Identification of a 62-kDa major allergen from Artemisia pollen as a putative galactose oxidase.

BACKGROUND: Around 20 years ago, a 60- to 70-kDa protein was reported as a major allergen of mugwort (Artemisia vulgaris) pollen. This study was to identify and characterize its molecular properties. METHODS: Sera from 113 Chinese and 20 Dutch Artemisia-allergic/sensitized subjects (and pools thereof) were used to identify the 60- to 70-kDa allergen. Pollen extracts of seven Artemisia species were compared by immunoblotting. Transcriptomics and proteomics (mass spectrometry) of A. annua pollen were used to identify the putative 60- to 70-kDa Artemisia allergen. Both the natural purified and recombinant allergens were evaluated for IgE reactivity by ImmunoCAP. Fourteen Chinese Artemisia-allergic patients were tested intradermally with purified natural allergen. RESULTS: Immunoblots revealed two major bands at 12 and 25 kDa, and a weak band at 70 kDa for all seven Artemisia species. Using a combined transcriptomic and proteomic approach, the high molecular mass allergen in A. annua pollen was shown to be a 62-kDa putative galactose oxidase, with a putative N-glycosylation site. More than 94% of Artemisia pollen-allergic patients had IgE response to this allergen. Although recognition of a nonglycosylated recombinant version was only confirmed in a minority (16%) and at much lower IgE levels, this discrepancy cannot be explained simply by reactivity to the carbohydrate moiety on the natural allergen. Intradermal testing with the natural allergen was positive in five of nine sensitized patients. CONCLUSIONS: The previously reported 60- to 70-kDa allergen of Artemisia pollen is most likely a 62-kDa putative galactose oxidase here designated Art an 7.

Allergic sensitization is associated with inadequate antioxidant responses in mice and men.

BACKGROUND: Allergies arise from aberrant Th2 responses to allergens. The processes involved in the genesis of allergic sensitization remain elusive. Some allergens such as derived from house dust mites have proteolytic activity which can induce oxidative stress in vivo. A reduced capacity of the host to control oxidative stress might prime for allergic sensitization. METHODS: Two different strains of mice were compared for their antioxidant and immune response to HDM. Protease activity of the HDM extract was reduced to investigate its role in oxidative stress induction in the airways and whether this induction could determine allergic sensitization and inflammation. The role of oxidative stress in allergic sensitization was also investigated in humans. An occupational cohort of animal workers was followed for the development of sensitization to rodent urinary proteins. Levels of oxidative stress in serum and antioxidant responses by PBMCs were determined. RESULTS: Susceptibility to allergic sensitization to mite allergens in mice was highly dependent on host genetic background and was associated with oxidative stress in the lungs before allergen exposure and poor antioxidant response after allergen exposure. Reduction in mite protease activity limited its capacity to induce oxidative stress and allergic inflammation in mice. We showed that also in human subjects, oxidative stress before allergen exposure and poor antioxidant responses were associated with predisposition to occupational allergy. CONCLUSION: Our study indicates that oxidative stress condition before allergen exposure due to an inadequate antioxidant response may prime for allergic Th2 responses.

The prevalence and distribution of food sensitization in European adults.

BACKGROUND: Complaints of 'food allergy' are increasing. Standardized surveys of IgE sensitization to foods are still uncommon and multicountry surveys are rare. We have assessed IgE sensitization to food-associated allergens in different regions of Europe using a common protocol. METHODS: Participants from general populations aged 20-54 years in eight European centres (Zurich, Madrid, Utrecht, Lodz, Sophia, Athens, Reykjavik and Vilnius) were asked whether they had allergic symptoms associated with specific foods. Weighted samples of those with and without allergic symptoms then completed a longer questionnaire and donated serum for IgE analysis by ImmunoCAP for 24 foods, 6 aeroallergens and, by allergen microarray, for 48 individual food proteins. RESULTS: The prevalence of IgE sensitization to foods ranged from 23.6% to 6.6%. The least common IgE sensitizations were to fish (0.2%), milk (0.8%) and egg (0.9%), and the most common were to hazelnut (9.3%), peach (7.9%) and apple (6.5%). The order of prevalence of IgE sensitization against different foods was similar in each centre and correlated with the prevalence of the pollen-associated allergens Bet v 1 and Bet v 2 (r = 0.86). IgE sensitization to plant allergen components unrelated to pollen allergens was more evenly distributed and independent of pollen IgE sensitization (r = -0.10). The most common foods containing allergens not cross-reacting with pollens were sesame, shrimp and hazelnut. DISCUSSION: IgE sensitization to foods is common, but varies widely and is predominantly related to IgE sensitization to pollen allergens. IgE sensitization to food allergens not cross-reacting with pollens is rare and more evenly distributed.

Impact of Food Matrices on Digestibility of Allergens and Poorly Allergenic Homologs.

Background: Protease resistance is considered a risk factor for allergenicity of proteins, although the correlation is low. It is nonetheless a part of the weight-of-evidence approach, proposed by Codex, for assessing the allergenicity risk of novel food proteins. Susceptibility of proteins to pepsin is commonly tested with purified protein in solution. Objective: Food proteins are rarely consumed in purified form. Our aim was to evaluate the impact of experimental and endogenous food matrices on protease susceptibility of homologous protein pairs with different degrees of allergenicity. Methods: Porcine and shrimp tropomyosin (ST) were subjected to sequential exposure to amylase, pepsin, and pancreatin in their respective endogenous matrix (pork tenderloin/boiled shrimp) and in three different experimental matrices (dessert mousse [DM], soy milk [SM], and chocolate bar [CB]). Digestion was monitored by immunoblotting using tropomyosin-specific antibodies. Recombinant peach and strawberry lipid transfer protein were biotinylated, spiked into both peach and strawberry fruit pulp, and subjected to the same sequential digestion protocol. Digestion was monitored by immunoblotting using streptavidin for detection. Results: Chocolate bar, and to a lesser extent SM, had a clear protective effect against pepsin digestion of porcine tropomyosin (PT) and to a lesser extent of ST. Increased resistance was associated with increased protein content. Spiking experiments with bovine serum albumin (BSA) confirmed the protective effect of a protein-rich matrix. The two tropomyosins were both highly resistant to pepsin in their protein-rich and lean native food matrix. Pancreatin digestion remained rapid and complete, independent of the matrix. The fat-rich environment did not transfer protection against pepsin digestion. Spiking of recombinant peach and strawberry lipid transfer proteins into peach and strawberry pulp did not reveal any differential protective effect that could explain differences in allergenicity of both fruits. Conclusions: Protein-rich food matrices delay pepsin digestion by saturating the protease. This effect is most apparent for proteins that are highly pepsin susceptible in solution. The inclusion of food matrices does not help in understanding why some proteins are strong primary sensitizers while homologs are very poor allergens. Although for induction of symptoms in food allergic patients (elicitation), a protein-rich food matrix that may contribute to increased risk, our results indicate that the inclusion of food matrices in the weight-of-evidence approach for estimating the potential risks of novel proteins to become allergens (sensitization), is most likely of very limited value.

Multiplicity of cross-reactive epitopes on Bet v I as detected with monoclonal antibodies and human IgE.

Six monoclonal antibodies against Bet v I, the major cross-reactive allergen of birch pollen (Betula verrucosa), were obtained. Four did not react with fruits, but two monoclonal antibodies (mAbs) (5H8 and 9C11) were reactive with apple and other fruits. These two cross-reactive antibodies reacted with identical or overlapping sites, but differed in their relative degree of cross-reactivity toward various fruits and hazelnut. Cross-reactive human IgE antibodies reacted with a nonoverlapping epitope, as indicated by results of a two-site radioimmunoassay (RIA) with the fruit-reactive mAb 9C11. By isoelectric focusing (IEF) in conjunction with immunoblotting, a maximum of seven isoforms could be distinguished. Depletion of birch-pollen extract for Bet v I with the most reactive mAb (7F7) removed approximately 95% of the IgE cross-reactivity between birch pollen and apple extract. The remaining 5% cross-reactive material was still capable of inhibiting the binding of IgE to apple allergen completely, and was reactive with mAbs 5H8 and 3C4. By means of IEF/immunoblot, it was shown that these mAbs recognize an isoform of Bet v I that is poorly, if at all, recognized by mAb 7F7. These results illustrate the heterogeneity of Bet v I, both with respect to the cross-reactive sites as well as to the backbone structure. This type of heterogeneity has possible implications for the use of monoclonal antibodies in allergen standardization.

Vineyard snail allergy possibly induced by sensitization to house-dust mite (Dermatophagoides pteronyssinus).

A female patient experienced a severe allergic reaction after consumption of vineyard snails. The patient proved to be sensitized to house-dust mite (HDM) and demonstrated a positive skin test and specific IgE to snail (Eobania vermiculata, Lofarma). The snail RAST was > 80% inhibited by HDM, whereas the mite RAST was < 10% inhibited by snail extract. This is possibly another example of food allergy related to primary sensitization by an aeroallergen.

How far can we simplify in vitro diagnostics for Fagales tree pollen allergy? A study with three whole pollen extracts and purified natural and recombinant allergens.

BACKGROUND: Current diagnostic tests for Fagales tree pollen allergy are often composed of mixtures of pollen of birch, alder and hazel. Their complex composition hampers accurate standardization. OBJECTIVE: The aim of this study was to investigate whether mixtures of tree pollen extracts can be replaced by a single pollen species, and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens. METHODS: Sera (n = 1725) were selected on ground of a general suspicion for inhalant allergy, and tested in a RAST for birch, alder and hazel pollen. Sera with > 0.5 RU/mL for any of the three species were tested in a RAST for natural Bet v 1 and Bet v 2 as well as for recombinant versions of both allergens. RESULTS: Specific IgE antibodies (> 0.3 RU/mL) against birch, alder and hazel were found in 242, 298 and 292 sera, respectively. All sera with a positive RAST for alder and/or hazel and a negative RAST for birch were low-responder sera on alder and hazel, only five sera having a RAST value > 1.0 (all < 2.0). For all sera with a RAST > 0.5 RU/mL (n = 250), the mean of individual ratio's alder/birch and hazel/birch was 1.02 and 0.54, respectively. Of 223 of these sera, 63.2% had specific IgE against natural Bet v 1 and 63.7% against natural Bet v 2. When responses to both allergens were combined 93.7% were positive. The mean ratios Bet v 1 + 2/extract were 1.00, 1.04 and 2. 11 in case of birch, alder and hazel, respectively. For 211 sera the same analysis was performed with recombinant Bet v 1 and Bet v 2. Only six sera with Bet v 1-specific IgE (all < 0.5 RU/mL) were negative (< 0.3 RU/mL) on recombinant Bet v 1. For Bet v 2, 77/132 sera with specific IgE to the natural allergen did not react to the recombinant version. Twelve false-negatives had RAST values > 1.0 RU/mL. The mean of the individual recombinant/natural ratios was 0. 98 for Bet v 1 and 0.38 for Bet v 2 (P < 0.001). The mean ratio rBet v 1 + 2/birch was 0.75 with 17.5% false-negatives on the combination of recombinant allergens. CONCLUSION: Reliable in vitro diagnosis is possible with a single tree pollen extract (birch or alder). The same is true for purified natural Bet v 1 and Bet v 2. A combination of recombinant molecules is slightly less efficient.

Identification of a cross-reactive allergen (presumably tropomyosin) in shrimp, mite and insects.

A monoclonal antibody to Dermatophagoides pteronyssinus is described that cross-reacts with an IgE-binding antigen present in insects, Crustacea (e.g. shrimp) and other invertebrates. By means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration and immunofluorescence it was shown that this monoclonal antibody presumably recognizes tropomyosin. Tropomyosin was shown to be involved in cross-reactivity between mite, shrimp and insects in shrimp-allergic patients.

Possible induction of food allergy during mite immunotherapy.

Sera of 17 patients receiving immunotherapy for house-dust mite allergy were tested for IgE antibodies against snail and shrimp. Serum samples were taken at the start of immunotherapy and 14-20 months later. While the average IgE response to mite, Der p 1, and Der p 2 did not alter significantly, the average response to snail showed a significant increase. This included two conversions from negative to strongly positive. These novel IgE antibodies against snail were shown to be cross-reactive with mite. Three patients had a positive RAST for shrimp. For one of them, a strong increase of IgE against shrimp (and snail) was observed. In 2/3 snail/shrimp-positive sera, IgE antibodies against the cross-reactive allergen tropomyosin from mite, snail, and shrimp were demonstrated. A clear IgE response to snail (> 10% binding in a snail RAST) was confirmed by a positive skin prick test (SPT) for 6/10 patients. The two patients with antitropomyosin IgE also had a positive SPT for shrimp, and demonstrated the oral allergy syndrome (OAS) after eating shrimp. The observations in this study indicate that house-dust mite immunotherapy is accompanied by the induction of IgE against foods, including tropomyosin-reactive IgE. Food allergy (OAS) was observed in patients that had IgE antibodies against this cross-reactive allergen. In conclusion, induction of IgE during mite immunotherapy might occasionally cause allergy to foods of invertebrate animal origin.

Asthma after consumption of snails in house-dust-mite-allergic patients: a case of IgE cross-reactivity.

A group of 28 patients from Italy was studied who had asthma after consumption of snail. All patients also had asthma and/or rhinitis caused by house-dust mite. RAST analyses confirmed the combined sensitization to snail and mite. In a few sera, IgE antibodies reactive with other foods of invertebrate origin (mussel and shrimp) were detected. RAST inhibition showed that most IgE antibodies against snail were cross-reactive with house-dust mite. In contrast, the mite RAST was not significantly inhibited by snail. This indicates that house-dust mite was the sensitizing agent. Immunoblot analyses revealed multiple bands in snail extract recognized by IgE. In contrast to what has been described for cross-reactivity between shrimp and mite, tropomyosin played only a minor role as a cross-reactive allergen in these patients. The observations in this study indicate that snail consumption can cause severe asthmatic symptoms in house-dust-mite-allergic patients. It might, therefore, be advisable to screen mite-allergic asthma patients for allergy to snail and other invertebrate animal foods.

Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion.

BACKGROUND: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. OBJECTIVE: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. RESULTS: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. CONCLUSION: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion.

BACKGROUND: Lipid transfer proteins (LTPs) are stable and highly conserved proteins of around 10 kD. They have recently been identified as allergens in fruits of the Rosaceae family. OBJECTIVE: The aim of this study was to investigate whether the highly conserved structure of LTPs justifies a designation as a true pan-allergen, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive skin prick test to Rosaceae fruit extracts were characterized by interviews and skin prick tests. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified natural and recombinant LTPs. To address the role of protein stability in the allergenicity of LTP, fruit extracts and LTPs were digested with pepsin. RESULTS: IgE antibodies to Rosaceae LTPs cross-reacted with a broad range of non-Rosaceae vegetable foods. Inhibition studies with purified natural and recombinant LTPs confirmed the role of LTP in this cross-reactivity. Many of the patients with this type of cross-reactive IgE antibodies had a clinical food allergy. In contrast to the typical birch Rosaceae cross-reactive patients, the oral allergy syndrome was frequently accompanied by more severe and systemic reactions. IgE reactivity to LTP was shown to be resistant to pepsin treatment of the allergen. CONCLUSION: LTP is a true pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.

The range of minimum provoking doses in hazelnut-allergic patients as determined by double-blind, placebo-controlled food challenges.

BACKGROUND: The risk for allergic reactions depends on the sensitivity of individuals and the quantities of offending food ingested. The sensitivity varies among allergic individuals, as does the threshold dose of a food allergen capable of inducing an allergic reaction. OBJECTIVE: This study aimed at determining the distribution of minimum provoking doses of hazelnut in a hazelnut-allergic population. METHODS: Thirty-one patients with a history of hazelnut-related allergic symptoms, a positive skin prick test to hazelnut and/or an elevated specific IgE level, were included. Double-blind, placebo-controlled food challenges (DBPCFC) were performed with seven increasing doses of dried hazelnut (1 mg to 1 g hazelnut protein) randomly interspersed with seven placebo doses. RESULTS: Twenty-nine patients had a positive challenge. Itching of the oral cavity and/or lips was the first symptom in all cases. Additional gastrointestinal symptoms were reported in five patients and difficulty in swallowing in one patient. Lip swelling was observed in two patients, followed by generalized urticaria in one of these. Threshold doses for eliciting subjective reactions varied from a dose of 1 mg up to 100 mg hazelnut protein (equivalent to 6.4-640 mg hazelnut meal). Extrapolation of the dose-response curve showed that 50% of our hazelnut-allergic population will suffer from an allergic reaction after ingestion of 6 mg (95% CI, 2-11 mg) of hazelnut protein. Objective symptoms were observed in two patients after 1 and 1,000 mg, respectively. CONCLUSION: DBPCFCs demonstrated threshold doses in half of the hazelnut-allergic patients similar to doses previously described to be hidden in consumer products. This stresses the need for careful labelling and strategies to prevent and detect contamination of food products with hazelnut residues.