Co-sensitization to the three non-homologous major cashew allergens Ana o 1, Ana o 2 and Ana o 3 is caused by IgE cross-reactivity.

BACKGROUND: Cashew nuts often cause strong allergic reactions, even exceeding those of peanuts. Ana o 1 (vicilin), Ana o 2 (legumin) and Ana o 3 (2S albumin) are major cashew allergens. Co-sensitization to all three non-homologous cashew nut allergens has been observed. We hypothesize that this might be due to IgE cross-reactivity. METHODS: IgE cross-inhibitions were performed with Ana o 1-3 using sera from cashew nut allergic patients. Related hazelnut allergens Cor a 11, 9 and 14 were used as controls. For comparison, IgE cross-reactivity between the hazelnut allergens was investigated using sera from hazelnut allergic patients. RESULTS: Median percentages of cross-inhibitions between Ana o 1-3 were 84-99%. In comparison, medians of cross-inhibitions between hazelnut allergens were 33-62%. The IC50 values revealed the highest IgE affinity to Ana o 3 and Cor a 14. Hazelnut legumin Cor a 9 inhibited IgE-binding to Ana o 1, 2, and 3 with median percentages of 75%, 56%, and 48%, respectively. No cross-reactivity was observed between allergenic vicilins or between 2S albumins from cashew and hazelnut. In silico identified potentially cross-reactive peptides of Ana o 3 overlapped with previously reported IgE epitopes of all three allergens. CONCLUSIONS: IgE with high affinity to Ana o 3 that cross-reacts with the other two major non-homologous cashew nut allergens might be responsible for the high allergenic potency of cashew nut. These cross-reactive IgE comprises the major fraction of specific IgE in cashew allergic patients, and might be responsible for cross-reactivity between unrelated tree nuts.

Concomitant sensitization to legumin, Fag e 2 and Fag e 5 predicts buckwheat allergy.

BACKGROUND: Buckwheat (Fagopyrum esculentum) has become increasingly popular as a healthy food in Europe. However, for sensitized individuals, consumption can cause anaphylactic reactions. The aim of this study was to identify individual well-characterized buckwheat allergens for component-resolved diagnosis. METHODS: Patients were selected by positive skin prick test to buckwheat and divided into two groups: (1) sensitized to buckwheat without clinical symptoms and (2) buckwheat allergy. Buckwheat proteins were extracted from raw buckwheat seeds, purified applying a combination of protein precipitation and chromatographic methods, and analyzed by IgE immunoblotting and ELISA. RESULTS: Buckwheat-allergic patients had a significantly larger median skin prick test weal diameter for buckwheat than the sensitized group and the positive control. Also, IgE immunoblotting clearly showed a distinct pattern in sera from allergic patients when compared to sensitized individuals. Several IgE-reactive proteins were purified from crude buckwheat extract, namely legumin (Fag e 1 plus its large subunit), Fag e 2 (2S albumin), and newly identified Fag e 5 (vicilin-like) as well as hevein-like antimicrobial peptides, designated Fag e 4. All four allergens showed superior diagnostic precision compared to extract-based ImmunoCAP with high sensitivity as well as high specificity. CONCLUSIONS: Patients with clinical symptoms clearly show a distinct allergen recognition pattern. We characterized a buckwheat vicilin-like protein as a new relevant marker allergen, designated Fag e 5. Additionally, another new allergen, Fag e 4, potentially important for cross-reactivity to latex was added to the allergen panel of buckwheat. Further, our data show that the full-length legumin comprising both, large and small subunit should be applied for component-resolved diagnosis. Our data indicate that concomitant sensitization to legumin, Fag e 2 and Fag e 5, predicts buckwheat allergy.

IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome.

BACKGROUND: Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. METHODS: Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. RESULTS: Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. CONCLUSION: Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac.

Allergen chip diagnosis for soy-allergic patients: Gly m 4 as a marker for severe food-allergic reactions to soy.

BACKGROUND: Gly m 5 and Gly m 6 are known to induce severe reactions in soy-allergic patients. For birch pollen (BP)-allergic patients, the Bet v 1 homologous allergen Gly m 4 is also a potential trigger of generalized severe reactions upon soy consumption. Therefore, reliable component-resolved diagnosis of soy allergy is needed. METHODS: IgE reactivity from sera of 20 patients from a BP environment with reported soy allergy was assessed. Skin prick tests (SPT) with BP and soy drink were performed. Specific IgE for BP, soy, Bet v 1 and Gly m 4 was analyzed by ImmunoCAP. In addition, ISAC microarray profiling was performed. RESULTS: Nineteen of 20 patients were BP allergic (positive SPT and/or CAP results for BP extract and Bet v 1). Eighteen soy-allergic patients were tested positive with soy drink in SPT. Soy CAP results were negative in the majority of tests (15/20), whereas 19/20 sera had specific IgE to Gly m 4. In the microarray approach, 14/20 sera displayed Gly m 4-specific IgE, the additional 6 sera had IgE levels below 0.3 ISAC standardized units. The BP-negative serum had Gly m 5- and Gly m 6-specific IgE which correlated with positive soy ImmunoCAP. CONCLUSIONS: Soy sensitization detected by SPT and Gly m 4 ImmunoCAP were in good qualitative agreement with ISAC results. Soy ImmunoCAP was only specific for Gly m 5 and Gly m 6 sensitization. Gly m 4 ImmunoCAP has a higher sensitivity than ImmunoCAP ISAC. In this patient cohort, Gly m 4 sensitization was linked to the development of severe and generalized allergic reactions upon soy consumption.

¹H, ¹³C and ¹5N resonance assignments and second structure information of Gad m 1: a ß-parvalbumin allergen from Atlantic cod (Gadus morhua).

Gad m 1 is the major allergen from Atlantic cod. It belongs to ß-parvalbumin protein family and is characterized by the presence of two calcium-binding sites so called EF-hand motifs. ß-Parvalbumins such as Gad m 1 are the most important fish allergens and their high cross-reactivity is the cause of the observed polysensitization to various fish species in allergic patients. Despite extensive efforts, the complete elucidation of ß-parvalbumin-IgE complexes has not been achieved yet. Allergen structural studies are essential for the development of novel immunotherapy strategies, including vaccination with hypoallergenic derivatives and chimeric molecules. Here, we report for the first time the NMR study of a ß-parvalbumin: Gad m 1. This report includes: (1)H, (13)C and (15)N resonance assignments of Gad m 1 as well as the second structure information based on the (13)C chemical shifts.

The performance of a component-based allergen microarray for the diagnosis of kiwifruit allergy.

BACKGROUND: Allergy to kiwifruit is increasingly reported across Europe. Currently, the reliability of its diagnosis by the measurement of allergen-specific IgE with extracts or by skin testing with fresh fruits is unsatisfying. OBJECTIVE: To evaluate the usefulness of a component-based allergen microarray for the diagnosis of kiwifruit allergy in a large group of patients. METHODS: With an allergen microarray, we measured specific IgE and IgG4 levels to a panel of nine kiwifruit allergens in sera of 237 individuals with kiwifruit allergy. Sera from 198 allergic patients without kiwifruit allergy served as controls. Furthermore, we determined the extent of sensitization to latex. RESULTS: The panel of kiwifruit allergens showed a diagnostic sensitivity of 66%, a specificity of 56% and a positive predictive value of 73%. Sera from kiwifruit-allergic patients contained significantly more frequently Act d 1-specific IgE than sera from control patients. Furthermore, 51% of the positive sera contained IgE directed to a single allergen, namely Act d 1 (45%), Act d 9 (27%) or Act d 7 (13%). Within the control group, 36% sera recognized a single allergen. Out of those, 48% were positive to the cross-reactive glycoallergen Act d 7, 43% to the profilin Act d 9 and only 5% to Act d 1. Allergen-specific IgG4 levels did not differ between kiwifruit-allergic and -tolerant patients. Kiwifruit- and latex-allergic patients contained Hev b 11-specific IgE significantly more frequently than latex-allergic patients without kiwifruit allergy. CONCLUSIONS: Act d 1 can be considered a marker allergen for genuine sensitization to kiwifruit. We demonstrated that a component-based kiwifruit allergen microarray would improve the prognostic value of in vitro diagnostic tests.

Bet v 1 and its homologous food allergen Api g 1 stimulate dendritic cells from birch pollen-allergic individuals to induce different Th-cell polarization.

BACKGROUND: Bet v 1 is the most relevant sensitizing protein for birch pollen (BP)-allergic individuals. Its homologues from plant foods are mainly involved in allergic reactions caused by IgE cross reactivity. We aimed to evaluate the polarizing effect of dendritic cells (DCs) pulsed with Bet v 1, Mal d 1, Api g 1 or Dau c 1 on Th-cell responses. METHODS: Immature DCs were generated from peripheral blood monocytes of BP-allergic and healthy donors by culture with GM-CSF and IL-4 and subsequently pulsed with allergens in combination with maturation factors. Cell surface markers were analysed by FACS. Mature DCs were co-cultured with autologous Th cells and T-cell proliferation and cytokine profiles were determined. RESULTS: In co-culture, mature allergen-pulsed DCs induced autologous Th cells of BP-allergic donors to proliferate significantly more than those of healthy individuals. Exposure of DCs from BP-allergic donors to Bet v 1 resulted in a robust Th2 skewing with significantly higher quantities of IL-5 and elevated IL-13 compared to maturation factors. In contrast, Api g 1-primed DCs from BP allergics significantly enhanced the production of the Th1 cytokine IFN-γ and significantly down-regulated IL-13 compared to maturation factors. In healthy donors, no significant cytokine production could be detected. CONCLUSION: Bet v 1 in contrast to homologous food allergens seems to possess distinct molecular features that enable it to condition DCs from BP-allergic donors to induce allergen-specific T-cell proliferation and Th2 polarization.

Expression levels of parvalbumins determine allergenicity of fish species.

BACKGROUND: Parvalbumins are the most important fish allergens. Polysensitization to various fish species is frequently reported and linked to the cross-reactivity of their parvalbumins. Studies on cross-reactivity and its association to the allergenicity of purified natural parvalbumins from different fish species are still lacking. In addition, some studies indicate that dark muscled fish such as tuna are less allergenic. METHODS: Total protein extracts and purified parvalbumins from cod, whiff, and swordfish, all eaten frequently in Spain, were tested for their IgE-binding properties with 16 fish allergic patients' sera from Madrid. The extent of cross-reactivity of these parvalbumins was investigated by IgE ELISA inhibition assays. Additionally, the cDNA sequences of whiff and swordfish parvalbumins were determined. RESULTS: Extractable amounts of parvalbumins from cod were 20 times and from whiff 30 times higher than from swordfish. Parvalbumins were recognized by 94% of the patients in extracts of cod and whiff, but only by 60% in swordfish extracts. Nevertheless, a high cross-reactivity was determined for all purified parvalbumins by IgE inhibition. The amino acid sequence identities of the three parvalbumins were in a range of 62-74%. CONCLUSIONS: The parvalbumins of cod, whiff and swordfish are highly cross-reactive. The high amino acid sequence identity among cod, whiff and swordfish parvalbumins results in the observed IgE cross-reactivity. The low allergenicity of swordfish is due to the low expression levels of its parvalbumin.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Generation of allergen-enriched protein fractions of Hevea brasiliensis latex for in vitro and in vivo diagnosis.

BACKGROUND: The latex of Hevea brasiliensis trees contains a complex proteome that includes a range of allergenic proteins. Current latex extracts that are used for the diagnosis of latex allergy still lack important allergens. We aimed to devise a production process for an improved reagent that would ideally contain the complete latex allergome. METHODS: Latex C-serum was fractionated by ammonium sulfate precipitation, and B- and C-serum proteins were then separated by anion exchange chromatography. Proteins eluting within defined salt concentration ranges were pooled into six final fractions. Fractions were evaluated by two-dimensional electrophoresis and subsequent IgE immunoblot for their spectrum of allergens. The presence of the most important latex allergens in the fractions was checked by Western blot analyses. Each fraction was further evaluated by skin prick test (SPT). RESULTS: Reproducibility of the preparation method was demonstrated with two batches of latex. Comparison of latex B- and C-serum to the six fractions showed a remarkable increase in the number of detectable allergens in the fractions. The presence of the latex allergens Hev b 1-8 and Hev b 13 in the fractions was demonstrated. In SPTs, the fractions produced wheal-and-flare reactions comparable to commercial latex extracts. CONCLUSIONS: This method provides reproducible latex protein fractions of high allergen content for the diagnosis of latex allergy.