Roasted hazelnuts--allergenic activity evaluated by double-blind,placebo-controlled food challenge

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.     

A mutant of the major apple allergen, Mal d 1, demonstrating hypo-allergenicity in the target organ by double-blind placebo-controlled food challenge

BACKGROUND: Allergen-specific immunotherapy for food allergy has been hindered by severe side-effects in the past. Well-characterized hypo-allergenic recombinant food allergens potentially offer a safe solution. OBJECTIVE: To demonstrate hypo-allergenicity of a mutated major food allergen from apple, Mal d 1, in vitro and in vivo. METHODS: A mutant of the major apple allergen, Mal d 1, was obtained by site-directed mutagenesis exchanging five amino acid residues. Fourteen patients with combined birch pollen-related apple allergy were included in the study. Hypo-allergenicity of the mutant rMal d 1 (rMal d 1mut) compared with rMal d 1 was assessed by in vitro methods, i.e. RAST (inhibition), immunoblotting and basophil histamine release (BHR) and in vivo by skin prick test and double-blind placebo-controlled food challenge (DBPCFC). RESULTS: RAST analysis (n = 14) revealed that IgE reactivity to rMal d 1mut was twofold lower than that of the wild-type molecule (95% confidence interval (CI): 1.7-2.4). RAST inhibition (n = 6) showed a 7.8-fold decrease in IgE-binding potency (95% CI: 3.0-12.6). In contrast to this moderate decrease in IgE-binding potency, the biological activity of rMal d 1mut assessed by SPT and BHR decreased 10-200-fold. Hypo-allergenicity was confirmed by DBPCFC (n = 2) with both recombinant molecules. CONCLUSION: A moderate decrease in IgE-binding potency translates into a potent inhibition of biological activity. This is the first study that confirms by DBPCFC that a mutated recombinant major food allergen is clinically hypo-allergenic. This paves the way towards safer immunotherapy for the treatment of food-allergic patients.     

Polyphenol‐enriched apple extract attenuates food allergy in mice

Background The immune system may be modulated with nutrition to prevent the development or to treat the symptoms of allergy. Among other foods, consumption of apples has been linked to reduced incidence of atopic dermatitis and respiratory allergy. Objective We evaluated the efficacy and mechanisms of a polyphenol‐enriched apple extract in reducing symptoms of food allergy. Methods In a model of food allergy to ovalbumin (OVA), BALB/c mice were fed with an apple extract either during sensitization or just before the challenge. After the challenge, allergic symptoms were scored, OVA‐specific serum immunoglobulins were determined by ELISA, cytokine production by mesenteric lymph node (MLN) cells was measured by a multiplex assay and gene expression profiles in the intestine were addressed using quantitative real‐time PCR. Results Consumption of the apple extract reduced symptoms of food allergy upon challenge. This was paralleled by reduced levels of intestinal mast cell protease, diminished cytokine secretion by MLN cells and reduced local intestinal mRNA expression of various T‐helper type‐2 associated and pro‐inflammatory genes. Mechanistic studies suggested decrease of mediator release by effector cells and reduction of allergenicity by protein–polyphenol interaction as potential mechanisms responsible for protection. Conclusion Polyphenol‐enriched apple extract can attenuate food allergy symptoms in sensitized mice via two distinct possible mechanisms.     

Outcome of double-blind, placebo-controlled food challenge tests in 107 children with atopic dermatitis

BACKGROUND AND OBJECTIVE: Little is known about late phase clinical reactions during oral food challenges and the value of specific IgE in terms of sensitivity and specificity. METHODS: We therefore analysed retrospectively the clinical outcome of 387 oral provocations during double-blind, placebo-controlled food challenge tests in 107 children with atopic dermatitis. RESULTS: Eighty-seven (81%) children showed a positive clinical reaction to at least one challenge. The vast majority of children (94%) showed clinical symptoms to one or two allergens. One hundred and thirty-one of 259 (51%) of verum challenges and 1/128 (0.8%) placebo challenge were assessed as positive. Oral provocations with hen's egg showed the highest percentage of positive reactions (70%). Sensitivity of specific IgE to the four allergens tested was 90%, specificity 30%. Sensitivity of the parental history as a predictive factor was 48%, specificity 72%. Ninety-two of 131 (70%) children with positive verum provocations showed early reactions, 33 (25%) late and six (5%) combined early and late reactions. In 84/131 (64%) positive provocations one organ system was involved, while in 44 (34%) provocations two and in three (2%) challenges three organ systems were involved. Skin reactions were the most frequent clinical manifestation leading to positive reactions followed by gastro-intestinal and respiratory symptoms. There was no correlation between titration dose and specific IgE. The subgroup of non-sensitized children did not differ in terms of sex, age or titration dose from the total study population. CONCLUSION: Double-blind, placebo-controlled oral food challenges are helpful in distinguishing children with clinically manifested symptoms from those with food sensitization. Accurately identifying children with a clinical relevant food allergy may help to prescribe specific diets on a scientific basis, avoiding dietary limitations which may be unnecessary or even harmful.     

Role of oral food challenges in the diagnostic work-up of food allergy in atopic eczema dermatitis syndrome

The diagnosis of food allergy in infants and children is still a challenging task for the pediatrician. While immediate-type allergic reactions to foods can be diagnosed quite easily, late-phase reactions, e.g. in atopic dermatitis, often represent a diagnostic challenge. Once classical diagnostic procedures such as history, skin prick tests, atopy patch test, and specific immunoglobulin E in serum have been exhausted, double-blind, placebo-controlled food challenges represent the state of the art. After an oligo-allergenic diet, suspected foods or placebo are given in a titrated manner until a clear clinical reaction or the highest dose. The observation period should be 48 h in the case of atopic dermatitis. Constant clinical monitoring is mandatory. Dietetic recommendations are given for 12 months. The effort involved in such a procedure is justified because it can help to avoid clinically relevant food allergens in some cases and in others can prevent children from being exposed unnecessarily to diets that may be harmful to them.     

Pitfalls in double-blind, placebo-controlled oral food challenges

Although controlled oral food challenges are considered to be the gold-standard in the diagnosis of food related symptoms, especially if performed in a double-blind, placebo-controlled food challenges (DBPCFC) manner, there are still many unanswered questions and newer aspects, which may explain some pitfalls encountered during oral food challenges. For stopping an oral food challenge and declaring a challenge as positive or negative, symptoms should be objective and/or repetitive. The time interval between administering the food and observing the clinical reaction is an ambivalent factor. Possible reasons for false negative assessments include inadvertent drug use during oral challenges, and the fact that a short-term specific oral tolerance induction (SOTI) may be induced as increasing amounts of the offended food are administered during a titrated oral food challenge. Possible reasons for false positive assessments are the difficulty to maintain an appropriate strict diet throughout the oral challenge procedure, and that the elimination diet implemented before the oral food challenge in children with atopic eczema and suspected food related symptoms may itself be responsible for immediate type clinical symptoms, which had not been reported by the parents before. Finally augmentation factors are among the most plausible explanations for the inadequate reproducibility of an oral food challenge. Although a 100% standardization of the challenge procedure does not seem realistic, efforts should be made to improve the methodology used so far. On the contrary, the possible relation of DBPCFC and SOTI may offer potential advantages for future therapeutic approaches of food allergy.     

Clinical characteristics of soybean allergy in Europe: a double-blind, placebo-controlled food challenge study

BACKGROUND: Soybean is a relevant allergenic food, but little is known about individual threshold doses in soy allergy. OBJECTIVE: We sought to determine the clinical characteristics of soy allergy in Europe, including a dose-response curve. METHODS: Patients with a history of soy allergy underwent a titrated, double-blind, placebo-controlled food challenge. A statistical model was used to calculate the risk of allergic consumers to experience an allergic reaction to soy. Sera were analyzed for specific IgE to soy, peanut, Bet v 1, and Gly m 4. RESULTS: All patients but one responded primarily with subjective symptoms to the challenge followed by objective symptoms in 11 subjects, ranging from rhinitis up to a decrease in blood pressure. Cumulative threshold doses for allergic reactions ranged from 10 mg to 50 g for subjective symptoms and from 454 mg to 50 g for objective symptoms. The pattern of IgE reactivity against proteins with molecular weights of between approximately 10 and 70 kd was highly individual among the patients and did not correlate with the severity of symptoms. CONCLUSIONS: When data are fitted by using a normal distribution statistical model, they predict that 1% of patients with soy allergy would react subjectively and objectively with 0.21 and 37.2 mg of soy protein, respectively. CLINICAL IMPLICATIONS: Both the clinical and immunologic basis of soy allergy in Europe are highly complex, which affects the diagnosis of soy allergy and the advice given to patients with soy allergy in regard to risk management.     

Recombinant Mal d 1 facilitates sublingual challenge tests of birch pollen‐allergic patients with apple allergy

It is still unclear whether allergen‐specific immunotherapy (AIT) with birch pollen improves birch pollen‐related food allergy. One reason for this may be the lack of standardized tests to assess clinical reactions to birch pollen‐related foods, for example apple. We tested the applicability of recombinant (r) Mal d 1, the Bet v 1‐homolog in apple, for oral challenge tests. Increasing concentrations of rMal d 1 in 0.9% NaCl were sublingually administered to 72 birch pollen‐allergic patients with apple allergy. The dose of 1.6 μg induced oral allergy syndromes in 26.4%, 3.2 μg in 15.3%, 6.3 μg in 27.8%, 12.5 μg in 8.3%, 25 μg in 11.1%, and 50 μg in 4.2% of the patients. No severe reactions occurred. None of the patients reacted to 0.9% NaCl alone. Sublingual administration of 50 μg of rMal d 1 induced no reactions in three nonallergic individuals. Our approach allows straight forward, dose‐defined sublingual challenge tests in a high number of birch pollen‐allergic patients that inter alia can be applied to evaluate the therapeutic efficacy of birch pollen AIT on birch pollen‐related food allergy.     

Introducing chemists to food allergy

Adverse reactions to food may be toxic or non toxic, depending on the susceptibility to a certain food; non toxic reactions that involve immune mechanisms are termed allergy if they are IgE-mediated. If no immunological mechanism is responsible, it is termed intolerance . The following disorders are considered a consequence of food allergy: gastrointestinal reactions (oral allergy syndrome, vomiting, diarrhea, protein-induced enterocolitic syndrome, eosinophilic gastroenteritis); respiratory reactions (rhinitis, asthma, laryngeal edema); cutaneous reactions (urticaria-angioedema, atopic dermatitis); anaphylaxis. There is much recent evidence to consider celiac disease an immunological disorder. Food allergy diagnosis is based on history, SPT, specific IgE, food challenges. DBPCFC is fundamental for diagnosing true food allergy; patients who have had anaphylaxis to food must not undergo DBPCFC. Rapidly progressive respiratory reactions and anaphylactic shock are life-threatening reactions that can be caused by food allergy. The doses of food inducing anaphylaxis can be very low, therefore commercial cross-contamination with an unsuspected food during food processing can be risky for the food allergic patient. The prevention of severe anaphylactic food reactions may lie in interdisciplinary collaboration among allergologists, chemists, food technologists, and experts in food industry research.     

Investigation of the stability of apple allergen extracts

To determine optimal conditions for allergen preservation, we investigated the influence of different stabilizing additives and of storage temperature on the allergen activity of apple protein preparations, obtained by extraction in phosphate buffer or by precipitation in diacetone alcohol and resolubilization in phosphate buffer in the presence or absence of enzyme inhibitors. For this purpose, the extracts were stored for 6 months either in frozen state at −20° C or in lyophilized state at −20° C, 4° C, or room temperature and were characterized by SDS-PAGE, immunoblot, ELISA inhibition, and prick test. The highest stability revealed the extracts that were prepared by precipitation in the organic solvent in the presence of enzyme inhibitors, lyophilized, and stored at −20° C. For storage of extract solutions at 4° C, PBS/glycerol and cysteine/sodium citrate/glycerol were found to be the most effective stabilizing additives.     

When is an oral food challenge positive?

Oral food challenges still remain the gold standard in the diagnosis of food related symptoms and are performed to obtain a clear 'yes or no' response. However, this is often difficult to achieve, and so proposals may be appropriate for criteria on when to stop oral food challenges. In daily practice it makes sense to challenge until clear objective symptoms occur without harming the patient. Clinical symptoms should be objective and/or: (a) severe or (b) reproducible or (c) persisting. A sensitive parameter for a beginning clinical reaction is a general change of mood. The sooner symptoms appear, the more likely they are to represent a 'true' positive reaction and the more organ systems are involved the easier it is to assess an oral food challenge as positive. In the case of subjective symptoms, the number of placebo doses should be increased. In unclear situations, the observation time until the next dose should be prolonged or the same dose repeated. Transient objective clinical symptoms usually end up in a positive challenge result. There are a number of causes for false positive and false negative challenge results, which should be considered. The aim of all oral challenge testing should be to hold the balance between two conflicting aspects: on the one hand the need to achieve clear and justified results from oral food challenges in order to avoid unnecessary diets, and on the other hand to protect patients from any harm caused by high doses of a potentially dangerous food.     

Placebo reactions in double-blind, placebo-controlled food challenges in children

BACKGROUND: A cardinal feature of the double-blind, placebo-controlled food challenge (DBPCFC) is that placebo administration is included as a control. To date, the occurrence and diagnostic significance of placebo events have not extensively been documented. OBJECTIVE: To analyse the occurrence and features of placebo events in DBPCFCs and to assess their contribution to the diagnostic accuracy of the DBPCFC in children. METHODS: The study population consisted of 132 challenges in 105 sensitized children (age range 0.7-16.6 years, median 5.3 years), who underwent DBPCFCs with cow's milk, egg, peanut, hazelnut and soy. Placebo and active food challenges were performed on different days. RESULTS: A total number of 17 (12.9%) positive placebo events occurred, which could be classified as immediate (9/17), late-onset (8/17), objective (11/17) or subjective (6/17). Four of 74 (5.4%) positive active food challenges were revealed to be false positive by administration of a placebo challenge. This is 3% (4/132) of all challenges. When computed by a statistical model, the false positive rate was 0.129 (12.9% of all challenges). CONCLUSION: Placebo events with diverse clinical characteristics occur in DBPCFCs in a significant number of children. The diagnostic significance of the administration of a placebo challenge is first, to identify false positive diagnoses in DBPCFCs by refuting false positive tests in individual patients. Secondly, to allow for blinding of the active food challenge. Thirdly, applying a statistical model demonstrates that some positive challenges may be false positive and that the test may need to be repeated in selected cases.     

How long does the effect of birch pollen injection SIT on apple allergy last?

Background: Recent studies showed that injection specific immunotherapy (SIT) with birch pollen extract greatly reduces or cures the associated apple allergy in a large proportion of birch pollen-allergic patients. However, the long-term efficacy of SIT for apple allergy has not been assessed. Objective: To evaluate the duration of the effect of injection SIT with birch pollen extract on apple allergy in birch pollen-allergic patients. Methods: Thirty birch pollen-allergic patients showing both the clinical disappearance of apple allergy and a negative SPT with fresh apple at the end of their injection SIT course were followed-up at 12-month intervals from 6 months after SIT was stopped. Apple tolerance as well as SPT was assessed on all occasions. Fifty-seven birch pollen-allergic subjects without apple allergy and not submitted to SIT regularly followed-up for the onset of oral allergy syndrome (OAS) were used as controls. Results: The overall prevalence of OAS after 30 months of follow-up did not differ between patients and controls. Although most patients became re-sensitized to apple by SPT over time, >50% of them were still able to tolerate eating the fruit at the 30-month follow-up visit. Conclusion: Although most patients show a ‘natural’, gradual propensity to apple re-sensitization (a consequence of prolonged and repeated inhalation of birch pollen responsible for primary sensitization?), the clinical effects of injection SIT on food allergy seem rather long lasting.     

Risk factors of adverse reactions to food in German adults

Background Allergic diseases have been increasing during the last decades. Previous studies suggest an impact of a variety of risk factors on the prevalence of food hypersensitivity. Objective Data of a cross‐sectional population‐based survey were analysed to study the prevalence of food hypersensitivity in females and males adjusted for age and education. Methods A population aged 18–79 years from a representative, randomly sampled survey studying 13 300 inhabitants from Germany (Berlin) was analysed. The Berlin study data were weighted with regard to age, sex, education and allergic diseases such as atopic dermatitis, rhinoconjunctivitis and asthma. Instruments for evaluation included mailed questionnaires, structured telephone interviews, physical examinations, detection of total IgE, skin prick tests and double‐blind, placebo‐controlled food challenge tests (DBPCFC). Results Three thousand two hundred and twenty‐seven questionnaires were evaluated. The data show a significantly higher risk of self‐reported symptoms in the female group, among persons with higher education and in the age group of 18–39 years. Among individuals with clinical symptoms, females were at lower risk of having positive skin prick tests [aOR=0.32; 95% CI (0.21–0.50); P<0.01] and having a raised total IgE [aOR=0.37; 95% CI (0.24–0.56); P<0.01], but showed a higher risk of non‐IgE and IgE‐mediated food hypersensitivity [aOR=2.27; 95% CI (1.31–3.93); P<0.01] than males. Based on weighted data, the point prevalence of adverse reactions to food resulted in 3.3% [95% CI (2.4–4.5%)] for women and 1.8% [95% CI (1.2–2.7%)] for men after DBPCFC. Conclusion From a general population survey conducted in Germany, we determined that women are at greater risk of having symptoms of food allergy and also at greater risk of having DBPCFC‐confirmed symptomatic food allergy. However, among individuals with symptoms of food allergy, men have a higher prevalence of food‐specific IgE‐sensitization and of raised total IgE than women.