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This common statement of the American Academy of Allergy, Asthma and Immunology (AAAAI) and The European Academy of Allergy and Clinical Immunology (EAACI) provides an update of the 2012 published guidelines on food challenges. The guidelines equally address food challenges in the research and the clinical settings. They first address the diagnostic tests which can guide the decision to conduct a challenge. Safety of food challenges is prime, and the various procedures and safety issues as well as medications potentially involved in challenges are extensively discussed. Challenges are suggested to be conducted with semi-logarithmic incremental doses based on the protein content, typically for IgE-mediated food allergy with intervals of 20-30 min between doses. Specific protocols for other types of reactions such atopic dermatitis or gastrointestinal food allergy are detailed separately. Proper stopping criteria are essential in order to reduce the risk of false-positive diagnoses, but also severe reactions. The guidelines recommend criteria based on "go on," "stop," or "observation." These revised guidelines will clearly provide much needed guidance for food challenges in the research and clinical settings. They will continue to evolve with new diagnostic tests or new needs in the field of food allergy., (© 2024 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Quantitative risk assessment (QRA) for food allergens has made considerable progress in recent years, yet acceptability of its outcomes remains stymied because of the limited extent to which it has been possible to incorporate severity as a variable. Reaction severity, particularly following accidental exposure, depends on multiple factors, related to the allergen, the host and any treatments, which might be administered. Some of these factors are plausibly still unknown. Quantitative risk assessment shows that limiting exposure through control of dose reduces the rates of reactions in allergic populations, but its impact on the relative frequency of severe reactions at different doses is unclear. Food challenge studies suggest that the relationship between dose of allergenic food and reaction severity is complex even under relatively controlled conditions. Because of these complexities, epidemiological studies provide very limited insight into this aspect of the dose-response relationship. Emerging data from single-dose challenges suggest that graded food challenges may overestimate the rate of severe reactions. It may be necessary to generate new data (such as those from single-dose challenges) to reliably identify the effect of dose on severity for use in QRA. Success will reduce uncertainty in the susceptible population and improve consumer choice., (© 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either β-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: The conduct of oral food challenges as the preferred diagnostic standard for food allergy (FA) was harmonized over the last years. However, documentation and interpretation of challenge results, particularly in research settings, are not sufficiently standardized to allow valid comparisons between studies. Our aim was to develop a diagnostic toolbox to capture and report clinical observations in double-blind placebo-controlled food challenges (DBPCFC)., Methods: A group of experienced allergists, paediatricians, dieticians, epidemiologists and data managers developed generic case report forms and standard operating procedures for DBPCFCs and piloted them in three clinical centres. The follow-up of the EuroPrevall/iFAAM birth cohort and other iFAAM work packages applied these methods., Recommendations: A set of newly developed questionnaire or interview items capture the history of FA. Together with sensitization status, this forms the basis for the decision to perform a DBPCFC, following a standardized decision algorithm. A generic form including details about severity and timing captures signs and symptoms observed during or after the procedures. In contrast to the commonly used dichotomous outcome FA vs no FA, the allergy status is interpreted in multiple categories to reflect the complexity of clinical decision-making., Conclusion: The proposed toolbox sets a standard for improved documentation and harmonized interpretation of DBPCFCs. By a detailed documentation and common terminology for communicating outcomes, these tools hope to reduce the influence of subjective judgment of supervising physicians. All forms are publicly available for further evolution and free use in clinical and research settings., (© 2016 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Background: In contrast to other Bet v 1-related food allergens, the major carrot allergen, Dau c 1, has been suggested to induce food allergy independently from Bet v 1. As T cells are crucial in the sensitization process, we sought to characterize the T-cell response to Dau c 1 and its cross-reactivity with Bet v 1., Methods: Dau c 1-specific T-cell lines (TCL) and clones (TCC) established from PBMC of birch pollen-allergic patients with carrot allergy were analyzed for reactivity to Bet v 1, epitope specificity, allergen-induced cytokine secretion, and expression of integrins α4β7 and α4β1, critical for gut and lung homing, respectively. mRNA expression of GATA3 and Tbet was analyzed in sorted CD3 + CD4 + CFSE low cells proliferating upon stimulation of PBMC with Dau c 1 or Bet v 1. Dau c 1 was incubated with endolysosomal proteases, and the resulting fragments were identified by mass spectrometry., Results: Among 14 distinct T-cell-activating regions, Dau c 1 139-153 was recognized by 55% of the patients. Only 6 of 15 (40%) Dau c 1-specific TCL and 9 of 21 (43%) TCC reacted with Bet v 1. Bet v 1-nonreactive TCC were mainly Th1-like and showed a higher expression of the integrin β7 and a significantly lower expression of the integrin β1 than Bet v 1-positive TCC. A Th1-like response was also detected in Dau c 1-reactive CD3 + CD4 + CFSE low cells. Full-length Dau c 1 was still detectable after 48 h of endolysosomal degradation. Proteolytic fragments of Dau c 1 matched its T-cell-activating regions., Conclusion: Dau c 1 displays several characteristics of sensitizing allergens, namely a major T-cell-activating region, low susceptibility to endolysosomal degradation, and induction of a Bet v 1-independent T-cell response. These cellular insights confirm that the major carrot allergen has a special status among Bet v 1-related food allergens., (© 2016 The Authors. Allergy Published by John Wiley & Sons Ltd.)

Anaphylaxis has been defined as a 'severe, life-threatening generalized or systemic hypersensitivity reaction'. However, data indicate that the vast majority of food-triggered anaphylactic reactions are not life-threatening. Nonetheless, severe life-threatening reactions do occur and are unpredictable. We discuss the concepts surrounding perceptions of severe, life-threatening allergic reactions to food by different stakeholders, with particular reference to the inclusion of clinical severity as a factor in allergy and allergen risk management. We review the evidence regarding factors that might be used to identify those at most risk of severe allergic reactions to food, and the consequences of misinformation in this regard. For example, a significant proportion of food-allergic children also have asthma, yet almost none will experience a fatal food-allergic reaction; asthma is not, in itself, a strong predictor for fatal anaphylaxis. The relationship between dose of allergen exposure and symptom severity is unclear. While dose appears to be a risk factor in at least a subgroup of patients, studies report that individuals with prior anaphylaxis do not have a lower eliciting dose than those reporting previous mild reactions. It is therefore important to consider severity and sensitivity as separate factors, as a highly sensitive individual will not necessarily experience severe symptoms during an allergic reaction. We identify the knowledge gaps that need to be addressed to improve our ability to better identify those most at risk of severe food-induced allergic reactions., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The availability of allergen molecules ('components') from several protein families has advanced our understanding of immunoglobulin E (IgE)-mediated responses and enabled 'component-resolved diagnosis' (CRD). The European Academy of Allergy and Clinical Immunology (EAACI) Molecular Allergology User's Guide (MAUG) provides comprehensive information on important allergens and describes the diagnostic options using CRD. Part A of the EAACI MAUG introduces allergen molecules, families, composition of extracts, databases, and diagnostic IgE, skin, and basophil tests. Singleplex and multiplex IgE assays with components improve both sensitivity for low-abundance allergens and analytical specificity; IgE to individual allergens can yield information on clinical risks and distinguish cross-reactivity from true primary sensitization. Part B discusses the clinical and molecular aspects of IgE-mediated allergies to foods (including nuts, seeds, legumes, fruits, vegetables, cereal grains, milk, egg, meat, fish, and shellfish), inhalants (pollen, mold spores, mites, and animal dander), and Hymenoptera venom. Diagnostic algorithms and short case histories provide useful information for the clinical workup of allergic individuals targeted for CRD. Part C covers protein families containing ubiquitous, highly cross-reactive panallergens from plant (lipid transfer proteins, polcalcins, PR-10, profilins) and animal sources (lipocalins, parvalbumins, serum albumins, tropomyosins) and explains their diagnostic and clinical utility. Part D lists 100 important allergen molecules. In conclusion, IgE-mediated reactions and allergic diseases, including allergic rhinoconjunctivitis, asthma, food reactions, and insect sting reactions, are discussed from a novel molecular perspective. The EAACI MAUG documents the rapid progression of molecular allergology from basic research to its integration into clinical practice, a quantum leap in the management of allergic patients., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Food allergies are believed to be on the rise, and currently, management relies on the avoidance of the food. Hen's egg allergy is after cow's milk allergy the most common food allergy; eggs are used in many food products and thus difficult to avoid. A technological process using a combination of enzymatic hydrolysis and heat treatment was designed to produce modified hen's egg with reduced allergenic potential. Biochemical (SDS-PAGE, Size exclusion chromatography and LC-MS/MS) and immunological (ELISA, immunoblot, RBL-assays, animal model) analysis showed a clear decrease in intact proteins as well as a strong decrease of allergenicity. In a clinical study, 22 of the 24 patients with a confirmed egg allergy who underwent a double-blind food challenge with the hydrolysed egg remained completely free of symptoms. Hydrolysed egg products may be beneficial as low-allergenic foods for egg-allergic patients to extent their diet., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In older children, adolescents, and adults, a substantial part of all IgE-mediated food allergies is caused by cross-reacting allergenic structures shared by inhalants and foods. IgE stimulated by a cross-reactive inhalant allergen can result in diverse patterns of allergic reactions to various foods. Local, mild, or severe systemic reactions may occur already after the first consumption of a food containing a cross-reactive allergen. In clinical practice, clinically relevant sensitizations are elucidated by skin prick testing or by the determination of specific IgE in vitro. Component-resolved diagnosis may help to reach a diagnosis and may predict the risk of a systemic reaction. Allergy needs to be confirmed in cases of unclear history by oral challenge tests. The therapeutic potential of allergen immunotherapy with inhalant allergens in pollen-related food allergy is not clear, and more placebo-controlled studies are needed. As we are facing an increasing incidence of pollen allergies, a shift in sensitization patterns and changes in nutritional habits, and the occurrence of new, so far unknown allergies due to cross-reactions are expected., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens., Method: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1., Results: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation., Conclusion: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

Patients with Oral Allergy Syndrome (OAS) to fresh apple may tolerate low allergenic apple cultivars. We aimed to investigate if the low allergenic properties of Elise and Santana, as previously identified in a Dutch population, could be generalised within North West Europe within the birch pollen region with regard to both the prevalence and degree of sensitization. Prick-to-prick tests (PTP) were performed in eighty-five adult patients with OAS to fresh apple in Great Britain, Switzerland and Northern Italy, before the birch pollen season, using the putatively low allergenic apple cultivars Elise, Santana, Granny Smith, Modi and Mcintosh, as well as the putatively high allergenic apple cultivars Golden Delicious and Kanzi. No significant differences in percentages of negative responses of PTPs were found between the three countries. Negative responses did not differ from negative responses to the different apple cultivars we previously found in 2006/2007 in the Netherlands. The size of the PTPs of all apple cultivars tested were correlated to the size of the skin prick tests with birch pollen. These results add to the indications for the low allergenic properties of the low allergenic apple cultivars Santana and Elise, as the number of negative responses were reproducible in three countries within the birch pollen region and were similar to previous results in the Netherlands. These results justify oral challenge studies with Elise and Santana within the birch pollen region, to establish the low allergenic properties for the benefit for apple allergic consumers for definite conclusions.

Background: Kiwifruit is a common cause of food allergy. Symptoms range from mild to anaphylactic reactions., Objective: We sought to elucidate geographic differences across Europe regarding clinical patterns and sensitization to kiwifruit allergens. Factors associated with the severity of kiwifruit allergy were identified, and the diagnostic performance of specific kiwifruit allergens was investigated., Methods: This study was part of EuroPrevall, a multicenter European study investigating several aspects of food allergy. Three hundred eleven patients with kiwifruit allergy from 12 countries representing 4 climatic regions were included. Specific IgE to 6 allergens (Act d 1, Act d 2, Act d 5, Act d 8, Act d 9, and Act d 10) and kiwifruit extract were tested by using ImmunoCAP., Results: Patients from Iceland were mainly sensitized to Act d 1 (32%), those from western/central and eastern Europe were mainly sensitized to Act d 8 (pathogenesis-related class 10 protein, 58% and 44%, respectively), and those from southern Europe were mainly sensitized to Act d 9 (profilin, 31%) and Act d 10 (nonspecific lipid transfer protein, 22%). Sensitization to Act d 1 and living in Iceland were independently and significantly associated with severe kiwifruit allergy (odds ratio, 3.98 [P = .003] and 5.60 [P < .001], respectively). Using a panel of 6 kiwifruit allergens in ImmunoCAP increased the diagnostic sensitivity to 65% compared with 20% for skin prick tests and 46% ImmunoCAP using kiwi extract., Conclusion: Kiwifruit allergen sensitization patterns differ across Europe. The use of specific kiwifruit allergens improved the diagnostic performance compared with kiwifruit extract. Sensitization to Act d 1 and living in Iceland are strong risk factors for severe kiwifruit allergy., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: The pattern of contact sensitization to the supposedly most important allergens assembled in the baseline series differs between countries, presumably at least partly because of exposure differences. Objectives. To describe the prevalence of contact sensitization to allergens tested in consecutive patients in the years 2007 and 2008, and to discuss possible differences., Methods: Data from the 39 departments in 11 European countries comprising the European Surveillance System on Contact Allergy network (www.essca-dc.org) in this period have been pooled and analysed according to common standards., Results: Patch test results with the European baseline series, and country-specific or department-specific additions to it, obtained in 25 181 patients, showed marked international variation. Metals and fragrances are still the most frequent allergens across Europe. Some allergens tested nationally may be useful future additions to the European baseline series, for example methylisothiazolinone, whereas a few long-term components of the European baseline series, namely primin and clioquinol, no longer warrant routine testing., Conclusions: The present analysis points to 'excess' prevalences of specific contact sensitization in some countries, although interpretation must be cautious if only few, and possibly specialized, centres are representing one country. A comparison as presented may help to target in-depth research into possible causes of 'excess' exposure, and/or consideration of methodological issues, including modifications to the baseline series., (© 2012 John Wiley & Sons A/S.)

Background:   Double-blind placebo-controlled food challenge (DBPCFC) is the gold standard for diagnosing food allergy. Standardized materials and protocols are essential for comparing DBPCFC results for multicentre studies such as EuroPrevall. This required the development and piloting of a standardized vehicle and low-dose protocol for confirming food allergy and determination of minimum eliciting doses (MEDs)., Methods:   A low-dose DBPCFC protocol was developed, with eight titrated protein doses from 3 μg to 1 g. This was delivered using a simple, microbiologically stable food base incorporating allergenic food ingredients manufactured at three sites and centrally distributed to clinical centres. Allergen blinding was assessed by a professional sensory testing panel using a triangle test. Homogeneity and allergen content were confirmed by ELISA and clinical efficacy was assessed in a pilot study, using celeriac and hazelnut as exemplars., Results:   Celeriac and hazelnut ingredients were sufficiently blinded in the dessert. The dessert meals were successfully piloted with hazelnut in allergy clinics in Spain, the Netherlands and Italy and with celeriac and hazelnut in Zurich. The challenges elicited a range of subjective and objective reactions ranging in severity from mild itching of the oral mucosa to bronchospasm., Conclusions:   A standardized challenge vehicle proven to sufficiently blind processed, powdered hazelnut and celeriac ingredients and that can be reproducibly manufactured has been developed. This pilot study shows that the vehicle is promising for the confirmation of food allergy and determination of MEDs in adults and children with body weight >28.8 kg (approximately 7-11 years old)., (© 2011 John Wiley & Sons A/S.)

Scope: Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1., Methods and Results: Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein., Conclusion: Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Scope: Four Bet v 1 homologous food allergens from celeriac (rApi g 1), apple (rMal d 1), peach (rPru p 1) and hazelnut (rCor a 1), were used to probe the structural responsiveness of the Bet v 1 scaffold to gastric digestion conditions and its impact on allergenicity., Methods and Results: Low pH induced conformational changes of all homologues, which was reduced at physiological ionic strength for all except rPru p 1 as observed by circular dichroism (CD)-spectroscopy. The homologues were rapidly digested by pepsin, losing their IgE binding activity, although the kinetics and patterns of digestion varied subtly between homologues, rApi g 1 being the most stable. We have demonstrated for the first time that gastric phosphatidyl-choline (PC) induced conformational changes in all homologues but only rMal d 1 penetrated the PC vesicles as detected by fluorescence polarization, slowing its digestion and retaining more of its allergenic activity. PC enhanced basophil activation of all digested allergens except rApi g 1., Conclusion: The Bet v 1 scaffold is generally susceptible to low pH and pepsinolysis and interacts with PC vesicles, properties which can explain effects of the gastric environment on their allergenicity. These data show the importance of including surfactants in model digestion systems., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Published data on crustacean allergens are incomplete. The identification of tropomyosin (TM), arginine kinase (AK), sarcoplasmic Ca-binding protein (SCP) and myosin light chain (MLC) as shrimp allergens are all important contributions but additional allergens are required for the development of a complete set of reagents for component resolved diagnosis and the exploration of novel vaccination strategies., Methods: The North Sea shrimp (Crangon crangon), which is frequently consumed in Europe, served as a model organism in this study. TM and AK were directly cloned from mRNA based on sequence homology and produced as recombinant proteins. Additional IgE-reactive proteins were isolated by preparative SDS-PAGE and identified by mass spectrometry and corresponding cDNAs were cloned and expressed in E. coli. The relevance of the 6 cloned crustacean allergens was confirmed with sera of 31 shrimp-allergic subjects, 12 of which had a positive double-blind, placebo-controlled food challenge (DBPCFC) to shrimp and 19 a convincing history of food allergy to shrimp, including 5 cases of anaphylaxis. Quantitative IgE measurements were performed by ImmunoCAP., Results: Six recombinant crustacean proteins: TM, AK, SCP, a novel MLC, troponin C (TnC), and triosephosphate isomerase (TIM) bound IgE in ImmunoCAP analysis. Specific IgE to at least one of these single shrimp allergens was detected in 90% of the study population, thus the in vitro diagnostic sensitivity was comparable to that of shrimp extract (97%). In 75% of the subjects, the combined technical sensitivity was similar to or greater with single shrimp allergens than with natural shrimp extract., Conclusions: We identified six IgE-binding proteins from C. crangon, three of which have not before been described as allergens in crustaceans. This extensive panel of shrimp allergens forms a valuable asset for future efforts towards the identification of clinically relevant biomarkers and as a basis to approach patient-tailored immunotherapeutic strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut., Methodology/principal Findings: Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6., Conclusions/significance: Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Background: Patch testing detects delayed-type sensitizations and may show changes in prevalence over time., Objectives: To compare sensitization prevalence to our baseline series allergens during the years 2000-2004 to results from 1990 to 1994., Methods: We analysed baseline patch test series allergens from 2000 through 2004., Results: 4094 individuals were patch tested to the baseline series (mean age 45.4 years, males 41.7%). The highest sensitization rates were found for nickel sulfate (19.3%), fragrance mix (10.4%) and Myroxylon pereirae (7.8%). Compared to 1990-1994, methylchloroisothiazolinone (and) methylisothiazoline (MCI/MI) and neomycin sulfate sensitization decreased and lanolin alcohol, thiuram mix and ammoniated mercury sensitization increased., Conclusions: Current sensitization rates are similar to those across Europe. Reduced sensitizations to MCI/MI and neomycin sulfate and increased sensitizations to thiuram mix and ammoniated mercury chloride may well be because of changes in exposure. The current group comprises a higher proportion affected by leg dermatitis and may thus explain the higher sensitization to lanolin alcohol. Comparing patch results over time shows important changes in sensitization prevalence.

Introduction: Persistence of allergen and immunocompetent cells at sites of healed contact dermatitis has been reported. Flare-up reactions triggered by patch testing and after systemic provocation with allergen are well-known phenomena. To our knowledge, we report the first flare-up of a previous patch test site following casual cutaneous application of nickel in an individual with hitherto latent nickel sensitization., Case Report: Patch testing in a 23-year-old female patient was performed for dermatitis following application of various gels and adhesive bandages: positive delayed-type hypersensitivity reactions were noted for nickel sulfate and potassium dichromate. The patient had never noticed skin reactions to nickel-containing items before. Three weeks following these patch tests, the patient wore earrings which in the past had been well tolerated. She subsequently developed dermatitis of both earlobes within hours and dermatitis at the site of nickel patch testing within a day., Conclusions: Nickel exposure for 48 h in a patch test is sufficient to induce overt delayed-type hypersensitivity on re-exposure with a previously tolerated antigen in a previously clinically unresponsive individual. Antigen and/or antigen-specific effector cells at the site of previous positive patch testing can be recruited into a delayed-type hypersensitivity reaction for a prolonged period of time., (2009 S. Karger AG, Basel.)

Safety assessment for genetically-engineered crop plants includes assessment for allergic responses. To facilitate this assessment, serum banks should contain well-characterised sera from patients with confirmed food allergies. A serum is defined as well-characterised if it is taken from a patient who has a convincing history of allergic responses to a known allergen or an allergen-containing food, a positive skin prick test (or elevated IgE response), and a positive response in a clinical food challenge.

Thresholds constitute a critical piece of information in assessing the risk from allergenic foods at both the individual and population levels. Knowledge of the minimum dose that can elicit a reaction is of great interest to all food allergy stakeholders. For allergic individuals and health professionals, individual threshold data can inform allergy management. Population thresholds can help both the food industry and regulatory authorities assess the public health risk and design appropriate food safety objectives to guide risk management. Considerable experience has been gained with the double-blind placebo-controlled food challenge (DBPCFC), but only recently has the technique been adapted to provide data on thresholds. Available data thus vary greatly in quality, with relatively few studies providing the best quality individual data, using the low-dose DBPCFC. Such high quality individual data also form the foundation for population thresholds, but these also require, in addition to an adequate sample size, a good characterization of the tested population in relation to the whole allergic population. Determination of thresholds at both an individual level and at a population level is influenced by many factors. This review describes a low-dose challenge protocol developed as part of the European Community-funded Integrated Project Europrevall, and strongly recommends its wider use so that data are generated that can readily increase the power of existing studies.

Background: Foods not commonly consumed in the European Union must be proven safe before being brought to market, including an assessment of allergenicity. We present a three-stepwise strategy for allergenicity assessment of natural novel foods using three novel vegetables, namely, water spinach, hyacinth bean, Ethiopian eggplant., Methods: First, vegetable extracts were analyzed for the presence of pan-allergens [Bet v 1 homologous proteins, profilins, nonspecific lipid transfer proteins (LTP)] by immunoblot analysis with specific animal antibodies. Secondly, the IgE-binding of the food extracts was investigated by EAST (Enzyme-allergosorbent test) and immunoblot analysis using sera with IgE-reactivity to known pan-allergens or to phylogenetically related foods from subjects (i) allergic to birch, grass and mugwort pollen, (ii) with food allergy to soy, peanut, tomato, multiple pollen-related foods and (iii) sensitized to LTP. Thirdly, the clinical relevance of IgE-binding was assessed in vivo by skin prick testing (SPT) and open oral food challenges (OFC)., Results: Profilin and LTP were detected by animal antibodies in all vegetables, a Bet v 1 homologue selectively in hyacinth bean. IgE-binding to LTP, profilin and a Bet v 1 homologue was proven by immunoblot analysis and EAST. Positive SPT and OFC results were observed for all vegetables in pollen-allergic patients., Conclusions: Our stepwise procedure confirmed the presence and IgE-binding capacity of novel vegetable proteins homologous to known allergens in endemic vegetable foods. In vivo testing proved the potential of the novel vegetables to elicit clinical allergy. Hence, our described algorithm seems to be applicable for allergenicity testing of natural novel foods.

Background: Hazelnuts are a common cause of food allergic reactions. Most hazelnut allergic individuals in central and northern Europe are sensitized to Cor a 1, a member of the PR-10 protein family, while the lipid transfer protein Cor a 8 acts as a major allergen in the south of Europe. Other allergens, including profilin and seed storage proteins, may be important in subgroups of patients. Reliable detection of specific IgE in the clinical diagnosis of food allergy requires allergen reagents with a sufficient representation of all relevant allergen components. Some reported observations suggest that natural hazelnut extract may not be fully adequate in this respect., Methods: The capacity of immobilized natural hazelnut extract to bind Cor a 1-, Cor a 2- and Cor a 8-specific IgE and IgG antibodies was investigated by serum adsorption and extract dilution experiments and by the use of allergen specific rabbit antisera. All measurements were performed with the ImmunoCAP assay platform., Results: The experimental results revealed an incomplete capacity of immobilized hazelnut extract to capture IgE antibodies directed to the major allergen Cor a 1. Spiking of hazelnut extract with recombinant Cor a 1.04 prior to solid phase coupling gave rise to significantly enhanced IgE antibody binding from Cor a 1 reactive sera. The spiking did not negatively affect the measurement of IgE to extract components other than Cor a 1., Conclusion: A hazelnut allergen reagent with enhanced IgE detection capacity can be generated by supplementing the natural food extract with recombinant Cor a 1.04.

Background: Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osborne's three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat., Methods: Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osborne's protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition., Results: The most important wheat allergens were the alpha-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients., Conclusions: The alpha-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.

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

Background: In Europe, pollen-related food allergy is the most frequent form of food allergy in adults. Reliability of current diagnostic procedures, however, is poor and therapeutic options are not available., Objectives: In the present study, we created a panel of recombinant allergens from carrot and evaluated its potential in component-resolved in vitro diagnosis of carrot allergy., Methods: Recombinant (r) Dau c 1.0104, Dau c 1.0201 and Dau c 4 were cloned by a polymerase chain reaction strategy, expressed in Escherichia coli and purified. Carrot lipid transfer protein (LTP) was expressed in the yeast Pichia pastoris. Sera from 40 carrot-allergic patients were investigated. Twenty-one birch pollen-allergic subjects with negative open provocation to carrot and 20 non-allergic subjects were included as controls. IgE binding to recombinant allergens as well as to cross-reactive carbohydrate determinants (CCD) was measured by ELISA. Cross-reactivity between Dau c 1 isoforms and Bet v 1 was assayed by ELISA inhibition. Biological activity of the recombinant carrot allergens was assessed by histamine release assay and peripheral blood mononuclear cells stimulation., Results: Ninety-eight percent of the carrot-allergic patients were positive to at least one recombinant allergen; 98% reacted to rDau c 1.0104, 65% to rDau c 1.0201, 38% to rDau c 4 and 20% had IgE against CCD. Specificity using the recombinant allergens was high when compared with non-allergic controls, but low compared with birch-sensitized subjects without carrot allergy. Sensitization to Dau c 1.0201, however, proved to be highly specific for clinically relevant sensitization. Inhibition assays indicated the absence of LTP in carrot root extract, and epitope diversity between Dau c 1.0104, Dau c 1.0201 and Bet v 1., Conclusions: Our panel of recombinant allergens from carrot can provide a standardized tool for in vitro diagnosis of carrot allergy, and for epitope studies.

Peanuts (Arachis hypogaea) contain some of the most potent food allergens. In recent years an increasing prevalence of peanut allergies both in children and adults has been observed in the USA and in Europe. In vitro identification and characterization of allergens including those from peanut have been frequently performed by Western blotting. However this method may alter the immunoglobulin E (IgE) antibody reactivity since the proteins are denatured by detergent treatment and/or reduction of disulfide bonds by reducing reagents and does not answer the question how peanut allergens interact with the human digestive apparatus and immune system. Size exclusion chromatography of peanut extract shows that approximately 90% of the total protein content is eluted as one peak in the exclusion volume with a molecular mass of over 200 kDa. The proteins of this fraction were analyzed by blue-native polyacrylamide gel electrophoresis (PAGE), immunoblotting, two-dimensional PAGE and Western blotting. A complex of Ara h 1 (Acc. no. P43237), Ara h 3/4 (AAM46958), Ara h 3 (AAC63045), Ara h 4 (AF086821), Gly 1 (AAG01363) and iso-Ara h 3 (AAT39430) was identified using patients' IgE and allergen-specific monoclonal antibodies; N-terminal sequencing and matrix-assisted laser desorption/ionisation-time of flight analysis verified these findings. A comparison of the peanut allergen sequences of Ara h 3/4, Ara h 3, Ara h 4 and peanut trypsin inhibitor (AF487543) and the proteins Gly 1 and iso-Ara h 3, not yet described as allergens, leads to the conclusion that these proteins are isoallergens of each other. It was shown that these isoallergens are post-translationally cleaved and held together by disulfide bonds in accordance to the 11S plant seed storage proteins signature.

Background: Profilins are recognised by IgE of about 20% of patients allergic to birch pollen and plant foods. They are ubiquitous intracellular proteins highly cross-reactive among plant species. Therefore, they were called panallergens and are made responsible for cross-sensitisation between plant pollen and food., Objectives: The aim of the present study was to clone the cDNAs encoding profilins from bell pepper and tomato, to produce and purify the recombinant proteins and to compare their IgE-binding capacities to those of the natural proteins., Methods: cDNA clones coding for profilin were obtained by RT-PCR from total RNA of tomato and bell pepper fruits, sequenced and expressed as non-fusion proteins in ESCHERICHIA COLI. The recombinant profilins were subsequently purified and tested for IgE-binding and inhibition capacity with sera from 34 food-allergic patients. Possible oligomerisation of recombinant profilins was investigated by HPLC analysis and its influence on IgE binding assayed by ELISA., Results: The open reading frame from both profilins encompasses 393 bp with a predicted molecular mass of 14,184 kD and a pI of 4.44 for bell pepper profilin (Cap a 2) and 14,257 kD and a pI of 4.46 for the profilin from tomato (Lyc e 1). The two protein sequences display 91% identity, whereas tomato profilin from pollen shares only 75% identity with tomato fruit profilin. Eleven out of 34 food-allergic patients (32%) display IgE binding to both purified profilins. Preincubation of a serum pool with either purified rCap a 2 or rLyc e 1 nearly abolished IgE binding to natural Cap a 2 and Lyc e 1, respectively. In addition, purified recombinant Cap a 2 was able to inhibit IgE-binding to rLyc e 1 by approximately 50%, whereas rLyc e 1 completely blocked IgE-binding to rCap a 2 in cross-inhibition assays. HPLC analysis showed that in solution Cap a 2 and Lyc e 1 can be found predominantly as dimers, which can be partially reduced to monomers by addition of dithiothreitol (DTT). In ELISA DTT-treated Lyc e 1 displayed a clearly lower IgE-binding capacity than untreated profilin., Conclusions: Purified rCap a 2 and rLyc e 1 proved to be valuable tools for studying cross-reactivity to profilins in patients allergic to pollen and food., (Copyright 2003 S. Karger AG, Basel)

Background: High quality and stability are essential requirements of commercial allergen preparations. Recently we have demonstrated the very low stability of protein allergens in an extract of the ubiquitous mould Fusarium culmorum., Objective: The present study was performed to identify, isolate and characterise allergens of F. culmorum as a basis for a stable allergenic reference material. In addition, the significance of IgE binding to carbohydrate structures in the natural allergen source was investigated., Methods: Sera of 52 subjects with suspected mould allergy were used to determine the IgE binding capacity of a commercial F. culmorum extract and an in-house extract by immunoblotting and enzyme allergo sorbent test (EAST). Binding of IgE-antibodies to putative carbohydrate structures located on glycoproteins was verified by periodate treatment of blot strips prior to immunodetection. A complementary (c)DNA expression library of F. culmorum was prepared and screened for IgE-binding clones using sera from F. culmorum-sensitive individuals. Positive clones were isolated, and the open reading frames were subcloned into expression vectors to produce recombinant proteins in E. coli. The recombinant proteins were tested for their IgE reactivity by immunoblotting and EAST., Results: Using the in-house extract for EAST and immunoblot experiments 44% (23/52) of the sera were found to contain F. culmorum-specific IgE antibodies. Compared to the in-house extract, nearly all IgE-reactivties in the range of 15-30kD were lacking in the commercial preparation as examined by immunoblot analysis and only 10% (5/52) of the sera were found to contain F. culmorum-specific IgE by EAST. IgE binding to putative carbohydrate structures was observed in the high molecular weight range in approximately 50% (12/23) of the IgE-positive sera by both extracts. Three IgE binding clones were isolated from the cDNA-library. One clone (Fus c 1) is homologous to the highly conserved 60S acidic ribosomal protein P2 described as minor allergen in other moulds. The second (Fus c 2) shows high similarity (64%) to a respiratory allergen from the basidiomycete Coprinus comatus (Cop c 2). The third clone (Fus c 3) was not related to known proteins. With sera from 26 individuals sensitised to F. culmorum the IgE prevalence of recombinant proteins rFus c 1, rFus c 2 and rFus c 3 was found to be 35, 50, and 15%, respectively., Conclusions: F. culmorum may represent an underestimated source of aeroallergens. In contrast to highly labile and poorly standardised F. culmorum extracts, the new recombinant allergens may serve as stable allergenic reference material. A combination of rFus c 1 and rFus c 2 is suitable to diagnose 81% of F. culmorum-sensitised subjects. IgE reactivity to putative carbohydrate structures is relatively frequent, and can not be detected by these allergens.

Background: Allergy to hazelnuts is a common example of birch pollen related food allergy. Symptoms upon ingestion are often confined to the mouth and throat, but severe systemic reactions have been described in some patients. The aim of the study was to evaluate the reduction in allergenicity by roasting of the nuts., Methods: Double-blind, placebo-controlled food challenges (DBPCFC) with roasted hazelnuts (140 degrees C, 40 min) were performed in 17 birch pollen allergic patients with DBPCFC-confirmed food allergy to raw hazelnuts. The effect of roasting was further evaluated by skin prick test (SPT), histamine release (HR), measurement of specific IgE, and IgE-inhibition experiments., Results: In 5/17 patients the DBPCFC with the roasted nuts were positive. The symptoms were generally mild and included OAS (oral allergy syndrome) in all patients. Roasting of the nuts significantly reduced the allergenic activity evaluated by SPT, HR, specific IgE, and IgE-inhibition. Immunoblotting experiments with recombinant hazelnut allergens showed sensitization against Cor a 1.04 in 16/17 patients and against Cor a 2 in 7/17 patients. None of the patients were sensitized to Cor a 8. Challenge-positive patients did not differ from the rest in IgE-binding pattern., Conclusions: All the applied methods indicated that roasting of hazelnuts reduces the allergenicity, but since 5/17 birch pollen allergic patients were DBPCFC-positive to the roasted nuts, ingestion of roasted hazelnuts or products containing roasted hazelnuts can not be considered safe for a number of hazelnut allergic consumers. For patients with a history of severe allergic symptoms upon ingestion of hazelnuts, thorough and conscientious food labelling of hazelnuts and hazelnut residues is essential.

Pollen-allergic patients frequently present allergic symptoms after ingestion of several kinds of plant-derived foods. The majority of these reactions is caused by four distinct cross-reactive structures that are present in birch pollen. Proteins that share common epitopes with Bet v 1, the major birch pollen allergen, occur in pollens of several tree species: apples, stone fruits, celery, carrot, nuts, and soybeans. Approximately 70% of our patients who are allergic to birch pollen may experience symptoms after consumption of foods from these groups. In contrast, two minor allergenic structures-profilins and cross-reactive carbohydrate determinants (CCD)-that sensitize approximately 10-20% of all pollen-allergic patients are also present in grass pollen and weed pollen. Moreover, IgE-binding proteins related to the birch pollen minor allergen Bet v 6 have been found in many vegetable foods such as apple, peach, orange, lychee fruit, strawberry, persimmon, zucchini, and carrot. Frequently, the occurrence of cross-reactive IgE antibodies is not correlated with the development of clinical food allergy. In particular, the clinical relevance of sensitization to CCD is doubtful. Generally, pollen-related allergens tend to be more labile during heating procedures and in the digestive tract compared to allergens from classical allergenic foods such as peanut. However, recent DBPCFC studies have shown that both cooked celery and roasted hazelnuts still pose an allergenic risk for pollen-sensitized subjects. Since pathogenesis-related proteins share several common features with allergens and both the Bet v 1 and the Bet v 6-related food allergens are defense-related proteins, approaches to introduce such proteins as a measure to protect plants against diseases should be performed with caution as they may increase the allergenicity of these crops.

The aim of this study was to confirm allergy to celery tuber and to zucchini, for the first time, by DBPCFC, and to identify the allergens recognized by IgE from DBPCFC-positive patients. Therefore, raw vegetables were hidden in a broccoli drink, and a DBPCFC-procedure was developed that consisted of a spit and swallow protocol, making sure that the procedure was safe for the patients and that reactions strictly localized to the oral cavity as well as systemic reactions could be reproduced by DBPCFC. The allergens in celery and zucchini extract were identified by immunoblot inhibition using allergen extracts, recombinant allergens and purified N-glycans as inhibitors. Celery allergy was confirmed in 69% (22/32) of subjects with a positive case history. Four subjects with a history of allergic reactions to zucchini had a positive DBPCFC to this vegetable. During DBPCFC, systemic reactions were provoked in 50% (11/22) of the patients to celery, and in 3/4 of the zucchini-allergic patients. The Bet v 1-related major celery allergen was detected by IgE of 59% (13/22) of the patients. Cross-reactive carbohydrate epitopes (CCD) bound IgE of 55% (12/22) of the celery-allergic patients and in 2/4 of the subjects with zucchini allergy. Profilin was a food allergen in celery in 23% (5/22) and in zucchini in 2/4 of the cases. A zucchini-specific allergen was detected by IgE from one patient. We conclude that ubiquitous cross-reactive structures are important in allergy to both, celery and zucchini, and that a specific association to birch pollen allergy exists in allergy to celery (mediated by Api g 1), but not in zucchini allergy.

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

Background: Allergic reactions to carrot affect up to 25% of food-allergic subjects. Clinical manifestations of carrot allergy and IgE responses to carrot proteins, however, have never been studied in subjects with carrot allergy confirmed by means of double-blinded, placebo-controlled food challenge (DBPCFC)., Objective: The purposes of this investigation were to confirm clinically relevant sensitizations to carrot by means of DBPCFC, to validate current diagnostic methods, and to identify IgE-reactive carrot proteins in patients with true allergy., Methods: DBPCFCs were performed in 26 subjects with histories of allergic reactions to carrot. Patients underwent skin prick tests with carrot extract, fresh carrot, and various pollen extracts. Specific IgE to carrot, celery, birch, and mugwort pollen and to rBet v 1, rBet v 2, and rBet v 6 were measured through use of the CAP method. Carrot allergens were identified by means of immunoblotting and blotting inhibition., Results: Twenty of 26 patients had positive DBPCFC results. The sensitivity of the determination of carrot-specific IgE antibodies through use of the CAP method (> or =0.7 kU/L) was 90%, the sensitivity for skin prick testing with commercial extracts was 26%, and the sensitivity for prick-to-prick tests with raw carrot was 100%. The Bet v 1--related major carrot allergen Dau c 1 was recognized by IgE from 85% of patients; 45% were sensitized to cross-reactive carbohydrate determinants and 20% to carrot profilin. In 1 subject, a Bet v 6--related carrot allergen was recognized. In 4 patients, IgE binding to Dau c 1 was not inhibited or was weakly inhibited by rBet v 1 or birch pollen extract., Conclusion: This study confirmed the allergenicity of carrot by means of DBPCFC. DBPCFC-positive patients had exclusively specific IgE antibodies to birch pollen--related carrot allergens, Dau c 1 being the major allergen. The lack of inhibition of IgE binding to Dau c 1 by birch allergens in a subgroup of patients might indicate an secondary immune response to new epitopes on the food allergen that are not cross-reactive with Bet v 1.

Profilin is a panallergen which is recognised by IgE from about 20% of birch pollen- and plant food-allergic patients. Little is known about epitope diversity among these homologous proteins, and about the correlation between IgE-cross-reactivity and allergenic reactivity. Plant food profilins from pear (Pyr c 4) and cherry (Pru av 4) were cloned by polymerase chain reaction and produced in Escherichia coli BL21. The profilins were purified as non-fusion proteins by affinity chromatography on poly-(L-proline)-Sepharose and characterized by immunoblotting, IgE-inhibition experiments and histamine release assays. The coding regions of the cDNA of pear and cherry profilin were identified as a 393 bp open reading frame. The deduced amino acid sequences showed high identities with birch pollen profilin Bet v 2 (76-83%) and other allergenic plant profilins. Pyr c 4 and Pru av 4 were investigated for their immunological properties in comparison with profilins from celery (Api g 4) and birch pollen (Bet v 2). Fourty-three of 49 patients (88%), preselected for an IgE-reactivity with Bet v 2 showed specific IgE-antibodies to the recombinant pear protein, 92% of the sera were positive with the recombinant cherry allergen and 80% of the sera were reactive with the celery protein. Inhibition experiments showed a strong cross-reactivity of IgE with profilins from plant food and birch pollen. However, IgE binding profiles also indicated the presence of epitope differences among related profilins. All investigated profilins, Pyr c 4, Pru av 4, Api g 4 and Bet v 2, presented almost identical allergenic properties in cellular mediator release tests. Therefore, cross-reactivities between related profilins may explain pollen-related allergy to food in a minority of patients. The nucleotide sequences reported have been submitted to the Genbank database under accession numbers AF129424 (Pyr c 4) and AF129425 (Pru av 4).

Certirizine, a potent H1-blocking agent, is often recommended as an emergency drug in anaphylactic reactions because of its well documented fast onset of action. In this randomized, cross-over study we compared the onset of action after a single dose of two recently introduced antihistamines, acrivastine and fexofenadine, with that of cetirizine. The inhibition of the wheal-and-flare reaction produced by skin prick test with histamine in 20 healthy volunteers and with a relevant pollen allergen in 20 atopic patients, respectively, were measured before and at regular intervals up to 60 min after the ingestion of acrivastine (8 mg and 16 mg), fexofenadine (120 mg) and cetirizine (10 mg and 20 mg). Wheal-and-flare reaction were significantly inhibited 20 min after the intake of 16 mg acrivastine in atopic patients and 30 min after intake of 8 mg acrivastine in healthy volunteers, whereas cetirizine produced a significant inhibition of the wheal-and-flare reaction within 40-60 min. No significant inhibition could be observed within 60 min after fexofenadine intake. Therefore, in clinical settings when a fast onset of the H1-blocking action is mandatory (e.g., after insect stings or for short-term prophylaxis) we recommend acrivastine.

Background: Benzyl alcohol is a widely used preservative, solvent and fragrance material. According to published data, it is a rare sensitizer in humans. Objectives: To identify characteristics and sensitization patterns of patients with positive patch test reactions to benzyl alcohol and to check the reliability of the patch test preparation benzyl alcohol 1% pet. Patients and Methods: Retrospective analysis of data from the Information Network of Departments of Dermatology (IVDK), 2010–2019. Results: Of 70 867 patients patch tested with benzyl alcohol 1% pet., 146 (0.21%) showed a positive reaction, most of them (89%) only weakly positive. The number of doubtful and irritant reactions significantly exceeded the number of positive reactions. Reproducibility of positive test reactions was low. Among benzyl alcohol‐positive patients, compared to benzyl alcohol‐negative patients, there were significantly more patients with leg dermatitis (17.8% vs. 8.6%), more patients aged 40 years or more (81.5% vs. 70.5%) and more patients who were tested because of a suspected intolerance reaction to topical medications (34.9% vs. 16.6%). Concomitant positive reactions were mainly seen to fragrances, preservatives and ointment bases. Conclusions: Sensitization to benzyl alcohol occurs very rarely, mainly in patients with stasis dermatitis. In view of our results, benzyl alcohol cannot be regarded as a significant contact allergen, and therefore marking it as skin sensitizer 1B and labelling it with H 317 is not helpful. [ABSTRACT FROM AUTHOR]

Abstract: The accurate assessment and communication of the severity of acute allergic reactions are important to patients, clinicians, researchers, the food industry, and public health and regulatory authorities. Severity has different meanings to different stakeholders with patients and clinicians rating the significance of particular symptoms very differently. Many severity scoring systems have been generated, most focusing on the severity of reactions following exposure to a limited group of allergens. They are heterogeneous in format, none has used an accepted developmental approach, and none has been validated. Their wide range of outcome formats has led to difficulties with interpretation and application. Therefore, there is a persisting need for an appropriately developed and validated severity scoring system for allergic reactions that work across the range of allergenic triggers and address the needs of different stakeholder groups. We propose a novel approach to develop and then validate a harmonized scoring system for acute allergic reactions, based on a data‐driven method that is informed by clinical and patient experience and other stakeholders’ perspectives. We envisage two formats: (i) a numerical score giving a continuum from mild to severe reactions that are clinically meaningful and are useful for allergy healthcare professionals and researchers, and (ii) a three‐grade‐based ordinal format that is simple enough to be used and understood by other professionals and patients. Testing of reliability and validity of the new approach in a range of settings and populations will allow eventual implementation of a standardized scoring system in clinical studies and routine practice. [ABSTRACT FROM AUTHOR]

Background Contact allergy is a common condition and can severely interfere with daily life or professional activities. Due to changes in exposures, such as introduction of new substances, new products or formulations and regulatory intervention, the spectrum of contact sensitization changes. Objective To evaluate the current spectrum of contact allergy to allergens present in the European baseline series ( EBS) across Europe. Methods Retrospective analysis of data collected by the European Surveillance System on Contact Allergies ( ESSCA, ) in consecutively patch-tested patients, 2013/14, in 46 departments in 12 European countries. Results Altogether, 31 689 patients were included in the analysis. Compared to a similar analysis in 2004, the prevalence of contact allergy to methylisothiazolinone went up to around 20% in several departments. In comparison, contact allergy to the metals nickel, cobalt and chromium remained largely stable, at 18.1%, 5.9% and 3.2%, respectively, similar to mostly unchanged prevalence with fragrance mix I, II and Myroxylon pereirae (balsam of Peru) at 7.3%, 3.8% and 5.3%, respectively. In the subgroup of departments diagnosing (mainly) patients with occupational contact dermatitis, the prevalence of work-related contact allergies such as epoxy resin or rubber additives was found to be increased, compared to general dermatology departments. Conclusion Continuous surveillance of contact allergy based on network data offers the identification of time trends or persisting problems, and thus enables focussing in-depth research (subgroup analyses, exposure analysis) on areas where it is needed. [ABSTRACT FROM AUTHOR]

Background Conflicting results exist on the effect of allergen immunotherapy ( AIT) on pollen-related food allergy. We aimed to investigate the efficacy of one-year AIT with the folding variant ( FV) of recombinant (r) Bet v 1 on birch-related soya allergy. Methods Of 138 subjects with Bet v 1 sensitization, 82 were positive at double-blind placebo-controlled food challenge ( DBPCFC) with soya. A total of 56 of 82 were randomized in the ratio of 2:1 (active: placebo). Per-protocol population ( PPP) had received ≥150 μg of allergen or placebo preparation. Outcome measures: lowest observed adverse effect levels ( LOAEL), postinterventional occurrence of objective signs (objS) at any dose level, sIgE/IgG4 against Bet v 1 and Gly m 4. Between-group changes were investigated ( ancova, Mann-Whitney U-test, Fisher exact test). Results Baseline characteristics including LOAELs were comparable in both groups with objS and subjS occurring in 82% and 95% of active ( n = 38) vs 78% and 83% of placebo group ( n = 18). After AIT, objS occurred in 24% and 47%, respectively. LOAEL group differences showed a beneficial tendency ( P = 0.081) for LOAELobjective in PPP (30 active, 15 placebo). sIgG4 raised only in active group (Bet v 1: P = 0.054, Gly m 4: P = 0.037), and no relevant changes occurred for sIgE. Only 56% of the intended sample size was recruited. Conclusion For the first time, we present data on the effect of rBet v 1- FV on birch-related soya allergy. rBet v 1- FV AIT induced significant immunogenic effects. Clinical assessment showed a tendency in favour of the active group but did not reach statistical significance. [ABSTRACT FROM AUTHOR]