Diagnosis of <Emphasis Type="Italic">Chenopodium album</Emphasis> allergy with a cocktail of recombinant allergens as a tool for component-resolved diagnosis

Chenopodium album pollen is one of the main sources of pollen allergy in desert and semi-desert areas and contains three identified allergens, so the aim of this study is comparison of the diagnostic potential of C. album recombinant allergens in an allergenic cocktail and C. album pollen extract. Diagnostic potential of the allergenic cocktail was investigated in 32 individuals using skin prick test and obtained results were compared with the acquired results from C. album pollen extract. Specific IgE reactivity against the pollen extract and allergenic cocktail was determined by ELISA and western blotting tests. Inhibition assays were performed for the allergenic cocktail characterization. The exact sensitization profile of all patients was identified which showed that 72, 81 and 46% of allergic patients had IgE reactivity to rChe a 1, rChe a 2 and rChe a 3, respectively. Almost all of C. album allergic patients (30/32) had specific IgE against the allergenic cocktail. In addition, there was a high correlation between IgE levels against the allergenic cocktail and IgE levels against the pollen extract. The allergenic cocktail was able to completely inhibit IgE binding to natural Che a 1, Che a 2 and Che a 3 in C. album extract. In addition, positive skin test reactions were seen in allergic patients that tested by the allergenic cocktail. The reliable results obtained from this study confirmed that the allergenic cocktail with high diagnostic potential could be replaced with natural C. album allergen extracts in skin prick test and serologic tests.     

Allergenic pollen in the atmosphere of Rohtak city,Haryana (India): a pioneer study

An attempt has been made in the present study to assess the allergenicity of dominant pollen types recorded from the atmosphere of Rohtak city. Skin prick test was performed with the antigenic extracts of 22 pollen types on 150 local patients who visited Asthma Clinic, University of Health Sciences, Rohtak. Markedly positive skin reactions (2+ and above) varied from 4.6 to 20.6 % to various pollen antigens. Cenchrus ciliaria (20.6 %), Zea mays (20 %) and Pennisetum typhoides (19.3 %) were the pollen allergens exhibiting maximum sensitivity. Antigenic extract of Cassia occidentalis, Cynodon dactylon and Ricinus communis showed marked skin reactivity in 18.6 % of patients. Prosopis juliflora, Chenopodium murale, Amaranthus spinosus, Cassia fistula and Cassia siamea showed 2+ and above reactions in 16.6, 15.3, 14.6 and 14.0 % of the local patients, respectively. Least reactivity (4.6 %) was shown to the antigenic extract of Cyperus rotundus. Out of 52 sera screened for the presence of specific IgE antibodies against different antigenic extracts, only 5.5 % showed >60 % binding. About 30 % and above binding was shown to the antigenic extracts of Z. mays, A. spinosus, R. communis and Xanthium strumarium. The concordance between positive skin reaction and serum-specific IgE antibodies ranged from 15 to 69 %.     

A Protein Allergen Microarray Detects Specific IgE to Pollen Surface,Cytoplasmic, and Commercial Allergen Extracts

Background

Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.

Methodology/Principal Findings

We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts). To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.

Conclusions/Significance

These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.     

Micro-regional hypersensitivity variations to inhalant allergens in the city of Zagreb and Zagreb County

Depending on the geographic and climatic region and vegetation, allergenic plants are characteristic for certain areas and the pollen concentrations of various plant species depend on the fenophase of each single species and most of all on the climatic and meteorological conditions of a certain area. It is precisely because of these specific characteristics that the hypersensitivity of patients to various types of pollen allergens differs according to the geographic regions. The aim of this paper is to determine the frequency of inhalation allergies in adult population (> 19 years of age) caused by single types of allergens according to the defined space units (micro-regions) with special emphasis on the rural and urban areas. A number of 2,192 patients have been tested for allergy skin prick tests over a period of four years. Every patient was asked to fill in the questionnaire which contained 29 questions. The results of skin prick testing show that out of a total of 2,192 patients 86.72% were sensitized to pollen, 36.45% to mites, 2.46% to spores of fungi and mould and 5.1% of patients to other allergens which include the allergens of cockroaches, feathers and animal hair (p < 0.001). The largest number of poly-sensitized persons allergic to pollen allergens was sensitized to allergens from the pollen of plants that belong to the botanical family of grass. There were 25.36% patients mono-sensitized to individual allergens. In the northern and western parts of the city and the county, the majority of persons were sensitized to the birch pollen allergens, and this is statistically much more than the share of patients with the place of residence in the southern and eastern parts of the city and the county. In the southern and eastern locations prevails the share of sensitized persons to ambrosia which is statistically much more than the share of patients with the place of residence in the northern and western part of the city and the county.     

Aerobiology, allergenicity and biochemistry of three pollen types in Berhampore town of West Bengal, India

An aerobiological survey was performed in Berhampore town of West Bengal, India, to know the frequency of three common airborne pollen, namely Acacia auriculiformis, Eucalyptus citriodora and Madhuca indica using an ASTIR one day volumetric sampler. Acacia pollen showed its peak concentration in September, followed by Madhuca in April, while Eucalyptus showed its two peaks between September–October and January–April. Meteorological factors like temperature, RH, rainfall played an important role in release and dispersal of pollen. Skin prick tests with the antigenic extracts of the three pollen types, showed their allergenic potentialities. The highest markedly positive reactions were exhibited by Eucalyptus (34.04%), followed by Madhuca (22.93%) and Acacia (21.87%). 30–60% (NH₄)₂ SO₄ cut fraction (Fraction II) of each pollen type showed maximum positivity in skin prick test. Biochemical analysis showed that Acacia pollen was richer in protein and carbohydrate, than the other two types. The total protein component of the above types were studied by SDS-PAGE showing different protein bands with a range of molecular weight 29–110 KD. In isolated fraction II (allergenically most potent) of Eucalyptus and Madhuca different protein band of 43–200 KD were obtained, while a single protein band of 57 KD was obtained for Acacia. The IgE specific allergenic reactivity was confirmed by Dot-blotting technique. This revised version was published online in June 2006 with corrections to the Cover Date.     

Aerial pollen diversity in Punjab and their clinical significance in allergic diseases

The present study has been carried out to assess the allergenicity of dominant pollen types from the atmosphere of nine districts of the state of Punjab, India. During the study period (2012–2014), 30 different kinds of pollens belonging to 17 families were recorded. Antigenic extract of thirteen pollen types were selected for skin prick tests (SPT). The allergenic pollens selected for the present study belong to families: Asteraceae, Amaranthaceae, Brassicaceae, Cannabaceae, Chenopodiaceae, Meliaceae, Moraceae, Myrtaceae and Xanthorrhoeaceae. Total 165 sensitized patients having diagnosed with bronchoprovocation test were selected for SPT. Diagnostic tests were performed in ENT department, Rajindra hospital Patiala, and SPT were done in the clinic of Dr. H.S. Bedi at Patiala. Antigenic reactivity (3+ to above) to various allergens varied from 2.4 to 9.09 %. Patients showed maximum allergenic sensitivity to Parthenium hystrophorus (9.09 %) followed by Morus alba (8.48 %), Amaranthus spinosus (7.87 %) and Ageratum sp. (6.06 %). Least reactivity was shown by Brassica compestris and Eucalyptus sp. (2.4 %).     

Proteomic profiling of birch (Betula verrucosa) pollen extracts from different origins

Pollen of the European white birch is a major source of spring pollinosis in Europe. Pollen-allergy diagnosis and treatment by specific immunotherapy commonly rely on extracts of natural origin. To gain insight into the protein content and its variability, we evaluated the profile of allergenic and non-allergenic proteins in extracts of pollen from different origins by MS-based proteomics. Aqueous extracts prepared from commercially available Swedish birch pollen, pollen collected from Austrian trees and a commercial skin prick extract were analyzed by 1-DE, 2-DE, immunoblotting and mass spectrometry, resulting in a complete inventory of extractable, disease-relevant pollen proteins. A main focus of this study was on the isoform distribution of Bet v 1, the major allergen of birch pollen. Using a combination of intact mass determination and peptide sequencing, five isoforms (a, b, d, f and j) were unequivocally identified in Swedish and Austrian birch pollen extracts, while the skin prick extract contained only isoforms a, b and d. Using the same methods as for Bet v 1, divergencies in the sequence of birch profilin (Bet v 2), a plant panallergen, were solved. The molecular characterization of pollen extracts is relevant for standardization and development of new reagents for specific immunotherapy.     

Hypersensitivity to Ailanthus altissima (tree of heaven) pollen: identification of a major IgE-binding component

Ailanthus altissima or Ailanthus glandulosa (Simaroubaceae) is known as tree of heaven. It is a dioecious plant with staminate and pistillate flowers that grow on separate trees. In recent years, A. altissima has been frequently planted in numerous areas, especially in arid and semiarid lands of Iran and also used as an ornamental tree in several Iran cities including Kerman. The aim of this research was to identify IgE-binding proteins responsible for type I hypersensitivities of A. altissima pollen extract. In this study, pollen’s proteins were extracted and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Total protein content of pollen extracts was measured by Bradford assay. Allergenicity of pollen extract was evaluated by skin test and immunoblotting. Results showed that total protein concentration of A. altissima pollen was found to be 1.34 μg μl^?1. In A. altissima pollen extract, different protein fractions were identified by SDS-PAGE mostly at 42, 53.7, 63, 87.7, 100 and 120 kDa. Skin tests showed a delayed hypersensitivity. Immunoblotting of A. altissima pollen extract with pooled subject’s sera detected a major IgE-binding component of 42 kDa. Moreover, these results will provide a platform for cloning cDNA encoding allergenic protein from A. altissima pollen.     

Allergy against chironomids (non biting midges) in adult atopic patients

Summary The aim of this investigation was to evaluate the prevalence of atopic sensitization to chironomids (CHI) in patients with asthma and/or rhinitis (A/R), and to study concomitant sensitization to CHI and other allergens. Skin prick tests were performed with 3 different CHI extracts as well as with common inhalant allergens in 600 consecutive patients, 495 of which had A/R. Allergen specific IgE antibodies in the sera against CHI, shell fish and cockroaches were analyzed with Magic Lite.59 (12%) of the patients with A/R had a positive skin test with CHI. Positive skin tests with house dust mites and a storage mite were more common in CHI allergic patients than in other atopic patients. Nasal or conjunctival provocation tests, performed on 23 of the patients with positive skin test with CHI, were clearly positive in 7 cases (30%), questionable in 8 (35%) and negative in 8 cases (35%).Magic Lite, performed on sera from 50 of the patients with positive skin test against CHI, was positive with CHI in 39 cases (78%), with crayfish in 33 (66%), shrimp 20 (40%), cockroach 21 (40%) and with crab in 3 cases (6%).It is concluded that sensitization against CHI is common in patients with A/R. The clinical relevance of the positive test results is, however, unknown. Concomitant sensitization with CHI, crustaceans and cockroach is common.     

Allergenicity of Acroptilon repens and Juglans regia pollen in rats

Pollen proteins that are located in the cytoplasm or on the surface of the exine can function as allergens and evoke immune system responses in sensitive patients, leading to allergic rhinitis and asthma. In this research, the pollen allergenicity and ability to induce IgE response of the pollen of two plant species were studied in rats. Acroptilon repens is an herbaceous, invasive plant with entomophilous pollen, while Juglans regia which is a tree crop produces anemophilous pollen. Immunoblot analysis using sera of sensitised rats revealed IgE reactivity to three protein bands including the 70, 41 and 25.12 kDa bands present in the A. repens pollen extract, while only one single immunogenic band of 11 kDa was detected in J. regia pollen extract. Both pollen extracts increased the eosinophil content and caused some clinical signs of allergy in treated rats. The results showed that both entomophilous and anemophilous pollen can be allergenic.     

Prosopis juliflora pollen allergen induced hypersensitivity and anaphylaxis studies in guinea pigs

In vivo and in vitro allergenic activities of Prosopis juliflora pollen allergens were measured in guinea pigs. Intracutaneous skin test showed an early wheal flare response and a late erythema-redness, sensitized with various concentrations (100, 50, 25, 5 and 1.5 micrograms/ml) of Prosopis juliflora pollen extract after administration of a challenging dose. A 50 micrograms/ml sensitizing dose of Prosopis juliflora pollen allergen gave optimum skin response as both early and late effects. The nature of immunochemical reactivity between pollen allergens and reaginic antibodies were further characterized by histamine release test, gel diffusion test, radioallergosorbent test and passive cutaneous anaphylaxis test. These tests confirm allergenicity caused by Prosopis juliflora pollen allergens and showed the binding of allergens with reaginic antibody and its regulation in guinea pigs.     

Molecular and Immunological Characterization of the First Allergenic Lipocalin in Hamster: THE MAJOR ALLERGEN FROM SIBERIAN HAMSTER (PHODOPUS SUNGORUS)*

The most frequent pet allergy is to cat and dog, but in recent years, it has become increasingly popular to have other pets, and the risk of exposure to new allergens is more prevalent. The list of new pets includes hamsters, and one of the most popular hamsters is the Siberian hamster (Phodopus sungorus). The aim of this study was the characterization and cloning of the major allergen from this hamster. The study of its allergenicity and cross-reactivity could improve the specific diagnosis and treatment for hamster-allergic patients. Thirteen Siberian hamster-allergic patients were recruited at the outpatient clinic. Protein extracts were prepared from the hair, urine, and salivary glands of four hamster species (European, golden, Siberian, and Roborovski). IgE-binding proteins were detected by immunoblotting and identified by mass spectrometry. The recombinant protein was produced in Escherichia coli and then purified by metal chelate affinity chromatography. The allergenic properties of the recombinant protein were tested by ELISA and immunoblotting, and biological activity was tested according to capacity for basophil activation. Three IgE-binding proteins were identified in extracts obtained from Siberian hamster hair, urine, and salivary glands. All proteins corresponded to the same protein, which was identified as a lipocalin. This lipocalin had no cross-reactivity with common and golden hamsters. The recombinant allergen was cloned and purified, showing similar IgE reactivity in vitro to Siberian hamster protein extracts. Also, the recombinant allergen was capable of producing biological activation in vivo. The major Siberian hamster allergen was cloned, and allergenic properties were characterized, providing a new tool for specific diagnosis of allergy to Siberian hamster.     

The identification of allergen proteins in sugar beet (Beta vulgaris) pollen causing occupational allergy in greenhouses

Background

During production of sugar beet (Beta vulgaris) seeds in greenhouses, workers frequently develop allergic symptoms. The aim of this study was to identify and characterize possible allergens in sugar beet pollen.

Methods

Sera from individuals at a local sugar beet seed producing company, having positive SPT and specific IgE to sugar beet pollen extract, were used for immunoblotting. Proteins in sugar beet pollen extracts were separated by 1- and 2-dimensional electrophoresis, and IgE-reactive proteins analyzed by liquid chromatography tandem mass spectrometry.

Results

A 14 kDa protein was identified as an allergen, since IgE-binding was inhibited by the well-characterized allergen Che a 2, profilin, from the related species Chenopodium album. The presence of 17 kDa and 14 kDa protein homologues to both the allergens Che a 1 and Che a 2 were detected in an extract from sugar beet pollen, and partial amino acid sequences were determined, using inclusion lists for tandem mass spectrometry based on homologous sequences.

Conclusion

Two occupational allergens were identified in sugar beet pollen showing sequence similarity with Chenopodium allergens. Sequence data were obtained by mass spectrometry (70 and 25%, respectively for Beta v 1 and Beta v 2), and can be used for cloning and recombinant expression of the allergens. As for treatment of Chenopodium pollinosis, immunotherapy with sugar beet pollen extracts may be feasible.     

Usefulness of manufactured tomato extracts in the diagnosis of tomato sensitization: Comparison with the prick-prick method

Background

Commercial available skin prick test with fruits can be negative in sensitized or allergic patients due to a reduction in biological activity during the manufacturing process. Prick-prick tests with fresh foods are often preferred, but they are a non-standardized procedure. The usefulness of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method in the diagnosis of tomato sensitization has been analyzed. The objective of the study was to assess the potential diagnostic of freeze-dried extracts of Canary Islands tomatoes, comparing the wheal sizes induced by prick test with the prick-prick method.

Methods

Two groups of patients were analyzed: Group I: 26 individuals reporting clinical symptoms induced by tomato contact or ingestion. Group II: 71 control individuals with no symptoms induced by tomato: 12 of them were previously skin prick test positive to a tomato extract, 39 were atopic and 20 were non-atopic. All individuals underwent prick-prick with fresh ripe peel Canary tomatoes and skin prick tested with freeze-dried peel and pulp extracts obtained from peel and pulp of Canary tomatoes at 10 mg/ml. Wheal sizes and prick test positivity (≥ 7 mm²) were compared between groups.

Results

In group I, 21 (81%) out of 26 patients were prick-prick positive. Twenty patients (77%) had positive skin prick test to peel extracts and 12 (46%) to pulp extracts. Prick-prick induced a mean wheal size of 43.81 ± 40.19 mm² compared with 44.25 ± 36.68 mm² induced by the peel extract (Not significant), and 17.79 ± 9.39 mm² induced by the pulp extract (p < 0.01). In group II, 13 (18%) out of 71 control patients were prick-prick positive. Twelve patients (all of them previously positive to peel extract) had positive skin prick test to peel and 3 to pulp. Prick-prick induced a mean wheal size of 28.88 ± 13.12 mm² compared with 33.17 ± 17.55 mm² induced by peel extract (Not significant), and 13.33 ± 4.80 mm² induced by pulp extract (p < 0.05 with peel extract and prick-prick).

Conclusion

Canary peel tomato extract seems to be as efficient as prick-prick tests with ripe tomatoes to diagnose patients sensitized to tomato. The wheal sizes induced by prick-prick and peel extracts were very similar and showed a high correlation coefficient.     

Dead Sea medical tourism: an allergological point of view

A 1-year study was conducted, with the aim to investigate the airborne allergens around the Dead Sea (DS), identify and quantify airborne pollen and spores in the DS region, and determine the different sensitization prevalence among various population groups to these aeroallergens. According to results, we also aimed to define “safe seasons” when there are no or only few aeroallergens in the atmosphere that surrounds the Dead Sea. A Rotorod and a Hirst trap were used for continuous monitoring of pollen and spores which were then identified. Sensitization to aeroallergens was assessed by skin prick tests (SPT) in three groups of allergic residents: foreign tourists, Israeli tourists, and local workers from the hotel industry. Air around the DS is by no means free of aeroallergens, 50 pollen and 43 mold types were identified. Pollen was from native plants, imported ornamentals, and others transported by winds from long distances. Overall pollen concentrations were lower around the Dead Sea than in the Tel Aviv region, but in certain months, they were higher around the DS. Marked seasonal variations in pollen and spore dispersal were observed. By SPT, hypersensitivity to Chenopodiaceae, Amaranthaceae, Cupressus, Solidago, Poaceae, Olea, Artemisia as well as molds such as Alternaria and Aspergillus, was found. As assessed by SPT, some of tourists and permanent residents are allergic to pollen, molds, and house dust mites. The presented study enables one to denote “safe” seasons when the concentration of airborne allergens is below “allergenic risk”: June–August and November–February. These seasons are the most suitable for allergic medical tourists.     

The partial contribution of specific airborne pollen to pollen induced allergy

The air that we inhale contains simultaneously a multiple array of allergenic pollen. It is well known that such allergens cause allergic reactions in some 15 of the population of the Western World. However little is known about the quantitative aspect of this phenomenon. What is the lowest concentration of pollen that might trigger allergic responses? As people are exposed to heterogeneous and variable environments, clarification of the partial contribution of each of the major airborne pollen allergens and determination of its role in invoking allergy are of prime importance. Objectives: (1) Assessment of a possible correlation between the concentration of airborne pollen and incidence of allergy. (2) Estimation of the lowest average concentrations for various species of airborne pollen that elicit allergic symptoms when exceeded. (3) Determination of the extent of the variations in manifestation of allergy symptoms that can be explained by fluctuations in the concentration of individual species of airborne pollen. Methods: The study was conducted during 14?months with a rural population in Israel. The participants completed a detailed questionnaire and were skin prick tested with the common airborne allergens. The appearance of clinical symptoms, i.e. nasal, bronchial, ocular or dermal, were reported daily by the patients. Concentrations of the airborne pollen and spores were monitored in the center of activity of the residents during one day every week, using three ‘Rotorod’ pollen traps. The pollen grains were identified by light microscopy. Results: The pollen spectrum was divided into time-blocks presenting the main pollination periods of the investigated species. The correlation between the concentration of airborne pollen of the relevant species and the clinical symptoms of the patients was determined for each time block. The correlation differed for different clinical symptoms and for different pollen allergens. Highest correlation with airborne pollen counts was found for patients with nasal and bronchial symptoms. The onset of the clinical symptoms by sensitive patients started, in each of the relevant groups, once the weekly average concentration of the airborne pollen crossed a threshold level. Under the limitations of the present study, this level was estimated to be 2–4 pollen m^?3 air for olive, 3–5 pollen m^?3 air for grasses, 4–5 pollen m^?3 air for Artemisia, 10–20 pollen m^?3 air for pecan and 50–60 pollen m^?3 air for cypress. Conclusions: Fluctuations in specific airborne pollen grains explained up to 2/3 of the variation in clinical allergy responses. Those were: 69 of the variation for cypress (March–April), 66 for the grasses (March–April), 49 for the pecan (May–June) and 62 for Artemisia (Autumn).     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic componenets. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103 000 daltons. Xan VIa was a glycoprotein of molecular weight 17 000 daltons. The carbohydrate moiety of Xan Vla was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

Assignment of disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen

Par j 2.0101, a major allergen of the Parietaria judaica pollen, was expressed in E. coli, purified to homogeneity and fully characterised both at the structural and the functional level. The recombinant rPar j 2.0101 protein showed an allergenic activity in histamine release, skin prick tests and capacity to bind IgE, almost identical to that of the native allergens purified from aqueous pollen extract. The complete pattern of S-S bridges of rPar j 2.0101 was determined by enzymatic digestion with endoproteinase Lys-C followed by mass spectrometric analysis of the resulting peptide mixtures. The eight cysteines occurring in the allergenic protein were found to be paired into the following four disulphides: Cys35-Cys83, Cys45-Cys60, Cys61-Cys106 and Cys81-Cys121. This structural information probes Par j 2.0101 to attain a 3-D fold consistent with that of the non-specific lipid transfer protein (ns-LTP) family and it represents an effective molecular basis to develop modified antigens by selective site-directed mutagenesis for immunotherapy.     

The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

BACKGROUND: Beauveria bassiana is an important entomopathogenic fungus currently under development as a bio-control agent for a variety of insect pests. Although reported to be non-toxic to vertebrates, the potential allergenicity of Beauveria species has not been widely studied. METHODS: IgE-reactivity studies were performed using sera from patients displaying mould hypersensitivity by immunoblot and immunoblot inhibition. Skin reactivity to B. bassiana extracts was measured using intradermal skin testing. RESULTS: Immunoblots of fungal extracts with pooled as well as individual sera showed a distribution of IgE reactive proteins present in B. bassiana crude extracts. Proteinase K digestion of extracts resulted in loss of IgE reactive epitopes, whereas EndoH and PNGaseF (glycosidase) treatments resulted in minor changes in IgE reactive banding patterns as determined by Western blots. Immunoblot inhibitions experiments showed complete loss of IgE-binding using self protein, and partial inhibition using extracts from common allergenic fungi including; Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Candida albicans, Epicoccum purpurascens, and Penicillium notatum. Several proteins including a strongly reactive band with an approximate molecular mass of 35 kDa was uninhibited by any of the tested extracts, and may represent B. bassiana specific allergens. Intradermal skin testing confirmed the in vitro results, demonstrating allergenic reactions in a number of individuals, including those who have had occupational exposure to B. bassiana. CONCLUSIONS: Beauveria bassiana possesses numerous IgE reactive proteins, some of which are cross-reactive among allergens from other fungi. A strongly reactive potential B. bassiana specific allergen (35 kDa) was identified. Intradermal skin testing confirmed the allergenic potential of B. bassiana.     

Birch pollen-associated peanut allergies in children

Panallergens show structural similarities, and they are responsible for many cross-reactions between pollen and plant food sources. The aim of the present study was to investigate IgE reactivity to peanut allergen components in children with birch pollen allergy. Patients experienced symptoms of allergic asthma, allergic rhinitis, and urticaria, and they underwent a complete diagnostic evaluation, including skin prick test (SPT), specific IgE (sIgE) to birch pollen allergen (t3), peanut allergen (f13). In addition, measurement of sIgE to the major birch allergen components, Betula verrucosa (Bet v1, Bet v2), and to peanut allergen components, Arachis hypogaea (genuine componens: Ara h1, Ara h2, Ara h3, and cross-reactive Ara h8) was performed, by using a microarray technique (component resolved diagnosis, CRD). SPT to birch extract was positive in all children, and SPT to peanut extract was positive in 51 % of them. sIgE to both allergens was increased in 39 % of children, 55 % of them had increased sIgE (t3), and one child had increased sIgE (f13). CRD results confirmed that some children were sensitized to Bet v1 only, and some children to genuine Ara h only. Bet v1/Ara h8 cross-reactivity was found in 16 % of children. Results of the present study reveal that SPT, sIgE, and CRD may detect sensitization and co-sensitization with birch and peanut allergens/allergen components, and CRD may help to differentiate sensitization to genuine peanut components from sensitization to peanut cross-reactive component in birch-sensitive children. Diagnostic approach has to be individualized for each patient.