Characterization of a 7 kDa pollen allergen belonging to the gibberellin-regulated protein family from three Cupressaceae species.

BACKGROUND: Severe allergy to fruits mediated by a 7 kDa allergen belonging to the gibberellin-regulated protein (GRP) family is known to be associated with Cupressaceae pollinosis. OBJECTIVE: To identify and characterize Cupressaceae pollen allergens involved in GRP-related fruit allergy. METHODS: Pru p 7-related proteins from pollen of Cupressus sempervirens, Juniperus ashei and Cryptomeria japonica were identified using a rabbit anti-Pru p 7 antiserum, purified chromatographically and sequenced by mass spectrometry and bioinformatic comparisons. The C sempervirens protein was produced as a recombinant allergen in Pichia pastoris. IgE antibody binding to pollen GRP proteins was analysed in a peach allergic (n = 54) and a cypress pollen allergic (n = 88) patient population from southern France using ImmunoCAP. RESULTS: In each of the three Cupressaceae species studied, a 7 kDa pollen protein related to Pru p 7 was identified and found to comprise an amino acid sequence of 63 residues in length, 92%-98% identical to each other and 67%-68% identical to Pru p 7. The C sempervirens, J ashei and C japonica GRP allergens have been officially recognized by the WHO/IUIS Allergen Nomenclature Sub-Committee and named Cup s 7, Jun a 7 and Cry j 7, respectively. Recombinant Cup s 7 showed IgE antibody binding capacity comparable to that of the purified natural allergen. Among 51 peach allergic subjects sensitized to Pru p 7, substantially higher levels of IgE to Cup s 7 than to Pru p 7 were found. Further, the pollen protein was able to completely outcompete IgE binding to Pru p 7, while the reverse competition effect was modest, consistent with primary sensitization by the pollen allergen. CONCLUSION AND CLINICAL RELEVANCE: Pru p 7-related pollen allergens from three Cupressaceae species have been characterized and may become useful for the identification of pollinosis patients at risk of developing severe fruit allergy.

Allergen Recognition Patterns in Walnut Allergy Are Age Dependent and Correlate with the Severity of Allergic Reactions.

BACKGROUND: Walnut is an important elicitor of food allergy in children and adults with a high rate of severe reactions. Multicenter studies using a common clinical protocol and a comprehensive allergen are lacking. OBJECTIVE: To investigate potential correlations between molecular sensitization patterns and clinical characteristics of walnut-allergic patients. METHODS: A total of 91 walnut-allergic subjects and 24 tolerant controls from Switzerland, Germany, and Spain were included. Walnut allergy was established by food challenge in all but anaphylactic subjects. Specific IgE (sIgE) to walnut extract, rJug r 1 (2S albumin), rJug r 3 (nonspecific lipid transfer protein 1), nJug r 4 (11S globulin), rJug r 5 (PR-10 protein), 2 vicilin fractions, profiling, and cross-reactive carbohydrate determinant was determined by ImmunoCAP. A threshold of 0.10 kUA/L was used for positivity. RESULTS: Sensitivity of sIgE to walnut extract was 87% and increased to 96% for the sum of all walnut components. sIgE to walnut extract and all walnut components, except rJug r 5, was significantly higher in patients younger than 14 years at inclusion. Stratification by age at onset of walnut allergy led to similar results. All patients younger than 14 years had severe reactions, whereas 38% of patients 14 years or older were mild reactors. Severe reactors (n = 70) had higher sIgE levels than did mild reactors (n = 21) to walnut extract (P < .0001), rJug r 1 (P < .0001), nJug r 4 (P = .0003), and both vicilin fractions (P < .0001), but not to Jug r 3 and Jug r 5. CONCLUSIONS: Sensitization to walnut storage proteins is acquired in childhood and correlates with severe reactions. sIgE levels to storage proteins Jug r 1 and Jug r 4 and vicilin fractions, but not to nonspecific lipid transfer protein and PR-10 proteins, correlate with systemic reactions to walnut.

Pru p 7 sensitization is a predominant cause of severe, cypress pollen-associated peach allergy.

BACKGROUND: Peach is a common elicitor of food allergic reactions. Peach-induced immediate reactions may occur as benign pollen-food syndromes, usually due to birch pollen-related PR-10 cross-reactivity in temperate climates, and as potentially severe primary food allergies, predominantly related to nsLTP Pru p 3 in Mediterranean regions. The newly described peach allergen Pru p 7 has gained recent attention as a potential peach allergy severity marker. Sensitization to Pru p 7 and its allergenic homologues of the gibberellin-regulated protein family occurs in areas with high Cupressaceae tree pollen exposure. OBJECTIVE: We sought to investigate the distribution, clinical characteristics and molecular associations of Pru p 7 sensitization among subjects with suspected peach allergy in different regions of France. METHODS: Subjects with suspected peach allergy (n = 316) were included. Diagnostic work-up was performed according to current guidelines, including open food challenge when required. IgE antibody measurements and competition experiments were performed using the ImmunoCAP assay platform. RESULTS: Sensitization to Pru p 7 was present in 171 (54%) of all subjects in the study and in 123 of 198 (62%) diagnosed as peach allergic, more than half of whom were sensitized to no other peach allergen. Frequency and magnitude of Pru p 7 sensitization were associated with the presence of peach allergy, the clinical severity of peach-induced allergic reactions and the level of cypress pollen exposure. Cypress pollen extract completely outcompeted IgE binding to Pru p 7. Pru p 7 was extremely potent in basophil activation tests. CONCLUSION AND CLINICAL RELEVANCE: A subtype of Cupressaceae pollinosis, characterized by Pru p 7 sensitization, can be an underlying cause of severe peach allergy.

Identification and implication of an allergenic PR-10 protein from walnut in birch pollen associated walnut allergy.

SCOPE: English walnut (Juglans regia) belongs to the most important allergenic tree nuts. Co-sensitization with birch (Betula verrucosa) pollen has been reported. We aimed to identify a walnut allergen homologous to the major birch pollen allergen Bet v 1. METHODS AND RESULTS: A cDNA encoding a Bet v 1-homologous allergen (Jug r 5) in walnut kernels was cloned by RT-PCR. Jug r 5 was expressed in Escherichia coli, purified by column chromatography and characterized by circular dichroism spectroscopy. Specific IgE levels to walnut, Bet v 1, and Jug r 5 in birch pollen allergics (n = 16) with concomitant walnut allergy were measured by ImmunoCAP: 44% of the patients were tested positive to walnut while 94% were reactive to Jug r 5, and 100% to Bet v 1. Jug r 5 and Bet v 1 allergens showed bidirectional IgE cross-reactivity by competitive ELISA and were capable of inducing histamine release from effector cells. Immunoblot competition experiments demonstrated the presence of IgE-reactive Jug r 5 in walnut extract, but at low levels. CONCLUSION: A Bet v 1-like allergen was identified in walnut. Diagnostic use of Jug r 5 will compensate for the low sensitivity of walnut extract for patients with birch pollen associated walnut allergy.

Predominant Api m 10 sensitization as risk factor for treatment failure in honey bee venom immunotherapy.

BACKGROUND: Component resolution recently identified distinct sensitization profiles in honey bee venom (HBV) allergy, some of which were dominated by specific IgE to Api m 3 and/or Api m 10, which have been reported to be underrepresented in therapeutic HBV preparations. OBJECTIVE: We performed a retrospective analysis of component-resolved sensitization profiles in HBV-allergic patients and association with treatment outcome. METHODS: HBV-allergic patients who had undergone controlled honey bee sting challenge after at least 6 months of HBV immunotherapy (n = 115) were included and classified as responder (n = 79) or treatment failure (n = 36) on the basis of absence or presence of systemic allergic reactions upon sting challenge. IgE reactivity to a panel of HBV allergens was analyzed in sera obtained before immunotherapy and before sting challenge. RESULTS: No differences were observed between responders and nonresponders regarding levels of IgE sensitization to Api m 1, Api m 2, Api m 3, and Api m 5. In contrast, Api m 10 specific IgE was moderately but significantly increased in nonresponders. Predominant Api m 10 sensitization (>50% of specific IgE to HBV) was the best discriminator (specificity, 95%; sensitivity, 25%) with an odds ratio of 8.444 (2.127-33.53; P = .0013) for treatment failure. Some but not all therapeutic HBV preparations displayed a lack of Api m 10, whereas Api m 1 and Api m 3 immunoreactivity was comparable to that of crude HBV. In line with this, significant Api m 10 sIgG4 induction was observed only in those patients who were treated with HBV in which Api m 10 was detectable. CONCLUSIONS: Component-resolved sensitization profiles in HBV allergy suggest predominant IgE sensitization to Api m 10 as a risk factor for treatment failure in HBV immunotherapy.

Hazelnut allergy across Europe dissected molecularly: A EuroPrevall outpatient clinic survey.

BACKGROUND: Hazelnut allergy is birch pollen-driven in Northern/Western Europe and lipid transfer protein-driven in Spain and Italy. Little is known about other regions and other allergens. OBJECTIVE: Establishing a molecular map of hazelnut allergy across Europe. METHODS: In 12 European cities, subjects reporting reactions to hazelnut (n = 731) were evaluated and sensitization to 24 foods, 12 respiratory allergen sources, and latex was tested by using skin prick test and ImmunoCAP. A subset (124 of 731) underwent a double-blind placebo-controlled food challenge to hazelnut. Sera of 423 of 731 subjects were analyzed for IgE against 7 hazelnut allergens and cross-reactive carbohydrate determinants by ImmunoCAP. RESULTS: Hazelnut allergy was confirmed in 70% of those undergoing double-blind placebo-controlled food challenges. Birch pollen-driven hazelnut sensitization (Cor a 1) dominated in most cities, except in Reykjavik, Sofia, Athens, and Madrid, where reporting of hazelnut allergy was less frequent anyhow. In Athens, IgE against Cor a 8 dominated and strongly correlated with IgE against walnut, peach, and apple and against Chenopodium, plane tree, and mugwort pollen. Sensitization to seed storage proteins was observed in less than 10%, mainly in children, and correlated with IgE to nuts, seeds, and legumes. IgE to Cor a 12, observed in all cities (10% to 25%), correlated with IgE to nuts, seeds, and pollen. CONCLUSIONS: In adulthood, the importance of hazelnut sensitization to storage proteins, oleosin (Cor a 12), and Cor a 8 is diluted by the increased role of birch pollen cross-reactivity with Cor a 1. Cor a 8 sensitization in the Mediterranean is probably driven by diet in combination with pollen exposure. Hazelnut oleosin sensitization is prevalent across Europe; however, the clinical relevance remains to be established.

Oral exposure to Mal d 1 affects the immune response in patients with birch pollen allergy.

BACKGROUND: Antibodies and T cells specific for the major birch pollen allergen Bet v 1 cross-react with structurally related food allergens, such as Mal d 1 in apple. OBJECTIVE: We sought to evaluate the effects of oral uptake of Mal d 1 on the allergen-specific immune response in patients with birch pollen allergy. METHODS: Patients received 50 µg of rBet v 1 sublingually on 2 consecutive days outside of the birch pollen season. One year later, equal amounts of rMal d 1 were administered. Blood samples were collected before and after oral exposure, as well as before and after the intermediate birch pollen season. Allergen-specific IgE levels were determined by using ImmunoCAP. Proliferation of allergen-stimulated PBMCs was assessed, as well as the expression of IL-5, IL-13, IL-10, IFN-γ, and forkhead box protein 3 (Foxp3) in isolated T cells (real-time PCR). Allergen-specific T-cell lines were analyzed for epitope recognition. RESULTS: Orally administered Bet v 1 transiently reduced Bet v 1-specific serum IgE levels, as well as Bet v 1- and Mal d 1-induced T-cell proliferation, and enhanced the expression of IL-5, IL-10, and Foxp3. Orally applied Mal d 1 significantly decreased Bet v 1- and Mal d 1-specific IgE levels and induced IL-5 and IL-10 but no Foxp3 expression. In contrast to Bet v 1, Mal d 1 triggered IFN-γ production and T cells with a different epitope repertoire. Inhalation of birch pollen significantly enhanced allergen-specific IgE levels, T-cell proliferation, and IL-5, IL-10, IL-13, and Foxp3 expression. CONCLUSION: Two sublingual administrations of 50 µg of Mal d 1 were well tolerated and induced transient immune responses seen during peripheral tolerance development. Thus recombinant Mal d 1 might be suitable and relevant for sublingual treatment of birch pollen-related apple allergy.

Birch pollen-related food allergy: clinical aspects and the role of allergen-specific IgE and IgG4 antibodies.

BACKGROUND: Patients with birch pollen allergy often develop allergic reactions to plant foods. OBJECTIVE: To evaluate the prevalence, main symptoms, and triggers of birch pollen-related food allergy and the role of food-specific IgG(4) antibodies in food tolerance. METHODS: Food-induced symptoms were evaluated in 225 individuals with birch pollen allergy by using a standardized questionnaire. IgE and IgG(4) levels specific for the major birch pollen allergen Bet v 1 and birch profilin Bet v 2 and the Bet v 1 homologs in apple (Mal d 1) and hazelnut (Cor a 1) were quantified by ImmunoCAP. Mock-treated and IgG-depleted sera from patients tolerating hazelnuts in food challenges were compared for their inhibitory activity for binding of Cor a 1-IgE complexes to B cells. RESULTS: In total, 73% of the study population experienced food allergy, which was perennial in 86% of the affected individuals. The oral allergy syndrome was the main clinical manifestation. However, more than 58% of the patients also experienced food-induced rhinoconjunctivitis. Apples and hazelnuts were identified as the most frequent triggers. Food allergy correlated with IgE reactivity to Bet v 1 but not to Bet v 2. Mal d 1-specific and Cor a 1-specific IgG(4)/IgE ratios were significantly higher in food-tolerant individuals than individuals with food allergy. Sera from IgG(4)-positive food-tolerant patients possessed IgG-dependent IgE-inhibitory activity. CONCLUSION: Birch pollen-related food allergy is highly prevalent and often perennial. High food allergen-specific IgG(4)/IgE ratios seem associated with food tolerance, potentially because specific IgG(4) blocks IgE binding to food allergens. Thus, the presence of food allergen-specific IgG(4) antibodies is no diagnostic marker for birch pollen-related food allergy.

Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota).

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

Component-resolved diagnosis of kiwifruit allergy with purified natural and recombinant kiwifruit allergens.

BACKGROUND: Kiwifruit is one of the most common causes of food allergic reactions. Component-resolved diagnostics may enable significantly improved detection of sensitization to kiwifruit. OBJECTIVE: To evaluate the use of individual allergens for component-resolved in vitro diagnosis of kiwifruit allergy. METHODS: Thirty patients with a positive double-blind placebo-controlled food challenge to kiwifruit, 10 atopic subjects with negative open provocation to kiwifruit, and 5 nonatopic subjects were enrolled in the study. Specific IgE to 7 individual allergens (nAct d 1-5 and rAct d 8-9) and allergen extracts was measured by ImmunoCAP. RESULTS: The diagnostic sensitivities of the commercial extract and of the sum of single allergens were 17% and 77%, respectively, whereas diagnostic specificities were 100% and 30%. A combination of the kiwi allergens Act d 1, Act d 2, Act d 4, and Act d 5 gave a diagnostic sensitivity of 40%, whereas diagnostic specificity remained high (90%). Exclusion of the Bet v 1 homolog recombinant (r) Act d 8 and profilin rAct d 9 from this allergen panel reduced sensitivity to 50% but increased specificity to 40%. Kiwifruit-monosensitized patients reacted more frequently (P < .001) with Act d 1 than polysensitized patients, whereas the latter group reacted more frequently with rAct d 8 (P = .004). CONCLUSION: Use of single kiwifruit allergen ImmunoCAP increases the quantitative test performance and diagnostic sensitivity compared with the commercial extract. Bet v 1 homolog and profilin are important allergens in pollen-related kiwifruit allergy, whereas actinidin is important in monoallergy to kiwifruit, in which symptoms are often more severe.

Strong allergenicity of Pru av 3, the lipid transfer protein from cherry, is related to high stability against thermal processing and digestion.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) have been identified as major fruit allergens in patients from the Mediterranean area. Sensitization to nsLTPs is accompanied by severe reactions, possibly because of specific biophysical and biochemical properties of this allergen family. OBJECTIVE: To assess the protein stability and allergenic potency of nsLTP from fruits in comparison with birch pollen-related allergens from the same allergenic source. METHODS: Stability of natural and recombinant cherry allergens Pru av 3 (nsLTP), Pru av 1 (Bet v 1 homologue), and Pru av 4 (profilin) to pepsin digestion and to thermal processing and stability of allergens in skin prick test reagents was investigated by immunoblotting and/or circular dichroism spectroscopy. Moreover, allergenicity of processed and fresh fruits in regard to Pru av 1 and Pru av 3 was analyzed by histamine release assays. RESULTS: Lipid transfer proteins showed the highest resistance to digestion by pepsin (rPru av 3 > rPru av 1 > rPru av 4). Immunologically active Pru av 3 was detectable after 2 hours of digestion by pepsin, whereas IgE reactivity of Pru av 1 and Pru av 4 was abolished within less than 60 minutes. In contrast with Pru av 1, IgE reactivity to nsLTPs was not diminished in thermally processed fruits, and secondary structures of purified Pru av 3 were more resistant to heating. Moreover, nsLTPs were stable components in skin prick test reagents. Histamine release assays confirmed the strong allergenicity of nsLTPs, which was not affected by protease treatment or thermal processing of fruits. CONCLUSION: In contrast with birch pollen-related allergens, nsLTPs are highly stable to pepsin treatment and thermal processing and show higher allergenic potency. Therefore, nsLTPs have the potential to act as true food allergens, probably eliciting severe systemic reactions by reaching the intestinal mucosa in an intact and fully active form.

Identification of cross-reactive and genuine Parietaria judaica pollen allergens.

BACKGROUND: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. OBJECTIVE: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). METHODS: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n = 42; Scandinavia, n = 8; United States, n = 19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. RESULTS: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. CONCLUSION: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract.

Characteristics and immunobiology of grass pollen allergens.

Grass pollens are one of the most important airborne allergen sources worldwide. About 20 species from five subfamilies are considered to be the most frequent causes of grass pollen allergy, and the allergenic relationships among them closely follow their phylogenetic relationships. The allergic immune response to pollen of several grass species has been studied extensively over more than three decades. Eleven groups of allergens have been identified and described, in most cases from more than one species. The allergens range from 6 to 60 kD in apparent molecular weight and display a variety of physicochemical properties and structures. The most complete set of allergens has so far been isolated and cloned from Phleum pratense (timothy grass) pollen. Based on the prevalence of IgE antibody recognition among grass pollen-sensitized individuals, several allergens qualify as major, but members of two groups, groups 1 and 5, have been shown to dominate the immune response to grass pollen extract. Isoform variation has been detected in members of several of the allergen groups, which in some cases can be linked to observed genetic differences. N-linked glycosylation occurs in members of at least three groups. Carbohydrate- reactive IgE antibodies have been attributed to grass pollen sensitization and found to cross-react with glycan structures from other allergen sources, particularly vegetable foods. Another cause of extensive cross-reactivity are the group 12 allergens (profilins), which belong to a family of proteins highly conserved throughout the plant kingdom and present in all tissues. Members of eight allergen groups have been cloned and expressed as recombinant proteins capable of specific IgE binding. This development now allows diagnostic dissection of the immune response to grass pollen with potential benefits for specific immunotherapy.

Purification, structural and immunological characterization of a timothy grass (Phleum pratense) pollen allergen, Phl p 4, with cross-reactive potential.

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

Identification of an allergen related to Phl p 4, a major timothy grass pollen allergen, in pollens, vegetables, and fruits by immunogold electron microscopy.

Group 4 grass pollen allergens represent 60 kDa glycoproteins recognized by 70% of patients sensitive to these pollens. An antiserum against purified Phl p 4 from timothy grass pollen was used to investigate various pollens, fruits, and vegetables for Phl p 4-related allergens by immunogold electron microscopy. In timothy grass, mugwort, and birch pollens, allergens were located in the wall, and in timothy grass and birch pollens additionally in the cytoplasm. In peanut, apple, celery root, and carrot root, only cytoplasmic areas were labeled. Group 4-related allergens thus occur in pollens of unrelated plants and in plant food and may therefore contribute to crossreactivities in patients allergic to various pollens and plant food.