Clinical reactivity to hazelnut in children: association with sensitization to birch pollen or nuts?
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Adolescent↗
Biomedical subjects
Jaap H Akkerdaas
Publications and source records attributed to Jaap H Akkerdaas.
Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library.
The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA library of hazelnut was constructed. The library was screened with serum of six hazelnut allergic patients displaying different IgE-binding patterns on hazelnut immunoblot. Rapid amplification of cDNA ends (RACE) protocols were applied to obtain full-length clones. Expression experiments were carried out in Eschericchia coli. Expression was monitored by SDS-PAGE, protein staining and immunoblotting. A hazelnut cDNA library was constructed. IgE screening resulted in the cloning of two isoforms of a novel putative hazelnut allergen. The clones were identified as oleosins, with theoretical molecular masses of 16.7 and 14.7 kDa and pI of 10.5 and 10.0, respectively. The isoforms demonstrated only 37% amino acid sequence identity but contained the typical hydrophobic stretch in the middle of the protein (53% identity) with the characteristic oleosin proline knot region (11/12 amino acids identical). Expression in E. coli of the longer isoform resulted in a clear band on SDS-PAGE. The expressed protein was recognized on an immunodot blot by IgE from serum that was used for screening the cDNA library. Hazelnut contains multiple isoforms of oleosin. IgE binding of a hazelnut-allergic patient to a recombinant version suggest that hazelnut oleosin is an allergen, as has been described for peanut and sesame.
Amino Acid Sequence↗
IgE binding to pepsin-digested food extracts.
BACKGROUND: Pepsin resistance of allergens like lipid transfer protein and 2S albumin has been suggested as explanation for the severity of symptoms often induced by these allergens. Component-resolved diagnosis with purified labile and stable allergens has therefore been proposed to better characterize the risk involved in a positive in vitro IgE test. However, for many foods, purified allergens are not (yet) available. OBJECTIVE: It was the aim of this study to evaluate the potential of pepsin-digested whole-food extracts to distinguish between IgE responses to stable (potentially severe) and labile (mild) allergens. METHODS: Sera (n = 143) from Italian, Spanish and Dutch patients with hazelnut and/or apple ingestion-related symptoms were analyzed for residual IgE binding to pepsin-resistant hazelnut and/or apple allergens. Control and pepsin-digested hazelnut and apple extracts were used for radioallergosorbent test analysis and immunoblot analysis. RESULTS: Pepsin digestion of food extracts, like from hazelnut and apple used for in vitro diagnostic tests, provides a way to distinguish sensitization to pepsin-resistant allergens from that to pepsin-susceptible allergens. In this selected group of patients, IgE reactivity to pepsin-digested extracts correlated with sensitization to the stable allergen lipid transfer protein. The analysis further revealed that the use of soluble pepsin can result in false-positive in vitro tests (2/143). CONCLUSION: Pepsin-digested food extracts are a convenient tool to identify patients with IgE antibodies against potentially dangerous stable allergens, in particular for those foods where the relevant stable allergens have not yet been identified. This can increase the clinical prognostic value of food allergy serology.
Allergens↗
Lipid transfer proteins from fruit: cloning, expression and quantification.
BACKGROUND: Lipid transfer proteins (LTP) are stable, potentially life-threatening allergens in fruits and many other vegetable foods. The aim of this study was to clone and express recombinant apple LTP (Mal d 3), as has previously been done for peach LTP (Pru p 3) and set up quantitative tests for measuring fruit LTPs. METHODS: cDNA for Mal d 3 and Pru p 3 was cloned, expressed in the yeast Pichia pastoris and the resulting proteins were purified via cation exchange chromatography. The immune reactivity of rMal d 3 was compared to nMal d 3 by RAST (inhibition), immunoblotting and basophil histamine release testing. To obtain monoclonal and monospecific polyclonal antibodies, mice and rabbits were immunized with purified nMal d 3. RESULTS: The deduced amino acid sequence of Mal d 3 was identical to the published sequence, Pru p 3 differed at two positions (S9A and S76H). The rMal d 3 had an IgE-binding potency and biological activity close to its natural counterpart. One sandwich ELISA selectively detecting apple LTP and another cross-reactive with cherry, nectarine and hazelnut LTP were developed. In addition, a competitive RIA was developed with polyclonal rabbit antiserum and labeled nMal d 3. CONCLUSION: rMal d 3 (as shown before for rPru p 3) may be a useful tool for application in component-resolved diagnosis of food allergy. Assays for the measurement of LTP will increase the traceability of this potentially dangerous allergen.
Allergens↗
A novel approach for the detection of potentially hazardous pepsin stable hazelnut proteins as contaminants in chocolate-based food.
Contamination of food products with pepsin resistant allergens is generally believed to be a serious threat to patients with severe food allergy. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to measure pepsin resistant hazelnut protein in food products. Capturing and detecting rabbit antibodies were raised against pepsin-digested hazelnut and untreated hazelnut protein, respectively. The assay showed a detection limit of 0.7 ng/mL hazelnut protein or <1 microg hazelnut in 1 g food matrix and a maximum of 0.034% cross-reactivity (peanut). Chocolate samples spiked with 0.5-100 microg hazelnut/g chocolate showed a mean recovery of 97.3%. In 9/12 food products labeled "may contain nuts", hazelnut was detected between 1.2 and 417 microg hazelnut/g food. It can be concluded that the application of antibodies directed to pepsin-digested food extracts in ELISA can facilitate specific detection of stable proteins that have the highest potential of inducing severe food anaphylaxis.
Cacao↗
How accurate and safe is the diagnosis of hazelnut allergy by means of commercial skin prick test reagents?
BACKGROUND: Allergy to tree nuts, like hazelnuts, ranks among the most frequently observed food allergies. These allergies can start at early childhood and are, in contrast to other food allergies, not always outgrown by the patient. Tree nut allergy is frequently associated with severe reactions. Diagnosis partially relies on in vivo testing by means of a skin prick test (SPT) using commercially available SPT reagents. METHODS: Protein and allergen composition of nine commercial SPT solutions was evaluated using standard protein detection methods and specific immunoassays for measurement of five individual allergens. Diagnostic performance was assessed by SPT in 30 hazelnut-allergic subjects, of which 15 were provocation proven. RESULTS: Protein concentrations ranged from 0.2-14 mg/ml. SDS-PAGE/silver staining revealed clear differences in protein composition. The major allergen Cor a 1 was present in all extracts but concentrations differed up to a factor 50. An allergen associated with severe symptoms, Cor a 8 (lipid transfer protein), was not detected on immunoblot in three products, and concentrations varied by more than a factor 100 as was shown by RAST inhibition. Similar observations were made for profilin, thaumatin-like protein and a not fully characterized 38-kD allergen. Ratios of individual allergens were variable among the nine extracts. SPT showed significant difference, and 6/30 patients displayed false-negative results using 3/9 products. CONCLUSION: Variability in the composition of products for the diagnosis of hazelnut allergy is extreme. Sometimes, allergens implicated in severe anaphylaxis are not detected by immunoblotting. These shortcomings in standardisation and quality control can potentially cause a false-negative diagnosis in subjects at risk of severe reactions to hazelnuts.
Adolescent↗
In vitro and in vivo characterization of hazelnut skin prick test extracts.
RATIONALE: Hazelnut allergy ranks among the most frequently observed food allergies. Clinical symptoms range from the oral allergy syndrome to life threatening anaphylaxis. Diagnosis of hazelnut allergy partially relies on in vivo testing by means of skin prick testing (SPT). The aim of this study was to characterize hazelnut SPT extracts both in vitro and in vivo. METHODS: Hazelnut SPT extracts were investigated for protein concentration and composition. The major hazelnut allergen Cor a 1, lipid transfer protein (LTP) and thaumatin-like-protein (TLP) were monitored by competitive RIA and immunoblotting. SPT extracts (n = 6) were analyzed for skin reactivity and the correlation between the SPT extract protein concentration and the mean skin reactivity (HEIC) was determined in a group of hazelnut-allergic patients (n = 30). For one SPT extract, the threshold level for Cor a 1 was determined in Cor a 1-monosensitized patients (n = 5). RESULTS: Protein concentrations ranged from 0.2-14 mg/ml. Although some proteins were present in most extracts (bands at 10, 22-28, 32 and around 48 kDa), clear differences in composition were observed (both intra- and inter-variability). The concentration of the major hazelnut allergen Cor a 1 differed up to a factor 50 (0.6-32 micrograms/ml). LTP was virtually absent in 3/9 SPT extracts and variable quantities of TLP were detected by immunoblotting. Some patients (6/30) had a false-negative SPT with 3/6 SPT extracts. There was a clear correlation between the protein concentration and the mean HEIC (RPearson = 0.87). The threshold level for Cor a 1 was +/- 3.2 ng/ml as assessed with one of the products investigated. CONCLUSIONS: Heterogeneous protein concentration/composition of SPT extracts results in variable skin test responses. The absence of potentially severe allergens like LTP may lead to false-negative SPT results that jeopardize a patient's safety. From these results it can be concluded that there is a strong need for standardization of products for SPT.
Humans↗
IgE to Bet v 1 and profilin: cross-reactivity patterns and clinical relevance.
BACKGROUND: Individuals with pollen allergy often have IgE against plant-derived foods. This can be due to cross-reactive IgE against Bet v 1 and homologues, profilins, and/or cross-reactive carbohydrate determinants. OBJECTIVE: The aim of this study was to correlate sensitization to Bet v 1 and profilin with individual recognition patterns to plant foods and clinical relevance. METHODS: Fifty-two patients with pollen allergy and IgE against at least one plant-derived food were included in the study. Adverse reactions to plant-derived foods were documented by using standardized interviews. Skin prick tests were performed for pollen (grass, birch, and mugwort) and 14 plant-derived foods. In addition, recombinant (r) Bet v 1 and rBet v 2 (profilin) were tested intracutaneously. Specific IgE against the abovementioned allergens were determined by means of RAST. Cross-reactivity was studied by means of RAST inhibition. RESULTS: Eighty-five percent of patients were sensitized to Bet v 1, and 71% were sensitized to profilin. Profilin was associated with a higher number of positive RAST results to plant-derived foods than Bet v 1. In contrast, Bet v 1 was associated with more positive skin prick test responses and more food-related symptoms. Sensitization to Bet v 1 was associated with IgE against apple, hazelnut, and peach, whereas sensitization to profilin was associated with positive RAST results to all investigated plant-derived foods except apple, peach, and melon. CONCLUSIONS: IgE antibodies against Bet v 1 have a more limited spectrum of cross-reactivity than those against profilin, but they frequently give rise to clinically relevant cross-reactivities to food. In analogy to anticarbohydrate IgE, cross-reactive IgE against food profilins have no or very limited clinical relevance.
Adolescent↗