Natural History of IgE-Mediated Fish Allergy in Children.

BACKGROUND: Fish allergy is not uncommon, especially in countries with high fish consumption, it can frequently be severe and may affect dietetic and lifestyle choices. Nevertheless, data on its clinical course and natural history are scarce. OBJECTIVE: To describe the natural history of immunoglobulin E-mediated fish allergy and the potential differential reactivity to various fish species and identify prognostic markers in children with confirmed disease. METHODS: Clinical history, specific immunoglobulin E, and skin prick tests to various fish were recorded in 126 children with confirmed immunoglobulin E-mediated fish allergy. Immunoglobulin E reactivity was also evaluated by immunoblotting. Eligible participants proceeded to a series of food challenges to tuna, swordfish, and codfish. In total, 234 challenges were performed. RESULTS: Fifty-eight children (9.7 ± 3.9 years) were included in the analysis. Age at first reaction was 0.5 to 5 years (median, 1.3 years). Thirteen children (22%) tolerated all fish tested, including cod, 1 to 14 years (mean, 8.2 ± 4.2 years) following their first reported reaction. Complete fish tolerance increased with age, ranging from 3.4% in preschool children to over 45% in adolescents (95% confidence interval, 26.3%-79.7%). Most children were able to tolerate swordfish (94%) and tuna (95%). Prechallenge specific immunoglobulin E to cod greater than 4.87 kUA/L was the best positive predictive marker for fish allergy persistence (94%), followed by skin prick tests to sardine greater than 6.5 mm (92%). CONCLUSIONS: A considerable proportion of fish-allergic children develop tolerance around adolescence. Most fish-allergic children can consume tuna and swordfish, which, thus, provide safe alternatives for a balanced diet.

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.

Molecular characterization of Api g 2, a novel allergenic member of the lipid-transfer protein 1 family from celery stalks.

SCOPE: Celery represents a relevant cross-reactive food allergen source in the adult population. As the currently known allergens are not typical elicitors of severe symptoms, we aimed to identify and characterize a non-specific lipid transfer protein (nsLTP). METHODS AND RESULTS: MS and cDNA cloning were applied to obtain the full-length sequence of a novel allergenic nsLTP from celery stalks. The purified natural molecule consisted of a single isoallergen designated as Api g 2.0101, which was recombinantly produced in Escherichia coli Rosetta-gami. The natural and recombinant molecules displayed equivalent physicochemical and immunological properties. Circular dichroism revealed a typical α-helical fold and high thermal stability. Moreover, Api g 2 was highly resistant to simulated gastrointestinal digestion. As assessed by ELISA, thermal denaturation did not affect the IgE binding of Api g 2. Natural and recombinant Api g 2 showed similar allergenic activity in mediator release assays. Api g 2-specific IgE antibodies cross-reacted with peach and mugwort pollen nsLTPs. CONCLUSION: Based on our results, it can be anticipated that inclusion of recombinant Api g 2 in the current panel of allergens for molecule-based diagnosis will facilitate the evaluation of the clinical relevance of nsLTP sensitization in celery allergy and help clinicians in the management of food allergic patients.

Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.

SCOPE: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. METHODS: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. RESULTS: N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. CONCLUSION: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

Diminished response to grass pollen allergen challenge in subjects with concurrent house dust mite allergy.

INTRODUCTION: The clinical manifestation of allergic rhinitis is influenced by many factors; while different subpopulations are not well defined. Different combinations of allergic sensitization may lead to different clinical manifestations of allergic disease. METHODS: In a nasal allergen challenge model we compared allergic rhinitis symptoms between subjects mono-sensitized to grass pollen or house dust mite, poly-sensitized subjects, and healthy controls. We measured visual analogue scales of symptoms and peak nasal inspiratory flow. We also compared serum total IgE, allergen-specific IgE and IgG4, and basophil histamine release. RESULTS: Nasal challenge with grass pollen extract led to a significantly larger increase in subjective (p = 0.031) and objective (p = 0.001) nasal symptoms in grass pollen mono-sensitized subjects than in poly-sensitized subjects. No differences were found in serum levels of allergen-specific IgE and IgG4 or in biological activity of IgE (basophil histamine release) between mono-sensitized and poly-sensitized subjects. We found a strong inverse correlation between serum allergen-specific IgE and basophil histamine release (-0.789, p = 0.001). CONCLUSIONS: Grass pollen mono-sensitized subjects have a more severe clinical response to nasal challenge than poly-sensitized subjects. This cannot be explained by serum levels of IgE or its biological activity. The continuous allergen exposure in poly-sensitized subjects may alter local immuno-regulatory processes, leading to a reduced clinical response to allergen challenge.

Purification and characterisation of relevant natural and recombinant apple allergens.

Apple (Malus domestica) is the most widely cultivated fruit crop in Europe and frequently causes allergic reactions with a variable degree of severity. So far, four apple allergens Mal d 1, Mal d 2, Mal d 3 and Mal d 4 have been identified. Mal d 1, a Bet v 1 related allergen, and Mal d 4, apple profilin, are sensitive to proteolytic degradation, whereas Mal d 2, a thaumatin-like protein and Mal d 3, a nonspecific lipid transfer protein, are rather stable to proteolytic processes. Mal d 1 and Mal d 4 were purified after expression in Escherichia coli expression system, while Mal d 2 and Mal d 3 were purified from apple fruit tissue. All purified proteins were subjected to detailed physicochemical characterisation to confirm their structural integrity and maintained IgE binding capacity. Detailed investigations of carbohydrate moieties of Mal d 2 demonstrated their involvement in the overall IgE binding capacity of this allergen. It was concluded that the folded structure and IgE binding capacity of all four allergens were preserved during purification.

The prevalence of plant food allergies: a systematic review.

BACKGROUND: There is uncertainty regarding the prevalence of allergies to plant food. OBJECTIVE: To assess the prevalence of allergies to plant food according to the different subjective and objective assessment methods. METHODS: Our systematic search of population-based studies (since 1990) in the literature database MEDLINE focused on fruits, vegetables/legumes, tree nuts, wheat, soy, cereals, and seeds. Prevalence estimates were categorized by food item and method used (food challenges, skin prick test, serum IgE, parent/self-reported symptoms), complemented by appropriate meta-analyses. RESULTS: We included 36 studies with data from a total of over 250,000 children and adults. Only 6 studies included food challenge tests with prevalences ranging from 0.1% to 4.3% each for fruits and tree nuts, 0.1% to 1.4% for vegetables, and < 1% each for wheat, soy, and sesame. The prevalence of sensitization against any specific plant food item assessed by skin prick test was usually < 1%, whereas sensitization assessed by IgE against wheat ranged as high as 3.6% and against soy as high as 2.9%. For fruit and vegetables, prevalences based on perception were generally higher than those based on sensitization, but for wheat and soy in adults, sensitization was higher. Meta-analyses showed significant heterogeneity between studies regardless of food item or age group. CONCLUSION: Population-based prevalence estimates for allergies to plant products determined by the diagnostic gold standard are scarce. There was considerable heterogeneity in the prevalence estimates of sensitization or perceived allergic reactions to plant food.

Purification and structural stability of the peach allergens Pru p 1 and Pru p 3.

Pru p 1 (a Bet v 1 homologue) and Pru p 3 (a nonspecific lipid transfer protein; nsLTP) are major allergenic proteins in peach fruit, but differ in their abundance and stability. Pru p 1 has low abundance and is highly labile and was purified after expression as a recombinant protein in Escherichia coli. Pru p 3 is highly abundant in peach peel and was purified by conventional methods. The identities of the proteins were confirmed by sequence analysis and their masses determined by MS analysis. The purified proteins reacted with antisera against related allergens from other species: Pru p 1 with antiserum to Bet v 1 and Pru p 3 with antiserum to Mal d 3 (from apple). The presence of secondary and tertiary structure was demonstrated by circular dichroism (CD) and high field NMR spectroscopy. CD spectroscopy also showed that the two proteins differed in their stability at pH 3 and in their ability to refold after heating to 95 degrees C. Thus, Pru p 1 was unfolded at pH 3 even at 25 degrees C but was able to refold after heating to 95 degrees C at pH 7.5. In contrast, Pru p 3 was unable to refold after heating under neutral conditions but readily refolded after heating at pH 3.

Measurement of lipid transfer protein in 88 apple cultivars.

BACKGROUND: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. OBJECTIVE: Determination of the levels of LTP in a wide range of apple cultivars. METHODS: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). RESULTS: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson's correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4-253.2 (sELISA), 2.7-120.2 (cELISA) and 0.4-47.3 microg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. CONCLUSIONS: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens.

The prevalence of food allergy: a meta-analysis.

BACKGROUND: There is uncertainty about the prevalence of food allergy in communities. OBJECTIVE: To assess the prevalence of food allergy by performing a meta-analysis according to the method of assessment used. METHODS: The foods assessed were cow's milk, hen's egg, peanut, fish, shellfish, and an overall estimate of food allergy. We summarized the information in 5 categories: self-reported symptoms, specific IgE positive, specific skin prick test positive, symptoms combined with sensitization, and food challenge studies. We systematically searched MEDLINE and EMBASE for publications since 1990. The meta-analysis included only original studies. They were stratified by age groups: infant/preschool, school children, and adults. RESULTS: A total of 934 articles were identified, but only 51 were considered appropriate for inclusion. The prevalence of self-reported food allergy was very high compared with objective measures. There was marked heterogeneity between studies regardless of type of assessment or food item considered, and in most analyses this persisted after age stratification. Self-reported prevalence of food allergy varied from 1.2% to 17% for milk, 0.2% to 7% for egg, 0% to 2% for peanuts and fish, 0% to 10% for shellfish, and 3% to 35% for any food. CONCLUSION: There is a marked heterogeneity in the prevalence of food allergy that could be a result of differences in study design or methodology, or differences between populations. CLINICAL IMPLICATIONS: We recommend that measurements be made by using standardized methods, if possible food challenge. We need to be cautious in estimates of prevalence based only on self-reported food allergy.

Lipid transfer protein allergy: primary food allergy or pollen/food syndrome in some cases.

PURPOSE OF REVIEW: To summarize recent findings on non-specific lipid transfer proteins in food allergy, with a specific focus on the localization, stability and route of sensitization. RECENT FINDINGS: Plant non-specific lipid transfer proteins are major food allergens, especially in the Mediterranean area. They have been identified as allergens in a number of foods and the list grows ever longer. As non-specific lipid transfer proteins are considered to be "true" food allergens that sensitize directly via the gastrointestinal tract their stability during food processing and gastric digestion has been studied in more detail. In addition, several groups have tried to determine the sensitization patterns of lipid transfer protein-reactive patients, to determine and possibly clarify the observed geographical differences in sensitization. Different sensitization routes (via the respiratory tract or even transdermally) have been suggested. SUMMARY: As the structure and molecular properties of non-specific lipid transfer proteins are resolved and more purified non-specific lipid transfer proteins become available for diagnostic purposes, detailed studies on the sensitization pattern and route are becoming feasible. Continuing studies on the pattern of lipid transfer protein sensitization will give more insight into the development and possible treatment of lipid transfer protein-related food allergy.

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.

Severe immediate allergic reactions to grapes: part of a lipid transfer protein-associated clinical syndrome.

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

Effect of in vitro gastric and duodenal digestion on the allergenicity of grape lipid transfer protein.

BACKGROUND: Severe grape allergy has been linked to lipid transfer protein (LTP) sensitization. LTPs are known to be resistant to pepsin digestion, although the effect of gastroduodenal digestion on its allergenicity has not been reported. OBJECTIVE: We sought to investigate the effect of gastric and gastroduodenal digestion on the allergenic activity of grape LTP. METHODS: The proteolytic stability of grape LTP was investigated by using an in vitro model of gastrointestinal digestion. The allergenicity of LTP and its digesta was assessed in vitro by means of IgE immunoblotting, RASTs, and in vivo skin prick tests in the same patients with grape allergy. RESULTS: Grape LTP was resistant to gastric digestion, and yielded a 6000-d relative molecular mass C-terminally trimmed fragment after duodenal digestion. This fragment retained the in vitro IgE reactivity of the intact protein. Inclusion of phosphatidylcholine during gastric digestion protected the LTP to a limited extent against digestion. Digestion did not affect the in vivo (skin prick test) biologic activity of LTP. CONCLUSION: The allergenic activity of grape LTP was highly resistant to in vitro digestion. This property might facilitate sensitization through the gastrointestinal tract and might also potentiate the ability of LTPs to elicit severe allergic reactions in sensitized individuals. CLINICAL IMPLICATIONS: Purified natural allergens will facilitate the development of component-resolved diagnostic approaches, including allergen chips. This study contributes to our understanding of the role digestion plays in symptom elicitation in true food allergy.

Apple allergy across Europe: how allergen sensitization profiles determine the clinical expression of allergies to plant foods.

BACKGROUND: Allergy to a plant food can either result from direct sensitization to that food or from primary sensitization to pollen, latex, or another food. OBJECTIVE: We sought to investigate the primary sensitizers in apple allergy across Europe, the individual allergens involved, and whether these differences determine the clinical presentation. METHODS: Patients (n = 389) with positive case histories and skin prick test responses to fresh apple were selected in the Netherlands, Austria, Italy, and Spain. Skin prick tests and RASTs to a panel of pollens and plant foods were performed, as well as RASTs to Bet v 1 and the apple allergens Mal d 1, 2, 3, and 4. RESULTS: In the Netherlands, Austria, and Italy apple allergy is mild (>90% isolated oral symptoms) and related to birch pollinosis and sensitization to Bet v 1 and its apple homologue, Mal d 1, which has an odds ratio of local reactions of 2.85 (95% CI, 1.47-5.55). In Spain apple allergy is severe (>35% systemic reactions) and related to peach allergy and sensitization to Mal d 3 (nonspecific lipid transfer protein), which has an odds ratio of systemic reactions of 7.76 (95% CI, 3.87-15.56). CONCLUSION: The analysis of individual apple allergens in a clinical context has provided insight into the sensitization pathway and into the intrinsic risk an allergen bears to induce mild or severe food allergy. CLINICAL IMPLICATIONS: Information on the sensitization pathway is essential to develop preventive strategies in food allergy. The application of individual food allergens with a known intrinsic risk will improve the prognostic value of diagnostic tests.

Effect of postharvest storage on the expression of the apple allergen Mal d 1.

Consumption of fresh apples can cause allergy in susceptible individuals. A competitive enzyme-linked immunosorbent assay (ELISA) has been developed to determine Mal d 1 levels in apple pulp using a monoclonal antibody (BIP-1). The ELISA was able to rank ten cultivars according to their Mal d 1 content (between 3.8 and 72.5 mug/g pulp). For the first time, it has been demonstrated that growing conditions and postharvest storage, using three different treatments over a 5 month period in 2 consecutive years, increase Mal d 1 expression at a translational and transcriptional level (3.5- and 8.5-fold under controlled atmosphere storage). Expression of three major Mal d 1 isoforms was observed by real-time polymerase chain reaction over the 5 month storage period, and Mal d 1.02 was the most highly expressed isoform. In conclusion, Mal d 1 gene expression was significantly increased during modified atmosphere storage. Individuals suffering from birch pollen-apple allergy syndrome might experience fewer problems consuming freshly picked apples.

Maturity and storage influence on the apple (Malus domestica) allergen Mal d 3, a nonspecific lipid transfer protein.

Consumption of apples can provoke severe allergic reactions, in susceptible individuals, due to the presence of the allergen Mal d 3, a nonspecific lipid transfer protein, found largely in the fruit skin. Levels of Mal d 3 were determined in peel as a function of apple cultivar, position of the fruit growing on the tree, apple maturity, and postharvest storage by ELISA. As the apples mature, Mal d 3 levels increased, although the rate was dependent on cultivar and tree position. During storage, levels of Mal d 3 decreased in all cultivars (cvs. Cox, Jonagored, and Gala), the rate of overall decrease being greatest under controlled atmosphere conditions. There was no correlation between Mal d 3 levels and total apple peel protein, indicating specific alterations in Mal d 3 expression. Thus pre- and postharvest treatments (i.e., storage) can modify the allergen load in apple peel, the highest levels being found in overly mature and freshly harvested fruits.