Laurian Zuidmeer-Jongejan

Sensitization to Cor a 9 and Cor a 14 is highly specific for a hazelnut allergy with objective symptoms in Dutch children and adults

BACKGROUND Component-resolved diagnosis has been shown to improve the diagnosis of food allergy. OBJECTIVE We sought to evaluate whether component-resolved diagnosis might help to identify patients at risk of objective allergic reactions to hazelnut. METHOD A total of 161 hazelnut-sensitized patients were included: 40 children and 15 adults with objective symptoms on double-blind, placebo-controlled food challenges (DBPCFCs) and 24 adults with a convincing objective history were compared with 41 children and 41 adults with no or subjective symptoms on DBPCFCs (grouped together). IgE levels to hazelnut extract and single components were analyzed with ImmunoCAP. RESULTS IgE levels to hazelnut extract were significantly higher in children with objective than with no or subjective symptoms. In 13% of children and 49% of adults with hazelnut allergy with objective symptoms, only sensitization to rCor a 1.04 was observed and not to other water-soluble allergens. Sensitization to rCor a 8 was rare, which is in contrast to rCor a 1. Sensitization to nCor a 9, rCor a 14, or both was strongly associated with hazelnut allergy with objective symptoms. By using adapted cutoff levels, a diagnostic discrimination between severity groups was obtained. IgE levels to either nCor a 9 of 1 kUA/L or greater or rCor a 14 of 5 kUA/L or greater (children) and IgE levels to either nCor a 9 of 1 kUA/L or greater or rCor a 14 of 1 kUA/L or greater (adults) had a specificity of greater than 90% and accounted for 83% of children and 44% of adults with hazelnut allergy with objective symptoms. CONCLUSION Sensitization to Cor a 9 and Cor a 14 is highly specific for patients with objective symptoms in DBPCFCs as a marker for a more severe hazelnut allergic phenotype.

Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut

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.

Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity

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.

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Hazelnut allergy across Europe dissected molecularly: A EuroPrevall outpatient clinic survey.

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.

Peanut allergy is common among hazelnut-sensitized subjects but is not primarily the result of IgE cross-reactivity.

Hazelnut and peanut are botanically unrelated foods, but patients are often sensitized and allergic to both, for reasons that are not well understood.

Oral Delivery of a Probiotic Induced Changes at the Nasal Mucosa of Seasonal Allergic Rhinitis Subjects after Local Allergen Challenge: A Randomised Clinical Trial

Objective To determine effects of probiotic consumption on clinical and immunological parameters of seasonal allergic rhinitis (SAR) in an out-of-season single nasal allergen challenge. Methods In a study registered at ClinicalTrials.Gov (NCT01123252), a 16-week dietary intervention was undertaken in 60 patients with allergic rhinitis (>16 years old). Using a double-blinded, placebo-controlled anonymised design, the patients were divided equally into two groups. One group was given a dairy drink containing Lactobacillus casei Shirota to ingest daily while the other consumed a similar drink without bacteria. Participants attended the clinic on two consecutive days before the intervention and then again at the end of the study period. On the first day of each 2-day visit, following clinical examination, assessments were made of total nasal symptoms scores and peak nasal inspiratory flow. Nasal scrapings, nasal lavage and blood were collected for laboratory analyses of cellular phenotypes, soluble mediator release and in vitro responses to pollen allergen. These procedures were repeated 24 hours following nasal allergen challenge. Results Prior to and following intervention there were no detectable differences between study groups in measured clinical outcome. After intervention, there were differences between groups in their percentages of CD86+ epithelial cells (p = 0.0148), CD86+CD252+ non-epithelial cells (p = 0.0347), sIL-1RII release (p = 0.0289) and IL-1β (p = 0.0224) levels at the nasal mucosa. Delivery of probiotic also suppressed production of sCD23 (p = 0.0081), TGF-β (p = 0.0283) and induced increased production of IFN-γ (p = 0.0351) in supernatants of cultured peripheral blood. Conclusions & Clinical Relevance This study did not show significant probiotic-associated changes with respect to the primary clinical endpoint. An absence of overt clinical benefit may be due to an inability of single nasal challenges to accurately represent natural allergen exposure. Nevertheless, oral delivery of probiotics produced changes of the immunological microenvironment at the nasal mucosa in individuals affected by SAR. Trial Registration ClinicalTrials.Gov NCT01123252

Effect of Processing on the Allergenicity of Foods

Proteins involved in sensitizing or eliciting allergic reactions may have undergone extensive modification during food processing and be present within complex structures within food. These physicochemical changes will alter the way in which proteins are broken down during digestion and may modify the form in which they are taken up across the gut mucosal barrier and presented to the immune system. Data on how processing might affect sensitization to food are currently sparse. However, much more data are available, which are beginning to give insights into how food processing may affect elicitation of allergic reactions, particularly in affecting the binding of IgE, which lies at the heart of triggering an allergic reaction. The chapter summarizes current knowledge of the effects of processing on the major food allergens of plant and animal origin as well as the effects of processing of whole foods.

The choice of hypoallergens for fish and peach to develop food allergy specific immunotherapy (the FAST project)

Background Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with food extracts, may be effective but dangerous due to anaphylactic side-effects. The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting persistent and severe allergy to fish (cod) and fruit (peach). Both are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. FAST will apply hypo-allergenic recombinant major allergens for SIT.

Development of a hypoallergenic and immunogenic Pru p 3 proline variant for treatment of peach allergy

Methods We deployed an in-silico mutagenesis approach to design a fold variant of recombinant Pru p 3. Four stability hot spot residues were identified and proline was predicted as the most effective replacement amino acid. Pru p 3 C1 was produced in E.coli using a pET-based expression system. After refolding and purification by cation exchange chromatography, the protein was characterized by reducing and non-reducing gel electrophoresis, circular dichroism spectroscopy, size exclusion chromatography, dynamic light scattering and mass spectrometry (MS). An accelerated stability test was performed by storing the protein up to 6 month at temperatures ranging from -70°C to +40°C.The IgE binding capacity of Pru p 3 C1 and WT Pru p 3 was tested in ELISA using sera from peach allergic patients. Different adjuvants and adsorption conditions were evaluated for use in a mouse model. Mice were s.c. immunized with Pru p 3 C1 and WT Pru p 3 and sera were analyzed for IgG reactivity.