Relative reactivity to egg white and yolk or change upon heating as markers for baked egg tolerance.

BACKGROUND: Hen's egg food allergy is frequent in childhood and phenotypically heterogeneous. Some children can tolerate extensively heated egg. We investigated whether individual relative responses could differentiate children who tolerate baked egg. METHODS: Reactivities to raw, pasteurized or hard-boiled egg (E), egg white (EW), and egg yolk (EY) fractions were tested by skin prick test (SPT) in 54 egg-allergic children. IgE-sensitization to EW and EY was determined by ImmunoCAP and IgE-binding to EW and 8 EW proteins and to EY and 4 EY sub-fractions by ELISA. Population heterogeneity was assessed by hierarchical ascending classification upon individual variations of reactivity and links between classifications and clinical features by analyzing the contingency tables. RESULTS: All children had positive SPT to raw E and raw EW and 72% to raw EY. Heating decreased SPT-reactivity for some children, pasteurization being less effective than hard-boiling. Children were classed into three classes from relative SPT-reactivity to raw fractions, two from variations of SPT-reactivity with each thermal processing or EW/EY ratio of sensitization, and four from their sensitization pattern. Classifications according to heating were found independent of each other. SPT variations with hard-boiling, IgE-sensitization (ratio or pattern) were linked to allowance by the physicians of egg in baked products. CONCLUSIONS: Egg-allergic children were often both sensitized to EY and EW, and heterogeneous patterns of relative responses were evidenced. Irrespective of age and level of sensitization, a low EW/EY ratio or SPT getting null with hard-boiling was found in children allowed to eat baked egg.

Digestion differently affects the ability of native and thermally aggregated ovalbumin to trigger basophil activation.

Ovalbumin (OVA), a major allergen from hen's egg albumen, tends to aggregate when heated. Depending on the balance of attractive and repulsive interactions, heat-induced OVA aggregates have various morphologies, which differ in digestibility. In the context of food allergy to egg, we investigated the ability of native and thermally aggregated OVA as well as their digests to induce the degranulation of a humanized rat basophil leukemia (RBL) cell line, which was sensitized with a pool of sera from egg-allergic children. Native and two thermally aggregated OVA forms were digested in vitro using a gastrointestinal digestion model based on the INFOGEST harmonized protocol including a final degradation with jejunal brush border membranes (BBM) enzymes. The course of digestion was monitored by the OPA method and by RP-HPLC. Digestibility was OVA small aggregates>OVA large aggregates>>native OVA and BBM peptidases only significantly hydrolyzed small-sized peptides from gastro-duodenal digests of the aggregates. The degranulation ability of the native OVA slightly changed during the gastric phase but mostly decreased during the duodenal digestion with no further change with BBM digestion. The degranulation ability of aggregates, which was significantly lower than the ability of native OVA, was not significantly affected by digestion. Digestibility and ability to induce basophil degranulation can thus not be straightforward linked.

An observational cohort study of the use of five-grass-pollen extract sublingual immunotherapy during the 2015 pollen season in France.

BACKGROUND: Allergic rhinitis affects around one quarter of the Western European population. Prophylactic allergen immunotherapy may be useful to reduce the risk of acute symptomatic attacks (hayfever). A five-grass pollen extract sublingual immunotherapy (5GPE-SLIT) has been developed for the treatment of allergic rhinitis to grass pollen. The objective of this study was to describe real-world treatment patterns with 5GPE-SLIT in France with respect to the prescribing information. METHODS: This prospective cohort study was conducted by 90 community and hospital allergists. Adults and children (> 5 years old) starting a first treatment with 5GPE-SLIT prior to the 2015 pollen season were eligible. Data was collected at the inclusion visit and at the end of the pollen season. The primary outcome variable was compatibility of 5GPE-SLIT prescription with the prescribing information. This was determined with respect to four variables: (1) interval between 5GPE-SLIT initiation and onset of the pollen season ≥ 3 months, (2) age of patient ≥ 5 years, (3) intermittent symptoms or mild symptom severity (4) confirmatory diagnostic test. At study end, symptoms reported during the pollen season and any modifications to treatment or adverse events were documented. RESULTS: 280 adults and 203 children were enrolled. The prescribing information was respected for 82.5% of adults and 86.7% of children. A skin test was performed for all patients. 5GPE-SLIT was started 3-5 months before the pollen season for 85.3%. Treatment was discontinued before the start of the pollen season in 11.0% of patients overall, generally because of an adverse event (78.8% of discontinuations). The mean duration of treatment was 5.2 months in adults and 5.6 months in children. At the end of follow-up, symptoms during the pollen season were intermittent for 75.0% of adults and 85.7% of children, and severity was mild for 61.8 and 66.0% respectively. During 5GPE-SLIT, the following symptoms reported during the previous year were not reported again in > 50% of patients: nasal congestion, rhinorrhoea, repeated sneezing, conjunctivitis and nasal pruritus. CONCLUSIONS: 5GPE-SLIT use was generally consistent with prescribing recommendations and was associated with an improvement of AR severity, with resolution of the principal AR symptoms in around half the patients treated.Trial registration EUPAS9358. Registered 13 May 2015. Not prospectively registered. http://www.encepp.eu/encepp/viewResource.htm?id=16229.

Immediate Hypersensitivity to Contrast Agents: The French 5-year CIRTACI Study.

BACKGROUND: Iodinated and gadolinium-based contrast media (ICM; GBCM) induce immediate hypersensitivity (IH) reactions. Differentiating allergic from non-allergic IH is crucial; allergy contraindicates the culprit agent for life. We studied frequency of allergic IH among ICM or GBCM reactors. METHODS: Patients were recruited in 31 hospitals between 2005 and 2009. Clinical symptoms, plasma histamine and tryptase concentrations and skin tests were recorded. Allergic IH was diagnosed by intradermal tests (IDT) with the culprit CM diluted 1:10, "potentially allergic" IH by positive IDT with pure CM, and non-allergic IH by negative IDT. FINDINGS: Among 245 skin-tested patients (ICM = 209; GBCM = 36), allergic IH to ICM was identified in 41 (19.6%) and to GBCM in 10 (27.8%). Skin cross-reactivity was observed in 11 patients with ICM (26.8%) and 5 with GBCM (50%). Allergy frequency increased with clinical severity and histamine and tryptase concentrations (p < 0.0001). Cardiovascular signs were strongly associated with allergy. Non-allergic IH was observed in 152 patients (62%) (ICM:134; GBCM:18). Severity grade was lower (p < 0.0001) and reaction delay longer (11.6 vs 5.6 min; p < 0.001). Potentially allergic IH was diagnosed in 42 patients (17.1%) (ICM:34; GBCM:8). The delay, severity grade, and mediator release were intermediate between the two other groups. INTERPRETATION: Allergic IH accounted for < 10% of cutaneous reactions, and > 50% of life-threatening ones. GBCM and ICM triggered comparable IH reactions in frequency and severity. Cross-reactivity was frequent, especially for GBCM. We propose considering skin testing with pure contrast agent, as it is more sensitive than the usual 1:10 dilution criteria.

Comparison of two enzymatic immunoassays, high resolution mass spectrometry method and radioimmunoassay for the quantification of human plasma histamine.

Histamine (HA) is one of the main immediate mediators involved in allergic reactions. HA plasma concentration is well correlated with the severity of vascular and respiratory signs of anaphylaxis. Consequently, plasma quantification of HA is useful to comfort the diagnosis of anaphylaxis. Currently, radioimmunoassay (RIA) is the gold standard method to quantify HA due to its high sensitivity, but it is time consuming, implicates specific formations and cautions for technicians, and produces hazardous radioactive wastes. The aim of this study was to compare two enzymatic immunoassays (EIA) and one in-house liquid chromatography high-resolution mass spectrometry method (LC-HRMS) with the gold standard method for HA quantification in plasma samples of patients suspected of anaphylaxis reactions. Ninety-two plasma samples were tested with the 4 methods (RIA, 2 EIA and LC-HRMS) for HA quantification. Fifty-eight samples displayed HA concentrations above the positive cut-off of 10nM evaluated by RIA, including 18 highly positive samples (>100 nM). This study shows that Immunotech(®) EIA and LC-HRMS concentrations were highly correlated with RIA values, in particular for samples with a HA concentration around the positive cut-off. In our hands, plasma concentrations obtained with the Demeditec Diagnostics(®) EIA correlated less with results obtained by RIA, and an underestimation of plasma HA levels led to a lack of sensitivity. In conclusion, this study demonstrates that Immunotech(®) EIA and LC-HRMS method could be used instead of RIA to assess plasma HA in human diagnostic use.

Assessment of the Allergenic Potential of Transgenic Wheat (Triticum aestivum) with Reduced Levels of ω5-Gliadins, the Major Sensitizing Allergen in Wheat-Dependent Exercise-Induced Anaphylaxis.

The ω5-gliadins are the major sensitizing allergens in wheat-dependent exercise-induced anaphylaxis (WDEIA). In this study, two-dimensional immunoblot analysis was used to assess the allergenic potential of two transgenic wheat lines in which ω5-gliadin genes were silenced by RNA interference. Sera from 7 of 11 WDEIA patients showed greatly reduced levels of immunoglobulin E (IgE) reactivity to ω5-gliadins in both transgenic lines. However, these sera also showed low levels of reactivity to other gluten proteins. Sera from three patients showed the greatest reactivity to proteins other than ω5-gliadins, either high-molecular-weight glutenin subunits (HMW-GSs), α-gliadins, or non-gluten proteins. The complexity of immunological responses among these patients suggests that flour from the transgenic lines would not be suitable for individuals already diagnosed with WDEIA. However, the introduction of wheat lacking ω5-gliadins could reduce the number of people sensitized to these proteins and thereby decrease the overall incidence of this serious food allergy.

Structural Basis of IgE Binding to α- and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains.

Wheat products cause IgE-mediated allergies. The present study aimed to decipher the molecular basis of α- and γ-gliadin allergenicity. Gliadins and their domains, the repetitive N-terminal and the nonrepetitive C-terminal domains, were cloned and expressed in Escherichia coli. Their secondary structures and their IgE binding capacity were compared with those of natural proteins before and after reduction/alkylation. Allergenicity was evaluated with sera from patients who had a wheat food allergy or baker's asthma. The secondary structures of natural and recombinant proteins were slightly different. Compared with natural gliadins, recombinant proteins retained IgE binding but with reduced reactivity. Reduction/alkylation decreased IgE binding for both natural and recombinant gliadins. Although more continuous epitopes were identified in the N-terminal domains of α- and γ-gliadins, both the N-terminal and C-terminal domains contributed to IgE binding. As for other members of the prolamin superfamily, disulfide bonds appear to be of high importance for IgE binding.

Molecular and immunological characterization of wheat Serpin (Tri a 33).

SCOPE: Several wheat proteins are responsible for food and respiratory allergies. Due to their large polymorphism, the allergenic potential of a number of them has not yet been precisely established. The aim of this work was to perform a thorough assessment of serpin (Tri a 33) allergenicity. METHODS AND RESULTS: Recombinant wheat Serpin-Z2B isoform (rSerpin-Z2B) was expressed in Escherichia coli. Synchrotron radiation circular dichroism data indicated that the recombinant serpin contains slightly more ß-strands than α-helix structures. IgE reactivity of sera from 103 patients with food allergy and 29 patients with Baker's asthma was evaluated using ELISA, a model of basophil activation and linear epitope mapping (Pepscan). Twenty percent of patients with food allergy to wheat and 31% of those with Baker's asthma displayed rSerpin-Z2B-specific IgE in ELISA. The protein was able to induce IgE-dependent basophil degranulation. The Pepscan experiment identified four regions involved in IgE binding to serpin. Heating the protein induced its irreversible denaturation and impaired IgE binding, revealing the predominance of conformational epitopes. CONCLUSION: This study confirms wheat serpin allergenicity and shows that recombinant serpin may be a marker of a broad spectrum of sensitization to wheat proteins.

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine ß-lactoglobulin.

The whey protein ß-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41-K60; Y102-R124; L149-I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS-PAGE and RP-HPLC. Peptides released were identified by LC-MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3-5year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.

Effects of heating and glycation of beta-lactoglobulin on its recognition by IgE of sera from cow milk allergy patients.

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.