Les Oléacées

Famille	« Ole e 1-like »	Profilines	Polcalcine	LTP	β 1,3 glucanase
Olivier	Ole e 1	Ole e 2	Ole e 3	Ole e 7	Ole e 9
Frêne (F. excelsior)	Fra e 1	Fra e 2	Fra e 3**		Fra e 9
Troène	Lig v 1	*
Lilas	Syr v 1	*	Syr v 3
Forsythia	For s 1
* existe mais sans nom IUIS ** à ne pas confondre avec Fra a 3 qui est la LTP de fraise, Fragaria ananassa et non la polcalcine du frêne américain (F. americana)

[1] - Alaoui Yazidi A, Nejjari C, Bartal M. La sensibilisation cutanée aux pollens au Maroc. Étude multicentrique. Rev Mal Respir 2001;18:523-529

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[2] - Bousquet J, Cour P, Guerin B, Michel FB. Allergy in the Mediterranean area I. Pollen counts and pollinosis of Montpellier. Clin Allergy 1984;14:249-258

The climatic conditions of the Mediterranean area result in vegetation and pollen very different from that of the other parts of Europe. The pollen content of the atmosphere of Montpellier, southern France, was examined using a filter sampler which was shown to be more efficient than most of the current devices for air sampling. Pollen counts were subsequently compared with pollinosis of patients born and living in and around Montpellier. The mean annual pollen counts showed that grass pollens and Cupressaceae pollens (cypress and juniper) are the highest. Some Mediterranean pollens (Oleaceae, London plane, Parietaria) are also important. Plantain and oak pollens are also present in relatively large amounts. Grass pollen allergy was found to be present in 86.5% of pollen-allergic patients. It was followed by plantain, Parietaria, Oleaceae, London plane and Cupressaceae pollens which were allergenic in 13-36% of pollen-allergic patients. Oak and pine pollens were present in large quantities in the counts but few persons were sensitive to oak and none to pine. By contrast, some patients had positive skin tests to alfalfa, red clover, acacia and lime tree pollens though these pollens were almost absent from the counts. In a few cases local sources of these pollens could account for the positive skin tests but cross-sensitivities could also occur. In summary, pollinosis of the Northern Mediterranean area is intermediate between the southern part of the area and the other parts of Europe.

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[3] - EXPO: Determinacion de la prevalencia de sensibilizacion a polenes mediante tecnicas de diagnostico molecular. ALK-Abello Ed., 2009, 86pp.

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[4] - Laurent J, Guinnepain MT, Lafay M, Sauvaget J. La pollinose des arbres. Rev Fr Allergol Immunol Clin 1999;39:276-282

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[5] - Niederberger V, Purohit A, Oster JP, Spitzauer S, Valenta R, Pauli G. The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species. Clin Exp Allergy 2002;32:933-941

BackgroundAsh, a wind-pollinated tree belonging to the family Oleaceae, is distributed world-wide and has been suggested as a potent allergen source in spring time. ObjectiveThe aim of this study was to determine the profile of allergen components in ash pollen in order to refine diagnosis and therapy for patients with sensitivity to ash pollen MethodsThe IgE reactivity profile of 40 ash pollen-allergic patients was determined by immunoblotting. Antibodies raised to purified pollen allergens from tree and grass pollens were used to identify cross-reactive structures in ash pollen extract. IgE immunoblot inhibition studies were performed with recombinant and natural pollen allergens to characterize ash pollen allergens and to determine the degree of cross-reactivity between pollen allergens from ash, olive, birch, grasses and weeds. ResultsThe allergen profile of ash pollen comprises Fra e 1, a major allergen related to the major olive allergen, Ole e 1, and to group 11 grass pollen allergens, the panallergen profilin, a two EF-hand calcium-binding protein, a pectinesterase-like molecule and an allergen sharing epitopes with group 4 grass pollen allergens. Thus, the relevant allergens of ash are primarily allergens that share epitopes with pollen allergens from other tree, grass and weed species. ConclusionsAllergic symptoms to ash pollen can be the consequence of sensitization to cross-reactive allergens from other sources. The fact that ash pollen-allergic patients can be discriminated on the basis of their specific IgE reactivity profile to highly or moderately cross-reactive allergens has implications for the selection of appropriate forms of treatment.

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[6] - Gastaminza G, Bartolomé B, Bernedo N, Uriel O, Audicana MT, Etxenagusia M, et al. Allergy to Oleaceae pollen in an area whithout olive trees. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1381

Background: Ash tree belongs to the Oleaceae family; it has been recently described as a relevant allergen in some countries. The aim of this study is to demonstrate the importance of the ash pollen as a triggering factor of the allergic symptoms showed in early spring by a group of patients who live in the Basque Country, where ash are common trees and olive trees are not present. Methods: In accordance with their predominantly sensitisation we selected and classified 48 pollen-allergic-patients in three groups: oleaceae allergic patients (O), grass allergic patients (G) and oleaceae + grass allergic patients (M). Prick tests, specific IgE (EAST) to Olea, Fraxinus and Lolium, patient's scores of symptoms and rescue medication between February and July 2000, conjunctival challenge test with ash and olive pollen extracts, SDS-PAGE immunoblotting, and EAST-inhibition studies were performed. Results: 100% of O patients, 40 % of M patients and 16 % of G patients suffered from early symptoms, coinciding with the flowering of ash, when grass pollen is not present yet. Conjunctival challenge tests with ash and olive pollen extracts were positive in 70% and 100 % respectively in O patients, 50% and 78% in M patients and 31% and 58% in G patients. IgE ˆImmunoblotting showed that sera from most of the patients who suffered from early symptoms (5/7) and belong to the O group, but none of the sera from the remainder patients, recognised Fra e 1. EAST-inhibition studies with a pool of O patients sera who suffered from early symptoms showed no inhibition when ash pollen extract was used as solid phase and grass pollen extract as inhibitor phase. Whereas, using a pool of M patients, Olea was completely inhibited by grass pollen. Conclusion:Ash pollen can be considered as a potentially cause of hay fever in these areas where it is present in considerable amounts. Monosensitised patients to oleaceae pollen, present the symptoms during early spring when ash is flowering. Polisensitised patients could recognise ash pollen allergens because of its cross-reactivity with grass pollen.

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[7] - Rodríguez R, Villalba M, Batanero E, González EM, Monsalve RI, Huecas S, et al. Allergenic diversity of the olive pollen. Allergy 2002;57(suppl. 71):6-16

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

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[8] - Leduc V, Aparicio C, Guérin L, Ledent C, Mairesse M. Importance of species selection for in vivo and in vitro diagnosis of ash tree pollen allergy in Europe. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1280

Background: The ash tree is an underestimated source of pollen in Europe owed the possibility to the confusion caused by the birch sharing an almost identical pollinisation period. The aim of this work was to study the allergenic diversity of two species of ash pollen : Fraxinus excelsior (European ash tree) and Fraxinus americana (White ash tree). Their allergenic diversity was also compared with that of Olea europea (olive tree) pollen. Methods: Seventeen patients with documented sensitivity to ash tree pollen were selected according to discrepancies between the clinical findings and their in vitro specific IgE level. Specific IgE level to the three pollen extracts were evaluated by direct ELISA and allergenic diversity was studied by SDS-PAGE followed by immunoblotting. Results: Out of 17 patients, 10 showed comparable specific IgE level against F.ex et F.am. Seven showed more specific IgE level against F.ex than F.am and four of them were negative to F.am. In addition, 13/17 patients showed a higher level of specific IgE to O.eu than F.ex although olive pollen is not present in the country where patients were selected (BE). Patient IgE specificity was also studied by SDS-PAGE and western blotting. In olive tree pollen, the major allergen Ole e 1, located at 20 kDa, was recognized by 5/17 sera whereas a 30 kDa allergen was detected by 15/17 patients. Profilin, usually considered as a minor allergen in ash and olive tree pollen, was identified as a major allergen in this study because of its detection by 70% (12/17) of the patients. In F.ex pollen, Fra e 1, located at 20 kDa, is recognized by 23% of patients (4/17). This allergen is absent - or not detected - in F.am tree pollen extract. These four patients with monospecific IgE to Fra e 1, correspond to those showing low specific IgE to F. am extract by direct ELISA. Conclusions: This study shows that the selection of ash tree specie could have important consequences for in vitro and in vivo diagnosis. CAP RAST (t15) using Fraxinus americana extract as solid phase can also participate to the underestimation of ash tree pollen sensitization in Europe by producing false negative results. In accordance with the standardization of allergenic extracts, this study moreover confirms that the raw material inducing symptoms should be studied in relation with any given geographical area.

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[9] - Conde Hernández J, Conde Hernández P, Gónzalez Quevedo Tejerína MT, Conde Alcañiz MA, Conde Alcañiz EM, Crespo Moreira P, et al. Antigenic and allergenic differences between 16 different cultivars of Olea europaea. Allergy 2002;57(suppl. 71):60-65

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[10] - Castro AJ, Alché JD, Cuevas J, Romero PJ, Alché V, Rofriguez-Garcia MI. Pollen from Different Olive Tree Cultivars Contains Varying Amounts of the Major Allergen Ole e 1. Int Arch Allergy Immunol 2003;131:164-173

BACKGROUND: Commercial olive pollen from uncertain cultivar origin is the common material used for clinical and biological studies. We aimed to assess the putative heterogeneity of olive cultivars with regard to the presence of the major pollen allergen Ole e 1 and to determine whether these differences have clinical relevance . METHODS: The Ole e 1 content of several cultivars was determined by immunoblotting and ultrastructural immunocytochemistry and compared to that of a commercially available olive pollen extract designed for diagnosis. Reverse transcription-polymerase chain reaction analysis of Ole e 1 transcripts was also performed. Crude protein extracts were used to carry out skin prick tests (SPTs) on 30 allergic patients in order to evaluate the clinical importance of such differences . RESULTS: Ole e 1 was present in all cultivars, although significant quantitative differences were detected. Ole e 1 transcripts positively correlated with the amount of the allergen. Significant variations in the average reactivity of allergic patients to SPTs were observed depending on the cultivar considered . CONCLUSIONS: The presence of the Ole e 1 allergen in all the cultivars suggests that this allergen may play an essential biological role. The expression of the allergen is controlled at the transcriptional level. The significant differences in the Ole e 1 content are likely responsible for the different average reactivity exhibited by patients to the cultivars studied, although the role of other allergens cannot be excluded. Our results suggest that the use of the commercial pollen mixtures currently available may lead to mistakes in allergy diagnosis and to limited success in immunotherapy. Therefore, further standardization is strongly recommended.

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[11] - Carnés Sánchez J, Iraola VM, Sastre J, Florido F, Boluda L, Fernández-Caldas E. Allergenicity and immunochemical characterization of six varieties of Olea europaea. Allergy 2002;57:313-318

Background:The inhalation of Olea europaea pollen is one of the most important causes of allergic respiratory diseases in the Mediterranean basin. The objective of this study was to investigate the antigenic and allergenic composition of six different O.europaea varieties collected in southern Spain. Methods:The varieties included in the study were: Acebuche (wild olive), Carrasqueño, Nevado, Hojiblanco, Manzanillo and Picual. Extracts of these six varieties were prepared. Twenty-nine olive individuals with an immunoglobulin(Ig)E-mediated allergy to olive pollen were skin tested with these extracts. The antigenic profile of these extracts was evaluated by SDS-PAGE; the allergenic profile was investigated by immunoblotting using the serum of these 29 individuals. The Ole e 1 content was established by ELISA inhibition using purified Ole e 1 and rabbit polyclonal antibodies and by scanning densitometry. Results:The extracts that induced the smallest wheal size were Acebuche and Hojiblanco, being significantly different from the rest of the extracts. The antigenic and allergenic profiles of the extracts also varied. The Ole e 1 content ranged from 0.050 in Hojiblanco to 0.232 in Manzanillo, measured by ELISA inhibition and from 0.153 in Hojiblanco to 0.677 in Nevado, measured by scanning densitometry. Conclusions:The different varieties of O.europaea pollen studied demonstrated great differences in the in vivo and in vitro potency of the extracts. There were significant differences in the Ole e 1 content, while the protein content remained very similar in these extracts. This study confirms previous observations of a great variability in the antigenic and allergenic composition of O.europaea pollen extracts and establishes significant differences in Ole e 1 content.

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[12] - Carnés J, Fernández-Caldas E. Ole e 4 and Ole e 5, important allergens of Olea europaea. Allergy 2002;57(suppl. 71):24-28

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[13] - Villalba M, Batanero E, Lopez-Otin C, Sanchez LM, Monsalve RI, Gonzalez de la Pena MA, et al. The amino acid sequence of Ole e1, the major allergen from olive tree (Olea europea) pollen. Eur J Biochem 1993;216:863-869

The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.

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[14] - Salamanca G, Batanero E, Ledesma A, Palomares O, Marazuela E, Barral P, et al. Identification of a new allergen in olive pollen. Allergy Clin Immunol Int 2005;17(Suppl. 1):225

Background Olive tree (Olea europea) pollen is one of the main causes of type-I allergy in Mediterranean countries during pollination seasons. Ten allergens (Ole e 1- Ole e 10) have been described from the complex allergic pattern of olive pollen. The aim of this work was to identify high molecular mass allergens in olive tree pollen. Methods High-molecular weight proteins from olive pollen were separated by size exclusion, SDS-PAGE and 2D electrophoresis. Proteins were detected by silver staining or western blot after transfer to membranes. Sera from olive pollen allergic patients were used in ELISA and inmunoblotting in order to identify new IgE-binding proteins. Results 2D electrophoresis allowed to resolve the olive pollen allergogram separating Ole e1 dimer (40 kDa) and Ole e 9 ( 46 kDa), two abundant allergens in olive pollen, from the rest of proteins with a similar size. An important group of sera from patients allergic to olive pollen from Jaen (Spain), which had been showed to be negative in ELISA to Ole e 1 and Ole e 9 were tested against pollen extract by western blot. Results showed other positive high molecular mass bands in blots. A polymorphic protein with an apparent molecular mass of 37 kDa and a range of pI from 6.0 to 8.8 was recognized by IgEantibodies from patients allergic to olive pollen. Conclusions A new high molecular weight allergen from olive pollen has been detected by proteomic tools. Further analysis of this molecule could help to perform a more accurate diagnosis of patients allergic to olive pollen.

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[15] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[16] - Poncet P, Sénéchal H, Clément G, Godfrin D, Purohit A, Pauli G, et al. Allergome du pollen de frêne (Fraxinus excelsior): intérêt de l'étude individuelle. Rev Fr Allergol Immunol Clin 2006;46:357

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[17] - Rodríguez R, Villalba M, Batanero E, González EM, Monsalve RI, Huecas S, et al. Allergenic diversity of the olive pollen. Allergy 2002;57(suppl. 71):6-16

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

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[18] - Alché JD, Castro AJ, Jiménez-López JC, Morales S, Zafra A, Hamman-Khalifa AM, et al. Differential characteristics of olive pollen from different cultivars: biological and clinical implications. J Investig Allergol Clin Immunol 2007;17(suppl. 1):69-75

The olive tree is grown in many parts of the world. Its germplasm is very broad, with 250 varieties in Spain alone. Variations in the ability of pollen to germinate have been studied in detail and show conspicuous differences between varieties. However, commercial olive pollen from cultivars whose origin is unknown is the material that is commonly used for clinical and biological studies. We aim to assess the putative heterogeneity of olive cultivars with regard to the presence of several pollen allergens and to determine whether these differences have biological and clinical relevance. Previous studies show that most allergens isolated and characterized to date are highly polymorphic. Olive cultivars display wide differences in the expression levels of many allergens and in the number and molecular characteristics of the allergen isoforms expressed. These differences are maintained over the years, and are intrinsic to the genetics of each cultivar. Such broad polymorphism seems to be involved in the physiology of the olive reproductive system, which might include the adaptation of the plant to different environmental conditions, the establishment of the compatibility system, and pollen performance. The differences in allergen composition in cultivars, particularly in the Ole e 1 allergen, are responsible for the important differences in the allergenic potency of the extracts. These findings could have a number of implications for the diagnosis and therapy of olive pollen allergy. We discuss how cultivar differences affect extract quality, diagnostic and therapeutic efficacy and safety, and the development of new vaccines based on the use of recombinant allergens.

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[19] - Duffort O, Palomares O, Lombardero M, Villalba M, Barber D, Rodríguez R, et al. Variability of Ole e 9 Allergen in Olive Pollen Extracts: Relevance of Minor Allergens in Immunotherapy Treatments. Int Arch Allergy Immunol 2006;140:131-138

BACKGROUND: Clustered severe adverse reactions to immunotherapy with olive pollen extracts have been occasionally reported in areas where olive trees are extensively grown. Allergic patients from these areas, in addition to the major olive pollen allergen Ole e 1, frequently recognize a recently described allergen, Ole e 9 . OBJECTIVE: We aimed to develop an immunoassay to measure Ole e 9 concentration and to study the variability of this allergen in olive pollen extracts . METHODS: Monoclonal antibodies (mAb) to Ole e 9 were produced from mice immunized with the pure allergen. One of these mAbs was used to develop a sandwich ELISA with an anti-olive pollen extract rabbit serum as the tracer. Olive pollen batches from several suppliers were analyzed using this method. These batches were also analyzed for Ole e 1 content and biological activity . RESULTS: A 10-fold variation between the extreme values was found for the biological activity of the batches analyzed. Ole e 1 concentration showed a 25-fold variation. Variability of Ole e 9 concentration was extremely high, up to 161 times. The ratio Ole e 1/Ole e 9 varied in a range from 0.6 to 390.4 . CONCLUSION: The availability of a mAb-based ELISA for Ole e 9 made it possible for us to detect an important source of variability in olive pollen batches. This variability may be the cause of outbreaks of adverse reactions in the course of immunotherapy treatments, which have sometimes been observed among olive-allergic patients living in areas with very high levels of airborne olive pollen.

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[20] - Pérez Formoso J, Monteseirin J, Chacón P, Martínez A, Asturias J, Ventura I, et al. Quantification of Ole e1 and Ole e2 in extracts of Olea europaea for immunotherapy from different commercial sources. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°840

Background: Pollen of the olive (Olea europaea) is one of the most important causes of respiratory allergy in the southern half of this country. Its mean concentration is 400 grains/m3, although in areas where the olive is grown intensively, such as Jaén and Córdoba, it can reach 13500 grains/m3. Immunoblotting has revealed in olive pollen 20 bands that correspond to allergenic proteins, from which 10 allergens have been characterized, denominated Ole e1 to Ole e10. The allergen Ole e2, while minority in the general population, is found at a high rate in more-exposed populations. The aim of this work was to analyze the content of two important allergens in different extracts of olive pollen: Ole e1 and Ole e2. Materials: We obtained a lyophilized extract for immunotherapy from each of the 5 commercial firms in the study. The quantification of allergens was performed by means of specific ELISA assays, using mono- and polyclonal antibodies, and purified proteins (Ole e1 and Ole e2) as reference material. Results: The content of Ole e1 in extracts of O. europaea pollen ranged between 7.08 and 63.48 ug (dry weight), with a maximum variation of 9-fold among them. The allergen profilin (Ole e2) had a lesser presence in the various extracts, but a greater variability. Its values ranged between 0.002 and 0.12 ug (dry weight), with a maximum variation of 60-fold. Conclusions: There was a notable difference in content of both Ole e1 and Ole e2 between the various commercial products. Moreover, none of them takes into account the concentration of Ole e2 in the extracts, given the tiny amount contained; this would have to be considered in patients very sensitive to this protein.

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[21] - Quiralte J, Palacios L, Rodríguez R, Cárdaba B, Arias de Saavedra JM, Villalba M, et al. Modelling diseases: the allergens of Olea europaea pollen. J Investig Allergol Clin Immunol 2007;17(suppl. 1):76-82

This study analyzes the influence of the IgE response to certain olive pollen allergens in the modulation of the different clinical phenotypes of allergic disease and their relationship with the level of exposure to pollen and genetic factors. Patients from high-exposure areas had a complex IgE antibody response to allergens of Olea euroapea, which included 3 or more allergens in 75% of cases. The majority allergens were Ole e 1, Ole e 2 (profilin), Ole e 7 (lipid transporting protein), Ole e 9 (glucanase), and Ole e 10. The existence of the antigen HLA-DR2 (15) led to a higher risk of sensitization to Ole e 10 and a greater trend towards the development of severe asthma, which increased in the presence of an anti-profilin IgE. Thirty percent of patients suffering from pollinosis simultaneously presented allergy to vegetable foods. Anti-Ole e 7 IgE was significantly associated with fruit anaphylaxis and anti-profilin IgE was detected in 90% of patients with oral syndrome. Finally, we analyzed the role of glucanase and Ole e 10 as causes of the pollen-latex-fruit syndrome.

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[22] - Morales S, Jiménez-López JC, Castro AJ, Rodríguez-García MI, Alché JD. Olive pollen profilin (Ole e 2 allergen) co-localizes with highly active areas of the actin cytoskeleton and is released to the culture medium during in vitro pollen germination. J Microsc 2008;231:332-341

Pollen allergens offer a dual perspective of study: some of them are considered key proteins for pollen physiology, but they are also able to trigger allergy symptoms in susceptible humans after coming in contact with their tissues. Profilin (Ole e 2 allergen) has been characterized, to some extent, as one of the major allergens from Olea europaea L. pollen, a highly allergenic species in the Mediterranean countries. In order to obtain clues regarding the biological role of this protein, we have analyzed both its cellular localization and the organization of actin throughout pollen hydration and early pollen tube germination. The localization of the cited proteins was visualized by confocal laser scanning microscopy immunofluorescence using different antibodies. Upon pollen hydration and pollen germination, a massive presence of profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the 'effective' apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. After using a permeabilization-enhanced protocol for immunolocalization, profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends. Noticeable accumulations of actin were observed in the cytoplasm of the pollen tube; particularly, in both the apical region and the area immediately close to the aperture. Actin filaments were not observed, probably due to the need of further enhanced fixation procedures. The ultrastructural localization of profilin showed the presence of the protein in the cytoplasm of both the mature pollen grain and the pollen tube. The results shown here could be interpreted as signs of a massive dissociation of the actin-profilin complexes, mobilization of actin monomers, and therefore, an intense activity of the actin cytoskeleton. The extensive release of allergenic proteins from the pollen grain into the surrounding aqueous media, as described here for profilin, may help us to understand the mechanisms by which these allergens might come in contact with the human mucosa, therefore triggering the symptoms of allergy.

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[23] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[24] - Fernandez MC, Olmedilla A, Alche JD, Palomino P, Lahoz C, Rodriguez-Garcia MI. Immunogold probes for light and electron microscopic localization of Ole e I in several Oleaceae pollens. J Histochem Cytochem 1996;44:151-158

We investigated the immunolocalization of the olive major allergen Ole e I and Ole e I-like proteins in pollen from several Oleaceae species [olive (Olea europaea), ash (Fraxinus excelsior), privet (Ligustrum vulgaris), lilac (Syringa vulgare), and forsythia (Forsythia suspensa)]. Crossreactions among different pollens were found in enzyme immunoassays. For immunolocalization with light microscopy we used the silver enhancement technique with three monoclonal antibodies (1D8, 10H1, and 16G2) that recognize three different epitopes of the allergen Ole e I. Our findings show that the silver enhancement technique is very useful when several antibodies are to be used for rapid screening of different materials. MAb 10H1 gave the most precise results and was selected for further immunolocalization studies with transmission electron microscopy. The epitope recognized by this MAb was localized exclusively in the endoplasmic reticulum in olive pollen. In lilac, privet, and ash pollen, most of the reactivity was also s een in the endoplasmic reticulum; however, the 10H1 epitope was not detected in forsythia pollen

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[25] - Hamman-Khalifa A, Castro AJ, Jimenez-Lopez JC, Rodriguez-Garcia MI, Alche JD. Olive cultivar origin is a major cause of polymorphism for Ole e 1 pollen allergen. BMC Plant Biol 2008;8:10

ABSTRACT: BACKGROUND: Pollens from different olive (Olea euroapea L.) cultivars have been shown to differ significantly in their content in Ole e 1 and in their overall allergenicity. This allergen is, in addition, characterized by a high degree of polymorphism in its sequence. The purpose of this study is to evaluate the putative presence of divergences in Ole e 1 sequences from different olive cultivars. RESULTS: RNA from pollen individually collected from 10 olive cultivars was used to amplify Ole e 1 sequences by RT-PCR, and the sequences were analyzed by using different bioinformatics tools. Numerous nucleotide substitutions were detected throughout the sequences, many of which resulted in amino acid substitutions in the deduced protein sequences. In most cases variability within a single variety was much lower than among varieties. Key amino acid changes in comparison with "canonical" sequences previously described in the literature included: a) the substitution of C19 -relevant to the disulphide bond structure of the protein-, b) the presence of an additional N-glycosylation motif, and c) point substitutions affecting regions of Ole e 1 already described like relevant for the immunogenicity/allergenicity of the protein. CONCLUSIONS: Varietal origin of olive pollen is a major factor determining the diversity of Ole e 1 variants. We consider this information of capital importance for the optimal design of efficient and safe allergen formulations, and useful for the genetic engineering of modified forms of the allergen among other applications.

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[26] - Valenta R, Twaroch T, Swoboda I. Component-resolved diagnosis to optimize allergen-specific immunotherapy in the Mediterranean area. J Investig Allergol Clin Immunol 2007;17(suppl. 1):88-92

Allergen-specific immunotherapy (SIT) is the only allergen-specific treatment for allergy. It can prevent progression of the disease and has a long-lasting therapeutic effect. Since SIT is allergen-specific, the identification of the disease-eliciting allergen is an essential prerequisite for the accurate prescription of treatment. Diagnostic tests based on allergen extracts consist of mixtures of various allergens of which some are specific for the allergen source and others occur as cross-reactive allergens in various unrelated allergen sources. It may therefore be difficult and sometimes impossible to identify the disease-causing allergen with such tests, particularly in patients who are sensitized to more than one allergen source. Sensitization to pollens from olive, grasses, and Parietaria in the Mediterranean area is frequently treated with SIT. Here, we describe allergen molecules from these sources that can be used for component-resolved diagnosis of allergy to facilitate the selection of patients for SIT and monitor the immunological effects of treatment.

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[27] - Rodriguez R, Villalba M, Monsalve RI, Batanero E. The spectrum of olive pollen allergens. Int Arch Allergy Immunol 2001;125:185-195

Olive pollen is one of the most important causes of seasonal respiratory allergy in Mediterranean countries, where this tree is intensely cultivated. Among the high number of protein allergens detected in this pollen, 8 - Ole e 1 to Ole e 8 - have been isolated and characterized. Ole e 1 is the most frequent sensitizing agent, affecting more than 70% of the patients suffering of olive pollinosis, although others, such as Ole e 4 and Ole e 7, have also been shown to be major allergens. In this context, the prevalence of many olive pollen allergens seems to be dependent on the geographical area where the sensitized patients live. Some of the olive allergens have been revealed as members of known protein families: profilin (Ole e 2), Ca(2+)-binding proteins (Ole e 3 and Ole e 8), superoxide dismutase (Ole e 5) and lipid transfer protein (Ole e 7). No biological function has been demonstrated for Ole e 1, whereas Ole e 4 and Ole e 6 are new proteins without homology to known sequences from databases. cDNAs encoding for Ole e 1, Ole e 3 and Ole e 8 have been overproduced in heterologous systems. The recombinant products were correctly folded and exhibited the functional activities of the natural allergens. In addition to the Oleaceae family, other species, such as Gramineae or Betulaceae, contain pollen allergens structurally or immunologically related to those of the olive tree. This fact allows to detect and evaluate antigenic cross-reactivities involving olive allergens. The aim of this research is the development of new diagnostic tools for olive pollinosis and new approaches to improve the classical immunotherapy.

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[28] - Marknell DeWitt Å, Niederberger V, Lehtonen P, Spitzauer S, Sperr WR, Valent P, et al. Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11. Clin Exp Allergy 2002;32:1329-1340

BackgroundAllergy to grass pollen is typically associated with serum IgE antibodies to group 1 and/or group 5 allergens, and additionally often to one or several less prominent allergens. Most of the grass pollen allergens identified to date have been characterized in detail by molecular, biochemical and immunological methods, timothy grass being one of the most thoroughly studied species. However, a 20-kDa allergen frequently recognized by IgE antibodies from grass pollen allergics has so far escaped cloning and molecular characterization. ObjectiveTo clone and characterize the 20kDa timothy grass pollen allergen Phl p 11. MethodsPhl p 11 cDNA was cloned by PCR techniques, utilizing N-terminal amino acid sequence obtained from the natural allergen. Phl p 11 was expressed as a soluble fusion protein in Escherichia coli, purified to homogeneity and used for serological analysis and to study Phl p 11 specific induction of histamine release from basophils and skin reactivity in sensitized and control subjects. ResultsPhl p 11 cDNA defined an acidic polypeptide of 15.8kDa with homology to pollen proteins from a variety of plant species and to soybean trypsin inhibitor. The sequence contained one potential site for N-linked glycosylation. Serological analysis revealed that recombinant Phl p 11 shared epitopes for human IgE antibodies with the natural protein and bound serum IgE from 32% of grass pollen-sensitized subjects (n=184). Purified recombinant Phl p 11 elicited skin reactions and dose-dependent histamine release from basophils of sensitized subjects, but not in non-allergic controls. ConclusionAs the first representative of group 11 grass pollen allergens, Phl p 11 has been cloned and produced as a recombinant protein showing allergenic activity. One-third of grass pollen-sensitized subjects showed specific IgE reactivity to recombinant Phl p 11, corresponding in magnitude to a significant proportion of specific IgE to grass pollen extract.

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[29] - Rodríguez R, Villalba M, Batanero E, González EM, Monsalve RI, Huecas S, et al. Allergenic diversity of the olive pollen. Allergy 2002;57(suppl. 71):6-16

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

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[30] - Barderas R, Villaba M, Batanero E, Pascual CY, Rodriguez R. Role of profilin and polcalcin in chenopod pollen allergy. J Allergy Clin Immunol 2003;111:1132-1133

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[31] - Barderas R, Villalba M, Lombardero M, Rodríguez R. Identification and Characterization of Che a 1 Allergen from Chenopodium album Pollen. Int Arch Allergy Immunol 2002;127:47-54

Background: Pollinosis to Chenopodium album has been reported, but no data are available on its allergenic proteins. Methods: An allergen from C. album pollen has been isolated by means of gel permeation and reverse-phase high-performance liquid chromatography. Molecular characterization was achieved by concanavalin A reaction, mass spectrometry, Edman degradation and cDNA sequence. Antigenic analyses were performed by immunoblotting, ELISA, and ELISA inhibition, using sera from allergic patients, two Ole e 1-specific monoclonal antibodies and an Ole e 1-specific polyclonal antiserum. Results: The isolated allergen, Che a 1, is a glycoprotein of molecular mass 17.088 kD and 143 amino acid residues, whose sequence exhibits 27-45% identity with known members of the Ole e 1-like protein family. 77% of sera from patients allergic to chenopod pollen were reactive to Che a 1. No correlation was found between the IgE reactivities to Che a 1 and Ole e 1, the major allergens from olive pollen, and both allergens display low, although detectable, IgE and IgG cross-reactivities. Conclusions: Che a 1, a relevant allergen from chenopod pollen, is structurally related to the Ole e 1-like protein family, but exhibits significant differences on its polypeptide sequence that could explain its different antigenic behavior and limited cross-reactivity.

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[32] - Calabozo B, Diaz-Perales A, Salcedo G, Barber D, Polo F. Structural and antigenic similarity between Pla l 1 and Ole e 1, the major allergens of English plantain and olive pollens. AAAAI 57th Annual Meeting, New Orleans, 16-21 March, 2001, Poster n°51

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[33] - Palomares O, Swoboda I, Villalba M, Balic N, Spitzauer S, Rodríguez R, et al. The Major Allergen of Olive Pollen Ole e 1 Is a Diagnostic Marker for Sensitization to Oleaceae. Int Arch Allergy Immunol 2006;141:110-118

BACKGROUND: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen . METHODS: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1 . RESULTS: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae . CONCLUSIONS: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy.

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[34] - Castro AJ, Alché JD, Calabozo B, Rodríguez-García MI, Polo F. Pla 1 1 and Ole e 1 pollen allergens share common epitopes and similar ultrastructural localization. J Investig Allergol Clin Immunol 2007;17(suppl. 1):93-99

BACKGROUND: English plantain (Plantago lanceolata L.) and olive (Olea europaea L.) pollens are important causes of pollinosis in large areas of North America, Australia, and the Mediterranean basin. The major pollen allergens of both plants, Pla I 1 and Ole e 1, share 38.7% of their amino acid sequences. OBJECTIVE: To analyze putative cross-reactivity between these 2 proteins. METHODS: Several antibodies and patients' sera were used in immunoblot and immunocytochemistry experiments. RESULTS: Two anti-Pla I 1 antibodies were able to bind to 3 polypeptides from olive pollen protein extracts, which correspond to the 3 glycosylation isoforms of Ole e 1 (18-22 kDa) previously described. Moreover, Pla I 1 protein was found in the cytoplasm of both the vegetative and the generative cells of P lanceolata mature pollen. On olive pollen sections, these anti-Pla I 1 antibodies displayed significant labeling in the cytoplasm of the vegetative cell and in both the exine and the material adhering to this outer layer of the pollen wall. In addition, the anti-Ole e 1 antibody 10H1 was found to cross-react with proteins of similar masses (16-20 kDa) to Pla I 1 variants. In Plantago pollen sections, the 10H1 antibody recognized proteins located in the cytoplasm of both the vegetative and generative cells. Cross-reaction was confirmed using sera from patients allergic to either plant pollen. CONCLUSION: Both allergens share common epitopes, which can be cross-recognized by different antibodies and sera from different patients, although this antigenic similarity seems to have little clinical relevance.

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[35] - Rodríguez R, Villalba M, Batanero E, Palomares O, Quiralte J, Salamanca G, et al. Olive pollen recombinant allergens: value in diagnosis and immunotherapy. J Investig Allergol Clin Immunol 2007;17(suppl. 1):56-62

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

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[36] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[37] - Villalba M, Batanero E, Lopez-Otin C, Sanchez LM, Monsalve RI, Gonzalez de la Pena MA, et al. The amino acid sequence of Ole e1, the major allergen from olive tree (Olea europea) pollen. Eur J Biochem 1993;216:863-869

The complete primary structure of the major allergen from Olea europaea (olive tree) pollen, Ole e I (IUIS nomenclature), has been determined. The amino acid sequence was established by automated Edman degradation of the reduced and alkylated molecule as well as of selected fragments obtained by proteolytic digestions. Ole e I contains a single polypeptide chain of 145 amino acid residues with a calculated molecular mass of 16331 Da. No free sulfhydryl groups have been detected in the native protein. The molecule contains a putative glycosylation site. A high degree of microheterogeneity has been observed, mainly centered in the first 33% of the molecule. Comparison of Ole e I sequence with protein sequence databases showed no similarity with other known allergens. However, it has a 36% and 38% sequence identity with the putative polypeptide structures, deduced, respectively, from nucleotide sequences of genes isolated from tomato anthers and corn pollen, which have been suggested to be involved in the growing of the pollen tube. Therefore, the olive tree allergen may be a constitutive protein of the pollen involved in reproductive functions.

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[38] - Barderas R, Villalba M, Batanero E, Palomares O, Barral P, Rodriguez R. Fraxinus excelsior pollen allergy: cloning, expression and characterisation of the major allergen Fra e 1. EAACI 22th Congress, Paris, 7-11 June, 2003, Poster n°1360

Background: The pollen of ash (Fraxinus excelsior) has become an important cause of allergy in Centre Europe and Minor Asia. Previous studies reported Fra e 1 as a major inductor of IgE-mediated hypersensitivity affecting more than 65% of patients allergic to the whole extract of ash pollen. The aim of this work has been the cloning, expression in Pichia pastoris and further characterisation of recombinant Fra e 1. Methods: Cloning has been done by PCR amplification of Fraxinus excelsior cDNA, which was obtained from total RNA of the pollen using specific oligonucleotides. The expression of the specific-DNA was performed with pPICZa A vector and KM71 strain of Pichia pastoris. Purification of the recombinant protein was achieved using gel permeation column and reverse-phase HPLC. Immunoblotting and ELISA were used to analyse the capability of rFra e 1-binding to Ole e 1-specific polyclonal and monoclonal antybodies, as well as to IgE from 32 sera of patients allergic to olive pollen. Results: The nucleotide sequence of cDNA encoding Fra e 1 was 438 bp, and its deduced amino acid sequence 145 residues length, with a potential N-glycosylation site at Asn-111. High sequence similarity was found with Ole e 1, Syr v 1 and Lig v 1, major allergens from olive, lilac and privet pollen respectively, identity ranging between 86 to 91%. Recombinant production yield 50 mg of pure rFra e 1 per liter of culture. Immunological characterization of the protein indicated that rFra e 1 exhibit a high degree of cross-reactivity with Ole e 1. Conclusion: A new member of the Ole e 1-like family of pollen allergens has been identified and characterized. The obtained data indicate that the major allergen from ash, Fra e 1, share most of IgG and IgE epitopes with other members of the Oleaceae family.

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[39] - Hemmer W, Focke M, Wantke F, Götz M, Jarisch M, Jäger S, et al. Ash (Fraxinus excelsior)-pollen allergy in central Europe: specific role of pollen panallergens and the major allergen of ash, Fra e1. Allergy 2000;55:923-930

BACKGROUND: The role of ash (Fraxinus excelsior) pollen as a cause of spring pollinosis in central Europe has received little attention. It is not clear whether ash pollen is a primary cause of sensitization or whether it is implicated through cross-sensitization to other pollens. METHODS: Over a 22-month period, ash pollen was included in a screening series for inhalant allergies. Pollen data were documented from 1976 through 1999. The frequency of IgE-binding to the ash-specific allergen Fra e 1 and pollen panallergens, respectively, was compared by Western blot between mono- (n = 6), oligo- (n = 16), and polysensitized (n=25) patients. RESULTS: Of 5,416 consecutive patients sensitized to any pollen, 920 (17.6%) had a positive skin prick test to ash. Total pollen counts varied extensively between years (229-5,351) as did peak concentrations (23-837 grains/m3/24 h). Western blotting revealed Fra e 1 sensitization in 100% of monosensitized, 93% of oligosensitized, but only 44% of polysensitized patients. IgE against profilins (Fra e 2), Ca-binding proteins (Fra e 3), and carbohydrate epitopes in the three groups was found in 0/0/17%, 0/19/31%, and 32/72/60%, respectively. At least 50% of sera from patients with Fra e 1 sensitization did not bind with the protein in Western blots under reducing conditions. CONCLUSIONS: Ash pollen should be considered a relevant factor and distinct entity in spring pollinosis. In all, only 20% of positive skin tests to ash appear to result from cross-sensitization to pollen panallergens.

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[40] - Tejera ML, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 1999;104:797-802

Olive tree (Olea europaea) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. OBJECTIVE: The aim of this study was to identify and purify a new allergen of olive tree pollen. METHODS: Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. RESULTS: The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. CONCLUSIONS: A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen.

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[41] - Huecas S, Villalba M, Rodriguez R. Ole e 9, a major olive pollen allergen is a 1,3-beta-glucanase. Isolation, characterization, amino acid sequence, and tissue specificity. J Biol Chem 2001;276:27959-27966

Olive pollen allergy is a clinical disorder affecting the human population of Mediterranean areas. A novel major allergen, Ole e 9, has been isolated from olive pollen by gel permeation, hydrophobic affinity, and reverse-phase high performance liquid chromatographies. It is involved in the allergic responses of 65% of patients suffering olive pollinosis. Ole e 9 (molecular mass of 46.4 kDa) displays 1,3-beta-endoglucanase activity (38.9 +/- 5.6 mg of glucose released/min x micromol of protein at pH 4.5-6.0 using laminarin as substrate). It is the first 1,3-beta-glucanase, a member of the "pathogenesis-related" protein family, detected in pollen tissue. Seven tryptic peptides of the allergen were sequenced by Edman degradation and used for designing primers to clone the cDNA codifying the protein. Specific cDNA for Ole e 9 was synthesized from total RNA and amplified using the polymerase chain reaction. The allergen sequence showed an open reading frame of 460 amino acids comprising a putative signal peptide of 26 residues. It shows 39, 33, and 32% sequence identity including the catalytic residues when compared with 1,3-beta-glucanases from wheat, willow, and Arabidopsis thaliana, respectively. Northern blot analysis showed that Ole e 9 transcript is specifically expressed in the pollen tissue, and highly conserved counterparts were only detected in taxonomically related pollens.

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[42] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[43] - Ledesma A, Tejera M, Rodríguez R, Villalba M, Guardia P, Moreno C, et al. Prevalence and cross-reactivity of the olive pollen LTP, Ole e 7. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°998

Background: Ten allergens from olive tree pollen have been described so far. One of them, Ole e 7, belongs to the nsLTP family. No data on prevalence in different geographical areas and cross-reactivity with protein homologous from related and unrelated species are available. Method: The study of the prevalence of Ole e 7 has been carried out by using the quantitative ADVIA Centaur specific IgE assay using biotinylated Ole e 7. A total of 108 patient sera, belonging to two Spanish regions with different levels of exposure to olive pollen, were included in this study. The presence of homologous proteins to Ole e 7 was tested by ELISA inhibition of the IgE binding of a pool of sera from patients sensitized to this allergen, with pollens from related (Oleaceae family) and unrelated (birch, grasses, chenopod) species used as inhibitors. Results: Prevalence of Ole e 7 varies from 5% in patients from Sevilla to 50% in patients from Córdoba. In contrast, a major allergen as Ole e 1, which is present in very high levels in pollen, is recognized by more than 60% of the patients in both areas. ELISA inhibition showed that pollens from ash, lilac, privet, birch, chenopod and mugwort contain polypeptides that share allergenic determinants with Ole e 7, as they were able to inhibit the binding of the pool of sera to Ole e 7 at some extent. Inhibition values ranged from 18% to 42%. In contrast, grass pollen extracts were not able to inhibit this binding. Conclusion: Two features concerning Ole e 7 have been studied, its prevalence as well as its implication in cross-reactivity. The prevalence of Ole e 7 depends on the geographical area of the patients, being a major allergen in the regions with a high olive pollen charge. On the other hand, our study revealed the presence of proteins homologous to Ole e 7 in pollens from related and unrelated pollen sources, however the IgE cross-reactivity among them is limited probably because the low amino acid sequence similarity of the members of this family of proteins.

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[44] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[45] - Hemmer W, Focke M, Wantke F, Götz M, Jarisch M, Jäger S, et al. Ash (Fraxinus excelsior)-pollen allergy in central Europe: specific role of pollen panallergens and the major allergen of ash, Fra e1. Allergy 2000;55:923-930

BACKGROUND: The role of ash (Fraxinus excelsior) pollen as a cause of spring pollinosis in central Europe has received little attention. It is not clear whether ash pollen is a primary cause of sensitization or whether it is implicated through cross-sensitization to other pollens. METHODS: Over a 22-month period, ash pollen was included in a screening series for inhalant allergies. Pollen data were documented from 1976 through 1999. The frequency of IgE-binding to the ash-specific allergen Fra e 1 and pollen panallergens, respectively, was compared by Western blot between mono- (n = 6), oligo- (n = 16), and polysensitized (n=25) patients. RESULTS: Of 5,416 consecutive patients sensitized to any pollen, 920 (17.6%) had a positive skin prick test to ash. Total pollen counts varied extensively between years (229-5,351) as did peak concentrations (23-837 grains/m3/24 h). Western blotting revealed Fra e 1 sensitization in 100% of monosensitized, 93% of oligosensitized, but only 44% of polysensitized patients. IgE against profilins (Fra e 2), Ca-binding proteins (Fra e 3), and carbohydrate epitopes in the three groups was found in 0/0/17%, 0/19/31%, and 32/72/60%, respectively. At least 50% of sera from patients with Fra e 1 sensitization did not bind with the protein in Western blots under reducing conditions. CONCLUSIONS: Ash pollen should be considered a relevant factor and distinct entity in spring pollinosis. In all, only 20% of positive skin tests to ash appear to result from cross-sensitization to pollen panallergens.

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[46] - Purohit A, Niederberger V, Papanikolaou L, Oster JP, Bessot JC, De Blay F, et al. Caractérisation des allergènes du pollen de frène. Communication: 9èmes Journées Parisiennes d'Allergie - Paris 10-12 janvier 2001

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[47] - Poncet P, Senechal H, Clement G, Godfrin D, Wal JM, Peltre G, et al. Allergy to ash (Fraxinus excelsior) pollen: allergomic study. Allergy Clin Immunol Int 2005;17(Suppl. 1):217

Background : In Europe, sensitization to ash pollen (Oleaceae family) is underestimated because of a lack of precise pollinisation calendar, lack of standardized extract for diagnosis and wide cross reactivities with other pollens. Objective and methods : To identify the panel of ash pollen allergens and to decipher the IgE response, sixty two sera from patients diagnosed for ash, olive or grass allergies were screened by IgE immunoblot of an aquaeous extract of ash pollen separated by SDS-PAGE. Ten sera were selected for 2-D IgE immunoblot analysis followed by mass spectrometry for protein identification. Results : IgE reactive bands were detected in 44 sera. Thirty seven showed binding to proteins of Mr 18-20kDa, Fra e 1 (major ash pollen allergen). Twelve sera recognized a 14kDa band, Fra e 2 (profilin), 2 sera, a 10kDa protein, Fra e 3 and 12, proteins >30kDa. Specific and private patterns of several spots were observed for each of individually tested sera in 2-D IgE immunoblots. When overlapped, 5 zones of IgE reactivity were determined. (1) 15 acidic spots at 43kDa, (2) 10 neutro-basic spots at 45kDa, (3) 6 neutro-acidic spots at 32kDa, (4) a series of doublets, pI 4.8 to 8.5 at 20kDa and (5) one spot pI< 4.75 at 14kDa. Forty three spots (allergens and non-allergens) were collected for protein identification by mass spectrometry. MALDI-TOF analysis of trypsin in-gel-digested proteins revealed that proteins from zone 4 corresponded to isoforms of Fra e 1. The protein of zone 5 belongs to the profilin family and the spots of zone 2 are isoforms of a sugar transport protein. Besides these allergens, 10 proteins that did not exhibit IgE reactivity were identified with significant scores and sequence coverages. Conclusion : Ninety two percent of the ash-sensitized patients have IgE against Fra e 1 and 46% against Fra e 2. Cross-reactivities of IgE from grasssensitized patients does not involve Fra e 1. Using the highly resolving power of 2D electrophoresis coupled to the sensitivity of mass spectrometry we show that Fra e 1 exhibited a wider heterogeneity of pI than expected, and that IgE reactivities against high Mr allergens may be resolved in 3 regions. Our data contribute to a better delineation of ash pollen allergens and pattern of sensitization. The diagnostic and therapeutic relevance of these identified allergens may help to predict the evolution of symptoms and to refine the treatment of ash allergy.

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[48] - Poncet P, Sénéchal H, Clément G, Godfrin D, Purohit A, Pauli G, et al. Allergome du pollen de frêne (Fraxinus excelsior): intérêt de l'étude individuelle. Rev Fr Allergol Immunol Clin 2006;46:357

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[49] - Barderas R, Villalba M, Purohit A, Papanikolaou I, Pauli G, Rodriguez R. Spectrotypes des IgE spécifiques de patients ayant une franche positivité aux extraits de frêne. Rev Fr Allergol Immunol Clin 2004;44:357

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[50] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[51] - Poncet P, Senechal H, Clement G, Godfrin D, Wal JM, Peltre G, et al. Allergy to ash (Fraxinus excelsior) pollen: allergomic study. Allergy Clin Immunol Int 2005;17(Suppl. 1):217

Background : In Europe, sensitization to ash pollen (Oleaceae family) is underestimated because of a lack of precise pollinisation calendar, lack of standardized extract for diagnosis and wide cross reactivities with other pollens. Objective and methods : To identify the panel of ash pollen allergens and to decipher the IgE response, sixty two sera from patients diagnosed for ash, olive or grass allergies were screened by IgE immunoblot of an aquaeous extract of ash pollen separated by SDS-PAGE. Ten sera were selected for 2-D IgE immunoblot analysis followed by mass spectrometry for protein identification. Results : IgE reactive bands were detected in 44 sera. Thirty seven showed binding to proteins of Mr 18-20kDa, Fra e 1 (major ash pollen allergen). Twelve sera recognized a 14kDa band, Fra e 2 (profilin), 2 sera, a 10kDa protein, Fra e 3 and 12, proteins >30kDa. Specific and private patterns of several spots were observed for each of individually tested sera in 2-D IgE immunoblots. When overlapped, 5 zones of IgE reactivity were determined. (1) 15 acidic spots at 43kDa, (2) 10 neutro-basic spots at 45kDa, (3) 6 neutro-acidic spots at 32kDa, (4) a series of doublets, pI 4.8 to 8.5 at 20kDa and (5) one spot pI< 4.75 at 14kDa. Forty three spots (allergens and non-allergens) were collected for protein identification by mass spectrometry. MALDI-TOF analysis of trypsin in-gel-digested proteins revealed that proteins from zone 4 corresponded to isoforms of Fra e 1. The protein of zone 5 belongs to the profilin family and the spots of zone 2 are isoforms of a sugar transport protein. Besides these allergens, 10 proteins that did not exhibit IgE reactivity were identified with significant scores and sequence coverages. Conclusion : Ninety two percent of the ash-sensitized patients have IgE against Fra e 1 and 46% against Fra e 2. Cross-reactivities of IgE from grasssensitized patients does not involve Fra e 1. Using the highly resolving power of 2D electrophoresis coupled to the sensitivity of mass spectrometry we show that Fra e 1 exhibited a wider heterogeneity of pI than expected, and that IgE reactivities against high Mr allergens may be resolved in 3 regions. Our data contribute to a better delineation of ash pollen allergens and pattern of sensitization. The diagnostic and therapeutic relevance of these identified allergens may help to predict the evolution of symptoms and to refine the treatment of ash allergy.

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[52] - Sookrung N, Chaicumpa W, Tungtrongchitr A, Vichyanond P, Bunnag C, Ramasoota P, et al. Periplaneta americana arginine kinase as a major cockroach allergen among Thai patients with major cockroach allergies. Environ Health Perspect 2006;114:875-880

Periplaneta americana is the predominant cockroach (CR) species and a major source of indoor allergens in Thailand. Nevertheless, data on the nature and molecular characteristics of its allergenic components are rare. We conducted this study to identify and characterize the P. americana allergenic protein. A random heptapeptide phage display library and monoclonal antibody (MAb) specific to a the P. americana component previously shown to be an allergenic molecule were used to identify the MAb-bound mimotope and its phylogenic distribution. Two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, peptide mass fingerprinting, and BLAST search were used to identify the P. americana protein containing the MAb-specific epitope. We studied the allergenicity of the native protein using sera of CR-allergic Thai patients in immunoassays. The mimotope peptide that bound to the MAb specific to P. americana was LTPCRNK. The peptide has an 83-100% identity with proteins of Anopheles gambiae, notch homolog scalloped wings of Lucilia cuprina, delta protein of Apis mellifera; neu5Ac synthase and tyrosine phosphatase of Drosophila melanogaster, and a putative protein of Drosophila pseudoobscura. This finding implies that the mimotope-containing molecule of P. americana is a pan-insect protein. The MAb-bound protein of P. americana was shown to be arginine kinase that reacted to IgE in the sera of all of the CR-allergic Thai patients by immunoblotting, implying its high allergenicity. In conclusion, our results revealed that P. americana arginine kinase is a pan-insect protein and a major CR allergen for CR-allergic Thai patients.

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[53] - Barderas R, Purohit A, Papanikolaou I, Rodriguez R, Pauli G, Villalba M. Cloning, expression, and clinical significance of the major allergen from ash pollen, Fra e 1. J Allergy Clin Immunol 2005;115:351-357

BACKGROUND: Ash tree, an Oleaceae member, is considered an important source of pollen allergy in Central Europe. Fra e 1 is a protein of the Ole e 1-like family, which regulates pollen tube growth. It has been suggested to be a relevant allergen from ash pollen . OBJECTIVE: Cloning Fra e 1-cDNA and overproducing a properly folded recombinant allergen to analyze its clinical significance . METHODS: Fra e 1-encoding cDNA was amplified by PCR, cloned in Escherichia coli , and sequenced. The recombinant allergen was produced in Pichia pastoris and used in immunoblotting, ELISA, histamine release, and skin prick tests. Sera and blood cells from patients sensitized to ash pollen as well as anti-Ole e 1 monoclonal and polyclonal antisera were used . RESULTS: Recombinant Fra e 1 (rFra e 1) is a glycoprotein of 145 amino acids exhibiting 82%, 88%, and 91% identity with Syr v 1, Ole e 1, and Lig v 1, allergens of the Oleaceae family. It was secreted to the extracellular medium of the yeast cultures and purified by means of 3 chromatographic steps. IgG from Ole e 1-specific antibodies recognized rFra e 1. IgE antibodies from ash-sensitized patients bound to rFra e 1 with a prevalence of 75%. The recombinant allergen induced histamine release. Twenty-nine of 30 ash-sensitized patients were positive to rFra e 1 by skin prick tests . CONCLUSION: Fra e 1 is a relevant allergen in ash pollen sensitization. It has been efficiently produced in P pastoris and could be used in diagnosis.

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[54] - Asturias JA, Arilla MC, Gomez-Bayon N, Aguirre M, Martinez A, Palacios R, et al. Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen. Mol Immunol 1998;35:469-478

Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy. We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2. Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5%. Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen. Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen. High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants.

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[55] - Ledesma A, Rodriguez R, Villalba M. Olive-pollen profilin: molecular and immunologic properties. Allergy 1998;53:520-526

Olive-pollen profilin has been isolated and characterized as a significant allergen. Its molecular properties, such as a molecular mass of 15 kDa amino-acid composition and secondary repetitive structure percentages of 15% alpha-helix, 33% beta-strand, 20% beta-turn, and 32% random coil, have been determined. Its allergenic capability, a recognition frequency estimated at 24% of olive-hypersensitive patients, and high cross-reactivity with all the pollen used have been found. The presence of conformation epitopes in the olive profilin, as well as a high structural and immunologic similarity to other pollen sources such as birch and ash, can be established from these studies.

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[56] - Hayek B, Vangelista L, Pastore A, Sperr WR, Valent P, Vrtala S, et al. Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4: structural basis for calcium-modulated IgE recognition. J Immunol 1998;161:7031-7039

Serum IgE was used to isolate a cDNA coding for a 9.4-kDa two EF-hand calcium-binding allergen, Aln g 4, from a lambda gt11 expression cDNA library constructed from alder (Alnus glutinosa) pollen. rAln g 4 was overexpressed in Escherichia coli and purified to homogeneity. It reacted with serum IgE from 18% of pollen-allergic patients (n = 122); shared IgE epitopes with homologous allergens present in tree, grass, and weed pollens; and thus belongs to a family of highly cross-reactive pollen allergens. Exposure of two E. coli-expressed rAln g 4 fragments comprising amino acids 1-41 and 42-85 to patients' IgE Abs, as well as to a rabbit antiserum raised against purified rAln g 4, indicated that most of the B cell epitopes reside in the N-terminal portion of the protein. IgE recognition of Aln g 4 was strongly modulated by the presence or absence of calcium. Circular dichroism analysis of rAln g 4 revealed that the protein consisted mostly of alpha helical secondary structure and possessed a remarkable thermal stability and refolding capacity, a property that was greatly reduced after calcium depletion. Circular dichroism analysis of the calcium-bound and apo form of rAln g 4 indicated that calcium-induced modulation of IgE binding could be due to changes in the protein conformation. Purified rAln g 4 elicited dose-dependent basophil histamine release and immediate type skin reactions in sensitized patients. It may hence be useful for allergy diagnosis and for specific immunotherapy.

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[57] - Barderas R, Villalba M, Pascual CY, Batenero E, Rodriguez E. Profilin (Che a 2) and polcalcin (Che a 3) are relevant allergens of Chenopodium album pollen: Isolation, amino acid sequences, and immunologic properties. J Allergy Clin Immunol 2004;113:1192-1198

Background Little is known about the molecular properties of chenopod allergens. Recently, profilin and 2 EF-hand calcium-binding protein (polcalcin) have been shown to play a role in chenopod pollinosis. Objective : We sought to analyze these panallergens in chenopod pollen and to evaluate their involvement in the allergy to this biologic source. Method s : Profilin and polcalcin were purified to homogeneity and characterized by using spectrometric and chemical methods. Immunologic analyses were performed by means of immunoblotting, ELISA, and competitive inhibition assays with olive profilinˆ and polcalcin-specific rabbit polyclonal antibodies and sera from patients with chenopod allergy. cDNAs encoding these proteins were cloned by means of PCR and sequenced. Result s : Purified Che a 2 (profilin) and Che a 3 (polcalcin) exhibited prevalences of 55% and 46%, respectively, in patients (n=104) hypersensitive to chenopod pollen. Both purified allergens individually inhibited the IgE binding to the whole pollen extract and showed strong cross-reactivity with the corresponding olive pollen profilin (Ole e 2) and polcalcin (Ole e 3). Chenopod profilin consists of a 131-amino-acid chain that displays identities of approximately 75% and 82% with pollen and food profilins, respectively. Che a 3 (86 amino acids) displays similarity (65% to 82% identity) with polcalcins from pollens of olive, birch, alder, rapeseed, and timothy. Conclusion : Profilin and polcalcin are relevant panallergens in chenopod pollen and good candidates to be involved in IgE cross-reactivity with other pollen sources, thus explaining the highly frequent polysensitization of patients allergic to chenopod.

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[58] - Poncet P, Sénéchal H, Clément G, Godfrin D, Purohit A, Pauli G, et al. Allergome du pollen de frêne (Fraxinus excelsior): intérêt de l'étude individuelle. Rev Fr Allergol Immunol Clin 2006;46:357

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[59] - Martinez A, Asturias JA, Palacios R, Sanz ML, Sanchez G, Oehling A, et al. Identification of a 36-kDa olive-pollen allergen by in vitro and in vivo studies. Allergy 1999;54:584-592

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.

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[60] - Ledesma A, Rodriguez R, Villalba M. Olive-pollen profilin: molecular and immunologic properties. Allergy 1998;53:520-526

Olive-pollen profilin has been isolated and characterized as a significant allergen. Its molecular properties, such as a molecular mass of 15 kDa amino-acid composition and secondary repetitive structure percentages of 15% alpha-helix, 33% beta-strand, 20% beta-turn, and 32% random coil, have been determined. Its allergenic capability, a recognition frequency estimated at 24% of olive-hypersensitive patients, and high cross-reactivity with all the pollen used have been found. The presence of conformation epitopes in the olive profilin, as well as a high structural and immunologic similarity to other pollen sources such as birch and ash, can be established from these studies.

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[61] - Martínez A, Asturias JA, Monteseirín J, Moreno V, García-Cubillana A, Hernández M, et al. The allergenic relevance of profilin (Ole e 2) from Olea europaea pollen. Allergy 2002;57(suppl. 71):17-23

Many works have dealt with the study of the allergenic relevance of profilin from allergenic extracts, mainly derived from pollens and vegetable foods. Olive pollen extracts also contain a profilin allergen (Ole e 2). This protein has been characterized in detail, so the aminoacid sequence of three isoforms and the structural model of one of them are already known. The prevalence of Ole e 2 for olive allergenic patients has been evaluated by different in vivo and in vitro methods, and the results compared with those obtained for another pollen profilins.

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[62] - Purohit A, Niederberger V, Papanikolaou L, Oster JP, Bessot JC, De Blay F, et al. Caractérisation des allergènes du pollen de frène. Communication: 9èmes Journées Parisiennes d'Allergie - Paris 10-12 janvier 2001

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[63] - Quiralte J, Llanes E, Barral P, Arias de Saavedra JM, Saenz de San Pedro B, Villalba M, et al. Ole e 2 and Ole e 10: new clinical aspects and genetic restrictions in olive pollen allergy. Allergy 2005;60:360-365

BACKGROUND: The clinical characteristics in olive pollen allergy are dependent on the antigenic load, the allergens profile, and the genetic restrictions. Our objective was to determine specific response pattern in Ole e 2 and Ole e 10 sensitization at those levels . METHODS: We studied 146 patients with seasonal rhinitis and/or asthma and positive prick test to Olea europaea pollen. IgE against Ole e 2 and Ole e 10 were detected by skin prick test and ELISA. HLA-DRB1 and HLA-DQB1 loci were typed by polymerase chain reaction sequence-specific primers method . RESULTS: A total of 102 (69.9%) and 79 (54.0%) patients showed significant IgE antibody response against Ole e 2 and Ole e 10, respectively. There was a significant association between Ole e 2 (OR 2.2, P = 0.04) and Ole e 10 reactivities (OR 2.8, P = 0.007) with asthma. In addition, total and specific IgE antibody levels significantly correlated with asthma (P < 0.05). Patients who reacted to both allergens reached the highest asthma risk factor (OR 4.3, P = 0.002). Phenotypic frequency of DR7 (OR 5.4, Pc = 0.003) and DQ2 (OR 3.6, Pc = 0.02) were increased in positive Ole e 2 patients compared with control subjects. DR2(15) phenotypic frequency was significantly increased (OR 5.6, Pc = 0.02) in positive Ole e 10 patients compared with control subjects . CONCLUSIONS: Our data suggest an association of Ole e 2 and Ole e 10 with bronchial asthma. Also, we found a genetic control of Ole e 2 and Ole e 10 IgE-specific responses that could be relevant to clinical disease in olive pollen allergy.

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[64] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[65] - Quiralte J, Palacios L, Rodríguez R, Cárdaba B, Arias de Saavedra JM, Villalba M, et al. Modelling diseases: the allergens of Olea europaea pollen. J Investig Allergol Clin Immunol 2007;17(suppl. 1):76-82

This study analyzes the influence of the IgE response to certain olive pollen allergens in the modulation of the different clinical phenotypes of allergic disease and their relationship with the level of exposure to pollen and genetic factors. Patients from high-exposure areas had a complex IgE antibody response to allergens of Olea euroapea, which included 3 or more allergens in 75% of cases. The majority allergens were Ole e 1, Ole e 2 (profilin), Ole e 7 (lipid transporting protein), Ole e 9 (glucanase), and Ole e 10. The existence of the antigen HLA-DR2 (15) led to a higher risk of sensitization to Ole e 10 and a greater trend towards the development of severe asthma, which increased in the presence of an anti-profilin IgE. Thirty percent of patients suffering from pollinosis simultaneously presented allergy to vegetable foods. Anti-Ole e 7 IgE was significantly associated with fruit anaphylaxis and anti-profilin IgE was detected in 90% of patients with oral syndrome. Finally, we analyzed the role of glucanase and Ole e 10 as causes of the pollen-latex-fruit syndrome.

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[66] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[67] - Tejera ML, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 1999;104:797-802

Olive tree (Olea europaea) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. OBJECTIVE: The aim of this study was to identify and purify a new allergen of olive tree pollen. METHODS: Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. RESULTS: The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. CONCLUSIONS: A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen.

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[68] - Ledesma A, González E, Pascual CY, Quiralte J, Villalba M, Rodríguez R. Are Ca2+-binding motifs involved in the immunoglobin E-binding of allergens ? Olive pollen allergens as model of study. Clin Exp Allergy 2002;32:1476-1483

BackgroundSeveral Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. ObjectiveTo study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. MethodsTwo olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunobloting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. ResultsThirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. ConclusionEF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.

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[69] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[70] - Purohit A, Niederberger V, Papanikolaou L, Oster JP, Bessot JC, De Blay F, et al. Caractérisation des allergènes du pollen de frène. Communication: 9èmes Journées Parisiennes d'Allergie - Paris 10-12 janvier 2001

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[71] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[72] - Ledesma A, Barderas R, Westritschnig K, Quiralte J, Pascual CY, Valenta R, et al. A comparative analysis of the cross-reactivity in the polcalcin family including Syr v 3, a new member from lilac pollen. Allergy 2006;61:477-484

BACKGROUND: Polcalcins are pollen-specific allergens with two EF-hand calcium-binding sites that exhibit strong cross-reactivity. Our objective was to isolate and express the cDNA coding of the EF-hand calcium-binding allergen from lilac pollen and to study cross-reactivity with other polcalcins from related and nonrelated pollen sources with different specific antibodies and sera from two different populations . METHODS: Specific cDNA was amplified by PCR, cloned and expressed in Escherichia coli. Purification was achieved by gel permeation and ion exchange chromatographies. ELISA titration and inhibition assays were performed using the recombinant forms of Syr v 3, Ole e 3, Che a 3 and Phl p 7 with sera from two Spanish regions with different sensitization profiles, as well as Phl p 7- and Ole e 3-specific polyclonal rabbit antisera, and an Ole e 3-specific monoclonal antibody . RESULTS: Syr v 3 displays two EF-hand consensus sites and 8863 Da of theoretical molecular mass. The allergen consists of 80 residues with identities ranging from 66 to 87% with polcalcins included in this study. Syr v 3, Ole e 3, Che a 3 and Phl p 7 showed a similar IgG- and IgE-binding capacity although differences at quantitative level were observed depending on the population of patients' sera . CONCLUSION: Syr v 3 is a polcalcin with structural and antigenic similarities to the members of this family. Diagnosis of polcalcin-sensitized patients could be performed whatever polcalcin used, whereas for immunotherapy, primary sensitization to a particular allergenic source should be considered.

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[73] - Ledesma A, Villalba M, Batanero E, Rodriguez R. Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy. Eur J Biochem 1998;258:454-459

A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

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[74] - Valenta R, Twaroch T, Swoboda I. Component-resolved diagnosis to optimize allergen-specific immunotherapy in the Mediterranean area. J Investig Allergol Clin Immunol 2007;17(suppl. 1):88-92

Allergen-specific immunotherapy (SIT) is the only allergen-specific treatment for allergy. It can prevent progression of the disease and has a long-lasting therapeutic effect. Since SIT is allergen-specific, the identification of the disease-eliciting allergen is an essential prerequisite for the accurate prescription of treatment. Diagnostic tests based on allergen extracts consist of mixtures of various allergens of which some are specific for the allergen source and others occur as cross-reactive allergens in various unrelated allergen sources. It may therefore be difficult and sometimes impossible to identify the disease-causing allergen with such tests, particularly in patients who are sensitized to more than one allergen source. Sensitization to pollens from olive, grasses, and Parietaria in the Mediterranean area is frequently treated with SIT. Here, we describe allergen molecules from these sources that can be used for component-resolved diagnosis of allergy to facilitate the selection of patients for SIT and monitor the immunological effects of treatment.

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[75] - Boluda L, Alonso C, Fernández-Caldas E. Purification, characterization, and partial sequencing of two new allergens of Olea europaea. J Allergy Clin Immunol 1998;101:210-216

BACKGROUND: The inhalation of olive (Olea europaea) pollen is an important cause of allergic respiratory diseases in southern Europe and California. OBJECTIVE: The aims of this study were to characterize the allergenic composition of O. europaea pollen collected in California and to purify two important allergens. METHODS: One hundred grams of O. europaea pollen was extracted dialyzed in 10 kd cut-off membranes and lyophilized. Allergen characterization was done by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Two allergens were isolated by gel filtration, ion exchange, and hydrophobic interaction chromatography and sequenced. RESULTS: Ole e 4 has an apparent molecular weight, under reducing conditions, of 32 kd and pIs between 4.65 and 5.1. The N-terminal was blocked and the analysis of the amino acid sequence of two internal regions revealed no homology with other known proteins. Ole e 5 has a molecular weight of 16 kd and pIs between 5.1 and 6.5. The amino acid sequence of the N-terminal showed a high degree of homology with superoxide dismutase of several plant species. Ole e 4 and Ole e 5 had an IgE binding frequency by immunoblot of 80% and 35%, respectively. CONCLUSIONS: Olive pollen extracts have a heterogeneous composition, with several important allergens. One of these allergens showed a high degree of homology with a superoxide dismutase.

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[76] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[77] - Boluda L, Alonso C, Fernández-Caldas E. Purification, characterization, and partial sequencing of two new allergens of Olea europaea. J Allergy Clin Immunol 1998;101:210-216

BACKGROUND: The inhalation of olive (Olea europaea) pollen is an important cause of allergic respiratory diseases in southern Europe and California. OBJECTIVE: The aims of this study were to characterize the allergenic composition of O. europaea pollen collected in California and to purify two important allergens. METHODS: One hundred grams of O. europaea pollen was extracted dialyzed in 10 kd cut-off membranes and lyophilized. Allergen characterization was done by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Two allergens were isolated by gel filtration, ion exchange, and hydrophobic interaction chromatography and sequenced. RESULTS: Ole e 4 has an apparent molecular weight, under reducing conditions, of 32 kd and pIs between 4.65 and 5.1. The N-terminal was blocked and the analysis of the amino acid sequence of two internal regions revealed no homology with other known proteins. Ole e 5 has a molecular weight of 16 kd and pIs between 5.1 and 6.5. The amino acid sequence of the N-terminal showed a high degree of homology with superoxide dismutase of several plant species. Ole e 4 and Ole e 5 had an IgE binding frequency by immunoblot of 80% and 35%, respectively. CONCLUSIONS: Olive pollen extracts have a heterogeneous composition, with several important allergens. One of these allergens showed a high degree of homology with a superoxide dismutase.

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[78] - Butteroni C, Afferni C, Barletta B, Iacovacci P, Corinti S, Brunetto B, et al. Cloning and Expression of the Olea europaea Allergen Ole e 5, the Pollen Cu/Zn Superoxide Dismutase. Int Arch Allergy Immunol 2005;137:9-17

BACKGROUND: Recombinant DNA technology does provide pure, well-defined and reproducible products to be used for clinical purposes, by cloning and expressing the cDNA of allergens present in a specific extract. Ole e 5 is a pollen allergen of Olea europaea with an IgE-binding frequency of about 35%, which has been identified as a superoxide dismutase (SOD). The aim of this study was to clone the cDNA of Ole e 5, to express Ole e 5 in Escherichia coli and to characterize its immunoreactivity . METHODS: cDNA of Ole e 5 was amplified by nested 3'-RACE PCR and cloned in pGEX vector 6P expression vector. After sequencing of some clones and homology analysis, the rOle e 5 was produced in an E. coli strain as a fusion protein with GST and purified. Then, the protein immunoreactivity was evaluated by patients' IgE binding (ELISA, ELISA inhibition, and immunoblotting) and by rabbit anti-rOle e 5 binding (immunoblotting and immunoblotting inhibition) . RESULTS: The sequence analysis of Ole e 5 cDNA confirmed that Ole e 5 is a Cu/Zn SOD, with an identity from 90 to 80% with SOD from other species. rOle e 5 was recognized by IgE from 39% of olive pollen-allergic patients tested; moreover, this binding was inhibited by the olive pollen extract. An anti-rOle e 5 antiserum raised in rabbit strongly reacted with a natural component of about 16-kDa molecular weight present in the olive pollen extract; moreover, this binding was inhibited by the recombinant protein . CONCLUSIONS: Ole e 5 is the first Cu/Zn SOD identified as an allergen in a pollen source. Due to the widespread presence of this enzyme, rOle e 5 allergen, cloned and expressed in a complete form in E. coli, could represent a good tool to investigate the allergen cross-reactivity between O. europaea pollen and other allergenic sources, such as plant foods and other pollens

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[79] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[80] - Guarneri F, Guarneri C, Guarneri B, Benvenga S. In silico identification of potential new latex allergens. Clin Exp Allergy 2006;36:916-919

BACKGROUND: Allergy to latex of Hevea brasiliensis is a frequent problem. In spite of the significant progress of research, the identity and cross-reactivity of some latex allergens are unknown . OBJECTIVE: To identify, among the fully characterized latex proteins, those with a higher probability to be allergenic . METHODS: We used in silico techniques (amino acid sequence comparison and molecular modelling) to identify potential new allergens among the known proteins of H. brasiliensis . RESULTS: Cu/Zn superoxide dismutase, heat shock protein and calmodulin of H. brasiliensis show highly significant (E < 10(-9)) amino acid sequence homologies with known allergens . CONCLUSION: On the basis of our data, Cu/Zn superoxide dismutase, heat shock protein and calmodulin are the most probable allergens among fully characterized proteins of H. brasiliensis, and could potentially explain, at least in part, the multiple cross-reactivities of latex with vegetable foods and other plant-derived products. Consequently, we think that the above proteins should be particularly considered in the future laboratory and clinical research.

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[81] - Trevino MA, Garcia-Mayoral MF, Barral P, Villalba M, Santoro J, Rico M, et al. NMR solution structure of Ole e 6, a major allergen from olive tree pollen. J Biol Chem 2004;279:39035-39041

Ole e 6 is a pollen protein from olive tree (Olea europaea) which exhibits allergenic activity with a high prevalence among olive allergic individuals. The three-dimensional structure of Ole e 6 has been determined in solution by NMR methods. This is the first experimentally determined structure of an olive tree pollen allergen. The structure of this 50 residue protein is based on 486 upper limit distance constraints derived from NOEs and 24 phi torsion angle restraints. The global fold of Ole e 6 consists of two nearly antiparallel alpha-helices spanning residues 3-19 and 23-33, which are connected by a short loop and followed by a long, unstructured C-terminal tail. Viewed edge-on, the structured N-terminus has a dumbbell-like shape with the two helices on the outside and the hydrophobic core, mainly composed of three aromatic and six cysteine residues, on the inside. All the aromatic rings lie on top of and pack against the three disulfide bonds. The lack of thermal unfolding, even at 85 masculine C, indicates a high conformational stability. Based on the analysis of the molecular surface, we propose five plausible epitopes for IgE recognition. The results presented here provide the structural foundation for future experiments to verify the antigenicity of the proposed epitopes, as well as to design novel hypoallergenic forms of the protein suitable for diagnosis and treatment of type-I allergies. In addition, 3D structure features of Ole e 6 are discussed to provide a basis for future functional studies.

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[82] - Tejera ML, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 1999;104:797-802

Olive tree (Olea europaea) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. OBJECTIVE: The aim of this study was to identify and purify a new allergen of olive tree pollen. METHODS: Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. RESULTS: The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. CONCLUSIONS: A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen.

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[83] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[84] - Rodríguez R, Villalba M, Batanero E, Palomares O, Quiralte J, Salamanca G, et al. Olive pollen recombinant allergens: value in diagnosis and immunotherapy. J Investig Allergol Clin Immunol 2007;17(suppl. 1):56-62

Olive pollen has a complex allergenic profile, from which more than 10 allergens have been identified and characterized. Some of these belong to well-known protein families and others cannot be included in reported biochemical types. Most of these allergens have been produced by recombinant technology, mainly in Escherichia coli or in Pichia pastoris, and they are good candidates for diagnostic and therapeutic purposes. Diagnosis and immunotherapy of allergy currently use extracts prepared from homogenates of natural sources, which only allow us to detect sensitivity to the complete source. These extracts can be successfully replaced by mixtures with controlled amounts of specific allergenic proteins obtained by recombinant technology in order to define the sensitization profile of individual patients. Recombinant Ole e 1 can be used as a marker for sensitization to Oleaceae. Recombinants Ole e 2 (profilin) and Ole e 3 (polcalcin) can serve as markers of polysensitivity. Finally, recombinant forms of Ole e 6, Ole e 10, and the carboxy-terminal and amino-terminal domains of Ole e 9 would help to detect sensitization to these minority allergens that could be overlooked in the complete olive pollen extract. These recombinant molecules can help provide an accurate diagnosis of sensitivity to individual allergens and, therefore, improve the design of more efficacious allergen-based immunotherapy strategies.

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[85] - Barber D, Moreno C, Ledesma A, Serrano P, Galán A, Villalba M, et al. Degree of olive pollen exposure and sensitization patterns. Clinical implications. J Investig Allergol Clin Immunol 2007;17(suppl. 1):63-68

BACKGROUND: Very high levels of exposure to olive pollen in the south of Spain lead to differential allergen sensitization profiles. Therefore, new approaches to allergen standardization, diagnosis, and vaccination are necessary. METHODS: Quantification of minor allergens in extracts, component-resolved patient diagnosis, and IgG4 individual allergen responses were used to evaluate new strategies in the management of olive pollen allergy. RESULTS: Allergen variability observed between different olive cultivars can be used to identify suitable allergen sources that can be combined to yield consistent allergen extracts for diagnosis and immunotherapy. Component-resolved diagnosis can provide a better patient classification. IgG4 levels to major allergens increase significantly, whereas specific IgG4 to minor allergens does not seem to increase, at least during the early phases of immunotherapy. CONCLUSION: Patients exposed to extreme olive pollen levels display a different severity of allergy from those exposed to normal levels, which makes it necessary to follow a different clinical approach. The use of component-resolved diagnosis, better standardized allergen extracts, and new efficacy monitoring techniques will lead to a significant improvement in the management of olive allergy disease.

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[86] - Tejera ML, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 1999;104:797-802

Olive tree (Olea europaea) pollen is an important cause of pollinosis in countries of the Mediterranean area and California. OBJECTIVE: The aim of this study was to identify and purify a new allergen of olive tree pollen. METHODS: Detection of a pollen allergen was done with individual allergic sera by immunoblotting and ELISA tests. Two allergenic fractions were isolated from olive pollen extract by using gel filtration and reverse-phase HPLC. Molecular characterization was achieved by acid hydrolysis and amino acid analysis, as well as by mass spectrometry. Sequencing of the N-terminal end of the allergen was carried out by Edman degradation of the polypeptide chain. Allergenic characterization was performed with sera from subjects with olive allergy by means of ELISA and immunoblotting after SDS-PAGE. RESULTS: The new allergen Ole e 7 exhibits a high degree of polymorphism. Its molecular mass is in the range of 9875 d to 10,297 d. Twenty-one amino acid residues from the N-terminal end of 2 isoforms of the allergen have been sequenced revealing no homology with proteins contained in database banks. Ole e 7 has an average frequency of about 47% in patients with olive allergy. The strategy of purification of Ole e 7 can be useful on the isolation of new allergens. CONCLUSIONS: A new olive pollen allergen of clinical significance has been purified and characterized, contributing to the study of the complete allergogram of the olive tree pollen.

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[87] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[88] - Ledesma A, Tejera M, Rodríguez R, Villalba M, Guardia P, Moreno C, et al. Prevalence and cross-reactivity of the olive pollen LTP, Ole e 7. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°998

Background: Ten allergens from olive tree pollen have been described so far. One of them, Ole e 7, belongs to the nsLTP family. No data on prevalence in different geographical areas and cross-reactivity with protein homologous from related and unrelated species are available. Method: The study of the prevalence of Ole e 7 has been carried out by using the quantitative ADVIA Centaur specific IgE assay using biotinylated Ole e 7. A total of 108 patient sera, belonging to two Spanish regions with different levels of exposure to olive pollen, were included in this study. The presence of homologous proteins to Ole e 7 was tested by ELISA inhibition of the IgE binding of a pool of sera from patients sensitized to this allergen, with pollens from related (Oleaceae family) and unrelated (birch, grasses, chenopod) species used as inhibitors. Results: Prevalence of Ole e 7 varies from 5% in patients from Sevilla to 50% in patients from Córdoba. In contrast, a major allergen as Ole e 1, which is present in very high levels in pollen, is recognized by more than 60% of the patients in both areas. ELISA inhibition showed that pollens from ash, lilac, privet, birch, chenopod and mugwort contain polypeptides that share allergenic determinants with Ole e 7, as they were able to inhibit the binding of the pool of sera to Ole e 7 at some extent. Inhibition values ranged from 18% to 42%. In contrast, grass pollen extracts were not able to inhibit this binding. Conclusion: Two features concerning Ole e 7 have been studied, its prevalence as well as its implication in cross-reactivity. The prevalence of Ole e 7 depends on the geographical area of the patients, being a major allergen in the regions with a high olive pollen charge. On the other hand, our study revealed the presence of proteins homologous to Ole e 7 in pollens from related and unrelated pollen sources, however the IgE cross-reactivity among them is limited probably because the low amino acid sequence similarity of the members of this family of proteins.

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[89] - Ledesma A, González E, Pascual CY, Quiralte J, Villalba M, Rodríguez R. Are Ca2+-binding motifs involved in the immunoglobin E-binding of allergens ? Olive pollen allergens as model of study. Clin Exp Allergy 2002;32:1476-1483

BackgroundSeveral Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. ObjectiveTo study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. MethodsTwo olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunobloting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. ResultsThirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. ConclusionEF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.

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[90] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[91] - Ledesma A, González E, Pascual CY, Quiralte J, Villalba M, Rodríguez R. Are Ca2+-binding motifs involved in the immunoglobin E-binding of allergens ? Olive pollen allergens as model of study. Clin Exp Allergy 2002;32:1476-1483

BackgroundSeveral Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. ObjectiveTo study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. MethodsTwo olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunobloting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. ResultsThirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. ConclusionEF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.

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[92] - Ledesma A, González E, Pascual CY, Quiralte J, Villalba M, Rodríguez R. Are Ca2+-binding motifs involved in the immunoglobin E-binding of allergens ? Olive pollen allergens as model of study. Clin Exp Allergy 2002;32:1476-1483

BackgroundSeveral Ca2+-binding proteins, which possess EF-hand sites with a high sequence similarity, have been found to be able to induce Type-I allergy. ObjectiveTo study whether the common EF-hand sequential motifs can be involved in the IgE-reactivity of these proteins, thus being responsible of a degree of cross-reactivity among different Ca2+-binding proteins. MethodsTwo olive pollen allergens, Ole e 3 and Ole e 8, have been used in the study. Parvalbumin and calmodulin were included in immunological analyses. Sera from patients allergic to olive pollen, as well as Ole e 3- and Ole e 8-specific rabbit antisera were used in indirect enzyme-linked immunosorbent assay (ELISA), ELISA inhibition assays and immunobloting. Conformational analyses (circular dichroism spectra and thermal stability) and specific immunodetection assays were performed in the presence and the absence of Ca2+. Chemical breakdown and high-performance liquid chromatography (HPLC) was used to obtain fragments from Ole e 3 containing a single EF-hand motif. ResultsThirty-four (17%) and 16 (8.2%) out of 195 sera from patients allergic to olive pollen contained specific IgE against Ole e 3 and Ole e 8, respectively. The IgE-binding of 12 allergic sera diminished up to 22% for Ole e 3 and to 82% for Ole e 8, when depleted Ca2+. A pool of these sera recognized the two olive allergens and parvalbumin, but at very different extent. Inhibition of the IgE-binding was only achieved between two olive allergens. No structural relationships between Ole e 3 and Ole e 8 were established when specific polyclonal antisera against both proteins were used. ConclusionEF-hand Ca2+-binding sites can not be considered as general allergenic motifs responsible for the cross-reactivity between Ca2+-binding allergens. Different families of Ca2+-binding allergens have specific epitopes that could be involved in the cross-reactivity among members of the same family.

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[93] - Ledesma A, Villalba M, Vivanco F, Rodríguez R. Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8-like proteins in different pollens. Allergy 2002;57:40-43

In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli. We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8). Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen. Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens. Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other. Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.

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[94] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[95] - Ledesma A, Villalba M, Rodriguez R. Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity. FEBS Lett 2000;466:192-196

A novel allergenic member of the family of Ca(2+)-binding proteins has been cloned from olive tree pollen. The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF-hand sequence motifs. The encoded protein was overproduced in Escherichia coli and purified. The protein (18: omitted:795 Da), which binds Ca(2+) and IgE antibodies from patients allergic to olive pollen, undergoes Ca(2+)-dependent conformational changes. It is retained on a phenyl-Sepharose column, which indicates the existence of regulatory EF-hand domains. This fact suggests its involvement in Ca(2+)-dependent signal transduction events of the pollen grain. This allergen could be considered as a member of a new subfamily of EF-hand Ca(2+)-binding proteins since it displays a low amino acid sequence similarity with the so far known proteins.

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[96] - Ledesma A, Villalba M, Vivanco F, Rodríguez R. Olive pollen allergen Ole e 8: identification in mature pollen and presence of Ole e 8-like proteins in different pollens. Allergy 2002;57:40-43

In a first approach, Ole e 8, a novel Ca2+-binding protein from olive pollen, was cloned and produced in Escherichia coli. We have obtained the natural form of Ole e 8 (nOle e 8) from the pollen and examined its immunologic equivalence with its recombinant form (rOle e 8). Size exclusion chromatography and a phenyl-Sepharose CL-4B affinity column were used to obtain nOle e 8 from the olive pollen. Inhibition assays by immunoblotting, using rOle e 8-specific rabbit antiserum, were performed to analyze the immunologic equivalence between the natural and the recombinant allergen, as well as to detect its presence in other pollens. Recombinant and natural Ole e 8 resulted immunologically equivalents, since they completely inhibited the IgG binding of the polyclonal antiserum to each other. Ole e 8-like proteins were detected in Oleaceae and Juniperus communis pollen, and might contribute to cross-reactivity processes between taxonomically related pollens.

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[97] - Huecas S, Villalba M, Rodriguez R. Ole e 9, a major olive pollen allergen is a 1,3-beta-glucanase. Isolation, characterization, amino acid sequence, and tissue specificity. J Biol Chem 2001;276:27959-27966

Olive pollen allergy is a clinical disorder affecting the human population of Mediterranean areas. A novel major allergen, Ole e 9, has been isolated from olive pollen by gel permeation, hydrophobic affinity, and reverse-phase high performance liquid chromatographies. It is involved in the allergic responses of 65% of patients suffering olive pollinosis. Ole e 9 (molecular mass of 46.4 kDa) displays 1,3-beta-endoglucanase activity (38.9 +/- 5.6 mg of glucose released/min x micromol of protein at pH 4.5-6.0 using laminarin as substrate). It is the first 1,3-beta-glucanase, a member of the "pathogenesis-related" protein family, detected in pollen tissue. Seven tryptic peptides of the allergen were sequenced by Edman degradation and used for designing primers to clone the cDNA codifying the protein. Specific cDNA for Ole e 9 was synthesized from total RNA and amplified using the polymerase chain reaction. The allergen sequence showed an open reading frame of 460 amino acids comprising a putative signal peptide of 26 residues. It shows 39, 33, and 32% sequence identity including the catalytic residues when compared with 1,3-beta-glucanases from wheat, willow, and Arabidopsis thaliana, respectively. Northern blot analysis showed that Ole e 9 transcript is specifically expressed in the pollen tissue, and highly conserved counterparts were only detected in taxonomically related pollens.

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[98] - Palomares O, Villalba M, Batanero E, Barderas R, Barral P, Rodríguez R. Recombinant production and characterization of a structural domain of the olive allergen Ole e 9. EAACI 21th Congress, Naples, 1-5 June, 2002, Poster n°888

Nine protein allergens have been characterized from olive pollen. Among them, Ole e 9 has been reported as a major inductor of IgE-mediated hypersensitivity, affecting more than 65% of the allergic patients. Ole e 9 (46 kDa) is constituted by a single polypeptide chain with 1,3-beta-glucanase enzymatic activity. Homology of Ole e 9 to other members of 1,3-beta-glucanase family allowed us to expect a conformation of two domains: I) a well-establish TIM barrel structure for the N-terminal 334 amino acid residues, and II) an unknown domain for the polypeptide C-terminal segment of 101 residues in length.The application of molecular cloning techniques for the recombinant production of proteins may facilitate the availability of whole molecules or their domains. The recombinant C-terminal domain (rCtD) has been produced in Pichia pastoris, isolated and characterized, being obtained as a monomeric and soluble form. The amino acid composition and N-terminal amino acid sequence of rCtD indicate its correct processing. As the natural protein, rCtD is glycosylated since it is reactive to concanavalin A. This is also in agreement with the molecular mass obtained for the recombinant product. The secondary structure of the protein was analysed by circular dichroism, and the spectrum showed a notable contribution of b-sheet to the conformation of the molecule, indicating that the recombinant product has a defined folding. rCtD was recognized by all of the sera sensitive to Ole e 9, although the individual responses reached different levels. This recombinant fragment of Ole e 9 was able to inhibit at 65% the binding of IgE from allergic sera to the whole allergen coating wells. On the other hand, an inhibition of 90% was reached for the IgE-binding to rCtD-coated wells, when Ole e 9 was used as inhibitor. In conclusion, the recombinant production in P. pastoris of a C-terminal fragment of the major olive pollen allergen Ole e 9 rendered a well-defined structure, which conserves allergenic properties of the whole allergen.

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[99] - Palomares O, Villalba M, Quiralte J, Rodriguez R. Allergenic contribution of the IgE-reactive domains of the 1,3-beta-glucanase Ole e 9: diagnostic value in olive pollen allergy. Ann Allergy Asthma Immunol 2006;97:61-65

BACKGROUND: Designing of methods for an accurate diagnosis is a main goal of allergy research. Olive pollen allergy is currently diagnosed using commercially available pollen extracts that do not allow identification of the molecules that elicit the disease. OBJECTIVE: To analyze the suitability of using the N- and C-terminal domains (NtD and CtD, respectively) of the 1,3-beta-glucanase Ole e 9, a major allergen from olive pollen, for in vitro diagnosis. METHODS: Serum samples from 55 olive-allergic patients were assayed using enzyme-linked immunosorbent assay to study hypersensitive patients with IgE reactivity to Ole e 9. The specific IgEs to NtD and CtD, obtained by recombinant technology, were determined by means of immunoblotting, enzyme-linked immunosorbent assay, and inhibition assays. RESULTS: Thirty-one of 33 serum samples from Ole e 9-allergic patients were IgE reactive to recombinant NtD (rNtD) (n = 26 [79%]), recombinant CtD (rCtD) (n = 22 [67%]), or both (n = 17 [52%]). Nine patients (27%) were exclusively reactive to rNtD and 5 (15%) to rCtD. Inhibition assays of IgE binding to Ole e 9 with a mixture of both domains abolished 90% of the binding, whereas 44% and 45% were abolished when rNtD and rCtD were used, respectively. CONCLUSIONS: Because sensitization to NtD or CtD of Ole e 9 could be correlated to vegetable food-latex-pollen cross-reactivity processes or to the exacerbation and persistence of asthma, respectively, these molecules could be used in vitro as markers of disease to classify patients and to design a patient-tailored immunotherapy approach.

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[100] - Receveur-Brechot V, Czjzek M, Barre A, Roussel A, Peumans WJ, Van Damme EJ, et al. Crystal structure at 1.45-A resolution of the major allergen endo-beta-1,3-glucanase of banana as a molecular basis for the latex-fruit syndrome. Proteins 2006;63:235-242

Resolution of the crystal structure of the banana fruit endo-beta-1,3-glucanase by synchrotron X-ray diffraction at 1.45-A resolution revealed that the enzyme possesses the eightfold beta/alpha architecture typical for family 17 glycoside hydrolases. The electronegatively charged catalytic central cleft harbors the two glutamate residues (Glu94 and Glu236) acting as hydrogen donor and nucleophile residue, respectively. Modeling using a beta-1,3 linked glucan trisaccharide as a substrate confirmed that the enzyme readily accommodates a beta-1,3-glycosidic linkage in the slightly curved catalytic groove between the glucose units in positions -2 and -1 because of the particular orientation of residue Tyr33 delimiting subsite -2. The location of Phe177 in the proximity of subsite +1 suggested that the banana glucanase might also cleave beta-1,6-branched glucans. Enzymatic assays using pustulan as a substrate demonstrated that the banana glucanase can also cleave beta-1,6-glucans as was predicted from docking experiments. Similar to many other plant endo-beta-1,3-glucanases, the banana glucanase exhibits allergenic properties because of the occurrence of well-conserved IgE-binding epitopes on the surface of the enzyme. These epitopes might trigger some cross-reactions toward IgE antibodies and thus account for the IgE-binding cross-reactivity frequently reported in patients with the latex-fruit syndrome.

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[101] - 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.

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[102] - Barber D, de la Torre F, Feo F, Florido F, Guardia P, Moreno C, et al. Understanding patient sensitization profiles in complex pollen areas: a molecular epidemiological study. Allergy 2008;63:1550-1558

BACKGROUND: Allergy diagnosis in patients exposed to multiple pollen species is complex and misdiagnosis is often a cause for unsuccessful specific immunotherapy . OBJECTIVE: We studied the sensitization profile of individual allergens (major, minor and pan-allergens) in pollen-sensitized patients in a region with high exposure to olive pollen by investigating the influence of minor allergens on allergic disease and the association between pan- and minor allergen sensitizations . METHODS: A panel of 13 purified allergens, which included the most relevant allergens in the area, as well as minor olive allergens and pan-allergens, were screened using a high-capacity technology (ADVIA-Centaur) in 891 patients . RESULTS: Olive allergy as measured by specific IgE to Ole e 1 was the leading pollinosis in the area. The minor olive allergens Ole e 7 and Ole e 9 were markers of more severe allergic illness. Profilin sensitization was associated mainly with grass allergy, the second most prevalent pollinosis. Salsola kali pollen allergy was the third most common cause of pollinosis in the area. The prevalence of sensitization to the peach allergen Pru p 3, a nonspecific lipid-transfer protein, was notable . CONCLUSION: Epidemiological analysis by component-resolved diagnosis is a new method, which elucidates the interaction between allergen exposure gradient and patient sensitization. High exposure leads to differential sensitization profiles some of which are associated with more severe allergic conditions. Profilin sensitization, related mainly to grass pollinosis, was a marker of more severe grass pollen sensitization.

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[103] - Palomares O, Fernandez-Nieto M, Villalba M, Rodriguez R, Cuesta-Herranz J. Occupational allergy in a researcher due to Ole e 9, an allergenic 1,3-b-glucanase from olive pollen. Allergy 2008;63:784-785

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[104] - Barral P, Batanero E, Palomares O, Quiralte J, Villalba M, Rodriguez R. A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies cross-reactivity. J Immunol 2004;172:3644-3651

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

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[105] - Barral P, Suarez C, Batanero E, Alfonso C, Alche Jde D, Rodriguez-Garcia MI, et al. An olive pollen protein with allergenic activity, Ole e 10, defines a novel family of carbohydrate-binding modules and is potentially implicated in pollen germination. Biochem J 2005;390:77-84

CBMs (carbohydrate-binding modules) are the most common non-catalytic modules associated with enzymes active in plant cell-wall hydrolysis. They have been frequently identified by amino acid sequence alignments, but only a few have been experimentally established to have a carbohydrate-binding activity. A small olive pollen protein, Ole e 10 (10 kDa), has been described as a major inducer of type I allergy in humans. In the present study, the ability of Ole e 10 to bind several polysaccharides has been analysed by affinity gel electrophoresis, which demonstrated that the protein bound 1,3-beta-glucans preferentially. Analytical ultracentrifugation studies confirmed binding to laminarin, at a protein/ligand ratio of 1:1. The interaction of Ole e 10 with laminarin induced a conformational change in the protein, as detected by CD and fluorescence analyses, and an increase of 3.6 degrees C in the thermal denaturation temperature of Ole e 10 in the presence of the glycan. These results, and the absence of alignment of the sequence of Ole e 10 with that of any classified CBM, indicate that this pollen protein defines a novel family of CBMs, which we propose to name CBM43. Immunolocalization of Ole e 10 in mature and germinating pollen by transmission electron microscopy and confocal laser scanning microscopy demonstrated the co-localization of Ole e 10 and callose (1,3-beta-glucan) in the growing pollen tube, suggesting a role for this protein in the metabolism of carbohydrates and in pollen tube wall re-formation during germination.

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[106] - Barral P, Batanero E, Palomares O, Quiralte J, Villalba M, Rodriguez R. A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies cross-reactivity. J Immunol 2004;172:3644-3651

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

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[107] - Barral P, Batanero E, Palomares O, Quiralte J, Villalba M, Rodriguez R. A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies cross-reactivity. J Immunol 2004;172:3644-3651

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

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[108] - Barral P, Batanero E, Palomares O, Quiralte J, Villalba M, Rodriguez R. A major allergen from pollen defines a novel family of plant proteins and shows intra- and interspecies cross-reactivity. J Immunol 2004;172:3644-3651

Olive tree (Olea europaea) pollen is a main cause of allergy associated with extensive areas of Europe and North America. Ole e 10, a small (10.8 kDa) and acidic (pI 5.8) protein, has been identified as a major allergen from the olive pollen, isolated, and characterized. Circular dichroism analysis gave 17% alpha helix, 33% beta sheet, and 21% beta turn for its secondary structure. Based on amino acid sequences of tryptic peptides, the protein was cloned and sequenced. The allergen consists of a single polypeptide chain of 102 aa, with a signal peptide of 21 residues. Ole e 10 showed homology with the C-terminal domain of another olive allergen, Ole e 9 (1,3-beta-glucanase, 53% identity), with deduced sequences from Arabidopsis thaliana genes (42-46% identity) and with polypeptide segments (Cys boxes) of proteins involved in yeast development (Epd1/Gas-1p/Phr2 families; 42-43% similarity). Ole e 10 showed 55% prevalence for olive-allergic patients and exhibited an IgE response dependent on its conformation. Remarkable IgE cross-reactivity was detected with Ole e 9, but no correlation was observed between the individual IgE responses to both allergens. Ole e 10 shares IgE B cell epitopes with proteins from Oleaceae, Gramineae, Betulaceae, Chenopodiaceae, Cupressaceae, Ambrosia, and Parietaria pollens, latex, and vegetable foods, such as tomato, kiwi, potato, and peach. These data indicate that Ole e 10 is a new pan-allergenic plant protein that shows notable intra- and interspecie IgE cross-reactivity and is a powerful candidate to be involved in pollen-latex-fruit syndrome.

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[109] - Salamanca G, Ledesma A, Palomares O, Castro L, Batanero E, Marazuela E, et al. Recombinant production of a new olive pollen allergen. EAACI 25th Congress, Vienna, 10-14 June, 2006, Poster n°1002

Background: Pollen from the olive tree (Olea europaea) is a major cause of allergy in countries where this tree is extensively cultivated. Ten allergens (Ole e 1 to Ole e 10) have been isolated and characterized from this biological source so far. However, when IgE-reactive proteins were resolved by twodimensional (2D) electrophoretic techniques, a new significant allergenic component appeared at approximately 40 kDa. The aim of this study is the recombinant production and characterization of this new olive pollen allergen, Ole e 11. Methods: 2D-analysis and MALDI-TOF MS analysis were used to obtain internal peptide sequences of the 40 kDa IgE-reactive protein. From them several oligonucleotides were designed for cloning. Recombinant Ole e 11 (rOle e 11) was produced in the KM71 Pichia pastoris cells using pPICZa-A. rOle e 11 was purified by aninon exchange and size-exclusion chromatographies. Molecular characterization was perfomed by Edman degradation, amino acid composition analysis and carbohydrate detection. Immunoblotting, ELISA and immunoblotting inhibition experiments using sera of patients allergic to olive pollen with IgE-reactivity to high-molecular weight allergens were carried out. Results: rOle e 11 was produced in Pichia pastoris with a yield of 6 mg/L of cell culture. The protein was obtained as a monomeric soluble form. The correct processing of the allergen was identified by N-terminal determination and by SDS-PAGE. rOle e 11 was glycosylated. Eleven out of the 17 sera from allergic patients to olive pollen were reactive against rOle e 11. Inhibition experiments showed that IgE epitopes of rOle e 11 were contained in IgEallergenic bands profile of olive pollen extract. Conclusion: A new high-molecular weight olive pollen allergen (Ole e 11, 40 kDa) has been produced as a recombinant protein retaining IgE-binding capability.

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[110] - Bistoni O, Emiliani C, Agea E, Russano AM, Mencarelli S, Orlacchio A, et al. Biochemical and Immunological Characterization of Pollen-Derived ß-Galactosidase Reveals a New Cross-Reactive Class of Allergens among Mediterranean Trees. Int Arch Allergy Immunol 2005;136:123-133

BACKGROUND: The most potent allergens in the Spermatophytae family exhibit significant homology with enzymes. Some of these are though to be involved in pectin metabolism, recognition of compatible stigma and delivery of sperm cells to the ovule . OBJECTIVE: To test if glycohydrolase activities from some Mediterranean tree pollens could act as allergens in sensitized hosts . METHODS: Freshly collected Cupressus and Olea pollens were investigated for their glycohydrolase activities by means of synthetic fluorogenic substrates and isoenzymes characterized by DEAE-cellulose ion-exchange chromatography. Binding of specific IgE was investigated by immunoblotting in 30 tree-sensitive subjects, as well as in 20 atopic non-tree-sensitive and 15 healthy controls. The enzymes were also adopted to stimulate proliferation of allergen-specific T cell clones. Finally, they were tested in vivo in a cutaneous immediate wheal and flare reaction . RESULTS: beta-Galactosidase (beta-GAL) is present with different isoenzymatic patterns on both pollen extracts, could be recognized by circulating IgE, as well as immunoprecipitated by sera from allergic subjects. The enzyme could stimulate the proliferation of T cells from allergic subjects, and favor the emergence of CD4+ T cell clones with specific in vitro reactivity to beta-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects . CONCLUSIONS: Despite different isoenzymatic patterns, Olea-derived beta-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.

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[111] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[112] - Bistoni O, Emiliani C, Agea E, Russano AM, Mencarelli S, Orlacchio A, et al. Biochemical and Immunological Characterization of Pollen-Derived ß-Galactosidase Reveals a New Cross-Reactive Class of Allergens among Mediterranean Trees. Int Arch Allergy Immunol 2005;136:123-133

BACKGROUND: The most potent allergens in the Spermatophytae family exhibit significant homology with enzymes. Some of these are though to be involved in pectin metabolism, recognition of compatible stigma and delivery of sperm cells to the ovule . OBJECTIVE: To test if glycohydrolase activities from some Mediterranean tree pollens could act as allergens in sensitized hosts . METHODS: Freshly collected Cupressus and Olea pollens were investigated for their glycohydrolase activities by means of synthetic fluorogenic substrates and isoenzymes characterized by DEAE-cellulose ion-exchange chromatography. Binding of specific IgE was investigated by immunoblotting in 30 tree-sensitive subjects, as well as in 20 atopic non-tree-sensitive and 15 healthy controls. The enzymes were also adopted to stimulate proliferation of allergen-specific T cell clones. Finally, they were tested in vivo in a cutaneous immediate wheal and flare reaction . RESULTS: beta-Galactosidase (beta-GAL) is present with different isoenzymatic patterns on both pollen extracts, could be recognized by circulating IgE, as well as immunoprecipitated by sera from allergic subjects. The enzyme could stimulate the proliferation of T cells from allergic subjects, and favor the emergence of CD4+ T cell clones with specific in vitro reactivity to beta-GAL. Finally, the enzyme induced in vivo a cutaneous wheal and flare reaction in clinically sensitive subjects . CONCLUSIONS: Despite different isoenzymatic patterns, Olea-derived beta-GAL cross-reacted with that from cypress pollen, suggesting that these enzymatic glycoproteins may represent major native allergens among these Mediterranean trees.

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[113] - Poncet P, Sénéchal H, Clément G, Godfrin D, Purohit A, Pauli G, et al. Allergome du pollen de frêne (Fraxinus excelsior): intérêt de l'étude individuelle. Rev Fr Allergol Immunol Clin 2006;46:357

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[114] - Martinez A, Asturias JA, Palacios R, Sanz ML, Sanchez G, Oehling A, et al. Identification of a 36-kDa olive-pollen allergen by in vitro and in vivo studies. Allergy 1999;54:584-592

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.

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[115] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[116] - Bousquet J, Guérin B, Hewitt B, Lim S, Michel FB. Allergy in the Mediterranean area. III: Cross reactivity among Oleaceae pollens. Clin Allergy 1985;15:439-448

Pollens of closely related species often share common antigens and allergens although this is not always the case. Four species of Oleaceae pollens commonly found in the Mediterranean area were investigated for cross-reactivity: olive (Olea europaea), ash (Fraxinus exselsior), privet (Ligustrum vulgare) and Phillyrea angustifolia, a common bush. Twenty individual sera of patients allergic to Oleaceae were investigated for specific IgE antibodies against the four species of Oleaceae pollens. The results indicated a high degree of correlation between RAST titres of Oleaceae pollens but three gave a particular emphasis on one species only. Cross-reactivity among these four pollen species was sought by means of RAST inhibition, iso-electric focusing and tandem cross-immunoelectrophoresis. All tests revealed a high degree of cross reactivity although there is no total identity among these four pollen species

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[117] - Gonzalez E, Villalba M, Rodriguez R. Immunological and molecular characterization of the major allergens from lilac and privet pollens overproduced in Pichia pastoris. Clin Exp Allergy 2001;31:313-321

The main allergens from privet and lilac pollens, Lig v 1 and Syr v 1, are proteins homologous to Ole e 1 and have been shown to be involved in cross-reactivity. To overproduce the correctly folded Lig v 1 and Syr v 1 allergens and to study their immunological properties in comparison with those of their natural counterparts. The yeast Pichia pastoris was used as an expression system to produce these recombinant allergens. The proteins were isolated by ion-exchange and size-exclusion chromatographies. Amino acid quantifying, Edman degradation, mass spectrometry and circular dichroism were carried out to obtain molecular properties of the recombinant proteins. Anti-Ole e 1 monoclonal and polyclonal antibodies, as well as sera from patients allergic to olive pollen, were used in immunoblotting and ELISA for immunological characterization. Recombinant Lig v 1 and Syr v 1 were secreted at high yield to the extracellular medium of the yeast. The purified proteins displayed the native conformation, as deduced from their spectroscopic properties and binding ability to an IgG monoclonal antibody. The recombinant allergens behaved similarly to their natural counterparts when they were analysed against Ole e 1-specific antibodies. IgE and IgG binding properties of lilac and privet allergens to olive allergic sera and Ole e 1-specific antibodies indicated that these molecules share common B-cell epitopes with Ole e 1. P. pastoris yeast is an appropriate system for the efficient production of Ole e 1-like allergens, which could be used as analogous allergens and predictors of clinical sensitization.

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[118] - Pajaron MJ, Vila L, Prieto I, Resano A, Sanz ML, Oehling AK. Cross-reactivity of Olea europaea with other Oleaceae species in allergic rhinitis and bronchial asthma. Allergy 1997;52:829-835

Cross-reactivity between pollen extracts of four species of Oleaceae was studied: olive (Olea europaea), ash (Fraxinus excelsior), privet (Ligustrum vulgare), and lilac (Syringa vulgaris). Thus, 51 patients and 13 atopic controls were studied, by means of intracutaneous skin tests, histamine-release tests against the four extracts, and specific IgE to O. europaea. The proteic content of the four extracts was assessed by SDS-PAGE and immunoblotting, and similarity of all the extracts studied was observed after electrophoresis and immunodetection. Six common bands were found to be responsible for the cross-reactivity, with apparent molecular weights of 49.6, 40, 36.7, 19.7, 16.7, and 14 kDa, respectively. The cross-reactivity was also corroborated by immunoblotting inhibition and FEIA inhibition. The patients had a similar response to the four allergenic extracts used, although the response to Olea was greatest. When the patients were compared by their geographic origin (northern or southern Spain, according to the distribution of areas of olive pollen influence), there were no significant differences between the two groups in skin reactivity, but a higher histamine release was observed for the four extracts in the southern group, although it was significant only for Fraxinus and Ligustrum. This work corroborated the practicality of the diagnostic methods used and the cross-reactivity between the four species studied, as demonstrated by the different methods used. Therefore, we suggest that only O. europaea extract be used in diagnosis and immunotherapy in Oleaceae pollen allergy

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[119] - Obispo TM, Melero JA, Carpizo JA, Carreira J, Lombardero M. The main allergen of Olea europaea (Ole e I) is also present in other species of the Oleaceae family. Clin Exp Allergy 1993;23:311-316

Three major pollen allergens from Fraxinus excelsior, Ligustrum vulgare and Syringa vulgaris belonging to the Oleaceae family were purified. Monoclonal antibodies previously raised against the main allergen of Olea europaea (Ole e I) were used for their purification by affinity chromatography. The three new purified allergens were able to bind human IgE from serum of olive-allergic patients in a way analogous to Ole e I. Crossed radioimmunoelectrophoresis of the four allergens, using anti-olive extract rabbit serum, showed a unique immunoprecipitation arc with the same characteristics. The four purified proteins had similar molecular weights on SDS-PAGE and the N-terminal sequences for the first 20 amino acids were identical. Furthermore, the concentration of the allergens could be determined using a two-side solid phase assay previously developed for the allergen Ole e I. Our results indicate that the four purified proteins share, to a great extent, antigenic and allergenic epitopes leading to cross-reactivities which could cause common clinical manifestations. We propose for the newly purified allergens the nomenclature of Fra e I, Lig v I and Syr v I.

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[120] - Lombardero M, Obispo T, Calabozo B, Lezaún A, Polo F, Barber D. Cross-reactivity between olive and other species. Role of Ole e 1-related proteins. Allergy 2002;57(suppl. 71):29-34

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[121] - Papanikolaou I, Barderas R, Thibaudon M, Pauli G. La pollinose au frêne : données palynologiques, description des allergènes et réactivité croisée. Rev Fr Allergol Immunol Clin 2005;45:395-405

La fréquence des sensibilisations cutanées au pollen de frêne commun (Fraxinus excelsior), constatée depuis quelques années pose de nombreuses difficultés diagnostiques aux allergologues, d'autant plus que cette sensibilisation est souvent mise en évidence dans un contexte de polysensibilisations vis-à-vis de nombreux pollens. Au fur et à mesure de la progression des connaissances, la réactivité croisée avec des espèces proches ou taxonomiquement éloignées est de mieux en mieux connue rendant le diagnostic de cette pollinose particulièrement difficile. Dans ce travail, nous portons un intérêt particulier aux caractéristiques botaniques, géographiques et palynologiques du frêne. Une mise au point sur la composition allergénique du pollen de frêne est ensuite réalisée au vu des connaissances récemment acquises.

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[122] - Wheeler AW. Hypersensitivity to the allergens of the pollen from the olive tree (Olea europaea) . Clin Exp Allergy 1992;22:1052-1057

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[123] - Pauli G, Papanikolaou I, Niederberger V. Sensibilisation au frêne: allergènes spécifiques et/ou allergènes croisants ? Rev Fr Allergol Immunol Clin 2003;43:120-124

Le frêne commun (Fraxinus excelsior) appartenant à la famille des oléacées est largement répandu dans l'ensemble du territoire européen. La fréquence des sensibilisations cutanées au pollen de frêne, constatée depuis quelques années, fait discuter la pertinence clinique de ces sensibilisations souvent observées chez des patients polliniques polysensibilisés. Une meilleure connaissance des allergènes des pollens, ainsi que la possibilité d'effectuer des expérimentations avec des allergènes purifiés ou recombinants a permis de différencier les allergènes spécifiques du pollen de frêne de ceux contenus dans d'autres pollens parfois taxonomiquement éloignés. Les auteurs présentent une revue de la littérature concernant les sensibilisations au frêne observées chez les patients polliniques et relèvent la rareté de la monosensibilisation aux allergènes de frêne. Ils décrivent également la pluralité des profils de sensibilisation observés chez les patients sensibilisés à un extrait de frêne, les sérums de ces patients pouvant réagir avec des familles moléculaires différentes : allergène majeur des oléacées, profiline, protéines liant le calcium, allergènes de haut poids moléculaire... Les sensibilisations vis-à-vis des allergènes « croisants » permettent de mieux interpréter des sensibilisations concomitantes à plusieurs extraits de pollens.

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[124] - Laurent J, Guinnepain MT, Lafay M, Sauvaget J. La pollinose des arbres. Rev Fr Allergol Immunol Clin 1999;39:276-282

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[125] - d'Amato G, Spieksma FTM, Liccardi G, Jäger S, Russo M, Kontou-Fili K, et al. Pollen-related allergy in Europe. Allergy 1998;53:567-578

The increasing mobility of Europeans for business and leisure has led to a need for reliable information about exposure to seasonal airborne allergens during travel abroad. Over the last 10 years or so, aeropalynologic and allergologic studies have progressed to meet this need, and extensive international networks now provide regular pollen and hay-fever forecasts. Europe is a geographically complex continent with a widely diverse climate and a wide spectrum of vegetation. Consequently, pollen calendars differ from one area to another; however, on the whole, pollination starts in spring and ends in autumn. Grass pollen is by far the most frequent cause of pollinosis in Europe. In northern Europe, pollen from species of the family Betulaceae is a major cause of the disorder. In contrast, the mild winters and dry summers of Mediterranean areas favor the production of pollen types that are rarely found in central and northern areas of the continent (e.g., the genera Parietaria, Olea, and Cupressus). Clinical and aerobiologic studies show that the pollen map of Europe is changing also as a result of cultural factors (e.g., importation of plants for urban parklands) and greater international travel (e.g., the expansion of the ragweed genus Ambrosia in France, northern Italy, Austria, and Hungary). Studies on allergen-carrying paucimicronic or submicronic airborne particles, which penetrate deep into the lung, are having a relevant impact on our understanding of pollinosis and its distribution throughout Europe.

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[126] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[127] - Kirmaz C, Yuksel H, Bayrak P, Yilmaz O. Symptoms of the olive pollen allergy: do they really occur only in the pollination season ? J Investig Allergol Clin Immunol 2005;15:140-145

BACKGROUND: Olive (Olea europaea; O.e.) pollen is a major cause of seasonal respiratory allergy. The O.e. pollination season lasts two months from the beginning of May till the end of June. It was expected that patients with allergic disease from O.e. sensitization were symptomatic only during this period. However, during the last few years, we have observed that the clinical symptoms appear not only during the O.e. pollination season but also during the rest of the year. OBJECTIVE: The aim of this study was to observe and document symptoms of respiratory allergic diseases in the O.e. sensitized patients during the O.e. pollination season and after it. METHODS: One hundred and twenty-seven patients with respiratory allergic disease were enrolled in the study. Allergenic sensitizations were shown by SPT. Finally, patients were split into two groups as monosensitized with O.e. (n=19) and polysensitized (n=108). Patients were assessed by using scores of respiratory allergic disease symptoms and percentage of peak expiratory flow rate values (PEFR %) (only for asthmatic patients) during the O.e. pollination season and after it. RESULTS: Of the patients with O.e. monosensitization, 13 had allergic rhinitis (AR) only while six had allergic asthma (AA) additionally. AR alone and accompanied by AA was present in 84 and 24 polysensitized patients respectively. Eleven patients with O.e. sensitization (57.9 %) and 86 patients with polysensitization (79.6 %) had AR symptoms throughout the year irrespective of the O.e. pollination season. Similarly, three of the O.e. monosensitized and ten of the polysensitized patients with AA had asthmatic symptoms during the O.e. pollination season and also after it. CONCLUSIONS: In the patient group sensitive to O.e. along with other pollen extracts, it was possible to observe symptoms outside the pollination season. However, patients with O.e. monosensitization also had symptoms to a great extent outside the season.

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[128] - d'Amato G, Spieksma FTM, Liccardi G, Jäger S, Russo M, Kontou-Fili K, et al. Pollen-related allergy in Europe. Allergy 1998;53:567-578

The increasing mobility of Europeans for business and leisure has led to a need for reliable information about exposure to seasonal airborne allergens during travel abroad. Over the last 10 years or so, aeropalynologic and allergologic studies have progressed to meet this need, and extensive international networks now provide regular pollen and hay-fever forecasts. Europe is a geographically complex continent with a widely diverse climate and a wide spectrum of vegetation. Consequently, pollen calendars differ from one area to another; however, on the whole, pollination starts in spring and ends in autumn. Grass pollen is by far the most frequent cause of pollinosis in Europe. In northern Europe, pollen from species of the family Betulaceae is a major cause of the disorder. In contrast, the mild winters and dry summers of Mediterranean areas favor the production of pollen types that are rarely found in central and northern areas of the continent (e.g., the genera Parietaria, Olea, and Cupressus). Clinical and aerobiologic studies show that the pollen map of Europe is changing also as a result of cultural factors (e.g., importation of plants for urban parklands) and greater international travel (e.g., the expansion of the ragweed genus Ambrosia in France, northern Italy, Austria, and Hungary). Studies on allergen-carrying paucimicronic or submicronic airborne particles, which penetrate deep into the lung, are having a relevant impact on our understanding of pollinosis and its distribution throughout Europe.

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[129] - Kirmaz C, Yuksel H, Bayrak P, Yilmaz O. Symptoms of the olive pollen allergy: do they really occur only in the pollination season ? J Investig Allergol Clin Immunol 2005;15:140-145

BACKGROUND: Olive (Olea europaea; O.e.) pollen is a major cause of seasonal respiratory allergy. The O.e. pollination season lasts two months from the beginning of May till the end of June. It was expected that patients with allergic disease from O.e. sensitization were symptomatic only during this period. However, during the last few years, we have observed that the clinical symptoms appear not only during the O.e. pollination season but also during the rest of the year. OBJECTIVE: The aim of this study was to observe and document symptoms of respiratory allergic diseases in the O.e. sensitized patients during the O.e. pollination season and after it. METHODS: One hundred and twenty-seven patients with respiratory allergic disease were enrolled in the study. Allergenic sensitizations were shown by SPT. Finally, patients were split into two groups as monosensitized with O.e. (n=19) and polysensitized (n=108). Patients were assessed by using scores of respiratory allergic disease symptoms and percentage of peak expiratory flow rate values (PEFR %) (only for asthmatic patients) during the O.e. pollination season and after it. RESULTS: Of the patients with O.e. monosensitization, 13 had allergic rhinitis (AR) only while six had allergic asthma (AA) additionally. AR alone and accompanied by AA was present in 84 and 24 polysensitized patients respectively. Eleven patients with O.e. sensitization (57.9 %) and 86 patients with polysensitization (79.6 %) had AR symptoms throughout the year irrespective of the O.e. pollination season. Similarly, three of the O.e. monosensitized and ten of the polysensitized patients with AA had asthmatic symptoms during the O.e. pollination season and also after it. CONCLUSIONS: In the patient group sensitive to O.e. along with other pollen extracts, it was possible to observe symptoms outside the pollination season. However, patients with O.e. monosensitization also had symptoms to a great extent outside the season.

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[130] - Wahl R, Schmid-Grendelmeier P, Cromwell O, Wüthrich B. In vitro investigation of cross-reactivity between birch and ash pollen allergen extracts. J Allergy Clin Immunol 1996;98:99-106

Allergenic cross-reactivity between members of the Fagales family (birch, alder, hazel, and beech) and between members of the Oleaceae family (ash, olive, lilac, and privet) is well known, but little is known about possible cross-reactivity between these two groups of trees, in particular between birch and ash, both of which flower in the spring. Various immunochemical methods including RAST inhibition, Western blot, and Western blot inhibition have been used in this study to show that there is partial cross-reactivity between birch and ash pollens. Enzyme allergosorbent test measurements were performed on sera from 35 patients with hay fever in spring by using birch and ash pollen allergen disks. The major allergen of birch, Bet v 1, was readily detectable in the birch pollen extract, but a homologous allergen in the ash pollen extract was barely detectable. Common allergens could be determined in the high molecular weight region. Ash pollen should be included in diagnostic procedures for spring pollinosis and should be considered for use in specific immunotherapy.

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[131] - Purohit A, Bardera R, Papanikolaou I, Vérot A, Villalba M, Rodriguez R, et al. Validation de l'allergène recombinant Fra e 1 par tests cutanés et histaminolibération chez les patients sensibilisés au frêne. Rev Fr Allergol Immunol Clin 2004;44:356

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[132] - Niederberger V, Purohit A, Oster JP, Spitzauer S, Valenta R, Pauli G. The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species. Clin Exp Allergy 2002;32:933-941

BackgroundAsh, a wind-pollinated tree belonging to the family Oleaceae, is distributed world-wide and has been suggested as a potent allergen source in spring time. ObjectiveThe aim of this study was to determine the profile of allergen components in ash pollen in order to refine diagnosis and therapy for patients with sensitivity to ash pollen MethodsThe IgE reactivity profile of 40 ash pollen-allergic patients was determined by immunoblotting. Antibodies raised to purified pollen allergens from tree and grass pollens were used to identify cross-reactive structures in ash pollen extract. IgE immunoblot inhibition studies were performed with recombinant and natural pollen allergens to characterize ash pollen allergens and to determine the degree of cross-reactivity between pollen allergens from ash, olive, birch, grasses and weeds. ResultsThe allergen profile of ash pollen comprises Fra e 1, a major allergen related to the major olive allergen, Ole e 1, and to group 11 grass pollen allergens, the panallergen profilin, a two EF-hand calcium-binding protein, a pectinesterase-like molecule and an allergen sharing epitopes with group 4 grass pollen allergens. Thus, the relevant allergens of ash are primarily allergens that share epitopes with pollen allergens from other tree, grass and weed species. ConclusionsAllergic symptoms to ash pollen can be the consequence of sensitization to cross-reactive allergens from other sources. The fact that ash pollen-allergic patients can be discriminated on the basis of their specific IgE reactivity profile to highly or moderately cross-reactive allergens has implications for the selection of appropriate forms of treatment.

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[133] - Lelong M, Castelain MC, Sulmont G. Existe-t-il, en France, une pollinose au frêne ? Rev Fr Allergol Immunol Clin 1992;32:41-42

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[134] - Laurent J, Guinnepain MT, Sauvaget J, Lafay M. La pollinose au frêne (Fraxinus excelsior) à Paris. Rev Fr Allergol Immunol Clin 1998;38:89-93

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[135] - Niederberger V, Purohit A, Oster JP, Spitzauer S, Valenta R, Pauli G. The allergen profile of ash (Fraxinus excelsior) pollen: cross-reactivity with allergens from various plant species. Clin Exp Allergy 2002;32:933-941

BackgroundAsh, a wind-pollinated tree belonging to the family Oleaceae, is distributed world-wide and has been suggested as a potent allergen source in spring time. ObjectiveThe aim of this study was to determine the profile of allergen components in ash pollen in order to refine diagnosis and therapy for patients with sensitivity to ash pollen MethodsThe IgE reactivity profile of 40 ash pollen-allergic patients was determined by immunoblotting. Antibodies raised to purified pollen allergens from tree and grass pollens were used to identify cross-reactive structures in ash pollen extract. IgE immunoblot inhibition studies were performed with recombinant and natural pollen allergens to characterize ash pollen allergens and to determine the degree of cross-reactivity between pollen allergens from ash, olive, birch, grasses and weeds. ResultsThe allergen profile of ash pollen comprises Fra e 1, a major allergen related to the major olive allergen, Ole e 1, and to group 11 grass pollen allergens, the panallergen profilin, a two EF-hand calcium-binding protein, a pectinesterase-like molecule and an allergen sharing epitopes with group 4 grass pollen allergens. Thus, the relevant allergens of ash are primarily allergens that share epitopes with pollen allergens from other tree, grass and weed species. ConclusionsAllergic symptoms to ash pollen can be the consequence of sensitization to cross-reactive allergens from other sources. The fact that ash pollen-allergic patients can be discriminated on the basis of their specific IgE reactivity profile to highly or moderately cross-reactive allergens has implications for the selection of appropriate forms of treatment.

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[136] - Guerra F, Galan Carmen C, Daza JC, Miguel R, Moreno C, Gonzalez J, et al. Study of sensitivity to the pollen of Fraxinus spp. (Oleaceae) in Cordoba, Spain. J Investig Allergol Clin Immunol 1995;5:166-170

The level of ash pollen grains (Fraxinus spp.) detected in the air in the city of Cordoba rarely surpasses the daily average of 8 g/m3 and is always detectable during the winter and the beginning of the spring. This fact, together with the knowledge of the presence of perennial symptoms in patients monosensitive to Olea in this area, allows us to suspect the possibility of cross-reactivity between both taxa. The skin tests carried out on a total of 1500 pollinotic patients with an extract of Fraxinus pollen offer us a global sensitization frequency of 59%. Furthermore, the Fx+ patients generally have a higher frequency of rural origin or they live in the most southerly district of the province. As for the age and sex of the patient, significant differences were found for the age groups between 5-10 years and over 25 but the frequency for both sexes was equal. As for the clinical features, differences in the family and personal antecedents of atopy were not observed, although rhinoconjunctival clinical antecedents seemed to prevail slightly over asthma antecedents. In relation to the duration of the symptoms, the patients presented a long evolution in their symptomatology. The great majority of the patients were polysensitive; only 8% were found to be monosensitive, with unimportant differences in their evolution after immunotherapeutic treatment. On the other hand, 92% of the Fx+ patients presented reactions to Olea pollen (but only 41% of the Fx- patients did), whereas 62% showed sensitivity to Cupressus, marking a strong contrast with the Fx-patients, who all tested negative for Cupressus pollen.

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[137] - Wahl R, Schmid-Grendelmeier P, Cromwell O, Wüthrich B. In vitro investigation of cross-reactivity between birch and ash pollen allergen extracts. J Allergy Clin Immunol 1996;98:99-106

Allergenic cross-reactivity between members of the Fagales family (birch, alder, hazel, and beech) and between members of the Oleaceae family (ash, olive, lilac, and privet) is well known, but little is known about possible cross-reactivity between these two groups of trees, in particular between birch and ash, both of which flower in the spring. Various immunochemical methods including RAST inhibition, Western blot, and Western blot inhibition have been used in this study to show that there is partial cross-reactivity between birch and ash pollens. Enzyme allergosorbent test measurements were performed on sera from 35 patients with hay fever in spring by using birch and ash pollen allergen disks. The major allergen of birch, Bet v 1, was readily detectable in the birch pollen extract, but a homologous allergen in the ash pollen extract was barely detectable. Common allergens could be determined in the high molecular weight region. Ash pollen should be included in diagnostic procedures for spring pollinosis and should be considered for use in specific immunotherapy.

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[138] - Kernerman SM, McCullough J, Green J, Ownby DR. Evidence of cross-reactivity between olive, ash, privet, and Russian olive tree pollen allergens . Ann Allergy 1992;69:493-496

In a clinical investigation, 103 Michigan residents with symptoms suggestive of allergic rhinitis or asthma were skin tested with olive (Olea europaea) pollen extract. Nineteen had positive reactions. Since the olive tree is not native to nor grown in Michigan, this study was undertaken to determine whether the skin test reactivity was the result of cross-reactivity among tree pollen allergens. ELISAs were developed to measure olive, ash (Fraxinus americana), privet (Ligustrum vulgare), and Russian olive (Elaeagnus angustifolia) specific IgE antibodies. Inhibition studies were performed to determine whether pollen extracts from each of these tree species could inhibit IgE antibody binding to olive extracts. Eleven of the 19 skin test-positive patients were olive-ELISA positive, eight either were ELISA-positive to ash, seven to privet and ten to Russian olive. There were significant correlations between the ELISA results to olive and each of the other three pollens. The inhibition studies demonstrated that all three of the tree pollens were capable of inhibiting the binding of IgE to olive extract in a dose-response fashion. IgE-immunoblot studies demonstrated several proteins common to olive, ash, and privet. Twelve of the olive skin test-positive patients were contacted and 75% were exposed to one or more of the studied trees in their yards. Five patients had traveled to areas where olive trees are grown. We conclude that there is a high degree of cross-reactivity among allergens from native Michigan trees and from olive trees. This cross-reactivity is the most likely reason for skin test reactivity to olive pollen extract in Michigan

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[139] - Sastre J, Lluch-Bernal M, Bustillo AMG, Carnés J, Maraçon F, Casanovas M, et al. Allergenicity and cross-reactivity of Russian olive pollen (Eleagnus angustifolia). Allergy 2004;59:1181-1186

BACKGROUND: The purposes of this study were: to determine the prevalence of sensitization and immunochemical characterization of Eleagnus angustifolia pollen (Russian olive) that belongs to the family Eleagnaceae . METHODS: A total of 134 patients with rhinoconjunctivitis and/or asthma were studied. Its allergenicity, cross-reactivity with olive pollen and the presence of Ole e 1 and Ole e 4-like molecules were evaluated . RESULTS: Eleagnus angustifolia pollen was detected from May to June. Seventy-three of 134 (30.5%) had positive skin test to E. angustifolia, all of them were positive to olive. There was a good correlation between specific immunoglobulin (Ig)E levels to E. angustifolia and Olea europaea (r = 0.77, P = 0.002). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots revealed major IgE-binding bands in the E. angustifolia extract of 43 and 63.7 kDa. The E. angustifolia extract was not able to inhibit olive, whereas O. europaea inhibited E. angustifolia up to 41%. The presence of Ole e 1- and Ole e 4-like allergens in E. angustifolia extract was confirmed by enzyme-linked immunosorbent (ELISA) inhibition assays. Nasal challenge with E. angustifolia was positive in three of six patients with positive skin test to both pollens and negative in five patients with positive skin test only to O. europaea . CONCLUSIONS: This study confirms that E. angustifolia is capable of sensitizing individuals in Madrid. A minimal-to-moderate cross-reactivity with olive pollen was established, suggesting some cross-reactivity but not excluding co-sensitization.

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[140] - Belver MT, Caballero MT, Contreras J, Cabañas R, Sierra E, Madero R et al. Associations Among Pollen Sensitizations From Different Botanical Species in Patients Living in the Northern Area of Madrid. J Investig Allergol Clin Immunol 2007;17:157-159

OBJECTIVE: To determinate the existence of associations among sensitizations to antigens produced by pollen grains of different botanical species as assessed by skin prick tests in patients with respiratory disorders . METHODS: Six hundred twenty nine consecutive patients living in the northern area of Madrid who underwent clinical evaluation because of rhinoconjunctivitis, and/or asthma were studied. All patients were tested with a skin prick test using a battery of inhalants including pollens, dust mites, molds and danders. The exploratory multivariate technique of Multiple Correspondence Analysis was used to compare the homogeneity of sensitizations between groups. Of the 629 patients, 459 (73.0%) had positive skin prick tests to pollen and were selected as the study group . RESULTS: The most prevalent pollen sensitization was to Gramineae pollen (83.7%) followed by Oleaceae sensitisation (75.8%). Multiple Correspondence Analysis revealed the existence of an association among pollen sensitizations, showing that they clustered two groups: sensitizations to Gramineae, Oleaceae, Cupressaceae, Chenopodiaceae, Plantaginaceae (group I), and sensitizations to Betulaceae, Platanaceae, Compositae (group II). Sensitization to Parietaria was not included in any of the sensitization groups and showed an independent behaviour . CONCLUSION: Pollen sensitizations in our area cluster into two association groups which have not previously been reported.

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[141] - Cuesta-Herranz J, Lazaro M, Figueredo E, Igea JM, Umpierrez A, De Las Heras M. Allergy to plant-derived fresh foods in a birch- and ragweed-free area. Clin Exp Allergy 2000;30:1411-1416

BACKGROUND: Allergy to plant-derived fresh foods has often been reported in geographical areas where birch or ragweed pollens are frequent and has been attributed to cross-reactivity to pollens. OBJECTIVE: The aim of this study has been to evaluate allergy to plant-derived fresh foods among pollen-allergic patients from a birch and ragweed-free area. METHODS: Ninety-five pollen-allergic patients took part in the study. The study consisted of a questionnaire, skin prick tests and challenge tests. Pollen skin tests to five grasses, eight trees and seven weeds were performed in duplicate. Prick tests (prick by prick) and challenge tests were carried out with the fresh foods. RESULTS: Most patients allergic to pollens were sensitized to grass (Lolium and Phleum; 97.9%), followed by tree (Olea; 82.1%) and weed pollens (Plantago; 64.2%). 35 of the 95 pollen-allergic patients had positive skin test responses to some plant-derived fresh foods, the highest percentage corresponding to several fruits in the Rosaceae family (peach and pear, 26.3%), followed by Cucurbitacea fruits (melon, 13.7%). The 21. 05% of the pollen-allergic patients were allergic to some type of plant-derived fresh food. Peach was the plant-derived fresh food which most frequently elicited allergy symptoms (12.6%), followed by melon (7.36%). The cluster of positive responses to Rosaceae fruits was higher for skin testing than for challenge testing. CONCLUSION: Peach was the most important allergy provoking fruit in a birch and ragweed free-area where apples were consumed at a rate of two times more than peaches and the patients allergic to pollen were principally sensitized to grass pollens.

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

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

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[143] - Corominas M, Lleonart R, Martin C, De la Torre F. Profile of allergen pollen reactivity in patients sensitised to Olea pollen. Allergy 2008;63(suppl. 88):104

Background: Previous studies performed in our area (with low levels of Olea pollen) showed that 38% of patients with pollinosis were sensitised to Olea, although only 7% of them are monosensitised. The aim of this study is to evaluate sensitisation to olive pollen as a consequence of cross reactivity with other pollen allergens. We studied the sensitisation pollen profile of patients with positive skin prick test (SPT) to Olea and the cross reactivity with other pollen allergens. Patients and methods: Fourteen adult patients (8 males and 6 females) whith a positive SPT to Olea pollen were included. We analyzed their clinical history and SPT to common airborne allergens and allergens with a high LTP, profilin or polcalcin content. Specific serum IgE against to recombinant allergens Ole e1, Ole e 9, Salk1, Par j 1, Phl p 1, Phl p 5, Art v 1, Cup s 1, Bet v 1, Pru p 3, Mal d 4, Che a 3 (ALK-Abello) were quantified in the ADVIA Centaur platform. Results: Ten patients presented rhinitis and four asthma and rhinitis. According to SPT, 78% were positive to grasses, 64% to Parietaria, 57% to Salsola, 57% to Plantago, 50% to Platanus, 43% to Artemisia, 36% to Cupressus, 36% to peach, 21% to polcalcin, 14% to profilin and 14% to Betula. When we analyzed specific IgE against recombinant allergens, we found that 11 of the 14 patients (78%) were reactive to Ole e1 and none to Ole e 9. Only one patient had specific IgE to Che a 3 (polcalcin), and another to Che a 3 and to Mal d 4 (profilin). Ole e 1 specific IgE levels correlated with IgE levels of Phl p 1, Phl p 5, Pru p 3, Cup s 1, Che a 3, Mal d4, Pla l 1 and Bet v 1 (P<0.002). Conclusions: Most of the patients sensitised to Olea had specific IgE to Ole e 1 allergen. Profilin and polcalcin allergens are not responsible for polysensitisation present in these patients. In this group, sensitisation to Olea pollen corresponds to the great number of sensitisation present, but it is not a result of cross reactivity.

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[144] - Cuesta-Herranz J, Lazaro M, Figueredo E, Igea JM, Umpierrez A, De Las Heras M. Allergy to plant-derived fresh foods in a birch- and ragweed-free area. Clin Exp Allergy 2000;30:1411-1416

BACKGROUND: Allergy to plant-derived fresh foods has often been reported in geographical areas where birch or ragweed pollens are frequent and has been attributed to cross-reactivity to pollens. OBJECTIVE: The aim of this study has been to evaluate allergy to plant-derived fresh foods among pollen-allergic patients from a birch and ragweed-free area. METHODS: Ninety-five pollen-allergic patients took part in the study. The study consisted of a questionnaire, skin prick tests and challenge tests. Pollen skin tests to five grasses, eight trees and seven weeds were performed in duplicate. Prick tests (prick by prick) and challenge tests were carried out with the fresh foods. RESULTS: Most patients allergic to pollens were sensitized to grass (Lolium and Phleum; 97.9%), followed by tree (Olea; 82.1%) and weed pollens (Plantago; 64.2%). 35 of the 95 pollen-allergic patients had positive skin test responses to some plant-derived fresh foods, the highest percentage corresponding to several fruits in the Rosaceae family (peach and pear, 26.3%), followed by Cucurbitacea fruits (melon, 13.7%). The 21. 05% of the pollen-allergic patients were allergic to some type of plant-derived fresh food. Peach was the plant-derived fresh food which most frequently elicited allergy symptoms (12.6%), followed by melon (7.36%). The cluster of positive responses to Rosaceae fruits was higher for skin testing than for challenge testing. CONCLUSION: Peach was the most important allergy provoking fruit in a birch and ragweed free-area where apples were consumed at a rate of two times more than peaches and the patients allergic to pollen were principally sensitized to grass pollens.

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[145] - Quiralte J, Florido F, Arias de Saavedra JM, Gómez A, Sáenz de San Pedro B, González E, et al. Olive allergen-specific IgE responses in patients with Olea europaea pollinosis. Allergy 2002;57(suppl. 71):47-52

BackgroundOlive tree (Olea europaea) pollen is an important cause of pollinosis in the countries of the Mediterranean area. ObjectiveThis work aimed to study the IgE-binding frequency of Ole e 1, Ole e 2, Ole e 3, Ole e 6 and Ole e 7 from O. europaea pollen in a large population of olive pollen-allergic patients. MethodsWe studied: 119 consecutive patients with seasonal rhinitis and/or asthma and a positive skin prick test to O. europaea pollen extract; 10 atopic patients without history of pollinosis and a negative skin prick test to O. europaea; and 10 healthy controls. Allergens were purified from O. europaea pollen extract by reverse phase HPLC and characterized by N-terminal amino acid sequencing, MALDI analysis, and specific IgE immunodetection. Skin prick tests and ELISA titration against above mentioned purified olive pollen allergens were performed in all pollinic patients and controls. ResultsOne-hundred and seven (90.7%) patients had a positive skin response to Ole e 1; 88 (74.6%) reacted to Ole e 2; 57 (47.9%) reacted to both Ole e 6 and Ole e 7; and 43 (37.8%) reacted to Ole e 3. The allergenic activity determined by ELISA to Ole e 1 was found in 84%; to Ole e 2 in 61.3%; to Ole e 3 in 31.9%; to Ole e 6 in 39.4%; and to Ole e 7 in 41.2% of patients. All patients had positive skin responses to at least one of the allergens tested, However, a combination of Ole e 1 and Ole e 2 together with a minor allergen Ole e 6 or Ole e 7, disclosed the same diagnostic value that was obtained with the use of crude olive pollen extract. The nonatopic and atopic control subjects did not react to any purified allergens on the skin prick test. ConclusionsThese results indicate that Ole e 1 and Ole e 2 are major allergens in patients with O. europaea pollinosis in our population. A combination of a few olive pollen allergens can substitute the crude extract for in vivo as well as in vitro diagnostic purposes.

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[146] - Pastorello EA, Pravettoni V, Farioli L, Rivolta F, Conti A, Ispano M, et al. Hypersensitivity to mugwort (Artemisia vulgaris) in patients with peach allergy is due to a common lipid transfer protein allergen and is often without clinical expression. J Allergy Clin Immunol 2002;110:310-317

Background: The observation of mugwort-specific IgE antibodies in patients with peach allergy suggests that mugwort sensitization might play a role in sensitization to peach. Objective: We sought to study the clinical manifestations of mugwort hypersensitivity in patients with peach allergy, identify the common allergens, and evaluate their IgE crossreactivity. Methods: Patients with oral allergy syndrome for peach and specific IgE antibodies to mugwort were investigated for respiratory symptoms during the mugwort season. Peach and mugwort allergens were identified by means of SDS-PAGE and IgE immunoblotting. Immunoblotting inhibition experiments were done to study cross-reactivity between peach and mugwort and other pollens. Results: Seventeen patients were studied, 10 with no seasonal respiratory symptoms and 7 with clear late summer respiratory symptoms. In IgE immunoblotting the 10 asymptomatic patients reacted only to a 9-kd allergen of both mugwort and peach, whereas the 7 patients with pollinosis reacted to other allergens. Ten patients with mugwort allergy, no history of allergy to peach, and negative results for peach-specific IgE antibodies were also studied. The mugwort 9-kd protein was identified as a lipid transfer protein (LTP) homologous to peach LTP. Immunoblotting inhibition showed that IgE binding to the peach 9-kd band was totally inhibited by 4 µg of peach LTP but only by 400 µg of mugwort LTP, whereas 4 µg of both mugwort and peach LTP totally inhibited the mugwort immunoblotting. The results were similar with other pollens. Conclusions: Patients sensitized only to the 9-kd LTP of mugwort do not present hay fever symptoms, and this sensitization is a consequence of the peach sensitization.

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[147] - Parra FM, Cuevas M, Lezaun A, Alonso MD, Beristain AM, Losada E. Pistachio nut hypersensitivity: identification of pistachio nut allergens. Clin Exp Allergy 1993;23:996-1001

Type I hypersensitivity to pistachio nut antigens was demonstrated in three patients by means of immediate skin-test reactivity, specific IgE determination by a fluoroimmunoassay (CAP), CAP-inhibition and leucocyte histamine release. Sensitization to other dried fruits and pollens was observed in the patients. The CAP-inhibition studies revealed significant crossreactivity between pistachio and cashew nut belonging to the Anacardiaceae family, and between pistachio nut and other dried fruits belonging to taxonomically unrelated botanical families. No relevant crossallergenicity was observed between pistachio nut and Lolium and Olea pollens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a pistachio nut extract followed by immunoblotting analysis identified four IgE-binding bands with molecular weights of 34, 41, 52 and 60 kD.

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[148] - Vassilopoulou E, Zuidmeer L, Akkerdaas J, Tassios I, Rigby NR, Mills ENC, et al. Severe Immediate Allergic Reactions to Grapes: Part of a Lipid Transfer Protein-Associated Clinical Syndrome. Int Arch Allergy Immunol 2007;143:92-102

BACKGROUND: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected . OBJECTIVES: The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur . METHODS: Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens . RESULTS: All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa beta-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity . CONCLUSIONS: LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The 'LTP syndrome' is the appropriate term to describe this condition.

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[149] - Scheurer S, Haase T, Schocker F, Cisteró-Bahíma A, Enrique E, Becker WM, et al. Prevalence of IgE-Sensitization and Cross-Reactivity of Pru av 3, the Lipid Transfer Protein From Cherry. AAAAI 58th Annual Meeting, New York, 1-6 March, 2002, Poster n°940

In Central and Northern Europe, food allergy to Rosaceae fruits is highly associated with birch pollinosis, owing to the existence of IgE cross-reactive homologous allergens in birch pollen and food. By contrast, in the Mediterranean area, severe allergic reactions to these fruits frequently are not related to birch pollen allergy, predominantly elicted by lipid transfer proteins (LTP). Recently, Pru av 3, the LTP from cherry has been identified as major allergen in Italian cherry-allergic subjects. The aim of this study was to evaluate the sensitization to Pru av 3 in a group of cherry allergic patients recruitated in Spain and to investigate the IgE-cross-reactivity of Pru av 3 with hazelnut LTP (Cor a 8) and with pollen extracts. All cherry-allergic patients (n=8) were allergic to plane tree pollen (Platanus acerifolia), and 4 out of all subjects investigated were allergic to grass pollen. The prevalence of IgE sensitization was investigated by EAST: Six out of 8 cherry-allergic patients were sensitized to rPru av 3, none was positive to the cherry profilin Pru av 4. Only one patient was sensitized to birch-related Pru av 1, identified as major allergen in the middle- and north European population. Two out of 8 cherry-allergic patients reported systemic reactions (angioedema) after ingestion of the corresponding food. IgE-Immunoblot and/or IgE-EAST (Enzyme Allergosorbent Test) inhibition experiments indicated a partial cross-reactivity between rPru av 3 and extracts from plane tree, ragweed, mugwort, and Parietaria pollen. No Inhibition of IgE-binding was found with the recombinant (r) hazelnut LTP Cor a 8, birch pollen and olive pollen extract as inhibitors. By contrast, IgE binding to recombinant hazelnut LTP was almost completely inhibited by rPru av 3. Preincubation of patient sera with cherry extract showed that natural and recombinant Pru av 3 shared identical IgE-epitopes. LTPs are potentially servere food allergens. The IgE cross-reactivity between food LTPs and pollen may indicate the presence of homologus pollen LTPs and their potential relevance in triggering food allergies by a primary sensitization by the inhalative route. Moreover, epitope similarities and differences of LTP-epitopes have to be taken into consideration in studies of cross-reactions in patients with multiple food allergies

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

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

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[151] - EXPO: Determinacion de la prevalencia de sensibilizacion a polenes mediante tecnicas de diagnostico molecular. ALK-Abello Ed., 2009, 86pp.

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

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

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[153] - Batanero E, Villalba M, Monsalve RI, Rodriguez R. Cross-reactivity between the major allergen from olive pollen and unrelated glycoproteins: evidence of an epitope in the glycan moiety of the allergen. J Allergy Clin Immunol 1996;97:1264-1271

Ole e 1, the major allergen from olive pollen, is a glycoprotein containing a single Asn-linked glycan moiety. Rabbit antiserum against this protein has been obtained; and its immunologic cross-reactivities in Western blotting with ascorbate oxidase, horseradish peroxidase, bromelain, ovalbumin, and honeybee venom phospholipase A2 have been studied. Ascorbate oxidase, peroxidase, and bromelain are recognized by the Ole e 1 antiserum. When these three proteins are deglycosylated by periodate treatment, such an immunologic reaction does not occur. The relative affinities of these proteins have been analyzed by direct and inhibition ELISA experiments. A commercially available antibody against horseradish peroxidase has also been considered in these studies. This antibody reacts with Ole e 1 but not with the periodate-deglycosylated allergen. Horseradish peroxidase, bromelain, and ascorbate oxidase are recognized by the IgE of sera from patients who are hypersensitive to olive tree pollen. This binding is also abolished by periodate treatment. The results are interpreted in terms of the presence of an epitope in the carbohydrate moiety of Ole e 1, which would contain a xylose involved in recognition by both IgE and IgG antibodies.

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[154] - Rodríguez R, Villalba M, Batanero E, González EM, Monsalve RI, Huecas S, et al. Allergenic diversity of the olive pollen. Allergy 2002;57(suppl. 71):6-16

A great number of allergenic proteins have been detected in olive pollen extracts. To date, nine allergens have been isolated and characterized, which have been called Ole e 1 to Ole e 9. The most prevalent olive allergen is Ole e 1, which affects more than 70% of patients hypersensitive to olive pollen, but others, such as Ole e 2, Ole e 8, and Ole e 9, have been demonstrated to be major allergens, and Ole e 6 or Ole e 7 reach high values of clinical incidence. Many of these allergens, such as Ole e 2 (profilin) and Ole e 3 (polcalcin), are involved in cross-reactivities, which agrees with their adscription to panallergenic families. Among the many olive allergens of high molecular mass, only Ole e 9 (46 kDa) has been characterized. The allergen is a polymorphic and glycosylated beta-1,3-glucanase, which belongs to a pathogenesis-related (PR-2) protein family. In addition to the polypeptide epitopes, Ole e 1 also exhibits IgE-binding determinants in the carbohydrate, which are recognized by more than 60% of the sera from patients sensitive to the whole allergen, although the level of such glycan-specific IgE seems not to be clinically relevant in the overall content of the sera. Recent advances in the elucidation of the structure of the Ole e 1-oligosaccharide component allows us to explain the antigenicity of the molecule. Finally, the recombinant production of several allergens from olive pollen in both bacterial and eukaryotic cells has allowed us to resolve problems derived from the polymorphism and scarcity of the natural forms of these allergens. The biological equivalence between the natural and recombinant forms lets us initiate studies on the design of mixtures for clinical purposes, in which hypoallergenic derivatives of these allergens could play a definitive role.

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[155] - van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, et al. beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 2000;275:11451-11458

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.

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[156] - Hemmer W, Focke M, Kolarich D, Wilson IBH, Altmann F, Wöhrl S, et al. Antibody binding to venom carbohydrates is a frequent cause for double positivity to honeybee and yellow jacket venom in patients with stinging-insect allergy. J Allergy Clin Immunol 2001;108:1045-1052

Background: Up to 50% of patients with stinging-insect allergy have double-positive RAST results to honeybee and yellow jacket (YJ) venom. True double sensitization and crossreactivity through venom hyaluronidases are considered main reasons for this multiple reactivity. OBJECTIVE: We investigated the role of antibodies against cross-reactive carbohydrate determinants in venom double positivity. METHODS: CAP inhibition experiments were performed with crude oilseed rape (OSR) and timothy grass pollen extracts and a neoglycoprotein construct displaying a MUXF glycan, as present in pineapple-stem bromelain (MUXF-BSA). CAP to OSR was used as a rough measure for carbohydrate-specific IgE in individual sera. RESULTS: CAP results to OSR pollen were positive in 2 of 14 single-positive honeybee venom sera, 2 of 16 single-positive YJ venom sera, and 33 (80.5%) of 41 double-positive sera (P < .00001, 2 test). CAP inhibition was performed in 16 selected patients with a CAP class of 3 or higher to both venoms. In 9 of 11 patients with a highly positive CAP result to OSR (CAP score to OSR > CAP score to second venom), pollen extracts, MUXF-BSA, or both were able to completely inhibit IgE binding to one of the venoms, whereas this was not the case in 5 patients with a negative or weakly positive CAP result to OSR (CAP score to OSR < CAP score to second venom). CONCLUSIONS: The data suggest that carbohydrate-specific IgE is a major cause for the double positivity to honeybee and YJ venom seen in patients with Hymenoptera allergy. Because these antibodies may have low clinical relevance, they may severely impede the correct diagnosis of Hymenoptera venom allergy

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[157] - Batanero E, Crespo JF, Monsalve RI, Martin-Esteban M, Villalba M, Rodriguez R. IgE-binding and histamine-release capabilities of the main carbohydrate component isolated from the major allergen of olive tree pollen, Ole e1. J Allergy Clin Immunol 1999;103:147-153

BACKGROUND: Pollen from olive trees (Olea europaea ) is a cause of pollinosis and an aggravating of asthma in Mediterranean regions. Recently, Ole e 1, the major allergen from olive tree pollen, has been isolated and its amino acid sequence has been elucidated. It is a glycoprotein whose carbohydrate moiety is involved in an IgE-binding epitope responsible for cross-reactivity among plant glycoproteins. However, the allergenicity of the free carbohydrate side chains remains to be clarified. OBJECTIVE: The purpose of this study was to isolate the main carbohydrate component of Ole e 1 allergen and analyze its IgE-binding and histamine-release capabilities. METHODS: Deglycosylation treatment of Ole e 1 with PNGase F and gel exclusion chromatography were used to isolate the main sugar component of the allergen. Sera of patients who are allergic to olive pollen and sera sensitive to Ole e 1 have been used in dot blotting assays of IgE binding to the isolated carbohydrate. Heparinized whole blood obtained from patients sensitive to Ole e 1 were stimulated by the free carbohydrate; the resulting histamine release was measured. RESULTS: The main sugar component of Ole e 1 has been isolated. Free carbohydrate was able to bind IgE from sera of patients allergic to olive pollen; the sera of 65% of these patients contained anticarbohydrate reacting IgE, and 100% of those patients were sensitive to Ole e 1. The free carbohydrate promoted in vitro histamine release from basophils of sensitized patients. CONCLUSION: The carbohydrate moieties of allergenic glycoproteins can constitute significant determinants on the binding to IgE of the sera from patients who are hypersensitive and can be responsible for inducing histamine release from blood cells.

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[158] - Batanero E, Villalba M, Rodriguez R. Glycosylation site of the major allergen from olive tree pollen. Allergenic implications of the carbohydrate moiety. Mol Immunol 1994;31:31-37

The electrophoretic analysis of purified Ole e I, the major allergen from Olea europaea pollen, reveals the presence of two main variants, glycosylated (20.0 kDa) and non-glycosylated (18.5 kDa) components. The glycosylated variant has been identified as a concanavalin A-binding glycoprotein. Its carbohydrate moiety has a molecular mass of about 1.3 kDa (5% weight of the glycosylated allergen), based on mass spectrometry analysis. Enzymatic treatment of native Ole e I with the specific glycosidase PNGase F accounts for an oligosaccharide N-linked to the polypeptide chain. This treatment does not sensibly modify the secondary structure of the protein but diminishes the affinity of the allergen for specific IgE antibodies. Tryptic digestion of Ole e I reveals the presence of a single carbohydrate-containing peptide. This peptide was recognized by the sera of hypersensitive individuals. The amino acid sequence of this peptide is Phe-Lys-Leu-Asn-Thr-Val-Asn-Gly-Thr-Thr-Arg, asparagine at the seventh being the carbohydrate attaching site. The obtained data are discussed in terms of the potential role of the sugar moiety in the allergenic activity of Ole e I.

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[159] - Gonzalez E, Villalba M, Lombardero M, Aalbers M, van Ree R, Rodriguez R. Influence of the 3D-conformation, glycan component and microheterogeneity on the epitope structure of Ole e 1, the major olive allergen. Use of recombinant isoforms and specific monoclonal antibodies as immunological tools. Mol Immunol 2002;39:93-101

Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area. Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia pastoris by recombinant methods, have been used as tools to determine the role of the three-dimensional (3D)-folding, the glycan component and several point changes of the amino acid sequence in the binding of murine IgG mAbs and human IgE to the olive allergen. Seven mAb families (F1-F7) were established, two of which (F1 and F2) recognize continuous epitopes. The carbohydrate moiety of Ole e 1 was involved in the binding to F2 and F4, whereas F3 and F7 were able to bind to all Ole e 1 variants. The remaining families of IgG murine antibodies exhibited different affinities for the antigens assayed in a native or denatured conformation. Although the binding of human IgE to Ole e 1 was not affected by heat treatment, it was shown to be strongly dependent on the integrity of the disulfide bridges and was partially inhibited by F3-F7 IgG antibodies, their individual values ranging from 12 to 31% and reaching 53% with their mixture. The IgE from sera of olive-allergic patients showed a significant diversity of binding capacity to the members of the Ole e 1-like family due to the microheterogeneity of their polypeptide sequences, in spite of their highly conserved primary structures. Whereas one of the isoforms of Ole e 1 exhibits a highly similar behavior to the natural form, being a putative molecule for diagnostic purposes, other ones can be considered as hypoallergenic variants of this allergen and, thus, potential candidates to be used in immunotherapy

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[160] - Quiralte J, Gonzalez E, Arias De Saavedra JM, Villalba M, Florido JF, Saenz De San Pedro B, et al. Immunological activity of recombinant Ole e 1 in patients with Olea europaea pollinosis. Int Arch Allergy Immunol 2000;122:101-107

BACKGROUND: Recombinant allergens have potential advantages over conventional allergenic extracts. However, these recombinant allergens should be evaluated for their antigenic activity and compared with their natural counterparts before being used for clinical purposes. METHODS: We studied 33 patients with seasonal rhinitis and/or bronchial asthma and a positive skin prick test to Olea europaea pollen extract, 10 atopic patients with no history of pollinosis and a negative skin prick test to O. europaea extract and 10 healthy controls. Skin prick tests and determination by ELISA of specific IgE to natural Ole e 1 (nOle e 1) and recombinant Ole e 1 (rOle e 1) expressed in Pichia pastoris were performed in all patients and controls. Inhibition assays were performed between nOle e 1 and rOle e 1 by ELISA. RESULTS: All patients with O. europaea pollinosis had positive skin test responses to both commercial O. europaea extract and nOle e 1 allergen, and all reacted to rOle e 1 on the skin prick test. The nonatopic and atopic control subjects with negative olive pollen skin test results did not react to rOle e 1 on the skin prick test, even at the highest concentrations, confirming the specificity of this test. We found a weak correlation between the wheal surface area produced by the prick test with nOle e 1 and the wheal surface area produced by rOle e 1 at 10 microgram/ml (r = 0.42, p < 0.05). Comparison of specific IgE against both nOle e 1 and rOle e 1 in the patients did not reveal any significant difference. There was a strong correlation between the amount of specific IgE against nOle e 1 and rOle e 1 (r = 0.99, p < 0.01). The two proteins displayed the same extent of binding inhibition to IgE antibodies in ELISA inhibition experiments. CONCLUSIONS: These results confirm the immunological activity of rOle e 1 expressed in P. pastoris and indicate that Ole e 1 is one of the major allergens in O. europaea pollinosis as evaluated by skin prick test and serological methods. The correlation between rOle e 1 and nOle e 1 in skin test results and serologic data indicates the potential of recombinant allergens for clinical applications and diagnosis of O. europaea pollen allergy.

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[161] - van Ree R, Foetisch K, Focke-Tejkl M, van Leeuwen A, Aalbers M, Valenta R, et al. Comparison of IgE-binding potency and biological activity of natural and recombinant major allergens from birch, grass and olive pollen and house dust mite, using sera from eight European countries. EAACI 23th Congress, Amsterdam, 12-16 June, 2004, Poster n°26

Background: Major allergens of the most important inhalant allergen sources have been purified and produced as recombinant allergens. These molecules are candidates to be used as reference materials for allergen standardisation. Objective: To be able to make a choice between natural (nat) and recombinant (rec) molecules as reference materials, both versions of Bet v 1, Phl p 1, Phl p 5, Ole e 1, Der p 1 and 2 and Der f 1 and 2 were produced to allow characterisation of physico-chemical properties and immune reactivity. In this study, the aim was to compare IgE-binding potency and biological activity. Methods: Sera (n=975) from patients with respiratory allergy to birch, grass, olive pollen and/or house dust mite were collected at 11 clinical centres in Europe. Each serum with a SPT > 5mm for any of the 4 allergen sources was tested by RAST for specific IgE against nat and rec major allergen from the respective allergen sources. A selection of 25 sera with IgE against these major allergens was used in a histamine release test (stripped basophil protocol) and a dot-blot to further compare nat and rec allergens. Results: Frequencies of recognition of all eight major allergens were > 80% with Spearman-rank correlations between nat and rec >0.9. IgE-binding to nat and rec Bet v 1 was close to identical (mean ratio: 1.0; p>0.2). For Phl p 1, binding to the rec was 0.6 times weaker (p<0.001). This is caused by incorrect folding of the recombinant molecule. For Phl p 5, two rec isoforms were tested, Phl p 5a (ratio to nat: 0.7) and Phl p 5b (ratio:0.5). A significant number of sera with IgE antibodies to the nat allergen were negative for Phl p 5b. This was not observed for Phl p 5a. For Ole e 1 the ratio rec/nat was 0.6. For both house dust mite group 1 allergens this was 0.5 and for Der p 2 and Der f 2, 0.7 and 0.8 respectively (p<0.001). Dotblot analyses gave very similar results with especially for rec Phl p 1 lower recognition intensities. Assessment of biological activity confirmed differences between nat and rec allergens. Interestingly, a two-fold lower IgE-binding potency in RAST translated into a 100-fold lower biological activity. Conclusion: Both nat and rec major allergens have good IgE-binding potencies, although differences were observed (£ two-fold). These minor differences in IgE-binding potency, however translates into a much stronger decrease in biological activity.

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[162] - Barderas R, Purohit A, Papanikolaou I, Rodriguez R, Pauli G, Villalba M. Cloning, expression, and clinical significance of the major allergen from ash pollen, Fra e 1. J Allergy Clin Immunol 2005;115:351-357

BACKGROUND: Ash tree, an Oleaceae member, is considered an important source of pollen allergy in Central Europe. Fra e 1 is a protein of the Ole e 1-like family, which regulates pollen tube growth. It has been suggested to be a relevant allergen from ash pollen . OBJECTIVE: Cloning Fra e 1-cDNA and overproducing a properly folded recombinant allergen to analyze its clinical significance . METHODS: Fra e 1-encoding cDNA was amplified by PCR, cloned in Escherichia coli , and sequenced. The recombinant allergen was produced in Pichia pastoris and used in immunoblotting, ELISA, histamine release, and skin prick tests. Sera and blood cells from patients sensitized to ash pollen as well as anti-Ole e 1 monoclonal and polyclonal antisera were used . RESULTS: Recombinant Fra e 1 (rFra e 1) is a glycoprotein of 145 amino acids exhibiting 82%, 88%, and 91% identity with Syr v 1, Ole e 1, and Lig v 1, allergens of the Oleaceae family. It was secreted to the extracellular medium of the yeast cultures and purified by means of 3 chromatographic steps. IgG from Ole e 1-specific antibodies recognized rFra e 1. IgE antibodies from ash-sensitized patients bound to rFra e 1 with a prevalence of 75%. The recombinant allergen induced histamine release. Twenty-nine of 30 ash-sensitized patients were positive to rFra e 1 by skin prick tests . CONCLUSION: Fra e 1 is a relevant allergen in ash pollen sensitization. It has been efficiently produced in P pastoris and could be used in diagnosis.

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[163] - Barderas R, Purohit A, Rodriguez R, Pauli G, Villalba M. Isolation of the main allergen Fra e 1 from ash (Fraxinus excelsior) pollen: comparison of the natural and recombinant forms. Ann Allergy Asthma Immunol 2006;96:557-563

BACKGROUND: Fra e 1 is a major allergen for ash pollen-sensitized individuals in northern and central Europe. It belongs to the Ole e 1-like family and displays high cross-reactivity with taxonomically related members. OBJECTIVES: To isolate and characterize natural Fra e 1 (nFra e 1) from ash pollen and to compare its structural, antigenic, and allergenic properties with those of its recombinant form (rFra e 1). METHODS: The allergen was isolated by means of gel permeation chromatography and reverse-phase high-performance liquid chromatography columns. Molecular characterization was performed by means of Edman degradation, mass spectrometry, circular dichroism, concanavalin A lectin reaction, and anti-horseradish peroxidase polyclonal antibody. Immunologic characterization was performed using immunoblotting and enzyme-linked immunosorbent assay, inhibition experiments, and histamine release assays with serum samples from allergic patients with well-known reactivity to Fra e 1 or Ole e 1 and with polyclonal antiserum and monoclonal antibodies against Ole e 1. The protein used as a reference was rFra e 1, which was produced in the yeast Pichia pastoris. RESULTS: Purified nFra e 1 appeared as 5 variants with different glycosylation degrees. Both nFra e 1 and rFra e 1 were equivalently folded as deduced from the spectroscopic analysis using circular dichroism. Both molecules share the antigenic and allergenic epitopes after the purification process, and the glycan group of nFra e 1 is a potential epitope. Natural Fra e 1 displayed strong cross-reactivity with Ole e 1. CONCLUSIONS: Natural Fra e 1 is a heterogeneously glycosylated protein with high allergenic relevance. It displays structural, antigenic, and allergenic similarity with rFra e 1. Both proteins could be used for clinical purposes.

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[164] - Purohit A, Bardera R, Papanikolaou I, Vérot A, Villalba M, Rodriguez R, et al. Validation de l'allergène recombinant Fra e 1 par tests cutanés et histaminolibération chez les patients sensibilisés au frêne. Rev Fr Allergol Immunol Clin 2004;44:356

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[165] - Gonzalez E, Villalba M, Lombardero M, Aalbers M, van Ree R, Rodriguez R. Influence of the 3D-conformation, glycan component and microheterogeneity on the epitope structure of Ole e 1, the major olive allergen. Use of recombinant isoforms and specific monoclonal antibodies as immunological tools. Mol Immunol 2002;39:93-101

Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area. Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia pastoris by recombinant methods, have been used as tools to determine the role of the three-dimensional (3D)-folding, the glycan component and several point changes of the amino acid sequence in the binding of murine IgG mAbs and human IgE to the olive allergen. Seven mAb families (F1-F7) were established, two of which (F1 and F2) recognize continuous epitopes. The carbohydrate moiety of Ole e 1 was involved in the binding to F2 and F4, whereas F3 and F7 were able to bind to all Ole e 1 variants. The remaining families of IgG murine antibodies exhibited different affinities for the antigens assayed in a native or denatured conformation. Although the binding of human IgE to Ole e 1 was not affected by heat treatment, it was shown to be strongly dependent on the integrity of the disulfide bridges and was partially inhibited by F3-F7 IgG antibodies, their individual values ranging from 12 to 31% and reaching 53% with their mixture. The IgE from sera of olive-allergic patients showed a significant diversity of binding capacity to the members of the Ole e 1-like family due to the microheterogeneity of their polypeptide sequences, in spite of their highly conserved primary structures. Whereas one of the isoforms of Ole e 1 exhibits a highly similar behavior to the natural form, being a putative molecule for diagnostic purposes, other ones can be considered as hypoallergenic variants of this allergen and, thus, potential candidates to be used in immunotherapy

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[166] - Palomares O, Swoboda I, Villalba M, Balic N, Spitzauer S, Rodríguez R, et al. The Major Allergen of Olive Pollen Ole e 1 Is a Diagnostic Marker for Sensitization to Oleaceae. Int Arch Allergy Immunol 2006;141:110-118

BACKGROUND: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen . METHODS: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1 . RESULTS: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae . CONCLUSIONS: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy.

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[167] - Gonzalez-Quintela A, Garrido M, Gude F, Campos J, Linneberg A, Lojo S, et al. Sensitization to cross-reactive carbohydrate determinants in relation to alcohol consumption. Clin Exp Allergy 2008;38:152-160

BACKGROUND: Alcohol consumption is associated with increased serum IgE of unknown specificity . OBJECTIVE: To investigate the prevalence of specific IgE to cross-reactive carbohydrate determinants (CCDs) in adults, and its relation to alcohol consumption . METHODS: Population-based survey of 457 adults (218 abstainers, 195 light-to-moderate drinkers, 44 heavy drinkers). Specific IgE determinations included a CCD (MUXF(3), the N-glycan of bromelain), pollens (Lolium perenne and Olea europaea), Hymenoptera venoms (Apis mellifera and Vespula spp.), and a mite (Dermatophagoides pteronyssinus). We replicated these studies in an additional sample of alcoholics (n=138). Inhibition assays were performed in selected cases . RESULTS: In the general population, 5.6% of individuals (95% confidence interval 3.5-7.6%) showed positive (>/=0.35 kU/L) CCD-specific IgE. The levels of CCD-specific IgE were particularly high in heavy drinkers, who also showed a high prevalence of positive IgE to pollens and Hymenoptera venoms, doubling (at least) the prevalence found in alcohol abstainers and light-to-moderate drinkers. The presence of IgE to pollens and Hymenoptera venoms was closely correlated with the presence of CCD-specific IgE. These features were confirmed in the additional sample of alcoholics. Inhibition studies indicated a role of CCD interference in IgE positivity to pollen and Hymenoptera allergens in alcoholics . CONCLUSIONS: CCD-specific IgE is prevalent in heavy drinkers, and is associated with positive IgE to pollens and Hymenoptera venoms. Specific IgE results should be interpreted with caution in heavy drinkers.

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[168] - Fernández-Rivas M. Alergia a frutas y hortalizas. in "Alergia a alimentos", Sociedad Española de Alergología e Inmunología Clínica (SEAIC) Ed., Euromedice, Badalona 2004, pp.143-67

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[169] - Azofra J. Olive allergy. Allergy 2004;59:559

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[170] - Hefle SL, Taylor SL. Allergenicity of edible oils. Food Technol 1999;53:62-68

Allergenicity studies on vegetable oils derived from commonly allergenic foods are described. Aspects considered include: allergenic foods; mechanisms of food allergies; difficulties in avoiding allergenic food proteins due to limited consumer knowledge, inadequate food labelling or poor cleaning practices in food preparation establishments; potentially allergenic oils (peanut (groundnut), soybean, tree nut, sunflower, sesame, cottonseed and (rarely) corn and coconut oils); methods of oil processing (solvent extraction/refining or cold pressing); labelling of oils; lower levels of allergenic proteins in solvent extracted/refined than cold pressed oils and levels required to cause allergic reactions; diagnostic tests for allergenicity (oral challenge, skin testing, radioallergosorbent assay, basophil histamine release, Prausnitz-Kustner test and immunoblotting); and studies on allergenicity of refined and cold pressed soybean, peanut, tree nut, cottonseed, sesame and sunflower oils (providing information concerning experimental techniques employed, shortcomings of the studies, levels of allergenic proteins present in the oils and oil allergenicity). It is concluded that refined oils contain virtually no protein and pose little or no hazard to allergic individuals; crude or cold pressed oils are less commonly available and may contain small amounts of protein and pose some risk

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[171] - Frémont S, Errahali Y, Bignol M, Metche M, Nicolas JP. What about the Allergenicity of Vegetable Oils ? Internet Symp Food Allergens 2002;4(2):111-118

Cases of allergy to peanut, sunflower seed, soybean and sesame seed oils have been reported in the literature, although some authors have claimed that these oils are not allergenic. The aim of this article is to review this subject, to describe the processing methods to which the seeds are subjected during the extraction of their oil, to recall that oils do not consist solely of triglycerides and to describe the findings of our studies. The allergenicity of oils is a frequent subject of controversy and the bibliography constantly produces contradictory examples. This can be explained by the variability of the processes used in industry, and by the conditions under which proteins are extracted in the laboratory.

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[172] - Hidalgo FJ, Alaiz M, Zamora R. Determination of peptides and proteins in fats and oils. Anal Chem 2001;73:698-702

A method for the determination of proteins in fats and oils is described. Proteins were sequentially precipitated with acetone and hydrolyzed, and the produced amino acids were fractionated and quantificated. This analysis protocol afforded a method of high sensitivity and specificity which was fully evaluated and validated. The data obtained showed good accuracy and linearity with excellent reproducibility and recovery. When the method was applied to 40 olive oils, all of them contained proteins in the range 10-50 microg/100 g of oil, suggesting that proteins are nonpreviously described minor components of these oils. In addition, the proteins precipitated were almost exclusively composed by one polypeptide of apparent 4600 molecular weight, which was isolated from olive drupes and partially characterized by amino acid analysis. Similar polypeptides were also detected in other seeds, suggesting that they may constitute a new class of polypeptides in plants with oleosin-like characteristics. Furthermore, the method was also applied to different fats and oils, and all the samples analyzed contained proteins, suggesting that natural fats and oils always contain polypeptides and/or proteins as minor components. These results also suggest that some peptides are soluble in lipid matrixes, where they might be playing unknown functions. The developed procedure provides a methodology for the determination of these components.

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[173] - Hidalgo FJ, Zamora R. Peptides and proteins in edible oils: Stability, allergenicity, and new processing trends. Trends Food Sci Technol 2006;17:56-63

Oilseeds are rich in bioactive compounds. However, a major portion of these components is not present in the refined oils because either they are not extracted or they are lost during refining. Nowadays, new processing trends are being explored to increase the nutritional value of edible oils. These procedures are increasing the contents of bioactive compounds, but they are also modifying the contents of other minor components. Among them, peptides and proteins are usually not considered. This review discusses the different types of peptides and proteins characterized in edible oils, the standardized and validated methodology existing for their determination, and the advantages and risks of increasing their contents in the edible oils in relation to oil stability and allergenicity.

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[174] - Hidalgo FJ, Zamora R. Peptides and proteins in edible oils: Stability, allergenicity, and new processing trends. Trends Food Sci Technol 2006;17:56-63

Oilseeds are rich in bioactive compounds. However, a major portion of these components is not present in the refined oils because either they are not extracted or they are lost during refining. Nowadays, new processing trends are being explored to increase the nutritional value of edible oils. These procedures are increasing the contents of bioactive compounds, but they are also modifying the contents of other minor components. Among them, peptides and proteins are usually not considered. This review discusses the different types of peptides and proteins characterized in edible oils, the standardized and validated methodology existing for their determination, and the advantages and risks of increasing their contents in the edible oils in relation to oil stability and allergenicity.

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Les allergènes des Oléacées

Réactions croisées entre pollens d’Oléacées

Réactions croisées entre Oléacées et d’autres pollens

Réactions croisées entre pollens d’Oléacées et aliments végétaux

Pollens d’Oléacées et CCD

Olive et huile d’olive

Rubriques

Dans la même rubrique

Les Cupressacées

Le bouleau

Les Fagales

Les Pinacées

Le platane