation with nasal provocation tests but a 45% correlation with a positive RAST result were observed. RAST inhibition experiments were carried out, and the results may suggest the presence of cross-reacting IgE to grass pollen, tomato, and peanut antigens. Clinical implications of these findings are discussed in the light of histories of food hypersensitivity, urticaria-angioedema, and atopic dermatitis in children with allergy to grass pollen.

↩

[51] - 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.

↩

[52] - 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.

↩

[53] - Castro L, Metz-Favre C, Villalba M, Purohit A, Rodrigez R, Pauli G et al. Incidence of profilin sensitisation in an ash allergic population with and without tomato sensitisation. Allergy 2009;64(Suppl. 90):106

Aim: To study the incidence of profilin sensitization in 74 rhinitic patients sensitized to ash, 26 of them being also sensitized to tomato. METHODS: 74 patients with Spring rhinitis were tested with ash pollen by prick tests. 30 of them had nasal challenge tests with the same extract (Stallergenes). Tomato allergy was detected by case history and cutaneous tests performed with tomato extract (ALK). IgE determination by ELISA was performed in all patients with tomato extract (in which the presence of profilin had been assessed by using polyclonal rabbit antibodies to profilin from chenopodium album) and recombinant profilin from chenopodium album (rChe a 2). Inhibition experiments with tomato extract and rChe a 2 were performed with pooled sera in immunoblot experiments and with individual sera in ELISA experiments. RESULTS: All patients had positive cutaneous tests to ash pollen and 25 out of 30 had positive nasal challenge tests (threshold <10 IR). Serological tomato sensitization was associated with clinical symptoms in half of the cases (8 OAS and 5 general reactions). The frequency of sensitization to profilin was 38 % in the patients sensitized to tomato and 4 % in the patients without sensitization to tomato. Inhibition experiments performed with the sera of patients with clinical symptoms to tomato showed partial inhibition of the IgE binding to rChe a 2 when tomato extract was used as inhibitor both in ELISA and immunoblot inhibition. CONCLUSIONS: This study confirms that prevalence of profilin sensitization is increased in patients sensitized to ash and tomato and that profilin may be a cross reacting allergen in these patients.

↩

[54] - Ferrer A, Andreu C, de Larramendi C, García-Abujeta J, Huertas J, Tella R, et al. Skin test evaluation of the allergenicity of seven tomato varieties, including peel, pulp, boiled and fried tomato extracts. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1541

Background: There is no evidence that different tomato varieties may express a different allergenicity in vivo. Therefore, we decided to analyse the allergenicity of several commonly consumed tomato varieties by skin testing in a group of tomato skin test positive individuals. Material & Methods: All consecutive individuals evaluated at the outpatient clinics of the participating Hospitals (located in different regions of the Mediterranean coast of Spain) for respiratory and/or cutaneous symptoms were skin tested with a battery of aeroallergens and with peel and pulp of the Canary variety tomato at 10 mg of freeze dried material/ml. Patients were also asked for any food related complaints, including oral allergy syndrome, contact urticaria, angioedema, anaphylaxis and/or gastrointestinal symptoms with the ingestion, or contact with tomato. All patients with a positive skin test and/or complaints related with tomatoes were asked to participate in the study and were skin tested with extracts of the following varieties: Canario, Pera, Kumato, Rambo, Raft, Cherry and Rama and canned fried tomato. All were purchased at a local market. The wheal sizes were measured and expressed in squared mm (± SD). Results: One hundred thirty six patients were recruited. The mean wheal sizes produced by the different extracts were: Canary peel: 33.6 ± 36.4; Canary pulp: 9.1 ± 13.2; Rama peel: 26.5 ± 36.7; Rama pulp: 9.8 ± 13.5; Cherry whole: 9.5 ± 14.7; Kumato peel: 26.9 ± 29.3; Kumato pulp: 17.7 ± 22.2; Rambo peel: 26.2 ± 26.9; Rambo pulp: 4.6 ± 8.6; Pera peel: 20.3 ± 26.7; Pera pulp: 13 ± 16.3; Raft peel: 25.3 ± 33; Raft pulp: 7.3 ± 13.5; Canned fried tomato: 11.4 ± 16.8; Rama boiled peel: 19.5 ± 29.5; Rama boiled pulp: 9.8 ± 16. Conclusions: The wheals skin test results obtained with all the peel extracts was, in all cases, greater than that obtained with the respective pulp extracts. The greatest mean skin test reaction induced by the peel extracts was obtained with the Canario and by the pulp extracts with the Kumato variety. Most of the Canario tomato positive patients were sensitised to all varieties studied. The Canario variety seems to be a good variety to diagnose in vivo sensitisation to tomatoes.

↩

[55] - Pravettoni V, Primavesi L, Farioli L, Brenna OV, Pompei C, Conti A et al. Tomato Allergy: Detection of IgE-Binding Lipid Transfer Proteins in Tomato Derivatives and in Fresh Tomato Peel, Pulp, and Seeds. J Agric Food Chem 2009;57:10749-10754

There is an increasing consumption of tomatoes worldwide: fresh in salads, cooked in household sauces, or industrially processed. Although many tomato allergens have been identified, there is no information in the literature on the allergenic components found in commercial tomato products. The primary aim of the study was to evaluate the allergenic profile of commercial tomato products by skin prick tests (SPTs) and IgE/immunoblotting in tomato-allergic subjects. The secondary end point was the study of the IgE-binding profile of tomato peel, pulp, and seeds. Forty tomato-allergic patients, reporting oral allergy syndrome (OAS) at different grades of severity for fresh and, in some cases, also for cooked tomato, were selected on the basis of positive tomato allergy history or open food challenge (OFC). They were evaluated by SPTs with different experimental tomato extracts. SDS-PAGE/immunoblotting was performed to detect tomato allergens, which were then identified by Edman degradation. Twenty-three patients (57.5%) presented first-grade OAS at the OFC, whereas 17 (42.5%) reported severe symptoms. Ten of these 17 patients (25%) reported allergic reactions to cooked tomatoes; in immunoblotting tests, their sera reacted only to lipid transfer protein (LTP). In commercial products, LTP was the only detectable allergen. In contrast to other LTP-containing fruits, in tomato, an IgE-binding LTP was identified not only in the peel but also in the pulp and seeds. This study demonstrates that, in fresh tomato, different LTP isoforms are present and allergenic. Industrial tomato derivatives still contain LTP, thus presenting a problem for LTP-allergic patients.

↩

[56] - Cadot P, Nelles L, Srahna M, Dilissen E, Ceuppens JL. Cloning and expression of the cyclophilin Bet v 7, and analysis of immunological cross-reactivity among the cyclophilin A family. Mol Immunol 2006;43:226-235

Allergic symptoms in sensitized individuals are caused by proteins named allergens. We report here the cloning and the production of the cyclophilin Bet v 7, one of the birch pollen allergens. Recombinant Bet v 7 was produced in bacteria and used to raise a rabbit anti-Bet v 7 antiserum. With this antiserum we detected cyclophilin A in several pollen species and we demonstrated immunological cross-reactivity among those plant cyclophilins A by immunoblot and ELISA inhibition experiments. However, we could not detect cyclophilins in extracts of animal or mould origin with our anti-Bet v 7 antiserum. By inhibition experiments with purified mould cyclophilins, we confirmed the absence of cross-reactivity between plant cyclophilins and non-plant cyclophilins. In addition, our results indicate that the level of immunological cross-reactivity correlates with the level of sequence identity among the cyclophilin A family. This allowed us to define the plant cyclophilin A sub-family as being immunologically distinct, which might have implications at the clinical level in the allergy practice.

↩

[57] - Poza P, Somoza ML, Crespo JF, Izquierdo G, Campo P, Rodriguez J. Diagnostic evaluation of 27 patients reporting adverse reactions to tomato. EAACI 21th Congress, Naples, 1-5 June, 2002, Communication n°59

Background: Sensitization to tomato has been reported in patients with other allergies, particularly latex. IgE inhibition studies have shown cross-reactive components shared by latex, potato and tomato. Nevertheless, less information is available on the clinical relevance of tomato sensitization objectively confirmed by DBPCFCs. The purpose of this investigation was to assess clinical and immunological features of adverse reactions to tomato. Methods: Twenty-seven adult patients (median π28 years) complaining of adverse reactions to tomato were included in the study. Skin prick tests and detection of specific IgE (CAP) were performed in all patients with tomato, latex, pollen and other offending foods. Clinical reactivity to foods was evaluated first by open food challenges (OFCs), unless a convincing history of a recent severe anaphylaxis. Positive OFCs were subsequently evaluated by the DBPCFC method. Results: Twelve and 13 patients had positive skin prick tests and CAP FEIA with tomato, respectively. Two patients reported severe systemic anaphylaxis, which excluded any challenge. Twenty-five patients underwent OFC with tomato. Ten of 25 initial OFCs were positive, but only four of the nine performed DBPCFCs elicited positive results. An OFC induced a severe reaction that precluded further provocations. Overall, seven reactions were considered positive in 16 patients with positive results of skin tests and/or CAP FEIA. The positive predictive value of a skin prick test was 46 and 43% for specific IgE. The most frequent symptom was the oral allergy syndrome. Thirty-two reactions to other foods were confirmed in the seven patients with actual allergy to tomato. The most common foods associated were melon (four patients), watermelon (three), banana (three), kiwi (three), avocado (two), peach (two), chestnut (two), hazelnut (two), and peanut (two). Moreover, three out of seven patients allergic to tomato were found to be sensitive to latex (one patient had anaphylactic episodes upon latex exposure and two had positive results of the used tests with latex). All patients have seasonal rhinitis and/or asthma and positive results of skin tests to several pollens. Conclusions: Tomato is an allergenic source that can induce actual clinical allergy, as established by DBPCFCs. Although most patients experienced oral allergy syndrome, severe anaphylactic reactions could be elicited. Isolated allergy to tomato seems to be infrequent, most patients having allergy to a number of foods, pollen and/or latex.

↩

[58] - Reche M, Pascual CY, Vicente J, Caballero T, Muñoz FM, Sanchez S, et al. Tomato allergy in children and young adults: cross-reactivity with latex and potato. Allergy 2001;56:1197-1201

Background: Several studies have shown that allergy to natural rubber latex is associated with cross-reactivity to certain foods such as tomato and potato. The objective was to investigate the clinical and immunologic differences between a group of patients with clinical allergy to tomato and latex and another which had only clinical allergy to tomato. We also aimed to assess, in vitro, the relationship of tomato and latex allergens, which could explain the cross-reactivity. Methods: Forty patients with histories of adverse reactions to tomato and IgE-mediated hypersensitivity were enrolled in the study. Tomato, latex, and potato components were analyzed by SDSˆPAGE immunoblotting. CAP and immunoblot inhibition were used to study allergen cross-reactivity. Results: Patients from group A had a mean age of 13.2 years, and in group B the mean age was 21.7 years. In group B, 9/10 patients belonged to the latex-fruits syndrome. All patients of both groups tolerated potato. Immunoblotting patterns obtained with patients' sera from pool A showed IgE-binding bands to tomato ranging from 44 to 46 kDa and a triple band at 67 kDa. For latex, there was a strong binding at 44 kDa, and potato showed a strong band of 44 kDa and a 67-kDa triple band. In pool B, the binding to the band of 44 kDa in latex and tomato was more intense than in pool A. In pool A, immunoblot inhibition with potato allergen showed an intense inhibition of the three allergens (potato, latex, and tomato); with latex, inhibition was partial and with tomato, a complete inhibition of tomato and latex was observed, and a partial inhibition of potato. In pool B, the inhibition pattern followed a similar tendency to pool A. The CAP inhibition confirmed the high rate of cross-reactivity between tomato, potato, and latex. Conclusions: In our study, tomato, potato, and latex showed a common band of 44ˆ46 kDa probably corresponding to patatin. This protein could be implicated in the high cross-reactivity between tomato, latex, and potato observed in the immunoblot and CAP inhibition.

↩

[59] - de Larramendi CH, Ferrer A, Huertas J, Pagan J, Bartra J, Andreu C et al. Cross-reactivity Between Tomato And Other Fruit Allergen Extracts. J Allergy Clin Immunol 2009;123:S30

RATIONALE: Cross-reactivity among fruits has been extensively demonstrated. The objective of this study was to analyze cross-reactivity between tomato and other common consumed fruits. METHODS: A serum pool from 18 individuals (9 males and 9 females; age 25.4 6 8.4) with positive skin prick test to peel tomato was prepared. Extracts from peach (peel and pulp), potato, carrot, kiwi, pineapple, banana, melon, avocado, nuts, (including chestnut, hazelnut and almond) and latex were prepared. Specific IgE to all extracts was measured by direct ELISA. Allergenic profile of different extracts was evaluated by Immunoblot. Percentage of cross-reactivity between extracts was analyzed by CAP inhibition and the allergens involved by immunoblot inhibition. RESULTS: Sensitization studies with fruits showed that 8 (44.4%) individuals were sensitized to nuts, 5 (27.8%) to peach, 4 (22.2%) to banana, 3 (16.7%) to kiwi and pineapple and 2 (11.1%) tomelon.Only 1 patient was sensitized to latex. Specific IgE to tomato and peach peelwas significantly higher than those obtained with the other allergen extracts. Tomato extract was inhibited 62.6% by peach, 46%by hazelnut, 45% by chestnut, 39%by avocado and 17% by melon. Other fruits and nuts tested inhibited less than 15%. No inhibition was detected with latex. Peach, chestnut and melon inhibited high molecularweight bands (between 32 and 45 kDa) and a band of about 10 kDa. CONCLUSIONS: Cross-reactivity between tomato and other fruits have been demonstrated, especially with peach, nuts and melon. Allergens with a molecular weight of 10, 32 and 45 KDa seem to be the responsible.

↩

[60] - 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.

↩

[61] - Huertas A, Ferrer A, Larramendi C, Lavin J, Andreu C, Garcia-Abujeta J, et al. Differences among tomato sensitised subjects according to their pattern of cosensitisation to fruits. Allergy 2008;63(suppl. 88):425-426

Background: Cross-reactivity between tomato and pollens or other fruits has been described. Several studies have demonstrated the implication of different families of panallergens as responsible for this phenomenon. Patients sensitised to crossreactive allergens may show positive skin prick test to a number of different extracts. Aim: To study the differences among tomato sensitised subjects, according their pattern of co-sensitisation to other fruits. Methods: The study included 192 individuals (94 Males/98 Females; mean age: 28.3±10.8 years, 81-42.2%- referring symptoms with tomato), with a positive skin prick test to at least one of 12 tomato manufactured extracts (peel and pulp of Canario, Rambo, Kumato, Raf, Pera and Rama tomato varieties at 10 mg/mL). All individuals were skin prick tested with common inhalants (mites, moulds, cat, dog, and pollen of grasses, Chenopodiaceae, P. judaica, A. vulgaris, P. lanceolata, O. europaea, C. arizonica, B. alba and P. dactylifera), latex and commercially available extracts of melon, peel peach, peel apple, melon, whole kiwi, and pineapple. Subjects were divided in three groups: Group I: negative to all five fruits. Group II: positive to 1 or 2 fruits and Group III: positive to 3 or more fruits. Chi2 for trends was used to compare the groups. Results: Group I: 54 subjects (34 M/20 F, 30.46±11.94 years), group II: 86 subjects (43 M/43 F, 28.1±11.1 years) and Group III: 52 subjects (17 M/35 F, 26.3±8.9 years). sensitisation to fruits in groups II and III respectively was: Peel peach: 72.11 and 98.1%; peel apple: 7 and 54.9%; melon: 32.6 and 69.2%, kiwi: 19.8 and 86.5% and pineapple: 9.3 and 63.5%. A positive tendency between women and polisensitisation to fruits was detected (P<0.01). A trend to report more frequently symptoms with tomato (P<0.05) was observed among fruit sensitised individuals, that were also more frequently sensitised to latex (P<0.05), Betula (P<0.0001) and Platanus (P<0.01) and less sensitised to Cupressus (P<0.05). Conclusions: Peach peel is the most frequently associated fruit to tomato in these individuals. Most individuals of group II (sensitised only to 1 or 2 fruits) were sensitised to peach. Tomato sensitised subjects with positivity to several fruits are more frequently women, report more frequently symptoms with tomato and may be sensitised to infrequent allergens in our area such as Betula, suggesting cross reactivity.

↩

[62] - Larramendi CH, Ferrer A, Huertas AJ, García-Abujeta JL, Andreu C, Tella R, et al. Sensitization to tomato peel and pulp extracts in the Mediterranean Coast of Spain: prevalence and co-sensitization with aeroallergens. Clin Exp Allergy 2008;38:169-177

BACKGROUND: Tomatoes (Lycopersicon esculentum) are consumed world-wide. The prevalence of sensitization to tomatoes remains unknown . OBJECTIVE: To determine the prevalence of skin test reactivity to tomato and to describe the characteristics of tomato-sensitized subjects . METHODS: Individuals attending for the first time during the period of the study to six Allergy centres, located along the Mediterranean coast of Spain, reporting respiratory and/or cutaneous symptoms, were included. All patients were skin prick tested with a battery of inhalant allergens and with peel and pulp of Canary tomato extracts . RESULTS: The study included 1734 individuals (757 males, 977 females; 31.9+/-17.8 years old). The prevalence of sensitization to tomato was 6.52% (113 patients; 65 males, 48 females; 29.5+/-13 years old). The peel extract was positive in 110 patients and the pulp extract in 47 patients; three patients were positive exclusively to pulp. Only 1.8% of individuals reported symptoms with tomato; 44% of them had skin test negative to both extracts. Among tomato-sensitized subjects, 16% reported symptoms with tomato, 97% were sensitized to inhalant aeroallergens, including 84% to pollens (mainly Artemisia vulgaris and Platanus hybrida), with differences between Northern and Southern centres . CONCLUSIONS: The prevalence found of skin test sensitivity to tomato is high. Peel extracts detected most of the sensitized subjects. Most of the sensitized subjects were asymptomatic and some patients reported symptoms without skin test sensitivity. Positive subjects were very frequently sensitized to pollens, suggesting allergen cross-reactivity. Regional differences may exist, possibly related to the pattern of sensitization to cross-reacting pollens.

↩

[63] - Ferrer A, Andreu C, de Larramendi C, García-Abujeta J, Huertas J, Tella R, et al. Skin test evaluation of the allergenicity of seven tomato varieties, including peel, pulp, boiled and fried tomato extracts. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1541

Background: There is no evidence that different tomato varieties may express a different allergenicity in vivo. Therefore, we decided to analyse the allergenicity of several commonly consumed tomato varieties by skin testing in a group of tomato skin test positive individuals. Material & Methods: All consecutive individuals evaluated at the outpatient clinics of the participating Hospitals (located in different regions of the Mediterranean coast of Spain) for respiratory and/or cutaneous symptoms were skin tested with a battery of aeroallergens and with peel and pulp of the Canary variety tomato at 10 mg of freeze dried material/ml. Patients were also asked for any food related complaints, including oral allergy syndrome, contact urticaria, angioedema, anaphylaxis and/or gastrointestinal symptoms with the ingestion, or contact with tomato. All patients with a positive skin test and/or complaints related with tomatoes were asked to participate in the study and were skin tested with extracts of the following varieties: Canario, Pera, Kumato, Rambo, Raft, Cherry and Rama and canned fried tomato. All were purchased at a local market. The wheal sizes were measured and expressed in squared mm (± SD). Results: One hundred thirty six patients were recruited. The mean wheal sizes produced by the different extracts were: Canary peel: 33.6 ± 36.4; Canary pulp: 9.1 ± 13.2; Rama peel: 26.5 ± 36.7; Rama pulp: 9.8 ± 13.5; Cherry whole: 9.5 ± 14.7; Kumato peel: 26.9 ± 29.3; Kumato pulp: 17.7 ± 22.2; Rambo peel: 26.2 ± 26.9; Rambo pulp: 4.6 ± 8.6; Pera peel: 20.3 ± 26.7; Pera pulp: 13 ± 16.3; Raft peel: 25.3 ± 33; Raft pulp: 7.3 ± 13.5; Canned fried tomato: 11.4 ± 16.8; Rama boiled peel: 19.5 ± 29.5; Rama boiled pulp: 9.8 ± 16. Conclusions: The wheals skin test results obtained with all the peel extracts was, in all cases, greater than that obtained with the respective pulp extracts. The greatest mean skin test reaction induced by the peel extracts was obtained with the Canario and by the pulp extracts with the Kumato variety. Most of the Canario tomato positive patients were sensitised to all varieties studied. The Canario variety seems to be a good variety to diagnose in vivo sensitisation to tomatoes.

↩

[64] - Doelle S, Fiedler EM, Schwarz D, Franken P, Eckhard G, Worm M. Impact of cultivation on the allergenicity of tomato. Allergy 2007;62(suppl. 83):360

Background: Food allergy is an increasing problem and in Europe a prevalence of 2-4% is estimated. Pollen-associated food allergies are especially frequent in adults. Tomato (Lycopersicon esculentum) belongs to the Solanaceae or nightshade family and is a widely produced and important crop plant. Worldwide, the prevalence of tomato allergy ranges from 1.5% to 20% in different populations of food-allergic patients. OBJECTIVES: The aim of this study was to analyse the impact of different cultivation methods and the grade of ripeness on the allergenicity of tomato. METHODS: Patients aged between 18-60 years with a history of symptoms like oral allergy syndrome (OAS), dyspnea, pruritus and urticaria after the intake of tomato were studied. Twenty-two different samples of tomatoes varying in terms of cultivation like species, intensity of UV-exposure, soil composition and grade of ripeness were examined. Total IgE as well as specific IgE towards tomato (commercial extract) were analysed (Pharmacia CAP System). Basophile activation were assessed by flow cytometry. RESULTS: In total, 18 patients with an average age of 43 (14 women and 4 men) with a positive history and skin prick test (SPT) were recruited. All patients were examined by an extensive SPT with 22 different samples of tomatoes. Unshaded variants from green (unriped) tomatoes caused stronger skin reactions than the shaded variants (p<0.05). The shaded as well as the unshaded variants of the red (riped) tomatoes resulted in much more skin reactions than the green species. Riped tomatoes may have an increased allergenicity compared to the green tomatoes. Also by applying basophile activation we confirmed the differences between the allergen extracts derived from different cultivations and with different stages of ripeness. Perspectives: This study shows that cultivating methods may influence the allergenicity of tomato. Whether it is possible to yield low allergenic tomatoes by the application of defined culture and cultivation methods is currently under investigation. Acknowledge: This project is financially supported by the Federal Ministry of Education and Research (BMBF - Bundesministerium für Bildung und Forschung; PTJ-BIO 0313350C).

↩

[65] - Armentia A, Callejo A, Diaz-Perales A, Martin-Gil F, Salcedo G. Enhancement of tomato allergenicity after treatment with plant hormones. Allergol Immunopathol (Madr) 2003;31:44-46

Background: Practical applications to enhance the productivity of agriculture by using plants with improved resistance to pathogens are expected to increase in the near future. Although tomato has been widely investigated for breeding purposes, there have been no studies on tomato allergenicity after plant hormones treatments. METHODS: Prick by prick tests were carried out with different tomato samples (fruits grown under biological conditions without addition of chemical products, and treated with ethylene and salicylic acid) in eight patients with ages between 12 and 27 years who suffered from anaphylaxis episodes after eating raw tomatoes. An immunoblot experiment with the different tomato extracts was performed using sera from these eight patients and controls.Results: The wheals obtained in prick tests were significantly higher with the extracts of tomato treated with ethylene and SAA (chi2 = 31.3, p < 0.0001) and the patients who presented higher wheal diameters in skin tests were those who had more severe episodes of anaphylaxis. Neither the protein stain nor the IgE immunodetection patterns clearly varied between the untreated and the hormone-treated samples.Conclusions: In the case of anaphylaxis induced by tomato, the treatment with plant hormones induced a higher cutaneous response than with non-treated tomato, but the "in vitro" response was similar.

↩

[66] - Le LQ, Mahler V, Lorenz Y, Scheurer S, Biemelt S, Vieths S, et al. Reduced allergenicity of tomato fruits harvested from Lyc e 1–silenced transgenic tomato plants. J Allergy Clin Immunol 2006;118:1176-1183

BACKGROUND: Profilin is a small actin-binding protein that contributes to the allergenic potency of many fruits and vegetables, including tomato. Two highly similar genes encoding tomato profilin have been isolated and designated as allergen Lyc e 1.01 and Lyc e 1.02 . OBJECTIVE: The aim of the study was to generate profilin-reduced hypoallergenic tomato fruits by silencing of both genes in transgenic tomato plants by means of RNA interference (RNAi) . METHODS: The efficiency of gene silencing was documented by means of Northern blotting, immunoblotting, and skin prick testing . RESULTS: Quantification of the remaining protein revealed that profilin accumulation in transgenic fruits was decreased 10-fold compared with that seen in untransformed controls. This decrease was sufficient to cause a reduced allergenic reactivity in patients with tomato allergy, as determined with skin prick tests. Because most patients with tomato allergy are not monosensitized to profilin, the IgE reactivity to the profilin-silenced tomato fruits in vivo varied widely between individuals tested . CONCLUSION: We could demonstrate the efficient silencing of both profilin genes in transgenic tomato plants using RNAi. This resulted in Lyc e 1-diminished tomato fruits, providing proof of concept and demonstrating that RNAi can be used to design allergen-reduced food. However, simultaneous silencing of multiple allergens will be required to design hypoallergenic tomatoes. CLINICAL IMPLICATIONS: Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This concept constitutes a novel approach to allergen avoidance.

↩

[67] - Lorenz Y, Le Quynh L, Retzek M, Fötisch K, Mahler V, Enrique E, et al. Gene Silencing is an Efficient Tool to Reduce the Allergenic Potential of Tomato Fruits. AAAAI 62nd Annual Meeting, Miami, 3-7 March 2006, Poster n°1166

RATIONALE: RNA interference (RNAi) is a powerful method for posttranscriptional down-regulation of protein expression. Our aim was to reduce certain allergens in tomato plants providing a proof of principle for this molecular approach METHODS: Expression of tomato (Lycopersicon esculentum) allergens Lyc e 1 (profilin) and Lyc e 3 (non-specific Lipid-Transfer-Protein) were suppressed in a fruit-specific or constitutive manner by RNAi. Transgenic tomato plants were generated by Agrobacterium-mediated gene transfer Gene silencing was analyzed by Northern - and Western Blotting. In vitro histamine release assays were performed to examine any remaining allergenic potency of tomato fruits RESULTS: Fruit-specific and constitutive gene suppression of Lyc e 1 resulted in a great reduction of protein expression in tomato plants; however, best results have been obtained using a constitutive promoter Constitutive gene silencing was accompanied by impaired plant growth and fruit yield. Therefore, a non-IgE reactive yeast profilin was selected to reconstitute RNAi plants to maintain their physiological function Fruit specific and constitutive gene silencing of Lyc e 3 resulted in neither IgG binding of an LTP-specific antibody nor IgE binding of Lyc e 3 positive tomato allergic patients` sera. Moreover, histamine release performed with basophils passively sensitized with sera from two LTPsensitized patients was drastically reduced after stimulation with extracts from transgenic fruits CONCLUSIONS: RNAi technology is an effective tool to reduce allergen expression in transgenic tomato fruits and hence to reduce allergenic potency. It might represent a versatile strategy to obtain hypoallergenic food Funding: Deutsche Forschungsgemeinschaft

↩

[68] - Petersen A, Vieths S, Aulepp H, Schlaak M, Becker WM. Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens. J Allergy Clin Immunol 1996;98:805-815

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.

↩

[69] - Westphal S, Kolarich D, Foetisch K, Lauer I, Altmann F, Conti A, et al. Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato. Eur J Biochem 2003;270:1327-1337

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Manalpha1-6(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Manalpha1-6(Manalpha1-3)(Xylbeta1-2)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

↩

[70] - Fötisch K, Westphal S, Lauer I, Retzek M, Altmann F, Kolarich D, et al. Biological activity of IgE specific for cross-reactive carbohydrate determinants. J Allergy Clin Immunol 2003;111:889-896

BACKGROUND: The clinical relevance of IgE specific for cross-reactive carbohydrate determinants (CCDs) has been a matter of controversy. Until now, no convincing experiments have been performed to test the biologic significance of individual multivalent allergens that carry multiple carbohydrate epitopes . OBJECTIVE: We sought to contribute to the understanding of the role of CCD-specific IgE antibodies and to study whether CCD-specific IgE antibodies are able to activate basophils using different multivalent glycoproteins as antigens . METHODS: Purified natural tomato beta-fructofuranosidase (nLyc e 2) and rLyc e 2.02 expressed in Escherichia coli were compared by means of histamine release tests. In addition, native and deglycosylated horseradish peroxidase and a neoglycoprotein consisting of a defined glycopeptide (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[Fucalpha1-3]GlcNAc) coupled to BSA were used in histamine release assays using stripped basophils from normal donors resensitized with IgE from CCD-reactive patients with food allergy to tomato . RESULTS: Ten CCD-positive and 2 CCD-negative sera from patients with tomato allergy underwent histamine release testing by using the glycoproteins and nonglycosylated controls as antigens, respectively. All sera induced histamine release with tomato extract (up to 100%), confirming the allergic status of the donors. Four of the CCD-positive sera induced releases ranging from 12% to 82% with all of the glycoproteins but not with the nonglycosylated or monovalent controls. All other sera showed no response or only very weak response to the glycoproteins . CONCLUSION: Approximately one third of the CCD-positive sera from patients with tomato allergy have biologically relevant CCD-specific IgE antibodies. Therefore the general claim that CCD-specific IgE is clinically irrelevant has to be reconsidered critically. Hence IgE specific for CCDs should be taken into consideration in the diagnosis and therapy of certain allergies. In the subgroup of patients sensitized to CCDs, the use of natural allergens should be preferred over the use of recombinant allergens expressed in prokaryotic organisms.

↩

[71] - van Ree R. Carbohydrate Epitopes and Their Relevance for the Diagnosis and Treatment of Allergic Diseases. Int Arch Allergy Immunol 2002;129:189-197

Allergenicity of plant and invertebrate N-glycans has been shown to be caused by the presence of two typical nonmammalian substitutions: an (1,3)-fucose linked to the proximal N-acetylglucosamine and a (1,2)-xylose linked to the core mannose. IgE antibodies against these carbohydrate structures are induced upon exposure to pollen or after insect stings, and result in extensive cross-reactivity to plant and invertebrate foods. These cross-reactive IgE antibodies have been shown to possess variable degrees of biological activity, but have never been convincingly shown to induce clinical food allergy. The most likely explanation for this lack of clinical relevance has to be sought in a combination of epitope valency and antibody affinity. In diagnostic tests, these antibodies are at the basis of many false-positive test results for food allergy. Recombinant technologies offer the possibility to produce allergens that do not carry IgE-binding glycans. Whether their absence or presence is of importance for the application of recombinant allergens in immunotherapy is still largely unknown.

↩

Les allergènes de tomate

Pulpe de tomate vs peau de tomate

Tomate et pollinose

Latex et tomate

Tomate et paramètres agronomiques

Tomate et CCD

Rubriques

Dans la même rubrique

Les Solanacées : généralités

La pomme de terre