IgE cross-reactivity measurement of cashew nut, hazelnut and peanut using a novel IMMULITE inhibition method.

Background Tree nut-allergic individuals are often sensitised towards multiple nuts and seeds. The underlying cause behind a multi-sensitisation for cashew nut, hazelnut, peanut and birch pollen is not always clear. We investigated whether immunoglobulin E antibody (IgE) cross-reactivity between cashew nut, hazelnut and peanut proteins exists in children who are multi-allergic to these foods using a novel IMMULITE®-based inhibition methodology, and investigated which allergens might be responsible. In addition, we explored if an allergy to birch pollen might play a role in this co-sensitisation for cashew nut, hazelnut and peanut. Methods Serum of five children with a confirmed cashew nut allergy and suffering from allergic symptoms after eating peanut and hazelnut were subjected to inhibition immunoassays using the IMMULITE® 2000 XPi. Serum-specific IgE (sIgE) to seed storage allergens and pathogenesis-related protein 10 (PR10) allergens were determined and used for molecular multicomponent allergen correlation analyses with observed clinical symptoms and obtained inhibition data. Results IgE cross-reactivity was observed in all patients. Hazelnut extract was a strong inhibitor of cashew nut sIgE (46.8%), while cashew nut extract was less able to inhibit hazelnut extract (22.8%). Peanut extract showed the least inhibition potency. Moreover, there are strong indications that a birch pollen sensitisation to Bet v 1 might play a role in the observed symptoms provoked upon ingestion of cashew nut and hazelnut. Conclusions By applying an adjusted working protocol, the IMMULITE® technology can be used to perform inhibition assays to determine the risk of sIgE cross-reactivity between very different food components.

Identification and characterization of major IgE binding of purified allergenic protein (11 kDa) from Buchanania lanzan.

Tree nut along with peanut are among the most potent food allergens, responsible for frequently inducing the IgE-mediated hypersensitivity reaction. Our aim was identification, purification of Buchanania lanzan (Bl-11 kDa) protein along with characterization and assessment of allergenic potential of clinically relevant allergen. Further study was executed in clinical samples of sensitive patients, BALB/c mice, and in-vitro. A major IgE binding 11-kDa protein from Buchanania lanzan was purified by anion exchange chromatography, reverse phase high pressure liquid chromatography (RP-HPLC) and characterized using peptide mass fingerprinting (PMF). Buchanania lanzan (Bl-11 kDa) protein shows the pepsin resistance and depicts IgE interacting capacity to Buchanania lanzan allergic patient's sera as well as sensitized mice sera. It also showed increase in the allergic mediator's like IgE, IgG1, histamine levels in sensitized mice sera. Further study was carried out in-vitro (RBL-2H3 cells) and increased release mast cell degranulation mediators such as ß-hexosaminidase, histamine, CysL and PGD2 in the culture supernatant was found. The activation of Th2 cytokines/transcription factors and expression of molecular markers in the downstream of mast cell signaling were up-regulated while the Th1 transcriptional factor (T-bet) was decreased in Bl-11 kDa protein treated mice. Conclusively, our study demonstrates Buchanania lanzan purified protein to be potential allergen that may generate an allergic reaction in sensitized individuals, and one of the most important IgE binding protein responsible for its allergenicity.

Analysis of Oral Food Challenge Outcomes in IgE-Mediated Food Allergies to Almond in a Large Cohort.

BACKGROUND: Although almond specific IgE-mediated food allergies have traditionally been equated with other tree nut allergies, outcomes of oral food challenges to almond and the utility of clinical testing to predict IgE-mediated almond hypersensitivity are not well known. OBJECTIVE: To describe almond oral challenge outcomes and assess the predictive value of clinical testing. METHODS: A total of 603 almond challenges performed for 590 patients, aged 1 to 66 years, were analyzed from Massachusetts General Hospital allergy practices. Reactions were graded using the Niggemann and Beyer allergic reaction grading system and the Sampson 2006 National Institute of Allergy and Infectious Diseases anaphylaxis definition. RESULTS: Almond challenges included 545 passes (92%), 15 (3%) indeterminates, and 30 (5%) failures, in contrast with 31% challenge failures for other foods. Most reactions were mild; 21 (4%) had grade 2/3 allergic symptoms, and 3 (0.5%) had anaphylaxis. Median almond specific IgE level was 0.89 kU/L (range, <0.35 to >100 kU/L), median skin prick test wheal diameter was 4.0 mm (range, 0-28 mm), and 475 subjects (81%) were sensitized to almond. Failure was associated with higher almond specific IgE level (P < .001), larger almond skin prick test wheal diameter (P = .001), higher peanut IgE level (P = .003), and a history of almond reaction (P < .029). Almond specific IgE level, almond skin prick test wheal diameter, and age at challenge combined demonstrated good predictive value for grade 2/3 allergic reactions by receiver-operating characteristic analysis (area under the curve, 0.83). CONCLUSIONS: The proportion of failed almond challenges (5%) was low in contrast with other allergens, suggesting that some almond challenges may be safely conducted with higher patient-to-staff ratios or potentially introduced at home. Although reactions are usually uncommon and mild, anaphylaxis is possible with high almond sensitization.

Association of tree nut and coconut sensitizations.

BACKGROUND: Coconut (Cocos nucifera), despite being a drupe, was added to the US Food and Drug Administration list of tree nuts in 2006, causing potential confusion regarding the prevalence of coconut allergy among tree nut allergic patients. OBJECTIVE: To determine whether sensitization to tree nuts is associated with increased odds of coconut sensitization. METHODS: A single-center retrospective analysis of serum specific IgE levels to coconut, tree nuts (almond, Brazil nut, cashew, chestnut, hazelnut, macadamia, pecan, pistachio, and walnut), and controls (milk and peanut) was performed using deidentified data from January 2000 to August 2012. Spearman correlation (ρ) between coconut and each tree nut was determined, followed by hierarchical clustering. Sensitization was defined as a nut specific IgE level of 0.35 kU/L or higher. Unadjusted and adjusted associations between coconut and tree nut sensitization were tested by logistic regression. RESULTS: Of 298 coconut IgE values, 90 (30%) were considered positive results, with a mean (SD) of 1.70 (8.28) kU/L. Macadamia had the strongest correlation (ρ = 0.77), whereas most other tree nuts had significant (P < .05) but low correlation (ρ < 0.5) with coconut. The adjusted odds ratio between coconut and macadamia was 7.39 (95% confidence interval, 2.60-21.02; P < .001) and 5.32 (95% confidence interval, 2.18-12.95; P < .001) between coconut and almond, with other nuts not being statistically significant. CONCLUSION: Our findings suggest that although sensitization to most tree nuts appears to correlate with coconut, this is largely explained by sensitization to almond and macadamia. This finding has not previously been reported in the literature. Further study correlating these results with clinical symptoms is planned.

Is Microarray Analysis Really Useful and Sufficient to Diagnose Nut Allergy in the Mediterranean Area?

BACKGROUND: Component-based diagnosis on multiplex platforms is widely used in food allergy but its clinical performance has not been evaluated in nut allergy. OBJECTIVE: To assess the diagnostic performance of a commercial protein microarray in the determination of specific IgE (sIgE) in peanut, hazelnut, and walnut allergy. METHODS: sIgE was measured in 36 peanut-allergic, 36 hazelnut-allergic, and 44 walnut-allergic patients by ISAC 112, and subsequently, sIgE against available components was determined by ImmunoCAP in patients with negative ISAC results. ImmunoCAP was also used to measure sIgE to Ara h 9, Cora 8, and Jug r 3 in a subgroup of lipid transfer protein (LTP)-sensitized nut-allergic patients (positive skin prick test to LTP-enriched extract). sIgE levels by ImmunoCAP were compared with ISAC ranges. RESULTS: Most peanut-, hazelnut-, and walnut-allergic patients were sensitized to the corresponding nut LTP (Ara h 9, 66.7%; Cor a 8, 80.5%; Jug r 3, 84% respectively). However, ISAC did not detect sIgE in 33.3% of peanut-allergic patients, 13.9% of hazelnut-allergic patients, or 13.6% of walnut-allergic patients. sIgE determination by ImmunoCAP detected sensitization to Ara h 9, Cor a 8, and Jug r 3 in, respectively, 61.5% of peanut-allergic patients, 60% of hazelnut-allergic patients, and 88.3% of walnut-allergic patients with negative ISAC results. In the subgroup of peach LTP-sensitized patients, Ara h 9 sIgE was detected in more cases by ImmunoCAP than by ISAC (94.4% vs 72.2%, P < .05). Similar rates of Cora 8 and Jug r 3 sensitization were detected by both techniques. CONCLUSIONS: The diagnostic performance of ISAC was adequate for hazelnut and walnut allergy but not for peanut allergy. sIgE sensitivity against Ara h 9 in ISAC needs to be improved.

Cor a 14 is the superior serological marker for hazelnut allergy in children, independent of concomitant peanut allergy.

BACKGROUND: Hazelnut is the most frequent cause of tree nut allergy, but up to half of all children with hazelnut allergy additionally suffer from peanut allergy. Our aim was to identify diagnostic values of the most promising serological markers (Cor a 9 and Cor a 14) and to address the influence of concomitant peanut allergy and PR10 sensitization. METHOD: We included 155 children suspected of hazelnut allergy and challenged according to the guidelines. Concomitant allergy to peanuts was verified or ruled out by challenge. Skin prick test, s-IgE and CRD to hazelnut, peanut, PR10 and LPT protein families were measured using ImmunoCAP. RESULTS: Sixty-five children had a positive hazelnut challenge, and 60% of these also had a concomitant peanut allergy. Children allergic to hazelnut were sensitized to Cor a 9 and Cor a 14; peanut-allergic children were sensitized to Ara h 2. Sensitization to PR10 protein components was seen in 45% of all included children, irrelevant to allergy to peanut or hazelnut. A cut-off >0.72 kU/L of IgE towards Cor a 14 diagnosed 87% correctly, making Cor a 14 the superior serology marker. However, nine hazelnut-allergic children were primarily sensitized to Cor a 9. CONCLUSION: Concomitant peanut allergy is common in hazelnut-allergic children, but decision points as well as diagnostic values for Cor a 14 are not affected. We found three independent and well-characterized serotypes; hazelnut-allergic children were sensitized to Cor a 14, peanut-allergic children were sensitized to Ara h 2, and independently of this were children sensitized to birch pollen (Bet v 1).

In vitro digestion of soluble cashew proteins and characterization of surviving IgE-reactive peptides.

SCOPE: The stability of food allergens to digestion varies. We characterized the stability of cashew allergens to digestion by pepsin and trypsin and identified IgE-binding epitopes that survive digestion. METHODS AND RESULTS: The ability of pepsin and trypsin to digest cashew allergens was assessed with an in vitro digestion model. Samples were evaluated by SDS-PAGE, MS, ELISA, and immunoblotting to compare IgE binding. Increasing amount of protease resulted in greater degradation of higher molecular weight cashew proteins. Among cashew proteins, the 2S albumin, Ana o 3, was most resistant to digestion by both pepsin and trypsin. MS identified digestion resistant Ana o 3 protein fragments that retained reported IgE-binding epitopes. Pretreatment of extracts or purified Ana o 3 with reducing agent increased the sensitivity of Ana o 3 to protease digestion. Circular dichroism revealed the structure of purified Ana o 3 was largely alphahelical and was disrupted following reduction. Ana o 3 reduction followed by protease digestion decreased binding of serum IgE from cashew allergic patients. Our results indicate that the Ana o 3 disulfide bond dependent structure protects the protein from proteolysis. CONCLUSION: Ana o 3 is the cashew allergen most likely to survive gastrointestinal digestion intact.

Isolated IgE reactivity to native walnut vicilin-like protein (nJug r 2) on ISAC™ microarray is due to cross-reactive carbohydrate epitopes.

BACKGROUND: The last version of the microarray-based testing ImmunoCAP ISAC 112™ includes the native walnut (Junglans regia) molecules 2S albumin (nJug r 1), vicilin (nJug r 2) and lipid transfer protein (nJug r 3). In view of the many unexpected cases of isolated positivity to nJug r 2 occurring in daily practice, we evaluated the association of these reactivities with clinical symptoms, as well as the relationship between sIgE and nJug r 2 and cross-reactive carbohydrate determinants (CCDs). METHODS: Sera from 320 consecutive allergic outpatients tested by ImmuoCAP ISAC™ 112 were considered. The medical records of all nJug r 2 positive patients were reviewed to assess clinical symptoms related to walnut allergy. A linear regression analysis was performed to evaluate the correlation between nJug r 2 and CCDs (nMUXF3) sIgE values, and a CAP inhibition assay was carried out to confirm the possible cross-reactivity between CCDs and nJug r 2. RESULTS: Thirty-seven out of 320 sera tested (11.6%) were positive to nJug r 2. Among them three (8.1%) and eight (21.6%) scored positive for nJug r 1 and nJug r 3 as well, respectively. Twenty-seven (73%) sera showed isolated nJug r 2 positivity. Only nJug r 1 reactors had symptoms referred to walnut allergy. Twenty-five/37 nJug r 2-positive sera (67.6%) showed a simultaneous positivity to nMUXF3 and a significant correlation (p<0.0001) between the IgE levels to nJug r 2 and nMUXF3 (r²=0.787). After incubation with nMUXF3 a complete inhibition of sIgE reactivity to both nMUXF3 and nJug r 2 was shown. CONCLUSIONS: The unexpected isolated sIgE reactivity to nJug r 2 found by ImmunoCAP ISAC™ 112 is frequently related to reactivity to cross-reactive carbohydrate epitopes and it is lacking clinical significance.

Modulation of mTOR effector phosphoproteins in blood basophils from allergic patients.

The mammalian target of rapamycin (mTOR) pathway contributes to various immunoinflammatory processes. Yet, its potential involvement in basophil responses in allergy remains unclear. In this pilot study, we quantified two key mTOR effector phosphoproteins, the eukaryotic initiation factor 4E (peIF4E) and S6 ribosomal protein (pS6rp), in blood basophils from nut allergy patients (NA, N = 16) and healthy controls (HC, N = 13). Without stimulation in vitro, basophil peIF4E levels were higher in NA than HC subjects (P = 0.014). Stimulation with nut (offending) but not chicken / rice (non-offending) extract increased basophil peIF4E and pS6rp levels (+32%, P = 0.018, and +98%, P = 0.0026, respectively) in NA but not HC subjects, concomitant with increased surface levels of CD203c and CD63, both known to reflect basophil activation. Pre-treatment with the mTOR inhibitor rapamycin decreased pS6rp and CD203c responses in nut extract-stimulated basophils in NA subjects. Thus, basophil responses to offending allergens are associated with modulation of mTOR effector phosphoproteins.

Diagnostic value of hazelnut allergy tests including rCor a 1 spiking in double-blind challenged children.

BACKGROUND: The diagnostic value of hazelnut allergy tests in double-blind challenged children is largely unknown. The aim of this study was to analyze the performance of current diagnostic tests for hazelnut allergy in children and the effect of spiking. METHODS: Data of 151 children who underwent a double-blind placebo-controlled food challenge for hazelnut were analyzed. The positive predictive value and negative predictive value (PPV/NPV) of level of specific IgE (sIgE) for hazelnut, the influence of rCor a 1 spiking of the ImmunoCAP, and size of the skin prick test (SPT) for hazelnut were determined, also in relation to the severity of the hazelnut allergy. Reported accidental ingestion leading to an allergic reaction to hazelnut was also analyzed in relation to hazelnut allergy. RESULTS: Specific IgE ≥0.35 kU(A) /l for hazelnut was a moderate predictor for hazelnut allergy. The spiking decreased the PPV from 41% to 38% and increased the NPV from 91% to 100% for sIgE ≥0.35 kU(A) /l. The maximum reached PPV was 73% for sIgE cutoff of 26 kU(A) /l. Level of sIgE before spiking was significantly different between different grades of severity and was lost after spiking. Skin prick test was a better predictor for hazelnut allergy and severity than the level of sIgE. A history of accidental ingestion leading to an allergic reaction to hazelnut had a predictive value of 59% for hazelnut allergy. CONCLUSIONS: This study showed a good NPV of diagnostic tests for hazelnut allergy in children which further improved by rCor a 1 spiking. However, the PPVs are moderate and decreased by spiking.

Young infants with atopic dermatitis can display sensitization to Cor a 9, an 11S legumin-like seed-storage protein from hazelnut (Corylus avellana).

Allergy to hazelnut (Corylus avellana) can be severe and occur at young age. Atopic dermatitis (AD) can involve sensitization to various foods. The objective is to investigate the pattern of hazelnut sensitization in infants with AD. Sera of 34 infants all under 1 year of age and suffering from AD were selected according to prior specific IgE results. Twenty-nine infants were sensitized to traditional food allergens, five were not. From the 29 infants with a sensitization to at least one food allergen, 20 demonstrated IgE reactivity to hazelnut. All sera were analyzed with the allergen microarray immunoassay (ImmunoCAP ISAC). Twelve (60%) of the children with IgE reactivity to hazelnut demonstrated sensitization to Cor a 9, the 11S legumin-like seed-storage protein from hazelnut. In these infants, no sensitization to Cor a 1, the homologue of the major birch pollen allergen Bet v 1 (Betula verrucosa), or the lipid transfer protein (Cor a 8) from hazelnut was demonstrable. Half of the children sensitized to Cor a 9 demonstrated IgE reactivity to its homologue in peanut (Arachis hypogaea; Ara h 3) from which five were also sensitized to Gly m 6 from soy (Glycine max). None of the infants with AD without IgE reactivity to hazelnut demonstrated sensitization to Cor a 1, 8, or 9. In conclusion, young infants with atopic dermatitis sensitized to hazelnut can already display IgE reactivity to Cor a 9, a potentially dangerous hazelnut component. The mechanism(s) of this early sensitization and its clinical significance remain elusive.

Influence of processing on the allergenic properties of pistachio nut assessed in vitro.

Pistachio (Pistacia vera) is a tree nut that has been reported to cause IgE-mediated allergic reactions. This study was undertaken to investigate the distinctions between different cultivars of pistachio nut and the influence of different processing on the IgE-binding capacity of whole pistachio protein extracts. The influence of different processes on allergenicity was investigated using competitive inhibition ELISA and Western blotting assays. The Western blotting results of extracts from pistachio cultivars showed no marked difference among them. The IgE-binding capacity was significantly lower for the protein extract prepared from steam-roasted than from raw and dry-roasted pistachio nuts. The results of sensory evaluation analysis and hedonic rating proved no significant differences in color, taste, flavor, and overall quality of raw, roasted, and steam-roasted pistachio nut treatments. The most significant finding of the present study was the successful reduction of IgE-binding by pistachio extracts using steam-roast processing without any significant changes in sensory quality of product.

Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.

SCOPE: 2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. METHODS: 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. RESULTS: N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. CONCLUSION: We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.

Linear IgE-epitope mapping and comparative structural homology modeling of hazelnut and English walnut 11S globulins.

Allergic reactions to walnuts and hazelnuts can be serious. The 11S globulins (legumins) have been identified as important allergens in these and other nuts and seeds. Here we identify the linear IgE-binding epitopes of walnut and hazelnut 11S globulins, and generate 3D 11S globulin models to map the locations of the epitopes for comparison to other allergenic homologues. Linear IgE-epitope mapping was performed by solid-phase overlapping 15-amino acid peptides probed with IgE from pooled allergic human sera. Several walnut (Jug r 4) and hazelnut (Cor a 9) 11S globulin peptides with reactivity to patient IgE were identified. Comparative alignment with cashew (Ana o 2), peanut (Ara h 3), and soybean G1 (Gly m 6.0101) and G2 (Gly m 6.0201) allergenic homologues revealed several shared allergenic 'hot spots'. Homology modeling was performed based on the atomic structure of the soybean glycinin. Surface map comparisons between the tree nut and peanut homologues revealed structural motifs that could be important for IgE elicitation and binding and show that, contrary to predictions, the reactive epitopes are widely distributed throughout the monomeric subunits, both internally and externally, including regions occluded by quaternary subunit association. These findings reveal structural features that may be important to allergenicity and cross-reactivity of this protein class.

Factors predicting anaphylaxis to peanuts and tree nuts in patients referred to a specialist center.

BACKGROUND: Although acute allergic reactions after ingestion of peanuts and tree nuts are common, fatalities are rare. Other than patients with coexisting asthma, it is currently not possible to predict which patients are most likely to develop severe reactions. OBJECTIVE: The aim of this study was to determine which clinical and laboratory parameters best predict the likelihood of severe allergic reactions. METHODS: From 1992 to 2004, we collected detailed information on the clinical severity and allergy test results of 1094 patients with peanut and tree nut allergy attending a regional allergy center. In a subgroup of 122 patients, sera were assayed for activity of enzymes involved in the catabolism of bradykinin. RESULTS: Severe pharyngeal edema was 3.8 (2.1-6.9) times more common in patients with severe rhinitis and 2.6 (1.8-3.7) more common after ingestion of tree nuts compared with peanuts. Patients with serum angiotensin-converting enzyme concentrations <37.0 mmol/L had a 9.6 (1.6-57)-fold risk of severe pharyngeal edema. Life-threatening bronchospasm was most likely in patients with severe asthma (relative risk, 6.8 [4.1-11.3]) and less so in patients with milder asthma (2.7 [1.7-4.0]). Altered levels of consciousness were more likely in patients with severe eczema (3.1 [1.1-8.4]). CONCLUSION: Severity of coexisting atopic diseases predicted which patients attending a tertiary referral clinic were most likely to develop life-threatening allergic reactions to peanuts and tree nuts. Patients with the lowest serum angiotensin-converting enzyme concentrations were more likely to develop life-threatening pharyngeal edema, suggesting that this complication may be partly mediated by bradykinin.

The diagnosis of Brazil nut allergy using history, skin prick tests, serum-specific immunoglobulin E and food challenges.

BACKGROUND: Allergy to Brazil nut is a relatively common nut allergy and can be fatal. However, the evidence is lacking regarding the best approach to its diagnosis. OBJECTIVE: We sought to determine the relative merits of history, skin prick testing, measurement of serum-specific IgE and challenge in the diagnosis of Brazil nut allergy. METHODS: Fifty-six children and adults with a history of an allergic reaction to Brazil nut or evidence of sensitization were investigated by questionnaire (n=56), skin prick tests (SPTs) (n=53), measurement of serum-specific IgE to Brazil nut (n=54) and double-blind, placebo-controlled labial, and if necessary oral, challenges (n=19). RESULTS: Brazil nut allergy occurred in highly atopic individuals of any age with a strong family history of atopy. In 24 of 56 (43%), the history of an immediate reaction was sufficient to make a diagnosis with confidence and an oral challenge was considered unsafe. Of the 19 subjects undertaking the 'gold standard' test of a double-blind, placebo-controlled, food challenge, all six subjects with a SPT of at least 6 mm had a positive challenge and all three subjects with a SPT of 0 mm had a negative challenge. In the remaining 10 (53%) subjects, where SPT was between 1 and 5 mm and serum-specific IgE was less than 3.5 kU/L, an oral challenge was performed resulting in three positive and seven negative challenges. CONCLUSION: A combination of history, SPT and serum-specific IgE was adequate in achieving a diagnosis in the majority (77%) patients with suspected Brazil nut allergy. However, a doubtful history with SPT between 1 and 5 mm, or a serum-specific IgE less than 3.5 kU/L may require an oral challenge to help determine the risk of a Brazil nut allergic reaction.