Toll-like receptor 4 and Syk kinase shape dendritic cell-induced immune activation to major house dust mite allergens.

Background: House dust mite (HDM) is a major cause of respiratory allergic diseases. Dendritic cells (DCs) play a central role in orchestrating adaptive allergic immune responses. However, it remains unclear how DCs become activated by HDM. Biochemical functions of the major HDM allergens Der p 1 (cysteine protease) and Der p 2 (MD2-mimick) have been implicated to contribute to DC activation. Methods: We investigated the immune activating potential of HDM extract and its major allergens Der p 1 and Der p 2 using monocyte-derived DCs (moDCs). Maturation and activation markers were monitored by flow cytometry and cytokine production by ELISA. Allergen depletion and proteinase K digestion were used to investigate the involvement of proteins, and in particular of the major allergens. Inhibitors of spleen tyrosine kinase (Syk), Toll-like receptor 4 (TLR4) and of C-type lectin receptors (CLRs) were used to identify the involved receptors. The contribution of endotoxins in moDC activation was assessed by their removal from HDM extract. Results: HDM extract induced DC maturation and cytokine responses in contrast to the natural purified major allergens Der p 1 and Der p 2. Proteinase K digestion and removal of Der p 1 or Der p 2 did not alter the immune stimulatory capacity of HDM extract. Antibodies against the CLRs Dectin-1, Dectin-2, and DC-SIGN did not affect cytokine responses. In contrast, Syk inhibition partially reduced IL-6, IL-12 and completely blocked IL-10. Blocking TLR4 signaling reduced the HDM-induced IL-10 and IL-12p70 induction, but not IL-6, while endotoxin removal potently abolished the induced cytokine response. Conclusion: Our data strongly suggest that HDM-induced DC activation is neither dependent on Der p 1 nor Der p 2, but depend on Syk and TLR4 activation, which might suggest a crosstalk between Syk and TLR4 pathways. Our data highlight that endotoxins play a potent role in immune responses targeting HDM.

IgE cross-inhibition between Ara h 1 and Ara h 2 is explained by complex formation of both major peanut allergens.

BACKGROUND: Surprisingly, IgE cross-reactivity between the major peanut allergens Ara h 1, 2, and 3 has been reported despite very low sequence identities. OBJECTIVE: We investigated the unexpected cross-reactivity between peanut major allergens. METHODS: Cross-contamination of purified natural Ara h 1, 2, 3, and 6 was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot test, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and sandwich enzyme-linked immunosorbent assay (ELISA). IgE cross-reactivity was studied with sera of peanut-allergic patients (n = 43) by ELISA and ImmunoCAP inhibition using both intact natural and recombinant allergens and synthetic peptides representing postulated Ara h 1 and Ara h 2 cross-reactive epitopes. RESULTS: Both purified nAra h 1 and nAra h 3 were demonstrated to contain small but significant amounts of Ara h 2 and Ara h 6 (<1%) by sandwich ELISA, SDS-PAGE/Western blot analysis, and LC-MS/MS. IgE cross-inhibition between both 2S albumins and Ara h 1 and Ara h 3 was only observed when using natural purified allergens, not recombinant allergens or synthetic peptides. Apparent cross-reactivity was lost when purified nAra h 1 was pretreated under reducing conditions, suggesting that Ara h 2 and Ara h 6 contaminations may be covalently bound to Ara h 1 via disulfide interactions. CONCLUSION: True cross-reactivity of both peanut 2S albumins with Ara h 1 and Ara h 3 could not be demonstrated. Instead, cross-contamination with small quantities was shown to be sufficient to cause significant cross-inhibition that can be misinterpreted as molecular cross-reactivity. Diagnostic tests using purified nAra h 1 and nAra h 3 can overestimate their importance as major allergens as a result of the presence of contaminating 2S albumins, making recombinant Ara h 1 and Ara h 3 a preferred alternative.

Peach allergen Pru p 1 content is generally low in fruit but with large variation in different varieties.

BACKGROUND: Pru p 1 is a major allergen in peach and nectarine, and the different content in varieties may affect the degree of allergic reactions. This study aimed to quantify Pru p 1 levels in representative peach varieties and select hypoallergenic Pru p 1 varieties. METHODS: To obtain monoclonal and polyclonal antibodies, mice and rabbits, respectively, were immunized with recombinant Pru p 1.01 and Pru p 1.02. The Pru p 1 levels in fruits from 83 representative peach varieties was quantified by sandwich enzyme-linked immunosorbent assay (sELISA). nPru p 1 was obtained through specific monoclonal antibody affinity purification and confirmed by Western blot and mass spectrometry. The variable Pru p 1 content of selected varieties was evaluated by Western blot and the expression level of encoding Pru p 1 genes by quantitative polymerase chain reaction. RESULTS: A sELISA method with monoclonal and polyclonal antibodies was built for quantifying Pru p 1 levels in peach. Pru p 1 was mainly concentrated in the peel (0.20-73.44 µg/g, fresh weight), being very low in the pulp (0.05-9.62 µg/g) and not detected in wild peach. For the 78 peach and nectarine varieties, Pru p 1 content varied widely from 0.12 to 6.45 µg/g in whole fruit. We verified that natural Pru p 1 is composed of 1.01 and 1.02 isoallergens, and the Pru p 1 expression level and Pru p 1 band intensity in the immunoblots were in agreement with protein quantity determined by ELISA for some tested varieties. In some cases, the reduced levels of Pru p 1 did not coincide with low Pru p 3 in the same variety in whole fruit, while some ancient wild peach and nectarines contained low levels of both allergens, and late-ripening yellow flesh varieties were usually highly allergenic. CONCLUSION: Pru p 1 content is generally low in peach compared to Pru p 3. Several hypoallergenic Pru p 1 and Pru p 3 varieties, "Zi Xue Tao," "Wu Yue Xian," and "May Fire," were identified, which could be useful in trials for peach allergy patients.

Variation in IgE binding potencies of seven Artemisia species depending on content of major allergens.

BACKGROUND: Artemisia weed pollen allergy is important in the northern hemisphere. While over 350 species of this genus have been recorded, there has been no full investigation into whether different species may affect the allergen diagnosis and treatment. This study aimed to evaluate the variations in amino acid sequences and the content of major allergens, and how these affect specific IgE binding capacity in representative Artemisia species. METHODS: Six representative Artemisia species from China and Artemisia vulgaris from Europe were used to determine allergen amino acid sequences by transcriptome, gene sequencing and mass spectrometry of the purified allergen component proteins. Sandwich ELISAs were developed and applied for Art v 1, Art v 2 and Art v 3 allergen quantification in different species. Aqueous pollen extracts and purified allergen components were used to assess IgE binding by ELISA and ImmunoCAP with mugwort allergic patient serum pools and individual sera from five areas in China. RESULTS: The Art v 1 and Art v 2 homologous allergen sequences in the seven Artemisia species were highly conserved. Art v 3 type allergens in A. annua and A. sieversiana were more divergent compared to A. argyi and A. vulgaris. The allergen content of Art v 1 group in the seven extracts ranged from 3.4% to 7.1%, that of Art v 2 from 1.0% to 3.6%, and Art v 3 from 0.3% to 10.5%. The highest IgE binding potency for most Chinese Artemisia allergy patients was with A. annua pollen extract, followed by A. vulgaris and A. argyi, with A. sieversiana significantly lower. Natural Art v 1-3 isoallergens from different species have almost equivalent IgE binding capacity in Artemisia allergic patients from China. CONCLUSION AND CLINICAL RELEVANCE: There was high sequence similarity but different content of the three group allergens from different Artemisia species. Choice of Artemisia annua and A. argyi pollen source for diagnosis and immunotherapy is recommended in China.

Protease resistance of food proteins: a mixed picture for predicting allergenicity but a useful tool for assessing exposure.

BACKGROUND: Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. AIM: To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. METHODS: Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2-2.5-4.0) and pepsin-to-protein ratio (PPR: 10-1-0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. RESULTS: At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. CONCLUSIONS: Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair.

Abnormal Responses to Local Esophageal Food Allergen Injections in Adult Patients With Eosinophilic Esophagitis.

Skin tests and measurement of serum levels of immunoglobulin E do not accurately identify foods for elimination from the diets of patients with eosinophilic esophagitis (EoE). We investigated whether an esophageal prick test, in which the esophageal mucosa is challenged by local injection of allergen extracts, could identify individuals with esophageal sensitization. During endoscopy, 6 allergens were injected in the esophagus of 8 patients with EoE and 3 patients without EoE (controls). A second endoscopy was performed after 24 hours to evaluate delayed responses. Five of the 8 patients with EoE had evidence for an acute response (luminal obstruction and mucosal blanching); 2 other patients had a delayed wheal or flare reaction. No responses were observed in controls. We conclude that esophageal mucosal food allergen injections induce acute and/or delayed responses in patients with EoE but not controls. The esophageal prick test deserves further exploration because it may guide elimination diets.

Development of a strategy for the total chemical synthesis of an allergenic protein: the peach LTP Pru p 3.

The possibility to obtain allergenic proteins by means of total chemical synthesis would be a big step forward in the development of cures to food allergy and in the study of the mechanism of allergic reactions, because this would allow to achieve control at the molecular level over the structure of the product and to study its relationship with the allergenic activity in fine details. This is instead not possible by using allergens produced by extraction from natural sources or by recombinant DNA techniques. In this work, we aimed to test for the first time the feasibility of the total chemical synthesis of an allergenic protein. Pru p 3, the most studied member of the family of lipid transfer proteins, relevant plant food pan-allergens, was used as model target. Strategies for the convergent assembly of the target protein, starting from five peptide fragments to be bound by means of either native chemical ligation or peptide hydrazide ligation, followed by desulfurization, to achieve ligations at alanine, were developed and tested. All the reaction conditions were set up and optimized. Two large peptides covering the two halves of the protein sequence were synthesized and structurally characterized by means of circular dichroism, and their immunogenicity was proved by means of immunoblot, using antibodies against Pru p 3, and immunoCAP inhibition tests. Finally, the five peptides were bound together to produce the whole protein stretch. The obtained results demonstrate the feasibility of total chemical synthesis as a new way to obtain pure allergens. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

IgE-binding potencies of three peach Pru p 1 isoforms.

SCOPE: Pru p 1, the Bet v 1 homologue from peach, has been identified as a clinically relevant allergen. Three isoforms have been described, two in peach fruit (Pru p 1.0101 and Pru p 1.0201) and one in pollen (Pru p 1.0301). The present study aimed to compare their IgE-binding potencies. METHODS AND RESULTS: Three Pru p 1 isoforms were cloned and expressed as soluble proteins with His-tags in Escherichia coli. Protein identity was confirmed by MS, circular dichroism, and RNAse activity. IgE-binding capacity using ELISA and ImmunoCAP was compared. Three Pru p 1 isoforms had quite similar IgE-binding potencies for 60% of the sera, but more than twofold between any two isoforms among 40% of the 47 sera. The mean IgE binding of Pru p 1.0201 was slightly higher than other two isoforms. In a sera pool, homologous ImmunoCAP inhibition was higher than other two heterologous isoforms. Individual serum with diverse IgE values of three isoforms demonstrated the higher IgE inhibition of specific isoform with higher IgE value. CONCLUSION: A similar and variable pattern of IgE recognition was observed among three Pru p 1 isoforms. The two new isoforms can be used as more accurate diagnostic reagents.

Development of a hypoallergenic recombinant parvalbumin for first-in-man subcutaneous immunotherapy of fish allergy.

BACKGROUND: The FAST (food allergy-specific immunotherapy) project aims at developing safe and effective subcutaneous immunotherapy for fish allergy, using recombinant hypoallergenic carp parvalbumin, Cyp c 1. OBJECTIVES: Preclinical characterization and good manufacturing practice (GMP) production of mutant Cyp (mCyp) c 1. METHODS: Escherichia coli-produced mCyp c 1 was purified using standard chromatographic techniques. Physicochemical properties were investigated by gel electrophoresis, size exclusion chromatography, circular dichroism spectroscopy, reverse-phase high-performance liquid chromatography and mass spectrometry. Allergenicity was assessed by ImmunoCAP inhibition and basophil histamine release assay, immunogenicity by immunization of laboratory animals and stimulation of patients' peripheral blood mononuclear cells (PBMCs). Reference molecules were purified wild-type Cyp c 1 (natural and/or recombinant). GMP-compliant alum-adsorbed mCyp c 1 was tested for acute toxicity in mice and rabbits and for repeated-dose toxicity in mice. Accelerated and real-time protocols were used to evaluate stability of mCyp c 1 as drug substance and drug product. RESULTS: Purified mCyp c 1 behaves as a folded and stable molecule. Using sera of 26 double-blind placebo-controlled food-challenge-proven fish-allergic patients, reduction in allergenic activity ranged from 10- to 5,000-fold (1,000-fold on average), but with retained immunogenicity (immunization in mice/rabbits) and potency to stimulate human PBMCs. Toxicity studies revealed no toxic effects and real-time stability studies on the Al(OH)3-adsorbed drug product demonstrated at least 20 months of stability. CONCLUSION: The GMP drug product developed for treatment of fish allergy has the characteristics targeted for in FAST: i.e. hypoallergenicity with retained immunogenicity. These results have warranted first-in-man immunotherapy studies to evaluate the safety of this innovative vaccine.

Oil body-associated hazelnut allergens including oleosins are underrepresented in diagnostic extracts but associated with severe symptoms.

BACKGROUND: Oil body-associated allergens such as oleosins have been reported for important allergenic foods such as peanut, sesame and hazelnut. Here we investigate whether oil body associated proteins (OAPs) are linked with specific clinical phenotypes and whether they are represented in skin prick test (SPT) reagents. METHODS: A hazelnut OAP fraction was characterized by mass-spectrometry (MS) to identify its major constituents. Polyclonal rabbit antibodies were generated against hazelnut OAPs. The presence of OAPs in commercially available hazelnut SPTs was studied by immunoblot and spiking experiments. OAP-specific IgE antibodies were measured in sera from patients with a convincing history of hazelnut allergy by RAST (n = 91), immunoblot (n = 22) and basophil histamine release (BHR; n = 14). RESULTS: Hazelnut OAPs were analysed by MS and found to be dominated by oleosins at ~14 and ~17 kDa, and a 27 kDa band containing oleosin dimers and unidentified protein. In 36/91 sera specific IgE against hazelnut OAPs was detected, and confirmed to be biologically active by BHR (n = 14). The majority (21/22) recognized the oleosin bands at 17 kDa on immunoblot, of which 11 exclusively. These OAP-specific IgE responses dominated by oleosin were associated with systemic reactions to hazelnut (OR 4.24; p = 0.015) and negative SPT (χ2 6.3, p = 0.012). Immunoblot analysis using OAP-specific rabbit antiserum demonstrated that commercial SPT reagents are virtually devoid of OAPs, sometimes (3/9) resulting in false-negative SPT. Spiking of SPT reagents with OAP restored serum IgE binding of these false-negative patients on immunoblot at mainly 17 kDa. CONCLUSION: Hazelnut allergens found in oil bodies dominated by oleosin are associated with more severe systemic reactions and negative SPT. Defatted diagnostic extracts are virtually devoid of these allergens, resulting in poor sensitivity for detection of IgE antibodies against these clinically relevant molecules.

Peanut-specific IgE antibodies in asymptomatic Ghanaian children possibly caused by carbohydrate determinant cross-reactivity.

BACKGROUND: The prevalence of peanut allergy has increased in developed countries, but little is known about developing countries with high peanut consumption and widespread parasitic infections. OBJECTIVE: We sought to investigate peanut allergy in Ghana. METHODS: In a cross-sectional survey among Ghanaian schoolchildren (n = 1604), data were collected on reported adverse reactions to peanut, peanut sensitization (serum specific IgE and skin reactivity), consumption patterns, and parasitic infections. In a subset (n = 43) IgE against Ara h 1, 2, 3, and 9 as well as cross-reactive carbohydrate determinants (CCDs) was measured by using ImmunoCAP. Cross-reactivity and biological activity were investigated by means of ImmunoCAP inhibition and basophil histamine release, respectively. RESULTS: Adverse reactions to peanut were reported in 1.5%, skin prick test reactivity in 2.0%, and IgE sensitization (≥0.35 kU/L) in 17.5% of participants. Moreover, 92.4% of those IgE sensitized to peanut (≥0.35 kU/L) had negative peanut skin prick test responses. Schistosoma haematobium infection was positively associated with IgE sensitization (adjusted odds ratio, 2.29; 95% CI, 1.37-3.86). In the subset IgE titers to Ara h 1, 2, 3, and 9 were low (<1.3 kU/L), except for 6 moderately strong reactions to Ara h 9. IgE against peanut was strongly correlated with IgE against CCDs (r = 0.89, P < .0001) and could be almost completely inhibited by CCDs, as well as S haematobium soluble egg antigen. Moreover, IgE to peanut showed poor biological activity. CONCLUSIONS: Parasite-induced IgE against CCDs might account largely for high IgE levels to peanut in our study population of Ghanaian schoolchildren. No evidence of IgE-mediated peanut allergy was found.

Sensitization to Cor a 9 and Cor a 14 is highly specific for a hazelnut allergy with objective symptoms in Dutch children and adults.

BACKGROUND: Component-resolved diagnosis has been shown to improve the diagnosis of food allergy. OBJECTIVE: We sought to evaluate whether component-resolved diagnosis might help to identify patients at risk of objective allergic reactions to hazelnut. METHOD: A total of 161 hazelnut-sensitized patients were included: 40 children and 15 adults with objective symptoms on double-blind, placebo-controlled food challenges (DBPCFCs) and 24 adults with a convincing objective history were compared with 41 children and 41 adults with no or subjective symptoms on DBPCFCs (grouped together). IgE levels to hazelnut extract and single components were analyzed with ImmunoCAP. RESULTS: IgE levels to hazelnut extract were significantly higher in children with objective than with no or subjective symptoms. In 13% of children and 49% of adults with hazelnut allergy with objective symptoms, only sensitization to rCor a 1.04 was observed and not to other water-soluble allergens. Sensitization to rCor a 8 was rare, which is in contrast to rCor a 1. Sensitization to nCor a 9, rCor a 14, or both was strongly associated with hazelnut allergy with objective symptoms. By using adapted cutoff levels, a diagnostic discrimination between severity groups was obtained. IgE levels to either nCor a 9 of 1 kUA/L or greater or rCor a 14 of 5 kUA/L or greater (children) and IgE levels to either nCor a 9 of 1 kUA/L or greater or rCor a 14 of 1 kUA/L or greater (adults) had a specificity of greater than 90% and accounted for 83% of children and 44% of adults with hazelnut allergy with objective symptoms. CONCLUSION: Sensitization to Cor a 9 and Cor a 14 is highly specific for patients with objective symptoms in DBPCFCs as a marker for a more severe hazelnut allergic phenotype.

The purification and characterisation of allergenic hazelnut seed proteins.

A lipid transfer protein (LTP, Cor a 8) together with the 11S (Cor a 9) and 7S seed storage globulins (Cor a 11) are major food allergens present in hazelnut. Methods are described for their purification and characterisation using in-gel tryptic digestion mass spectrometry to confirm their identities and circular dichroism and Fourier-transform infrared spectroscopies to demonstrate that they are authentically folded. Preliminary immunochemical studies have also confirmed that the purified preparations retain their immunological properties in terms of immunoglobulin E binding, determined by immunoblotting using serum from hazelnut allergic patients. These preparations form a basis for development of improved methods of diagnosis of food allergy based on the concept of component-resolved diagnosis.

Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis.

Peanut is a major cause of type 1 hypersensitive reactions including anaphylaxis. This results from the presence of a number of protein allergens, six of which are being studied as part of the EU FP6 EuroPrevall programme. These are Ara h 1 (7S globulin), Ara h 2, Ara h 6 (2S albumins), Ara h 3/4 (11S globulins) and Ara h 8 (Bet v 1 homologue). Methods for the purification of Ara h 1, Ara h 3/4, Ara h 2 and Ara h 6 from peanut seeds and for the production of recombinant Ara h 8 in Escherichia coli are described with spectroscopic analyses being used to confirm that they are authentically folded. N-terminal sequencing of the proteins purified from peanut seeds also revealed details of the differences between isoforms and their generation by proteolytic processing within the seed. Preliminary IgE binding studies of the purified allergens confirmed that they retained their immunological properties indicating their suitability for use in allergy diagnosis.

Hazelnut allergy: from pollen-associated mild allergy to severe anaphylactic reactions.

PURPOSE OF REVIEW: Hazelnut allergy can vary between mild oral symptoms and potentially dangerous anaphylaxis. There is a need to predict which subjects are at risk for severe reactions. In this study, possibilities for 'component-resolved diagnosis', based on sensitization to different allergens in hazelnut, are discussed. RECENT FINDINGS: One type of hazelnut allergy can be associated with sensitization to homologues of pollen allergens, predominantly birch, in hazelnut: Cor a 1 (Bet v 1) and Cor a 2 (profilin). These allergens account for relatively mild symptoms. However, subjects can also be sensitized to several other allergens in hazelnut that are related to more severe symptoms. These allergens are homologues of allergens in other nuts and peanut: Cor a 8 (lipid transfer protein) and Cor a 9 (11S globulin) and perhaps Cor a 11 (7S globulin). The clinical relevance of these and other potential hazelnut allergens has to be further defined. The diagnosis of hazelnut has to be confirmed by oral double-blind placebo-controlled food challenge. SUMMARY: Sensitization to hazelnut can either be associated with mild oral symptoms, depending on sensitization to pollen, or with more serious allergic symptoms, related to sensitization to homologues of nut and peanut allergens.

Lipid transfer protein-linked hazelnut allergy in children from a non-Mediterranean birch-endemic area.

BACKGROUND: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8. OBJECTIVE: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area. METHODS: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed. RESULTS: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1. CONCLUSION: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.

Optimized techniques for the extraction of grape allergens appropriate for in vivo and in vitro testing and diagnosis.

Standardized allergen extracts are needed for diagnosis and therapy purposes. For grapes, standardization is hampered by low protein and high tannin and pectin concentrations. The aim of the current study was to develop an optimized method for the extraction of grape proteins and possibly extend this to other fruits. Several existing or modified extraction methods were compared by means of protein concentration determination, SDS-PAGE, immunoblotting and radioallergosorbent test (RAST). An optimized extraction protocol was obtained in which we combined a high concentration of plant tissue, a concentrated, enriched and neutral buffer able to remove sugars and keep proteins soluble and a bivalent buffer for pectin removal. Both the quantitative (protein concentration) and qualitative parameters (SDS-PAGE protein patterns and IgE reactivity) were compared to standard protocols and commercial extracts used as diagnostic tools in the clinical practice. This method proved to be the most efficient mainly compared to the standard Björksten protocol in extracting the low molecular weight proteins, including the major grape allergen (lipid transfer protein, Vit v 1). It proved to be an easy, low cost and reproducible method proposed to prepare grape extracts that could replace the commercially available ones, used for diagnosis and possibly extend the method to other fruits especially in extracting LTPs.