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Background: Allergic sensitisation towards cashew nut often happens without a clear history of eating cashew nut. IgE cross-reactivity between cashew and pistachio nut is well described; however, the ability of cashew nut-specific IgE to cross-react to common tree nut species and other Anacardiaceae, like mango, pink peppercorn, or sumac is largely unknown. Objectives: Cashew nut allergic individuals may cross-react to foods that are phylogenetically related to cashew. We aimed to determine IgE cross-sensitisation and cross-reactivity profiles in cashew nut-sensitised subjects, towards botanically related proteins of other Anacardiaceae family members and related tree nut species. Method: Sera from children with a suspected cashew nut allergy (n = 56) were assessed for IgE sensitisation to common tree nuts, mango, pink peppercorn, and sumac using dot blot technique. Allergen cross-reactivity patterns between Anacardiaceae species were subsequently examined by SDS-PAGE and immunoblot inhibition, and IgE-reactive allergens were identified by LC-MS/MS. Results: From the 56 subjects analysed, 36 were positive on dot blot for cashew nut (63%). Of these, 50% were mono-sensitised to cashew nuts, 19% were co-sensitised to Anacardiaceae species, and 31% were co-sensitised to tree nuts. Subjects co-sensitised to Anacardiaceae species displayed a different allergen recognition pattern than subjects sensitised to common tree nuts. In pink peppercorn, putative albumin- and legumin-type seed storage proteins were found to cross-react with serum of cashew nut-sensitised subjects in vitro. In addition, a putative luminal binding protein was identified, which, among others, may be involved in cross-reactivity between several Anacardiaceae species. Conclusions: Results demonstrate the in vitro presence of IgE cross-sensitisation in children towards multiple Anacardiaceae species. In this study, putative novel allergens were identified in cashew, pistachio, and pink peppercorn, which may pose factors that underlie the observed cross-sensitivity to these species. The clinical relevance of this widespread cross-sensitisation is unknown. [ABSTRACT FROM AUTHOR]

Background: Enhancing the quality and yield of protein production in heterologous expression systems is an important issue for developing new biopharmaceuticals. It has been shown that the dynamics of protein folding is influenced by codon frequencies. As codon usage frequencies are species specific, this can affect heterologous protein expression. In this respect, "codon harmonization," that is, the usage of synonymous codons with usage frequencies in the host resembling the usage frequencies in the native organism, is a promising strategy. As recombinant proteins are important tools in the area of allergy research, we investigated in this study the influence of codon harmonization on the production of the major birch pollen allergen Bet v 1.0101. Methods: To accomplish this task, parallel production of several batches of rBet v 1, BWT, together with a harmonized variant, BH, was applied. The expression yield of soluble and insoluble protein was assayed via densitometric analysis of -SDS-PAGEs for every batch. The quality of purified proteins was assessed with a variety of physicochemical methods including mass spectrometry, circular dichroism, dynamic light scattering, Fourier transform infrared spectroscopy, in vitro degradation, and 1-anilino-8-naphthalene sulfonate-binding assays. Patients' IgE reactivity was tested in enzyme-linked immunosorbent assays and rat basophil mediator release experiments. Results: No significant differences in the ligand-binding capacity and secondary structure elements, as well as, in immunological assays could be found; however, the production yield was drastically increased for BH. Conclusion: We could show that codon harmonization is a powerful method to enhance protein yields in heterologous expression systems and should be considered especially for difficult-to-express proteins. [ABSTRACT FROM AUTHOR]

Background: Google Trends (GT) searches trends of specific queries in Google, which potentially reflect the real-life epidemiology of allergic rhinitis. We compared GT terms related to ragweed pollen allergy in American and European Union countries with a known ragweed pollen season. Our aim was to assess seasonality and the terms needed to perform the GT searches and to compare these during the spring and summer pollen seasons. Methods: We examined GT queries from January 1, 2011, to January 4, 2017. We included 15 countries with a known ragweed pollen season and used the standard 5-year GT graphs. We used the GT translation for all countries and the untranslated native terms for each country. Results: The results of “pollen,” “ragweed,” and “allergy” searches differed between countries, but “ragweed” was clearly identified in 12 of the 15 countries. There was considerable heterogeneity of findings when the GT translation was used. For Croatia, Hungary, Romania, Serbia, and Slovenia, the GT translation was inappropriate. The country patterns of “pollen,” “hay fever,” and “allergy” differed in 8 of the 11 countries with identified “ragweed” queries during the spring and the summer, indicating that the perception of tree and grass pollen allergy differs from that of ragweed pollen. Conclusions: To investigate ragweed pollen allergy using GT, the term “ragweed” as a plant is required and the translation of “ragweed” in the native language needed. [ABSTRACT FROM AUTHOR]

Background: Plant food allergies associated with lipid transfer protein (LTP) have been widely described in the Mediterranean Basin. Objective: The aim of this work was to describe the clinical profile and pollen sensitization of plant foodallergic patients sensitized to LTP in a non-Mediterranean area. Methods: Patients with clear IgE-mediated symptoms associated with plant foods and a positive skin prick test (SPT) to Pru p 3 were included in a prospective study in the north of Spain. Reported symptoms were analyzed together with a battery of food and pollen SPTs and specific IgE components by ISAC microarray. Cross-inhibition studies were performed by ImmunoCAP with plane tree, mugwort and rPru p 3. Results: Among the 72 patients included, the most frequent food allergy reported was to peaches (69%) followed by nuts (walnuts 55%, peanuts 54% and hazelnuts 43%). Most patients suffered from symptoms with multiple plant foods (a median of 6 foods per patient). Regarding the patients' pollen sensitization, 36% were sensitized to mugwort pollen (72% showing sIgE to Art v 3), 33% to grass pollen and 24% to plane tree pollen (94% with sIgE to Pla a 3). Inhibition studies showed that specific IgEs against mugwort and plane tree pollen are inhibited by Pru p 3 in a strong manner, whereas Pru p 3 was less inhibited by pollen extracts. Conclusions: LTP syndrome occurs in a non-Mediterranean area and is related to multiple sensitizations to foods and pollens such as plane tree and mugwort. In these pollen sensitizations, Pru p 3 seems to be the primary sensitizer. [ABSTRACT FROM AUTHOR]

Background: Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods: Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-assay coefficient of variation for the BaGP-allergic serum pool was 6.92%. Conclusions: Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Pollutants and other stressing factors like mold infection might increase the production of pathogen-related proteins in plants. Since this is invoked as one of the causes for the high prevalence of allergic diseases in devel-oped countries, we aimed to determine the potential effect of environmental pollution, with or without mold Infection of the trees, on the allergenic potency of pine pollen (Pinus radiata). Methods: Pine pollen samples were recovered from three selected areas: low polluted (A), highly polluted (B) and highly polluted and infected with fungi (Spheropsis sapinea) (C).The allergenic potency of pollen from areas A, B or C were compared in vivo in 35 pine pollen-allergic patients by skin prick test and specific IgE (slgE) quantification. Pollen was also analyzed in vitro by SDS-PAGE immunoblotting, RAST inhibition and cDNA-AFLP (amplified fragment length poly-morphism) to compare differences in proteins and mRNA ex-pression. Results:The allergenic potency measured by prick test, slgE and RAST inhibition was greater in pollen A, which was exposed to smaller amounts of NOx, PM10 and S02 but greater amounts of 03. No differences were found in IgE-binding bands in immunoblotting or densitometry of the bands. In cDNA-AFLP, three homologous transcript-derived fragments were expressed in samples B only, with an ex-pressed sequence tag related with stress-regulated gene ex-pression. Conclusions: A greater allergenic potency, In terms of skin tests and slgE, is observed in pine pollen coming from unpolluted areas. We consider that this fact might be related to a higher exposure to ozone, resulting in a greater expres-sion of allergenic proteins. [ABSTRACT FROM AUTHOR]

Background and Objective: Reducing the allergenicity of food allergens can suppress the clinical symptoms of food allergy. The objective of the present study was to investigate the effects of processing on the allergenic properties of hen's egg white proteins. Methods: Eggs were processed by traditional Chinese cooking, including steaming, water boiling, frying, spicing and tea boiling. The contents of processed egg protein were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis; the allergenicity was evaluated by Western blotting, enzyme-linked immunosorbent assay and enzyme allergosorbent test inhibition. Circular dichroism spectrum analysis of four major egg allergens from various egg products was performed as well. A mouse model of food allergy was developed to test the allergenicity of processed egg protein in vivo. Results: Protein degradation was significant following tea boiling and spiced-tea boiling. The total allergenic potential of water-boiled egg and fried egg was relatively higher than that of steamed egg, spiced egg and tea-boiled egg. Challenge with proteins from raw egg, water-boiled egg and fried egg induced skewed T-helper 2 pattern responses (Th2 responses) in the intestine of mice sensitized to egg proteins; however, when the mice sensitized to egg proteins were challenged with proteins from steamed egg, spiced egg and tea-boiled egg, respectively, only weak Th2 responses were induced in their intestine. Conclusion: Processing by steaming, spicing, or tea boiling can weaken the allergenicity of egg proteins. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Patients with pollinosis develop symptoms after intake of plant food more often than the general population. In order to study the prevalence of the presentation of allergic symptoms to plant foods in pollinosis, we selected a representative sample of the population from our Mediterranean area. Methods: All patients completed a questionnaire, provided a blood sample and underwent a battery of skin and other complementary tests (prick-prick, oral challenge test) when necessary. The pollen counts were obtained from the Elche pollen station. In addition, sera from a subgroup of patients were checked with an allergen molecule panel on an Advia Centaur XP platform. Results: Of the final sample (n = 233), 39.9% of the patients with pollinosis were sensitized and 30.9% had clinical allergy to at least one of the plant foods studied. Regression analysis showed that age and sensitization to the extracts of Platanus acerifolia and Artemisia vulgaris were the most important variables for discriminating between groups. Patients with pollinosis at a risk of allergy to plant foods had significantly higher Pru p 3 values [odds ratio (OR) 3.3; 95% confidence interval (95% CI) 2.3-4.8], and the value increased according to the number of plant food sensitizations. Conclusion: Plant food allergy is more common in patients with pollinosis than in the general population. The use of the London plain tree (P. acerifolia) and mugwort (A. vulgaris) in the skin tests may help identify such patients in our Mediterranean area, but determination of rPru p 3 could also be very useful in patients suspected of having plant food allergy. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4+ T cell epitope peptides of the major BaGP allergen, Pas n 1. Methods: Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. Results: Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. Conclusions: The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. Methods: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. Results: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. Conclusions: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Only a few randomized controlled trials have been carried out to evaluate various complementary treatments for allergic disorders. This study assessed the effects of the preseasonal use of birch pollen honey (BPH; birch pollen added to honey) or regular honey (RH) on symptoms and medication during birch pollen season. Methods: Forty-four patients (59% female, mean age 33 years) with physician-diagnosed birch pollen allergy consumed either BPH or RH daily in incremental amounts from November 2008 to March 2009. Seventeen patients (53% female, mean age 36 years) on their usual allergy medication served as the control group. From April to May, patients recorded daily rhinoconjunctival and other symptoms and their use of medication. Fifty patients completed the study. Results: During birch pollen season in 2009, BPH patients reported a 60% lower total symptom score (p < 0.01), twice as many asymptomatic days (p < 0.01), and 70% fewer days with severe symptoms (p < 0.001), and they used 50% less antihistamines (p < 0.001) compared to the control group. The differences between the BPH and RH groups were not significant. However, the BPH patients used less antihistamines than did the RH patients (p < 0.05). Conclusions: Patients who preseasonally used BPH had significantly better control of their symptoms than did those on conventional medication only, and they had marginally better control compared to those on RH. The results should be regarded as preliminary, but they indicate that BPH could serve as a complementary therapy for birch pollen allergy. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) are accepted to be important molecules in the development and maintenance of allergic diseases. Although several types of histamine H1 receptor antagonist (antihistamine) have been developed and used for the treatment of allergic diseases, the influence of antihistamines on TARC and MDC production is not well understood. Objective: The present study was undertaken to examine the influence of antihistamines on TARC and MDC production from CD14+ cells after antigenic stimulation in vitro. Methods: CD14+ cells prepared from patients with pollinosis to Japanese cedar pollen were stimulated with specific allergen extracted from Japanese cedar pollen (Cry j 1) in the presence of azelastine (AZE), ketotifen (KET), fexofenadine (FEX) and oxatomide (OXA) for 6 days. TARC and MDC levels in culture supernatants were examined by ELISA. We also examined the influence of FEX on TARC and MDC mRNA expression, phosphorylation of mitogen-activated protein kinases (MAPKs) and transcription factor activation in CD14+ cells after Cry j 1 stimulation. Results: FEX at 250 ng/ml, which is almost equal to therapeutic blood levels, caused a significant inhibition of TARC and MDC production.However, AZE, OXA and KET required higher concentrations than their therapeutic blood levels to suppress production of these factors. FEX at 250 ng/ml also suppressed NF-κB activation, phosphorylation of p38 MAPK and extracellular signal-regulated kinases 1 and 2 and expression of mRNA for TARC and MDC. Conclusions: These results suggest that antihistamines, especially FEX, suppress CC chemokine production from CD14+ cells through interference with antigen-mediated signaling and result in favorable modification of allergic disease states or conditions. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background:Ascaris lumbricoides induces a Th2 response and specific IgE synthesis in humans. This confers antiparasite immunity but could modify the natural history of allergic diseases in the tropics, justifying the study of its allergenic composition. We analyzed the allergenic properties of Ascaris tropomyosin and the frequency of sensitization in subjects exposed to the parasite. Methods: cDNA was obtained by reverse transcription PCR, cloned into pQE30-UA and purified as a 6× His-tagged protein. Equivalence with its natural counterpart was analyzed by cross-inhibition and liquid chromatography-tandem mass spectrometry. Specific IgE was measured by ELISA in 175 asthmatics and 170 nonasthmatics naturally exposed to the parasite and sensitized to the Ascaris extract. Results: The cDNA encoded 287 residues with high sequence identity with other invertebrate tropomyosins. The 40-kDa protein was recognized by human serum and affinity-purified anti-rBlo t 10 IgE. Specific IgE to tropomyosin could represent approximately 50% of the total IgE response to the extract. Ascaris tropomyosin induced wheal and flare in skin prick tests and histamine release from basophils. Although the prevalence of IgE to Ascaris tropomyosin was higher in asthmatic patients, logistic regression analysis suggested that this result was biased by sensitization to mites. Conclusions:A. lumbricoides tropomyosin (Asc l 3) is a new allergen that binds specific IgE, induces mediator release from effector cells and is cross-reactive to mite tropomyosins. IgE reactivity to this allergen is very frequent in both asthmatic and normal subjects sensitized to Ascaris extract. The potential role of Ascaris tropomyosin in asthma pathogenesis in tropical regions should be further investigated. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Russian thistle (Salsola kali) pollen is an important cause of pollinosis in areas where rainfall is not abundant. Our aim was to develop an ELISA for quantification of the major allergen of S. kali extracts, Sal k 1, and to assess the correlation of this allergen content with the allergenic activity of extracts. Methods: Sal k 1 was purified by ion exchange and gel permeation chromatography and identified by mass spectrometry. Monoclonal antibody 4C11 was used for capture at 5 μg/ml and biotin-labeled specific antiserum at 0.25 μg/ml served for detection. The allergenic activity of the pollen extracts was measured by enzyme allergosorbent test inhibition. Results: Sal k 1 reacted to 85% of sera from 40 S. kali-allergic patients and was able to inhibit 92% of the IgE-binding capacity of patients’ serum pool to the whole extract. The ELISA had a lineal range between 1.25 and 20 ng/ml of purified Sal k 1. The intra- and interassay coefficients of variation were lower than 5 and 10%, respectively. The assay was very sensitive since it had a detection limit of 0.08 ng/ml. No reactivity was found outside the Amaranthaceae family where only Kochia and Salicornia sp. gave significant reactivity. A good correlation (Spearman’s ρ = 0.92) was obtained between Sal k 1 content of different S. kali extracts and their IgE-binding activity. Conclusions: The results proved the usefulness of the two-site sandwich ELISA for aeroallergen control and for the standardization of S. kali pollen extracts intended for clinical use. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background:Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. Objectives:We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. Methods:We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. Results: cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. Conclusions:We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Mouse models of asthma suffer from the necessity to prime the animals by injections before respiratory exposure. Our aim was to develop a mouse model that mimics the progression of human allergic disease upon low-dose inhaled allergen exposure. Methods: Mice were primed intraperitoneally to ovalbumin (OVA) before they were exposed repeatedly to aerosols of either OVA, ryegrass (Lolium perenne) pollen extract, or both concomitantly. The sensitization to ryegrass pollen proteins was evaluated by measurement of specific serum antibody, by the respiratory response to a challenge with ryegrass pollen extract and by lung cytokine production after challenge. Results: Inhalation of ryegrass pollen extract alone did not result in sensitization. Sensitization to inhaled ryegrass pollen proteins, however, did occur in mice that had been sensitized to OVA by intraperitoneal injections and were then exposed to inhaled ryegrass pollen extract and OVA simultaneously. T and B cell priming was ascertained by ryegrass pollen-specific IgG1 and IgE antibody production and by induction of airway inflammation and of Th2 cytokine mRNA transcripts in the lungs upon airway challenge with ryegrass pollen extract. A progressive spread of the IgE/IgG1 response to different ryegrass pollen proteins could be visualized in immunoblots by comparing antibody patterns at day 56 and 86. Conclusions: Low-dose inhalatory allergen exposure results in sensitization when airways are exposed at the same time to another allergen to which the animals are already sensitized. This model can help to unravel the mechanisms that underlie the development and progression of respiratory allergic diseases. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Employees in strawberry greenhouses are highly exposed to several (potential) allergenic agents. However, no occupational allergy in this branch has been described before. First, the presence of work-related allergic symptoms in strawberry workers was explored. Second, we aimed to prove the concept that an IgE-mediated allergy could be responsible for work-related symptoms. To test the possibility of an IgE response secondary to cross-reactivity to birch or grass pollen, inhibition experiments were performed. Methods: First, a questionnaire survey concerning work-related allergic symptoms among strawberry workers in the Netherlands was carried out. Second, 3 workers with work-related symptoms were investigated in detail. Skin tests, serum-specific IgE tests with home-made extracts of strawberry pollen and other possible allergenic agents of the strawberry greenhouse environment were executed. Furthermore, immunoblots and nasal provocations with strawberry pollen extract were performed. In addition, inhibition experiments were performed. Results: 29 of 75 questionnaire respondents (38.7%) reported work-related symptoms. Sensitization to strawberry pollen was found in skin tests in all 3 employees with work-related symptoms. ELISA and immunoblotting with strawberry pollen showed positive results in 2 employees. Birch and grass pollen failed to inhibit IgE binding to strawberry pollen in 1 of 2 employees. Partial inhibition was seen in the second employee. Nasal provocation validated clinically relevant allergy to these pollens in 2 of 3 subjects. Conclusions: Allergic symptoms attributable to the workplace are present among a proportion of strawberry greenhouse employees. An IgE-mediated occupational allergy to strawberry pollen may contribute to these symptoms. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The Poaceae family is considered one of the main causes of pollen allergy in industrialized countries. The aim of this study is to establish the dynamics of the Poaceae allergens and determine their distribution in the different-sized particles in the atmosphere. Methods: The air of Granada (southern Spain) was sampled during the pollination period of Poaceae using a cascade impactor and a Hirst-type volumetric collector simultaneously. The sampled airborne allergens were analyzed by indirect ELISA and field emission scanning electron microscopy. Airborne pollen was evaluated with the Spanish Aerobiological Network methodology. Results:Poaceae pollen and allergenic activity have parallel dynamics during the period of maximum pollination, which is reflected in the positive correlations between the 2 variables. In addition, the highest Lol p 1 concentrations were recorded in particle sizes lower than 3.3 μm (stage 4-F). The Spearman correlation test showed that airborne allergens are not dependent on meteorological factors, such as humidity, wind direction or sunshine, however, Lol p 1 allergen correlated positively with Poaceae pollen. Conclusions: The results of the present study confirm that the Lol p 1 allergen is detected more frequently with pollutants than with coarse particles with similar dynamics and a positive correlation between airborne pollen and aeroallergens. Moreover, Lol p 1 is released in stable weather conditions without large changes in humidity or temperature. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The house dust mite allergen Der p 2 is one of the most important indoor allergens associated with allergic disease. Recombinant Der (rDer) p 2 with high IgE binding activity can be readily produced in Escherichia coli and Pichia pastoris, but the structure and IgE binding of the different methods of preparation have not been compared. Methods: Secondary structure was assessed by circular dichroism (CD). Intrinsic fluorescence and hydrophobic probe (1-anilinonaphthalene 8-sulphonic acid, ANS) were used to study the Der p 2 hydrophobic cavity. IgE binding was assessed by ELISA inhibition. Results: CD analysis showed the expected secondary structure for both nDer p 2 and refolded Der p 2 prepared from E. coli inclusion bodies but primarily random structure for Der p 2 secreted from P. pastoris. The secreted product, however, had disulphide bonding and could be refolded to a similar structure to natural Der (nDer) p 2 after precipitation with trichloro-acetic or ammonium sulphate. ANS binding and intrinsic Trp92 fluorescence showed that all recombinant proteins were different to nDer p 2 and that the allergen secreted from P. pastoris did not form a hydrophobic cavity. Despite the marked structural changes, all preparations of Der p 2 had similar IgE binding to nDer p 2. Conclusion: Despite almost identical IgE binding, rDer p 2 prepared from both E. coli and P. pastoris showed structural differences to nDer p 2. Der p 2 secreted from P. pastoris lacked most of the natural structure, but refolding could induce the natural structure. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The oral aspirin (ASA) provocation test is considered to be the gold standard in the diagnosis of ASA sensitivity. However, since it may be associated with severe adverse reactions, safer alternatives would be highly desirable. The basophil activation test has been proposed as such an alternative, but there is limited information about its usefulness. Our aim was to evaluate the clinical usefulness of flow cytometric basophil activation in the diagnosis of ASA sensitivity. Methods: Patients with ASA sensitivity (n = 18), patients with ASA tolerance (n = 12) and healthy volunteers (n = 12) were included in the study. A 2-day single-blind placebo-controlled oral ASA provocation test was performed on all patients. Basophil activation after lysine-ASA and diclofenac stimulation was measured by Flow-CASTTM (Bühlmann Laboratories) for CD63 and an allergenicity kit (Beckman Coulter) for CD203c. The results of CD63 and CD203c were compared within groups, and sensitivity and specificity of the assay were measured against oral ASA provocation. Results: The highest sensitivity and specificity of CD63 were 33.3 and 79.2%, respectively, and of CD203c were 16.7 and 100%, respectively, for ASA. The highest sensitivity and specificity of CD63 were 16.7 and 91.7%, respectively, and of CD203c were 22.2 and 100%, respectively, fordiclofenac. Neither the addition of CD203c to CD63 nor the addition of diclofenac improves the overall sensitivity and specificity of CD63 to ASA. Conclusion: At present, basophil activation using CD63 and CD203c does not seem to be optimally sensitive for the diagnosis of ASA sensitivity. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The stability of food allergens to proteolysis and thermal denaturation contributes considerably to their allergenicity. Methods: Mal d 2, an allergenic thaumatin-like protein (TLP) from apple, was isolated and purified by anion exchange chromatography. Its IgE reactivity was tested by ELISA and immunoblotting using sera from apple allergic patients. The proteolytic stability of Mal d 2 was investigated using two in vitro digestion models. Effects of thermal treatment at different pH values on the secondary structure of the protein were recorded by circular dichroism. The level and IgE reactivity of Mal d 2 present in industrially prepared foods were assessed. Results: Purified Mal d 2 consisted of two isoforms. Both harbored carbohydrate moieties and bound serum IgE from apple allergic individuals. Mal d 2 showed remarkable stability to proteolysis and thermal treatments. The allergen remained intact after 2 h each of gastric and subsequent duodenal digestion retaining its full IgE-binding capacity. Mal d 2 was unfolded at neutral and acidic pH at 70°C. Refolding after cooling was only observed at acidic pH. Mal d 2 detected by an anti-TLP antibody in cloudy apple juice did not bind IgE of a serum pool of apple allergic patients. Conclusion: Our findings suggest that Mal d 2 maintains its structure in the gastrointestinal tract, a feature essential for sensitizing the mucosal immune system and provoking allergic reactions. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: The increasing number of applications of sweet lupins in food is paralleled by an increase in immunoglobulin E (IgE)-mediated allergic reactions to lupin proteins. In particular, lupin allergy seems to appear in patients with an existing peanut allergy. In the present study, IgE-binding studies towards fractionated lupin seed proteins, and peanut and almond proteins were performed using sera from patients with confirmed lupin allergy. Methods:Immunoblotting and indirect ELISA were performed to investigate IgE binding to protein extracts. ELISA inhibition experiments were performed to investigate the presence of cross-reactive allergens in the protein extracts. Results: Immunoblotting and ELISA experiments demonstrated IgE binding to all lupin conglutins (α, β, γ and δ) as well as to peanut and almond proteins, with a unique IgE-binding profile for each patient. High IgE binding to α-conglutin was observed and IgE from the majority of patients similarly recognized two proteins within the α-conglutin-containing fraction, 40 and 43 kDa in size. Inhibition ELISA experiments showed that preincubation of sera with lupin conglutins, peanut and almond resulted in decreased IgE binding to lupin flour. Conclusions: Overall, these results indicate that α-, β-, γ- and δ-conglutins are candidate allergens in lupin and suggest a particularly strong allergenicity of α-conglutins. Furthermore, the results indicate the presence of cross-reactive allergens in lupin, peanut and almond. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Fruits are a major cause of food allergy in adults. Lipid transfer proteins (LTP) are implicated in severe allergic reactions to fruits, but little is known about LTP content in different cultivars. Objective: Determination of the levels of LTP in a wide range of apple cultivars. Methods: LTP was measured in apples from 53 cultivars grown in Italy and 35 grown in The Netherlands, using three different immunoassays: a competitive ELISA (cELISA), a sandwich ELISA (sELISA) and a RAST inhibition (RI). Selected cultivars were evaluated using the basophil histamine release test (BHR), skin prick test (SPT) and double-blind, placebo-controlled food challenge (DBPCFC). Results: LTP levels measured with the three immunoassays were significantly correlated, as judged by Pearson’s correlation (0.61 < Rp < 0.65; p < 0.0001), but differed with respect to the actual quantities: 3.4–253.2 (sELISA), 2.7–120.2 (cELISA) and 0.4–47.3 μg/g tissue (RI). Between cultivars, LTP titers varied over about a two-log range. Pilot in vitro and in vivo biological testing (BHR, SPT and DBPCFC) with selected cultivars supported the observed differences in LTP levels. Conclusions: Around 100-fold differences in LTP levels exist between apple cultivars. Whether the lowest observed levels of LTP warrant designation as hypo-allergenic requires more extensive confirmation by oral challenges. Determination of cultivar variation in LTP levels provides important information for growers and consumers. Comparison to earlier reported Mal d 1 levels in the same cultivars reveals that a designation as low allergenic does not always coincide for both allergens. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: In spite of being among the main foods responsible for allergic reactions worldwide, soybean (Glycine max)-derived products continue to be increasingly widespread in a variety of food products due to their well-documented health benefits. Soybean also continues to be one of the elected target crops for genetic modification. The aim of this study was to characterize the soya proteome and, specifically, IgE-reactive proteins as well as to compare the IgE response in soya-allergic individuals to genetically modified Roundup Ready soya® versus its non-transgenic control. Methods: We performed two-dimensional gel electrophoresis of protein extracts from a 5% genetically modified Roundup Ready flour sample and its non-transgenic control followed by Western blotting with plasma from 5 soya-sensitive individuals. We used peptide tandem mass spectrometry to identify soya proteins (55 protein matches), specifically IgE-binding ones, and to evaluate differences between transgenic and non-transgenic samples. Results: We identified 2 new potential soybean allergens – one is maturation associated and seems to be part of the late embryogenesis abundant proteins group and the other is a cysteine proteinase inhibitor. None of the individuals tested reacted differentially to the transgenic versus non-transgenic samples under study. Conclusion: Soybean endogenous allergen expression does not seem to be altered after genetic modification. Proteomics should be considered a powerful tool for functional characterization of plants and for food safety assessment. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Japanese cedar pollinosis is a severe allergic disease in Japan. The most effective means of decreasing allergic inflammation reactions is still avoidance of the aeroallergen. Recently, a novel air purification system using positively and negatively charged cluster ions was developed to create comfortable living environments. We aimed to assess the ability of existing technology to lower allergenicity of Japanese cedar pollen. Methods: A Japanese cedar pollen extract was nebulized from the top of a cylindrical container with 2 or 4 ion-generating devices. The extract in a mist was passed through the space filled with or without plasma cluster ions for 90 s, and the ion-treated or nontreated extract was then collected in a Petri dish at the bottom of the container. Results: The ion-exposed extract was significantly diminished in its reactivities to anti-Cry j 1 or anti-Cry j 2 antiserum and to human allergic sera IgE on ELISA. SDS-PAGE analysis revealed that ion exposure induced protein degradation in the pollen extract. Similarly, the ion treatment impaired about 80% of the binding to pooled sera IgE from patients allergic to Japanese cedar pollen on ELISA inhibition. Furthermore, intracutaneous and conjunctival reaction tests showed a remarkable diminution in the allergenicity of the ion-irradiated extract. Conclusion: Ion irradiation resulted in a remarkable decrease in in vitro and in vivo allergenicities of atomized Japanese cedar pollen extracts. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes. Methods: Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS). Results: Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by ∼1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen. Conclusions: Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The first genetically modified (GM) crops approved for food use (tomato and soybean) were evaluated for safety by the United States Food and Drug Administration prior to commercial production. Among other factors, those products and all additional GM crops that have been grown commercially have been evaluated for potential increases in allergenic properties using methods that are consistent with the current understanding of food allergens and knowledge regarding the prediction of allergenic activity. Although there have been refinements, the key aspects of the evaluation have not changed. The allergenic properties of the gene donor and the host (recipient) organisms are considered in determining the appropriate testing strategy. The amino acid sequence of the encoded protein is compared to all known allergens to determine whether the protein is a known allergen or is sufficiently similar to any known allergen to indicate an increased probability of allergic cross-reactivity. Stability of the protein in the presence of acid with the stomach protease pepsin is tested as a risk factor for food allergenicity. In vitro or in vivo human IgE binding are tested when appropriate, if the gene donor is an allergen or the sequence of the protein is similar to an allergen. Serum donors and skin test subjects are selected based on their proven allergic responses to the gene donor or to material containing the allergen that was matched in sequence. While some scientists and regulators have suggested using animal models, performing broadly targeted serum IgE testing or extensive pre- or post-market clinical tests, current evidence does not support these tests as being predictive or practical. Based on the evidence to date, the current assessment process has worked well to prevent the unintended introduction of allergens in commercial GM crops. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: In European countries, pine processionary caterpillar (Thaumetopoea pityocampa) is one of the species of Lepidoptera able to affect humans. Its allergenicity has been demonstrated in previous studies. However, in all of them larval extract in the fifth stage of development has been used as antigen. Therefore, allergenicity of other larval stages was unknown. The objective of our study was to detect allergenic changes of pine processionary caterpillar during larval development. Methods: Pine processionary caterpillar extracts in all larval stages were analyzed by SDS-PAGE. Immunoblotting was performed using patient sera with suggestive history of allergy and a positive prick test.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Numerous products based on soybean are available and various food technologies are applied for their production. The allergenicity of natural soybean may be modified by these treatments. Objectives: To compare the allergenicity of native soybean proteins with those of soy milk and texturized protein products. To show additional allergens. Methods: Three commercial products and two infant formulas were studied: Soybean flour, soy milk, texturized soy proteins, two infant formulas; the first containing total proteins and the second containing a soy protein hydrolysate. Sera from 9 patients allergic to soy protein were tested by immunoblotting (IB). IB inhibition was achieved by incubating sera with protein extract from soybean flour. Results: The SDS-PAGE profile of soybean flour protein and soy milk showed a difference in the proportions of the various protein fractions, with a higher concentration of 37-kD protein in flour and 33-kD protein in milk. Infant formula 1 contained proteins with a molecular weight below 28 kD. The texturized extract contained high proportions of 31- to 34- and 38-kD proteins. Immunoblotting revealed a lack of allergenicity in infant formula. Sera recognizing the 38- and 50-kD proteins in texturized soy protein also recognized the 37- and 49-kD proteins in soybean flour and in soy milk, suggesting a protein glycation by texturization processes. The 30- to 34-kD band in texturized proteins was devoid of any allergenicity. This study seems to indicate that the 30-kD allergen (Gly m Bd 30) disappears during the production of texturized soy protein. Conclusion: All technologies applied to soybean-based products induce striking variation in the protein profile and allergenicity. Texturized protein could lack the major allergen Gly m Bd 30. Further studies or texturization might generate modified technologies in order to create hypoallergenic texturized proteins.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Grass pollen of the Poaceae grasses are known to be highly allergenic. Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available. Methods: Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris. Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA). In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed. Results: Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids. Both mature isoforms were expressed in Pichia at a concentration of ∼15 mg/l. SDS-PAGE analysis showed that rDac g 5 had an apparent M[sub r] approximately 10 kD above nDac g 5. By mass spectrometry this difference was shown to be around 2.5 kD. Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in M[sub r] . Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5. RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5. A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02. Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA. Conclusions: Dac g 5 was successfully cloned and expressed in P. pastoris. Minor differences in primary structure between isoforms influence their immune reactivity.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Major respiratory allergens of dogs, mice, rats, horses and cows belong to the lipocalin group of proteins. The sequence identity of lipocalins is often less than 20%, but they contain between one and three structurally conserved regions and their three–dimensional structures are similar. Lipocalins share common biological functions, predominantly related to the transport of small hydrophobic molecules, such as vitamins and pheromones. Immune reactivity to lipocalin allergens is not well known. In Bos d 5, the IgE–binding epitopes are spread along the molecule, whereas in Bos d 2, the C terminus appears to contain the human B cell epitopes. Bos d 5 contains several murine T cell epitopes. No information is available on human T cell epitopes. The maximal number of epitopes an allergic patient’s T cells could recognize in Bos d 2 was five. Three of the epitopes were colocalized in the structurally conserved regions of lipocalins. Interestingly, one of the epitopes was recognized by the T cells of all patients and the computer predictions suggested that there would be an epitope in the corresponding parts of human endogenous lipocalins. The proliferative responses of peripheral blood mononuclear cells of Bos d 2–allergic subjects to Bos d 2 were weak. The T cell response was Th2–dominated. To explain these observations, we have proposed that the allergenicity of lipocalins may be a consequence of molecular mimicry between lipocalin allergens and endogenous lipocalins at the T cell level. [ABSTRACT FROM AUTHOR]