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Background: Specific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin-specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors., Methods: 2S albumin-binding B-cells were single-cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non-atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering., Results: The frequency of 2S albumin-binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non-atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non-specific mAbs (9/10) were mainly derived from non-atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non-atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances., Conclusions: Peanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs)., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Human monoclonal antibodies (mAbs) are valuable tools to link genetic information with functional features and to provide a platform for conformational epitope mapping. Additionally, combined data on genetic and functional features provide a valuable mosaic for systems immunology approaches. Strategies to generate human mAbs from peripheral blood have been described and used in several studies including single cell sequencing of antigen-binding B cells and the establishment of antigen-specific monoclonal Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCLs). However, direct comparisons of these two strategies are scarce. Hence, we sought to set up these two strategies in our laboratory using peanut 2S albumins (allergens) and the autoantigen anti-Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2, alternatively 'ARHGDIB') as antigen targets to directly compare these strategies regarding costs, time expenditure, recovery, throughput and complexity. Regarding single cell sequencing, up to 50% of corresponding V(D)J gene transcripts were successfully amplified of which 54% were successfully cloned into expression vectors used for heterologous expression. Seventy-five percent of heterologously expressed mAbs showed specific binding to peanut 2S albumins resulting in an overall recovery of 20.3%, which may be increased to around 29% by ordering gene sequences commercially for antibody cloning. In comparison, the establishment of monoclonal EBV-LCLs showed a lower overall recovery of around 17.6%. Heterologous expression of a mAb carrying the same variable region as its native counterpart showed comparable concentration-dependent binding abilities. By directly comparing those two strategies, single cell sequencing allows a broad examination of antigen-binding mAbs in a moderate-throughput manner, while the establishment of monoclonal EBV-LCLs is a powerful tool to select a small number of highly reactive mAbs restricted to certain B cell subpopulations. Overall, both strategies, initially set-up for peanut 2S albumins, are suitable to obtain human mAbs and they are easily transferrable to other target antigens as shown for ARHGDIB., Competing Interests: The research position of AE was partially funded by EUROIMMUN AG, Lübeck, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ehlers, den Hartog Jager, Kardol-Hoefnagel, Katsburg, Knulst and Otten.)

Background: The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy., Objectives: Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value., Methods: Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively., Results: Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity., Conclusions: Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1., (© 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

Background: Screening for specific IgE against 2S albumin proteins Ara h 2 and 6 has good positive predictive value in diagnosing peanut allergy. From the third 2S member Ara h 7, 3 isoforms have been identified. Their allergenicity has not been elucidated., Objective: This study investigated the allergenicity of Ara h 7 isoforms compared to Ara h 2 and 6., Methods: Sensitization of 15 DBPCFC-confirmed peanut-allergic patients to recombinant Ara h 2.0201, Ara h 6.01 and isoforms of recombinant Ara h 7 was determined by IgE immunoblotting strips. A basophil activation test (BAT) was performed in 9 patients to determine IgE-cross-linking capacities of the allergens. Sensitivity to the allergens was tested in 5 patients who were sensitized to at least 1 Ara h 7 isoform, by a concentration range in the BAT. 3D prediction models and sequence alignments were used to visualize differences between isoforms and to predict allergenic epitope regions., Results: Sensitization to Ara h 7.0201 was most frequent (80%) and showed to be equally potent as Ara h 2.0201 and 6.01 in inducing basophil degranulation. Sensitization to Ara h 7.0201 together with Ara h 2.0201 and/or 6.01 was observed, indicating the presence of unique epitopes compared to the other 2 isoforms. Differences between the 3 Ara h 7 isoforms were observed in C-terminal cysteine residues, pepsin and trypsin cleavage sites and 3 single amino acid substitutions., Conclusion & Clinical Relevance: The majority of peanut-allergic patients are sensitized to isoform Ara h 7.0201, which is functionally as active as Ara h 2.0201 and 6.01. Unique epitopes are most likely located in the C-terminus or an allergenic loop region which is a known allergenic epitope region for Ara h 2.0201 and 6.01. Due to its unique epitopes and allergenicity, it is an interesting candidate to improve the diagnostic accuracy for peanut allergy., (© 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.)

Background: Dietary non-digestible oligosaccharides (NDOs) have a protective effect against allergic manifestations in children at risk. Dietary intervention with NDOs promotes the colonization of beneficial bacteria in the gut and enhances serum galectin-9 levels in mice and atopic children. Next to this, NDOs also directly affect immune cells and low amounts may reach the blood. We investigated whether pre-incubation of whole blood from peanut-allergic patients with NDOs or galectin-9 can affect basophil degranulation., Methods: Heparinized blood samples from 15 peanut-allergic adult patients were pre-incubated with a mixture of short-chain galacto-oligosaccharides and long-chain fructo-oligosaccharides (scGOS/lcFOS), scFOS/lcFOS, or galectin-9 (1 or 5 µg/mL) at 37°C in the presence of IL-3 (0.75 ng/mL). After 2, 6, or 24 h, a basophil activation test was performed. Expression of FcεRI on basophils, plasma cytokine, and chemokine concentrations before degranulation were determined after 24 h., Results: Pre-incubation with scGOS/lcFOS, scFOS/lcFOS, or galectin-9 reduced anti-IgE-mediated basophil degranulation. scFOS/lcFOS or 5 µg/mL galectin-9 also decreased peanut-specific basophil degranulation by approximately 20%, mainly in whole blood from female patients. Inhibitory effects were not related to diminished FcεRI expression on basophils. Galectin-9 was increased in plasma after pre-incubation with scGOS/lcFOS, and both NDOs and 5 µg/mL galectin-9 increased MCP-1 production., Conclusion and Clinical Relevance: The prebiotic mixture scFOS/lcFOS and galectin-9 can contribute to decreased degranulation of basophils in vitro in peanut-allergic patients. The exact mechanism needs to be elucidated, but these NDOs might be useful in reducing allergic symptoms.

Scope: The growing world population is a key driver for the exploration of sustainable protein sources to ensure food security. Mealworm and other insects are promising candidates. Previously we found that shrimp allergic patients are at risk for mealworm allergy, and that mealworm can induce a primary allergy . This study set out to investigate the allergenic potential of edible insects, suggested for human consumption by agencies such as WHO/FAO, in both the shrimp (potentially cross-reactive) and primary mealworm allergic population. The following insects were studied: mealworm, house cricket, giant mealworm, lesser mealworm, African grasshopper, large wax moth, and black soldier fly., Methods and Results: Fifteen shrimp (mealworm sensitized or allergic) patients and four primary mealworm allergic subjects, who participated in previous studies, were included. All shrimp allergic patients were sensitized to multiple insects with similar response profiles for all insects tested. Primary mealworm allergic patients, showed IgE binding to proteins from only a few insects on immunoblot, although basophil activation test was positive for all tested insects., Conclusion: Shrimp allergic patients are most likely at risk of food allergy to mealworm and other insects. Primary mealworm allergy does not mean subjects are likely to react to all insects., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Specific immunotherapy for peanut allergy is associated with significant side-effects. Chemically modified allergens may provide a safer alternative., Objective: This study aimed to analyse the immunogenicity and allergenicity of modified peanut conglutin., Methods: Native peanut conglutin and two modifications thereof were generated (RA and RAGA). Conglutin-specific T cell lines from 11 peanut-allergic patients were analysed for proliferation and cytokine production. Sera from 14 patients were analysed for IgE/IgG1/IgG4 binding by immunoblot and ELISA. IgE reactivity was analysed by direct and indirect basophil activation test (BAT), in presence and absence of patient plasma or CD32-blocking antibodies., Results: T cell proliferation to RA was unchanged, and proliferation to RAGA was reduced compared to native conglutin. Cytokine profiles remained unchanged. IgE, IgG1 and IgG4 binding to RA and RAGA was significantly reduced. In the direct BAT, the relative potency of modified conglutin was decreased in 67% and increased/similar in 33% of the patients. In the indirect BAT, RA and RAGA were 10-100 times less potent than native conglutin. Addition of plasma to the indirect BAT increased the relative potency of modified conglutin in patients with high peanut-specific IgG levels. This was mediated via blocking of the response to native conglutin, most likely by soluble IgG, and not via CD32., Conclusion and Clinical Relevance: Chemical modification of peanut conglutin by RA retains immunogenicity and reduces allergenicity and may be a promising approach for development of a curative treatment for peanut allergy. In a subgroup of patients, where the reactivity of native conglutin is already partially blocked by IgG, the effect of the modification of conglutin is less pronounced., (© 2014 John Wiley & Sons Ltd.)

Background: Several studies investigated whether hydrolysed proteins can induce tolerance to cow's milk (CM) in children at risk of developing CM allergy. Due to methodological problems and inconsistent findings, the evidence for a tolerogenic effect is limited. A major problem is that different hydrolysates may give different outcomes due to variations in their production and composition., Objective: The aim of the study was to investigate the effect of the degree of hydrolysis on the allergenicity and immunogenicity of whey hydrolysates., Methods: The hydrolysis of whey was stopped at different time-points between 1 and 60 min. In 18 CM allergic patients, the allergenicity of the hydrolysates was determined by immunoblot and the basophil activation test. To test immunogenicity, CM-specific T cell lines were generated., Results: In most patients, increasing time of hydrolysis decreased IgE recognition and basophil activation. However, in five patients, hydrolysed proteins induced more basophil activation than non-hydrolysed proteins. The immunoblot data indicated that these patients recognized either a 25- to 30-kDa degradation product of casein or a 10-kDa degradation product of whey. Although T cell activation was decreased in all patients over time, half of them still showed a positive response to the proteins after 60 min of hydrolysis., Conclusion: Increasing the time of hydrolysis reduces both allergenicity and immunogenicity of whey hydrolysates in most but not all patients. This indicates that not the degree of hydrolysis is decisive but the presence and stability of IgE and T cell epitopes in the hydrolysate recognized by individual patients., (© 2013 John Wiley & Sons Ltd.)

Scope: Due to the imminent growth of the world population, shortage of protein sources for human consumption will arise in the near future. Alternative and sustainable protein sources (e.g. insects) are being explored for the production of food and feed. In this project, the safety of Yellow mealworms (Tenebrio molitor L.) for human consumption was tested using approaches as advised by the European Food Safety Authority for allergenicity risk assessment., Methods and Results: Different Yellow mealworm protein fractions were prepared, characterised, and tested for cross-reactivity using sera from patients with an inhalation or food allergy to biologically related species (House dust mite (HDM) and crustaceans) by immunoblotting and basophil activation. Furthermore, the stability was investigated using an in vitro pepsin digestion test. IgE from HDM- and crustacean allergic patients cross-reacted with Yellow mealworm proteins. This cross-reactivity was functional, as shown by the induction of basophil activation. The major cross-reactive proteins were identified as tropomyosin and arginine kinase, which are well known allergens in arthropods. These proteins were moderately stable in the pepsin stability test., Conclusion: Based on these cross-reactivity studies, there is a realistic possibility that HDM- and crustacean allergic patients may react to food containing Yellow mealworm proteins., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow's milk allergens the T cell epitopes have been described, the T cell epitopes in the major allergen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this study was to determine the T cell epitopes in α-LAC., Methods: Nineteen synthetic peptides spanning α-LAC were obtained. Cow's milk-specific T cell lines (TCLs) of 46 subjects were generated and tested for their specificity for α-LAC. The lines responding to α-LAC were subsequently tested to determine their activation in response to the peptides., Results: More than half of the TCLs generated did not respond to α-LAC or lost their responsiveness during subsequent experiments, which indicates that α-LAC has low immunogenicity. Only 8 TCLs recognized 1 or more peptides. The recognition of the peptides was diverse and no major epitopes could be defined., Conclusion: The immunogenicity of α-LAC is very low compared to other major allergens in cow's milk. Moreover, there seems to be no dominant epitope present in the protein. Therefore, it seems unlikely that peptides of this protein can be used for immunotherapy., (© 2014 S. Karger AG, Basel.)

Background: Prior exposure to partial whey hydrolysates has been shown to reduce the allergic response to whey in mice. This effect was more pronounced in combination with a diet containing non-digestible oligosaccharides (scGOS/lcFOS/pAOS). It is unknown which fractions/epitopes are responsible for this effect. Therefore, the prophylactic ability of synthetic peptides of β-lactoglobulin with/without a scGOS/lcFOS/pAOS-containing diet to reduce the allergic response in a mouse model for cow's milk allergy was investigated., Methods: Of 31 peptides, nine peptides were selected based on human T cell data. Mice were pre-treated orally with three peptide mixtures or single peptides for six consecutive days. During this period, they received a control or scGOS/lcFOS/pAOS-containing diet. Subsequently, mice were orally sensitized to whey and received an intradermal and oral challenge. After sacrifice, serum and mesenteric lymph nodes (MLN) were collected for further analysis., Results: Prior exposure to peptide mixtures 1 and 3 significantly reduced the acute allergic skin response to whey. Mixture 2 showed no effect. An additive effect of the scGOS/lcFOS/pAOS-containing diet was only observed for mixture 1. Of the peptides in mixture 1, one peptide (LLDAQSAPLRVYVEELKP) showed the strongest effect on the acute allergic skin response. This peptide also tended to decrease whey-specific antibody levels and to increase the percentages of CD11b+CD103+ dendritic cells and CD25+Foxp3+ T cells in the MLN., Conclusions: Prior exposure to specific peptides of β-lactoglobulin reduces the allergic response to whey, which may involve regulatory dendritic and T cells. Combining peptides with a sGOS/lcFOS/pAOS-containing diet enhances this effect., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Allergen-IgE complexes are more efficiently internalized and presented by B cells than allergens alone. It has been suggested that IgG Abs induced by immunotherapy inhibit these processes. Food-allergic patients have high allergen-specific IgG levels. However, the role of these Abs in complex formation and binding to B cells is unknown. To investigate this, we incubated sera of peanut- or cow's milk-allergic patients with their major allergens to form complexes and added them to EBV-transformed or peripheral blood B cells (PBBCs). Samples of birch pollen-allergic patients were used as control. Complex binding to B cells in presence or absence of blocking Abs to CD23, CD32, complement receptor 1 (CR1, CD35), and/or CR2 (CD21) was determined by flow cytometry. Furthermore, intact and IgG-depleted sera were compared. These experiments showed that allergen-Ab complexes formed in birch pollen, as well as food allergy, contained IgE, IgG1, and IgG4 Abs and bound to B cells. Binding of these complexes to EBV-transformed B cells was completely mediated by CD23, whereas binding to PBBCs was dependent on both CD23 and CR2. This reflected differential receptor expression. Upon IgG depletion, allergen-Ab complexes bound to PBBCs exclusively via CD23. These data indicated that IgG Abs are involved in complex formation. The presence of IgG in allergen-IgE complexes results in binding to B cells via CR2 in addition to CD23. The binding to both CR2 and CD23 may affect Ag processing and presentation, and (may) thereby influence the allergic response.

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., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: T cell responses involved in peanut allergy are poorly understood., Objective: To investigate T cell responses towards major peanut allergens in peanut-allergic (PA) subjects compared with peanut-sensitized (PS) non-allergic children and non-atopic (NA) controls., Methods: Eighteen PA children, seven non-allergic PS children and 11 NA adults were included. Peripheral blood mononuclear cells were stimulated with a crude peanut extract (CPE). Short-term T cell lines were generated and subsequently stimulated with CPE and purified Ara h 1, Ara h 2, Ara h 3 and Ara h 6. The proliferation and production of IL-13, IFN-gamma, IL-10 and TNF-alpha were analysed., Results: Proliferation to CPE and major allergens was enhanced in PA subjects. The primary response to CPE was comparable with PS subjects, with increased production of IL-13 and IFN-gamma compared with NA. Production of IL-10 was not observed. In short-term T cell lines, the response to CPE was stronger in PA than in PS and NA subjects. Only PA children had a detectable response to major peanut allergens, characterized by IL-13 production. The response was the highest after Ara h 3 stimulation, and the lowest after Ara h 2 stimulation. No significant correlation was observed between peanut-specific IgE levels and T cell responses to CPE., Conclusion: T cell responses to CPE in PA and PS children were characterized by Th1 and Th2 cytokines. Only PA children showed enhanced Th2 responses to Ara h 1, Ara h 3 and Ara h 6.

Background: Better understanding of the relationship between antibody response to peanut and clinical sensitivity might lead to more accurate prognostication., Objective: We sought to investigate peanut-specific IgE and IgG4 epitope diversity in relation to challenge-defined clinical sensitivity to peanut in a group of peanut-sensitized children., Methods: Clinical sensitivity was determined by means of double-blind, placebo-controlled peanut challenges in 24 sensitized children. Six atopic control subjects were included. Specific IgE and IgG4 binding to 419 overlapping 15-amino-acid peptides representing the sequence of recombinant Ara h 1, Ara h 2, and Ara h3 was analyzed by means of microarray immunoassay., Results: Peanut-sensitized patient sera bound significantly more IgE and IgG4 epitopes than control sera. This patient group reacted to the same Ara h 1, Ara h 2, and Ara h 3 epitopes as reported previously. There was a positive correlation between IgE epitope diversity (ie, number of epitopes recognized) and clinical sensitivity (r = 0.6), such that patients with the greatest epitope diversity were significantly more sensitive than those with the lowest diversity (P = .021). No specific epitopes were associated with severe reactions to peanut. IgG4 binding was observed to largely similar epitopes but was less pronounced than IgE binding and did not relate to the clinical sensitivity to peanut. IgE and IgG4 epitope-recognition patterns were largely stable over a 20-month period., Conclusion: Clinical sensitivity, as determined by means of double-blind, placebo-controlled peanut challenge, is positively related to a more polyclonal IgE response, which remains stable over time.

Background: Hazelnut allergy in birch pollen-exposed areas is usually due to cross-reactivity (Cor a 1 and 2) and is usually mild in nature (oral allergy). In areas without birches, severe reactions are more prevalent and linked to sensitization to the lipid transfer protein (LTP) Cor a 8., Objective: We sought to investigate whether sensitization to LTP plays a role in more severe (objective) hazelnut-induced symptoms in children from a birch-endemic area., Methods: Sensitization to Cor a 8, Cor a 2, Cor a 1, and Bet v 1 was determined by means of RASTs and immunoblotting in hazelnut-sensitized children with (n = 8) and without (n = 18) objective reactions during double-blind, placebo-controlled food challenges. Additionally, samples from 191 hazelnut-sensitized nonchallenged children were analyzed., Results: Children with objective reactions during double-blind, placebo-controlled food challenge had higher IgE titers to hazelnut (P < .001) and recognized more allergens on immunoblotting (P = .001) than those without such reactions. All children with objective symptoms were sensitized to Cor a 8 (0.51-23.3 IU/mL) compared with only 1 child without objective reactions (0.90 IU/mL). In a multivariate analysis only IgE against Cor a 8 remained as an independent risk factor (undefined odds ratio; P < .0001). In the group of nonchallenged children (n = 191), the prevalence of LTP sensitization was greater than 30%. Unexpectedly, sensitization to Cor a 1 was observed in children not sensitized to Bet v 1., Conclusion: Sensitization to hazelnut LTP is a risk factor for objective symptoms in children from a birch-endemic area.

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.

Background: Recognition of specific peanut allergens or the diversity of IgE binding to peanut allergens may play a role in the elicitation of severe allergic reactions., Objective: To investigate whether sensitization to individual allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 is correlated with clinical severity., Methods: The reactivity of purified peanut allergens was measured by skin prick test (SPT) and by IgE immunoblot in 30 patients. The results were related to the clinical reactivity by history, and in 25 of them to the eliciting dose (ED)., Results: The majority of patients recognized Ara h 2 and Ara h 6. Patients with severe symptoms had a higher SPT response to Ara h 2 and Ara h 6 at low concentrations (0.1 micro g/mL) and to Ara h 1 and Ara h 3 at higher concentrations (100 micro g/mL), compared with patients with mild symptoms. They also recognized a greater number of allergens and showed a higher cumulative SPT response compared with patients with mild symptoms. No significant differences were observed between patients with a low or high ED., Conclusions: Ara h 2 and Ara h 6 appeared to be more potent than Ara h 1 and Ara h 3. Both SPT reactivity to low concentrations of Ara h 2 and Ara h 6 and to higher concentrations of Ara h 1 and Ara h 3 were shown to be indicative of severe symptoms.

The prototypic receptor for IgA (FcalphaRI, CD89) is expressed on myeloid cells and can trigger phagocytosis, tumor cell lysis, and release of inflammatory mediators. The functions of FcalphaRI and activating receptors for IgG (FcgammaRI and FcgammaRIII) are dependent on the FcR gamma-chain dimer. This study increases our understanding of the molecular basis of the FcalphaRI-FcR gamma-chain transmembrane interaction, which is distinct from that of other activatory FcRs. FcalphaRI is unique in its interaction with the common FcR gamma-chain, because it is based on a positively charged residue at position 209, which associates with a negatively charged amino acid of FcR gamma-chain. We explored the importance of the position of this positive charge within human FcalphaRI for FcR gamma-chain association and FcalphaRI functioning with the use of site-directed mutagenesis. In an FcalphaRI R209L/A213H mutant, which represents a vertical relocation of the positive charge, proximal and distal FcR gamma-chain-dependent functions, such as calcium flux, MAPK phosphorylation, and IL-2 release, were similar to those of wild-type FcalphaRI. A lateral transfer of the positive charge, however, completely abrogated FcR gamma-chain-dependent functions in an FcalphaRI R209L/M210R mutant. By coimmunoprecipitation, we have demonstrated the loss of a physical interaction between FcR gamma-chain and FcalphaRI M210R mutant, thus explaining the loss of FcR gamma-chain-dependent functions. In conclusion, not only the presence of a basic residue in the transmembrane region of FcalphaRI, but also the orientation of FcalphaRI toward the FcR gamma-chain dimer is essential for FcR gamma-chain association. This suggests the involvement of additional amino acids in the FcalphaRI-FcR gamma-chain interaction.

A modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5-95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy induction.