The diagnostic accuracy of specific IgE to Ara h 6 in adults is as good as Ara h 2

Specific IgE (sIgE) to Ara h 2 is useful in diagnosing peanut allergy. Our aim was to assess the diagnostic value of sIgE to Ara h 6, another 2S albumin, in an adult population suspected of peanut allergy. Subjects with suspected peanut allergy between 2002 and 2013 were included if a diagnostic double‐blind, placebo‐controlled food challenge with peanut was performed. sIgE to Ara h 2 and Ara h 6 was measured by ImmunoCAP ISAC 112. Of 107 challenged subjects, 65 had a positive challenge (61%). The discriminative ability of sIgE to Ara h 2 and Ara h 6 was comparable: AUC 0.81 vs 0.82. Positive predictive value for both tests was 95% using a cutoff value ≥1 ISU/l with poor corresponding sensitivity values (58% for Ara h 2, 62% for Ara h 6), but good specificity values (95% for both tests). In conclusion, the diagnostic value of sIgE to Ara h 6 on population level was as good as sIgE to Ara h 2. On individual level, however, 5% of the subjects showed contradicting results between both tests using a cutoff of 0.3 ISU/l, leading to a risk of misdiagnosis if only one of both tests is used.     

Gastro-duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential

BACKGROUND: The process of gastro-duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro-duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release. METHODS: An in vitro model of gastro-duodenal digestion was used to investigate changes in the allergenic properties of Ara h 1 using in vitro assays monitoring T cell reactivity (proliferation, cytokine production) and histamine release of basophils from peanut allergic individuals. The digestion process was monitored using an SDS-PAGE gel. RESULTS: In vitro gastric digestion led to rapid degradation of Ara h 1 into small fragments M(r) L5600. Gastric digestion did not affect the ability of Ara h 1 to stimulate cellular proliferation. Gastro-duodenal digestion significantly reduced its ability to stimulate clonal expansion (P<0,05; Wilxocon's signed rank test). The Th-2 type cytokine polarization of T cells from peanut allergic donors (IFN-gamma/IL-13 ratio and IFN-gamma/IL-4 ratio of CFSE(low) CD4(+) T cells) remained unchanged regardless of the level of digestion. Histamine release of basophils from peanut allergic individuals was induced to the same extent by native Ara h 1 and its digestion products. CONCLUSION: Gastro-duodenal digestion fragments of Ara h 1 retain T cell stimulatory and IgE-binding and cross-linking properties of the intact protein.     

Effect of boiling on the structure and immunoreactivity of recombinant peanut protein Ara h 1

Ara h 1 is a key peanut allergen and its activity is significantly affected by protein intrinsic structure which is found to be regulated by heat treatment, although the molecular basis for this regulation has remained largely unknown. Here, we explored the effect of boiling on the structure and allergenicity of recombinant peanut protein Ara h 1 (rAra h 1). rAra h 1 was purified from E. Coli BL21(DE3) plysS cells and structurally studied. According to the results, rAra h 1 undergoes degradation during the heating process, and the aggregation of fragments happened after 20?min of heating. An increased surface hydrophobic index and a decreased content of α-helixes were found in rAra h 1, indicating a looser protein structure of rAra h 1 caused by heat treatment. Destroyed epitopes during protein degradation and aggregation could be a mechanism of reducing the allergenic nature of rAra h 1 by heat treatment.     

Children with peanut allergy recognize predominantly Ara h2 and Ara h6, which remains stable over time

BACKGROUND: In peanut-allergic adults, IgE is mainly directed to Ara h1 and Ara h2. More recently, a role for Ara h6 has been suggested. In contrast to adults, IgE in children can fluctuate over time. Therefore, children may have a more dynamic reactivity to peanut. OBJECTIVE: To examine the IgE reactivity to major peanut allergens in peanut-allergic children at two subsequent time-points. METHODS: Twenty children (3-15 years old) with peanut allergy, confirmed by a double-blind placebo-controlled food challenge (DBPCFC), were included. Just before and 20 months after DBPCFC, IgE reactivity to purified Ara h1, Ara h2, Ara h3 and Ara h6 was studied by immunoblots and skin prick tests (SPTs). RESULTS: Before DBPCFC, all peanut-allergic children showed IgE reactivity to Ara h2; Ara h6 was recognized by 16 children, and Ara h1 and Ara h3 by 10 children. After 20 months, peanut-specific IgE levels (median 23 kU/L) and the individual recognition of major allergens were comparable with the levels and recognition before challenge (median 28.2 kU/L). SPT with Ara h2 and Ara h6 was positive in most children, whereas SPT with Ara h1 and Ara h3 was positive in approximately half of the children. Ara h6 induced the largest weals. None of the parameters were related to the severity of peanut allergy. CONCLUSION: Ara h2 and Ara h6 are the most frequently recognized major peanut allergens in children. The individual reactivity to the major peanut allergens remained stable over time, despite DBPCFC.     

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays

Background Peanuts are often consumed after roasting, a process that alters the three‐dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. Objective We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). Methods Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20 min at 145 °C in the presence (R+g) or absence (R?g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well‐characterized peanut‐allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. Results Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE‐binding capacity of R+g and R?g Ara h 1 was decreased 9000‐ and 3.6‐fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE‐binding capacity and the degranulation capacity of R?g Ara h 2/6 were 600–700‐fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. Conclusions and Clinical Relevance Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component‐resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins. Cite this as: Y. M. Vissers, M. Iwan, K. Adel‐Patient, P. Stahl Skov, N. M. Rigby, P. E. Johnson, P. Mandrup Müller, L. Przybylski‐Nicaise, M. Schaap, J. Ruinemans‐Koerts, A. P. H. Jansen, E. N. C. Mills, H. F. J. Savelkoul and H. J. Wichers, Clinical & Experimental Allergy, 2011 (41) 1631–1642.     

Relevance of Ara h1, Ara h2 and Ara h3 in peanut-allergic patients, as determined by immunoglobulin E Western blotting, basophil–histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen

BACKGROUND: A number of allergenic proteins in peanut has been described and the relative importance of these allergens is yet to be determined. OBJECTIVES: We have investigated the relevance of previously identified peanut allergens in well-characterized peanut-allergic patients by in vitro, ex vivo and in vivo assays. METHODS: Thirty-two adult peanut-allergic patients were included based on careful and standardized patient history and the presence of peanut-specific IgE. The diagnosis peanut allergy was confirmed using double-blind placebo-controlled food challenges in 23 patients. Major peanut allergens Ara h1, Ara h2 and Ara h3 were purified from peanuts using ion-exchange chromatography. IgE immunoblotting was performed and IgE-cross-linking capacity was examined by measuring histamine release (HR) after incubating patient basophils as well as passively sensitized basophils with several dilutions of the allergens. Intracutaneous tests (ICTs) using 10-fold dilution steps of the purified allergens and crude peanut extract were performed. RESULTS: Ara h2 was recognized most frequently (26 out of 32) in all tests and induced both positive skin tests and basophil degranulation at low concentrations, whereas Ara h1 and Ara h3 were recognized less frequently and reacted only at 100-fold higher concentrations as analysed with HR and intracutaneous testing (ICT). Next to the three tested allergens, proteins with molecular weights of somewhat smaller than 15 kDa were identified as a IgE-binding proteins on immunoblot in the majority of the patients (20 out of 32). CONCLUSION: We conclude that Ara h2 is, for our patient group, the most important peanut allergen, and that previously unidentified peanut proteins with molecular weights of somewhat smaller than 15 kDa may be important allergens as well. ICT in combination with basophil-HR and IgE immunoblotting provides insight in the patient specificity towards the individual peanut allergens.     

Characterization of the T-cell epitopes of a major peanut allergen, Ara h 2

BACKGROUND: The development of safe and effective immunotherapy for peanut allergy has been complicated by the high anaphylactic potential of native peanut extracts. We sought to map the T-cell epitopes of the major peanut allergen, Ara h 2 in order to develop T-cell targeted vaccines. METHODS: A panel of eight peanut-specific CD4+ T-cell lines (TCL) was derived from eight peanut-allergic subjects and proliferative and cytokine responses to stimulation with a set of overlapping 20-mer peptides representing the entire sequence of Ara h 2 determined. Proliferation was assessed in 72 h assays via tritiated thymidine incorporation, while interleukin (IL)-5 and interferon (IFN)-gamma production were assessed via sandwich enzyme-linked immunosorbent assay (ELISA) of cell culture supernatants. RESULTS: Eight of the 17 Ara h 2 peptides were recognized by one or more subjects, with the two peptides showing highest reactivity [Ara h 2 (19-38) and Ara h 2 (73-92)] being recognized by three subjects each. Adjoining peptides Ara h 2 (28-47) and Ara h 2 (100-119) induced proliferative responses in two subjects. Each of these peptides was associated with a Th2-type cytokine response. CONCLUSION: Two highly immunogenic T-cell reactive regions of Ara h 2 have been identified, Ara h 2 (19-47) and Ara h 2 (73-119), providing scope for the development of safe forms of immunotherapy for peanut allergy.     

Digested Ara h 1 has sensitizing capacity in Brown Norway rats

Background Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion.
Objective Using Ara h 1 as a model allergen, the ability of digested protein to sensitize has been investigated.
Methods Ara h 1 was purified from whole roasted peanuts. Intact Ara h 1 was digested in an in vitro model, simulating the human gastrointestinal digestion process. Digestion products were analysed for peptide sizes and their ability to aggregate. Brown Norway (BN) rats, used as an animal model, were immunized with purified intact Ara h 1 or the gastrointestinal digestion products thereof. The sensitizing capacity was evaluated by analyses of specific antibody (IgG1, IgG2a and IgE) responses and ability to trigger mediator release of rat basophilic leukaemia (RBL)-2H3 cells.
Results The present study showed that Ara h 1 was broken down, resulting in peptide fragments of sizes <2.0 kDa, of which approximately 50% was in aggregated complexes of M _r up to 20 kDa. Ara h 1 digesta were shown to have sensitizing capacity in BN rats, being capable of inducing specific IgG and IgE antibodies. The IgE response was functional, having the capacity to induce specific degranulation of RBL cells.
Conclusion From this study, it can be concluded that lability of a food allergen to gastrointestinal digestion does not necessarily abrogate its allergenic sensitizing potential.     

Comparative potency of Ara h 1 and Ara h 2 in immunochemical and functional assays of allergenicity

To assess the relative potency of the major peanut allergens, Ara h 1 and Ara h 2, we examined the relative ability of purified proteins to bind IgE on immunoblots, to cross-link allergen specific IgE in an in vitro assay of degranulation based on RBL SX-38 cells, and to bind IgE in the ImmunoCap assay. Sera from 12 highly sensitive, peanut allergic patients were studied in all assays. IgE immunoblots with crude peanut extracts showed binding of IgE to multiple bands including the 63 kDa and 17-19 kDa bands that contain Ara h 1 and Ara h 2, respectively. In the functional assay, Ara h 2 was more potent than Ara h 1 in 11 of 12 sera tested with a median potency that was 52.5-fold more than Ara h 1 (P < 0.005). Contrary to findings with the functional assay, IgE immunoblots with purified Ara h 1 and Ara h 2 showed substantially lighter binding of IgE to Ara h 2 compared with Ara h 1 (P = 0.02). The ImmunoCap assay gave intermediate results with slightly more IgE binding to Ara h 2 than to Ara h 1 (P = 0.005). In conclusion, Ara h 2 is a very potent allergen and is much more potent than Ara h 1 for most sera using an in vitro assay of IgE cross-linking and cell activation. This finding is different from what was predicted based on immunoblots or with the ImmunoCap assay.