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Background: Art v 1, Amb a 4, and Par h 1 are allergenic defensin-polyproline-linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens., Methods: Recombinant defensin-polyproline-linked proteins were expressed in E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T-cell cross-reactivity were studied in vitro., Results: Despite variations in the proline-rich region, similar secondary structure elements were observed in the defensin-like domains. Seventy-four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross-reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin-like domain was limited to Art v 1 and no T-cell cross-reactivity with Art v 1 25-36 was observed., Conclusions: Despite structural similarity, different IgE-binding profiles and proteolytic processing impacted the allergenic capacity of defensin-polyproline-linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE-binding epitopes, we suggest inclusion in molecule-based allergy diagnosis., (© 2017 The Authors Allergy Published by John Wiley / Sons Ltd.)

Background: In early childhood, the allergen-specific IgG repertoire is mainly directed to animal and vegetable food molecules and infrequently to airborne molecules. It is unknown whether this early pattern is maintained throughout childhood., Objective: To investigate the evolution of IgG and IgE responses to a broad panel of allergenic molecules from birth to age 10 years., Methods: We examined the sera collected between birth and age 10 years from participants in the German Multicentre Allergy Study, a birth cohort born in 1990. The IgE (cutoff ≥0.30 ISU) and IgG (cutoff ≥0.10 ISU) responses to 35 genuine allergenic molecules were measured with a multiplex microarray approach (ImmunoCAP ISAC™)., Results: IgE responses were mostly directed against a restricted group of airborne molecules, with a sequence and prevalence hierarchy (Phl p 1> Bet v 1> Fel d 1> Phl p 5> Der p 2> Der p 1) largely maintained over time. Conversely, the IgG repertoire was much broader, starting with animal foodborne, then spreading to vegetable foodborne and finally to airborne molecules. A strong and persistent IgG response to a given airborne molecule almost invariably preceded or accompanied an IgE response to that molecule., Conclusions: The evolution of IgG and IgE responses throughout childhood differs widely at population level. IgG responses are mostly directed to animal food allergens, while IgE responses are dominated by airborne allergens. However, a strong IgG response almost invariably precedes or accompanies the appearance of IgE to the same molecule in specifically sensitized subjects., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Background: Pollution is considered as one main cause for the increase of allergic diseases. Air pollutants may cause and worsen airway diseases and are probably able to make pollen allergens more aggressive. Previous studies looked at traffic-related air pollution, but no data about the effects of polluted soils on pollen allergens are available. We aimed to assess the effects of plant exposure to cadmium-contaminated soil on allergenicity of the annual blue grass, Poa annua L, pollen., Methods: Poa plants were grown in soil contaminated or not contaminated (control) with cadmium. At flowering, mature pollen was analyzed by microscopy, to calculate the percentage of pollen grains releasing cytoplasmic granules, and by proteomic techniques to analyze allergen proteins. Allergens were identified by sera from grass pollen-allergic patients and by mass spectrometry., Results: Pollen from Cd-exposed plants released a higher amount of allergenic proteins than control plants. Moreover, Cd-exposed pollen released allergens-containing cytoplasmic grains much more promptly than control pollen. Group 1 and 5 allergens, the major grass pollen allergens, were detected both in control and Cd-exposed extracts. These were the only allergens reacting with patient's sera in control pollen, whereas additional proteins strengthening the signal in the gel region reacting with patient's sera were present in Cd-exposed pollen. These included a pectinesterase, a lipase, a nuclease, and a secretory peroxydase. Moreover, a PR3 class I chitinase-like protein was also immunodetected in exposed plants., Conclusion: Pollen content of plants grown in Cd-contaminated soils is more easily released in the environment and also shows an increased propensity to bind specific IgE.

Background: Immunoglobulin E (IgE)-mediated allergy belongs to common chronic disorders resulting from an interaction between both genetic and environmental factors. The gene encoding CD14 is a positional candidate gene for allergic diseases as it is localized on chromosome 5q31.1, a region that is linked to asthma and bronchial hyperresponsiveness. Recently, several polymorphisms in the promoter region of this gene have been associated with atopic phenotypes in various populations., Methods: We investigated relationship among atopic phenotypes and two polymorphisms [C(-159)T and G(-1359)T] in the promoter of the CD14 gene in the Czech population. Polymerase chain reaction with restriction fragment length polymorphism analyses was used to determine the CD14 genotypes in subjects with IgE-mediated allergic diseases (n = 562) and random controls (n = 320)., Results: The CD14 allele or genotype distributions were similar in patients and control group. However, the frequency of the C allele of the C(-159)T polymorphism was higher in patients with positive skin prick tests for moulds than in patients without reactivity to this antigen (P < 0.002, Pcorr<0.01). In addition, we found that patients with homozygous genotype (GG) of the G(-1359)T polymorphism had marginally lower percentage of positive skin prick tests compared with the other genotypes (P < 0.029, Pcorr > 0.05)., Conclusions: Our study supports the idea that CD14 gene variants may act as disease modifiers of IgE-mediated allergic diseases.

Background: Pine processionary caterpillars induce dermatitis by a toxic-irritative mechanism. The existence of true allergic reactions to allergens from these caterpillars has been recently demonstrated by positive immediate skin prick tests and specific IgE determination by immunoblotting using crude larval extracts. The aim of this work was to purify allergens from the crude larval extract in order to characterize IgE-binding proteins from these caterpillars., Methods: Allergens were separated by ethanol gradient fractionation and reversed phase HPLC. The N-terminal amino acid sequence of a selected allergen was obtained after SDS-PAGE and transfer. The clinical relevance of this allergen was measured using sera from patients allergic to caterpillar., Results: An allergen with a molecular weight close to 15 kDa was purified. It was recognized by 9 out of 11 allergic patients (82%). Its N-terminal amino acid sequence had no homologies to any other protein already described in data bases. For this reason, no information about its biological function could be obtained., Conclusions: This 15-kDa IgE-binding protein is a major caterpillar allergen and shows no homologies to other insect allergens already described.

Background: According to a hypothesis allergens induce Th2 responses in allergic patients, and microbes induce Th1 responses. We studied the kinetics of in vitro allergen-, tuberculin (PPD)- and tetanus toxin (TT)-induced IFN-gamma and IL-4 mRNA expression in peripheral blood mononuclear cell (PBMC) cultures of pollen-allergic patients and healthy controls., Methods: PBMC of 10 birch or timothy pollen-allergic patients and of 13 healthy controls were stimulated in vitro with allergen (birch or timothy), PPD or TT. Pellets and supernatants were collected at 24, 48, 72 and 96 h after stimulation. IFN-gamma and IL-4 production was measured by enzyme linked immunosorbent assay and mRNA expression using RT-PCR and time-resolved fluorometry., Results: Allergen induced IFN-gamma production and mRNA expression in PBMC more in allergic patients than in healthy controls. Also allergen induced IL-4 mRNA expression more in allergic patients than in healthy controls. PPD induced IFN-gamma mRNA expression both in allergic patients and healthy controls, whereas IFN-gamma production was induced only in healthy controls and IL-4 was not induced at all. TT induced IFN-gamma mRNA expression in both groups, IFN-gamma production in allergic patients, and IL-4 mRNA expression in both allergic patients and healthy controls., Conclusions: In vitro stimulation with allergen induced both IFN-gamma and IL-4 mRNA expression of PBMC in allergic patients. These observations challenge the clearcut division of microbe-specific Th1 and allergen-specific Th2 responses in peripheral blood.

Background: Clinical complaints in atopic subjects with asthma and rhinitis occur more frequently in the presence of high total and allergen-specific IgE serum levels. Here we report on the relationship between total and allergen-specific IgE serum levels and presence of symptoms in an unselected farmer population sensitized to Tetranychus urticae (TU)., Methods: Farmers were recruited as previously described. Total IgE and allergen-specific IgE were measured by immunoassay in TU-positive skin prick test (SPT) farmers (n = 58) and two control groups including Dermatophagoides pteronyssinus (Dp)-positive SPT subjects (n = 40) and non-atopic, TU-negative SPT healthy farmers (n = 25)., Results: Both TU+ and Dp+ subjects had significantly higher total IgE values (P < 0.001) than healthy non-atopic subjects. TU-specific IgE levels were significantly more elevated in symptomatic than non-symptomatic TU+ subjects (P = 0.028). Dp-specific IgE levels were higher in symptomatic than non-symptomatic Dp+ subjects (P = 0.003). Finally, total IgE levels were significantly higher in the symptomatic than non-symptomatic subgroups in both TU+ and Dp+ subjects (P < 0.0001 and P = 0.007, respectively). Logistic regression analysis showed that only total IgE concentrations were significant predictors of current symptoms in TU+ subjects., Conclusions: High total IgE and allergen-specific IgE levels are associated with symptoms in TU+ subjects. Definition of their predictive value requires further studies.

Background: Acidic oxidative potential water (AOPW) is strongly acidic, and contains active oxygen species and thus exhibits an oxidative potential to cause biochemical alterations of wheat proteins., Methods: A commercial wheat allergen extract was treated with AOPW, and then analysed by means of SDS-PAGE and the skin prick test in IgE-mediated wheat allergic patients., Results: The wheat extract comprised 70 kDa, 32 kDa and 30 kDa fractions, and the protein in the 70 kDa fraction was broken down into less than 14 kDa fractions by AOPW. AOPW mitigated 39% of the wheal reaction to the wheat extract in the skin prick test., Conclusion: AOPW may be useful for enhancing the digestibility and lowering the allergenicity of wheat proteins.

Background: The inhalation of Olea europaea pollen is one of the most important causes of allergic respiratory diseases in the Mediterranean basin. The objective of this study was to investigate the antigenic and allergenic composition of six different O. europaea varieties collected in southern Spain., Methods: The varieties included in the study were: Acebuche (wild olive), Carrasqueño, Nevado, Hojiblanco, Manzanillo and Picual. Extracts of these six varieties were prepared. Twenty-nine olive individuals with an immunoglobulin(Ig)E-mediated allergy to olive pollen were skin tested with these extracts. The antigenic profile of these extracts was evaluated by SDS-PAGE; the allergenic profile was investigated by immunoblotting using the serum of these 29 individuals. The Ole e 1 content was established by ELISA inhibition using purified Ole e 1 and rabbit polyclonal antibodies and by scanning densitometry., Results: The extracts that induced the smallest wheal size were Acebuche and Hojiblanco, being significantly different from the rest of the extracts. The antigenic and allergenic profiles of the extracts also varied. The Ole e 1 content ranged from 0.050 in Hojiblanco to 0.232 in Manzanillo, measured by ELISA inhibition and from 0.153 in Hojiblanco to 0.677 in Nevado, measured by scanning densitometry., Conclusions: The different varieties of O. europaea pollen studied demonstrated great differences in the in vivo and in vitro potency of the extracts. There were significant differences in the Ole e 1 content, while the protein content remained very similar in these extracts. This study confirms previous observations of a great variability in the antigenic and allergenic composition of O. europaea pollen extracts and establishes significant differences in Ole e 1 content.

Background: Several associations have been described between tree and plant pollens and certain foods. The objective of this study is to verify whether there is cross-reactivity between Platanus pollen and vegetable origin foods., Methods: We selected 56 patients allergic to vegetable foods and subjected them to cutaneous tests with aeroallergens and vegetable foods. A statistical analysis was performed to evaluate the association of Platanus pollen with foods and with other aeroallergens. Later, a specific IgE determination was performed as well as a RAST (radioallergosorbent) inhibition experiment, to verify the existence of cross-reactivity in vitro., Results: In the cutaneous tests we found a positive correlation between Platanus pollen and hazelnut, peanut, banana and celery. The results of the RAST inhibition experiment indicate an important cross-reactivity between the pollen of Platanus acerifolia and hazelnut and banana fruit, and an intermediate cross-reactivity with celery and peanut., Conclusion: We have described an association between the pollen of the Platanus tree and some vegetable foods such as hazelnut, banana, peanut and celery. This association could be explained by the in vitro IgE cross-reactivity detected.

IgE-antibody analysis is a major diagnostic procedure and a primary tool in allergological research. The determination of sensitization frequencies and antibody concentrations against allergens of defined sources provides critical information for the estimation of the relative importance of food and environment in clinical allergy. True quantitation is essential and requires assay designs providing allergen excess and mass unit calibration. Standardized and reproducible methods show geographic and culture dependent differences between patient populations and contribute to the quality of diagnosis and treatment of allergic disease.

Background: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens., Methods: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects., Results: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens., Conclusions: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.

Mugwort and birch pollen allergy are frequently associated with IgE-mediated hypersensitivity to celery and spices. We analyzed 22 sera from patients with the mugwort-birch-celery-spice syndrome for IgE binding to the spices pepper and paprika by immunoblotting. Immunoblot results revealed two major allergens of 28 and 60 kDa in pepper and a 23-kDa allergen together with allergens of higher molecular weight in paprika. In immunoblot-inhibition studies, crude mugwort, birch pollen, and celery extracts significantly reduced the IgE binding to pepper and paprika allergens. However, no inhibition was achieved with rBet v 1 and rBet v 2, suggesting that no homologs of these birch proteins act as allergens in pepper or paprika extracts. N-terminal sequence analysis of the 14- and 28-kDa pepper and 23-kDa paprika allergens revealed no homology to known allergens. The 28-kDa pepper allergen showed homology to a wheat germin protein, and the 23-kDa paprika allergen was identified as a homolog of a osmotin-like or pathogenesis-related protein in tomato. Therefore, we conclude that the IgE cross-reactivity in the mugwort-birch-celery-spice syndrome to the spices pepper and paprika is not caused by homologs of Bet v 1 and profilin. N-terminal amino acid sequence analysis of the main allergens in pepper and paprika indicate a relation to frequently occurring plant proteins.

ELISA is increasingly used to measure antibodies in new circumstances. Recently, it has been applied to the measurement of IgG subclass antibodies against common antigens in early childhood. These studies have raised concerns about the specificity of some of these assays. This paper details the results of experiments which have assessed the specificity of IgG1 binding to allergens of dust mite (Dermatophagoides pteronyssinus) and ryegrass (Lolium perenne) pollen by inhibition ELISA in the sera of 2-year-old children of atopic parents. Six sera which showed binding of IgG1 to D. pteronyssinus and six to L. perenne were used. All had IgG1 antibody against ovalbumin. In the children's sera, binding to D. pteronyssinus was substantially inhibited by preincubation with the homologous antigen, but not with ovalbumin, thereby confirming the specificity of the assay. However, suppression of IgG1 binding to L. perenne with the homologous antigen was comparatively small, and ovalbumin could cause an equivalent inhibition, indicating poor specificity. Furthermore, the level of IgG1 binding to L. perenne was closely correlated to the level of IgG1 binding to ovalbumin (r = 0.98; P < 0.001). When the assay was reversed, IgG1 binding to ovalbumin was only slightly inhibited by L. perenne, indicating that most antibody binding to ovalbumin was specific. Thus, binding IgG1 in both adult and child sera to D. pteronyssinus appeared to be specific, while child, but not adult, IgG1 binding to L. perenne showed poor specificity. This disparity may be due to differences in the affinities of the respective antibodies, and it illustrates the importance of determining assay specificity when making measurements in early childhood.

Two latex (Hevea brasiliensis) raw materials, one from the cytosolic fraction, the other from the total natural latex have been characterised by immunoblotting techniques. The two latex fractions A and B were studied by immunoprints using eight sera from patients who were allergic to latex. They gave different profiles though there were also allergens of common molecular weights (60, 35, 30, 25, 20, 18, 15, and 10 kD). This study confirms the importance of allergic reactions to latex, that may in some patients cause anaphylactic shock.