Basidiomycete allergens.
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Air Pollutants↗
Biomedical subjects
W E Horner
Publications and source records attributed to W E Horner.
Why are some proteins allergenic? Implications for biotechnology.
In recent years, a number of agricultural crops have been developed with recombinant DNA technology. Because the transferred genes code for proteins that are ordinarily not present in these particular foods, there is concern about the potential allergenicity of these new crop varieties. Foods contain many proteins; however, only a small fraction are allergens. Although the structural properties of proteins that cause allergic reactions have not been characterized completely, known food allergens in general have molecular weights between 10 and 70 kDa, stimulate the immune response (induce the production of allergen-specific IgE), and are stable molecules that are resistant to processing, cooking, and digestion. Although any type of food is potentially allergenic, the majority of food allergies are caused by a small group of foods (cows' milk, nuts, legumes, eggs, seafood). Cross-reactivities occur within a given food group and between foods and seemingly unrelated proteins. Even though most transgenic foods are considered safe, biotechnological manipulation can affect crop allergenicity. The safety evaluation of transgenic foods is relatively easy when the allergenicity of the gene sources are known. The recombinant food can be assayed using traditional in vitro inhibition assays. Recently, reduced allergen content of biotechnologically altered rice was shown. In contrast, increased allergenicity was demonstrated in transgenic soybeans after a methionine- and cystine-rich protein from Brazil nuts, identified as a major Brazil nut allergen, was expressed in soybean to increase its content of sulfur-rich amino acids. The most difficult issue regarding transgenic food allergenicity is the effect of transfer of proteins of unknown allergenicity. The challenge is to determine whether these proteins are allergenic as there is no generally accepted, established, definitive procedure to define or predict a protein's allergenicity. Comparing the structures of the transferred protein with known allergens and allergen epitopes could be one approach. Additionally, Th-2 cell stimulation, the production of IL-4, and IgE antibody induction in animal models may help to evaluate the potential allergenicity of a protein. In conclusion, there is no evidence that recombinant proteins in newly developed foods are more allergenic than traditional proteins. The evidence suggests that the vast majority of these proteins will be completely safe for the consumer. The concern is that if a few transgenic foods cause serious allergic reactions, this could undermine the public's confidence in such products. It is essential that proper guidelines are established and tests are developed to assure that this will not occur.
Allergens↗
Identification of important allergens in German cockroach extracts by sodium dodecylsulfate-polyacrylamide gel electrophoresis and western blot analysis.
BACKGROUND: Despite recent advances in the purification and characterization of cockroach allergens, identification of clinically important allergens and their source have not been completely elucidated. This study investigated the allergen content of German cockroach (Blattella germanica) whole body (GWBE) and fecal (GFE) extracts. METHODS: Sera from 37 subjects with asthma, with positive skin test results to cockroach, were used for RAST and Western blot (after sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] under reducing conditions); this serum panel is the largest used to date for cockroach allergen analysis. RESULTS: RAST reactivity to GWBE and GFE were highly correlated (r = 0.882, p < 0.001). SDS-PAGE and Western blotting showed that GWBE and GFE had similar patterns of IgE binding. Furthermore, Western blot inhibition investigations revealed that either GWBE or GFE could almost completely inhibit the reactivity of the other extract, SDS-PAGE and Western blotting demonstrated in both extracts numerous bands that displayed a high prevalence of IgE binding. Protein bands at 67, 50, 45, and 36 kd bound more than 50%, and the band at 60 kd bound approximately 80% of the sera tested. CONCLUSIONS: In summary, this investigation identified German cockroach allergens, established their relative importance by prevalence of reactivity to a large serum panel, and demonstrated that cockroach feces possess significant allergenic activity. Five allergens identified demonstrated reactivity with up to 50% to 80% of the 37 subjects' sera tested.
Allergens↗
Fungal allergens.
Airborne fungal spores occur widely and often in far greater concentrations than pollen grains. Immunoglobulin E-specific antigens (allergens) on airborne fungal spores induce type I hypersensitivity (allergic) respiratory reactions in sensitized atopic subjects, causing rhinitis and/or asthma. The prevalence of respiratory allergy to fungi is imprecisely known but is estimated at 20 to 30% of atopic (allergy-predisposed) individuals or up to 6% of the general population. Diagnosis and immunotherapy of allergy to fungi require well-characterized or standardized extracts that contain the relevant allergen(s) of the appropriate fungus. Production of standardized extracts is difficult since fungal extracts are complex mixtures and a variety of fungi are allergenic. Thus, the currently available extracts are largely nonstandardized, even uncharacterized, crude extracts. Recent significant progress in isolating and characterizing relevant fungal allergens is summarized in the present review. Particularly, some allergens from the genera Alternaria, Aspergillus, and Cladosporium are now thoroughly characterized, and allergens from several other genera, including some basidiomycetes, have also been purified. The availability of these extracts will facilitate definitive studies of fungal allergy prevalence and immunotherapy efficacy as well as enhance both the diagnosis and therapy of fungal allergy.
Antigens, Fungal↗
[Current aspects of fungal spores allergy].
In industrialized countries the prevalence of allergic inhalant diseases is some 15-20%. More than 10% of these individuals are sensitized to fungal allergens. Many fungal spores are less than 10 microns in size, which permits penetration into the smaller airways of the lung. Bronchial provocation tests have demonstrated that fungal spores and spore extracts can cause both an early and a late phase reaction in sensitive subjects. Over 80 genera of fungi have been associated with symptoms of respiratory tract allergy. Ascomycetes, basidiomycetes and zygomycetes are the major fungal groups that contain genera known to induce and elicit allergic reactions. These groups contribute most of the spores found in air. Although ascomycetes include the greatest number of any fungal group, only a few species, such as Aspergillus fumigatus, Alternaria alternata and Cladosporium herbarum, have been investigated in a scientific manner. In recent years spores of basidiomycetes have been tested for allergenicity and some species have been determined to be allergenic, such as Calvatia cyathiformis, Ganoderma applanatum, Pleurotus ostreatus, or Psilocybe cubensis. Compared to pollen-related allergies, diagnosis of fungal allergy is often difficult. Provocative challenge with specific fungal antigens can provide a definitive diagnosis. To date, only three controlled immunotherapy trials with standardized extracts of A. alternata and C. herbarum have shown clinical efficacy. In spite of these studies, immunotherapy with fungal antigens requires further investigations. Thus, the indication for immunotherapy with fungal extracts must be judged by an experienced allergist. Apart from pharmacological management, avoiding or minimizing exposure is the front-line measure.
Allergens↗
Basidiospore allergen release: elution from intact spores.
BACKGROUND: We sought to test the hypothesis that allergens are readily released by intact basidiospores in vitro and that different species release allergens in different patterns that are related to spore wall structure. METHODS: To assess basidiospore allergen releasability, basidiospore allergens were extracted from disrupted spores and eluted from intact spores, and the allergenic potency of the extracts and eluates was compared in Calvatia cyathiformis, Psilocybe cubensis, Lentinus edodes, and Pleurotus ostreatus. RESULTS: There was a significant species difference; on the basis of dry weight, the yield of extract and eluates from P1. ostreatus greatly exceeded the yield from C. cyathiformis extract and eluates. As measured by RAST inhibition, the allergenic potency of P1. ostreatus and L. edodes spore eluates reached the potency of disrupted spore extract in less than 4 hours. Allergen potency of Ps. cubensis and C. cyathiformis eluate approached the potency of disrupted spore extract only after 8 and 24 hours. Allergen staining intensity in sodium dodecyl sulfate polyacrylamide gel electrophoresis IgE immunoblot correlated with RAST activity. C. cyathiformis and Ps. cubensis spores have thick walls, whereas P1. ostreatus and L. edodes spore walls are thin and hyaline (nonpigmented). Thus spore allergen release may relate to spore wall characteristics. CONCLUSIONS: These studies indicate that intact basidiospores of all four species release allergens, but the release patterns differ by species, and these differences may be clinically significant.
Allergens↗
Comparison of Psilocybe cubensis spore and mycelium allergens.
BACKGROUND: Basidiospores are an important cause of respiratory allergy in mold-sensitive atopic subjects. Collection of the large amounts of spores required for extract preparation is tedious and difficult. A desirable alternative could be mycelium grown in vitro if it is allergenically similar to spores. METHODS: Therefore this study compared the allergen contents of Psilocybe cubensis spore and mycelium extracts by different techniques with the use of pooled sera from subjects who had skin test and RAST results that were positive to P. cubensis spores. RESULTS: Isoelectric focusing immunoprints revealed six common IgE-binding bands at isoelectric points 4.7, 5.0, 5.5, 5.6, 8.7, and 9.3. Two additional bands at isoelectric points 3.9 and 5.7 were detected only in the spore extract. Sodium dodecylsulfate-polyacrylamide gel electrophoresis immunoblots exhibited six common IgE-binding bands at 16, 35, 487, 52, 62, and 76 kd; 20 and 40 kd bands were present only in the spore extract. Although RAST and isoelectric focusing inhibition demonstrated that P. cubensis spore and mycelium extracts share many allergens, spores were allergenically more potent than mycelium. CONCLUSION: The results indicate that mycelium is a useful source of P. cubensis allergen, even though several spore allergens were not detected in mycelium.
Agaricales↗
Basidiomycete allergens: comparison of three Ganoderma species.
High atmospheric concentrations of basidiospores occur in various parts of the world. Ganoderma basidiospores are distinctive, easily identifiable in aeroallergen surveys, and widely abundant. Previous studies showed that Ganoderma basidiospores caused respiratory allergies. Thus, we investigated various extracts (spore, cap, and/or mycelial) of G. meredithae, G. lucidum, and G. applanatum for allergen components. Analyses included radioallergosorbent test (RAST) inhibition and IgE blots from isoelectric focusing (IEF) and SDS-PAGE. RAST inhibition with spores and caps of G. meredithae and G. lucidum showed that spores inhibited caps better than caps inhibited spores. Species differences were minor. Coomassie blue (CB) staining of IEF gels detected at least 23 protein bands (pI 3.6-6.6) in caps of G. meredithae and G. lucidum. G. meredithae spore extracts contained 17 of these (pI 3.6-5.0, 6.6). Spores and caps of G. meredithae contained 13 and 11 allergen bands, respectively, on IEF blots. SDS-PAGE of G. meredithae spore and cap showed one and four bands, respectively, by CB staining, but IgE blots showed 13 bands in cap and 17 in spore. Culture mycelia of G. lucidum and G. applanatum attained significant and essentially constant RAST activity by day 4. Activity was also present in culture supernatant by day 4. Blots of mycelium and supernatant detected a single allergen in day-8 mycelia and subsequently six allergen bands in day-16 mycelia and eight in day-16 supernatant (one appeared as a doublet). These data show that Ganoderma extracts contain a complex mixture of allergens. Differences among species were minor; spores and mycelia are apparently better sources of allergens than caps.
Allergens↗
Identification of Psilocybe cubensis spore allergens by immunoprinting.
Previous studies established that Psilocybe cubensis contains potent allergens, and that a significant percentage of atopic subjects were sensitized to P. cubensis spores. The objective of this study was to identify P. cubensis spore allergens using isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoprinting. Coomassie blue staining of IEF gels detected approximately 20 bands between pI 3.6 and 9.3. Immunoprints obtained with 15 P. cubensis skin test- and RAST-positive sera revealed 13 IgE-binding bands; the most reactive were at pI 5.0 (80%), 5.6 (87%), 8.7 (80%) and 9.3 (100%). SDS-PAGE resolved 27 proteins ranging from about 13 to 112 kD. SDS-PAGE immunoprints conducted with 11 skin test- and RAST-positive sera demonstrated 18 IgE-binding bands; most sera reacted to 16 (82%), 35 (100%) and 76 kD (91%) allergens. Both electrophoretic procedures demonstrated a single allergen (at pI 9.3 and 35 kD) that reacted with all sera tested. This study corroborates the allergenic significance of P. cubensis spores and identifies the allergens of greatest importance.
Allergens↗
Basidiospore aeroallergens.
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Air Microbiology↗
Comparison of cockroach allergenic activity in whole body and fecal extracts.
Previous studies have established cockroach allergens as important sensitizing agents in the induction/exacerbation of urban asthma. The present investigation compared saline extracts of American cockroach (Periplaneta americana) whole bodies and feces and German cockroach (Blattella germanica) whole bodies and feces as important sources of allergens. All extracts were tested before or after gel filtration on Sephadex G-75 columns (fraction 2) as previously described. Skin test studies of 69 subjects with asthma with extracts of American or German cockroaches demonstrated a significant correlation of reactivity to whole body and fecal extracts for both species. Direct RASTs of 13 sera from cockroach skin test-positive subjects were generally greater to both German whole body extracts (GWBEs) and German fecal extracts (GFEs) as compared to American whole body and fecal extracts. There was a good correlation of RAST reactivity to GWBE with GFE. RAST inhibition demonstrated that GFE contained most of the allergenic activity present in GWBE. These studies demonstrate the allergenic similarities of cockroach whole body and fecal extracts and suggest that cockroach feces are an important sensitizing agent in atopic asthma.
Allergens↗
Cockroach allergenic activity: analysis of commercial cockroach and dust extracts.
Previous investigations demonstrated that cockroach whole bodies and feces are important sources of allergens in the induction/exacerbation of bronchial asthma. The current study investigated different cockroach source materials, commercial extracts, and house dust extracts for cockroach allergenic activity. In general, extracts from four different sources of either American or German cockroaches contained similar amounts of allergenic activity by RAST inhibition. Three commercial American cockroach extracts compared by RAST inhibition had similar allergenic activity on an equal protein basis. Skin test results correlated house dust reactivity to both commercial and inhouse cockroach wholebody extracts and to fecal extracts. Six different samples of house dust obtained from vacuum cleaners in the New Orleans area and three commercially obtained house dust extracts contained varying quantities of cockroach allergenic activity by RAST inhibition. These studies demonstrate that commercial cockroach extracts vary in allergenic activity and that all house dust extracts tested contain cockroach allergens.
Allergens↗
Basidiomycete allergy: identification and characterization of an important allergen from Calvatia cyathiformis.
Basidiomycetes were not considered as major aeroallergen sources until spore traps revealed the prevalence of basidiospores, which were in some cases associated with epidemic outbreaks of asthma. More recently, we established that approximately one third of subjects with respiratory allergic disease were skin prick test positive to basidiospore extracts. Bronchial challenge with these extracts induced reversible immediate and late phase bronchospasm in sensitive subjects. Screening individual RAST-positive sera for reactivity to immunoprinted Calvatia cyathiformis spore extract indicated that a basic component with pI 9.3 reacted with 63% of test sera. Further analysis by RAST and immunoprint (IP) inhibition showed that this component, Cal c Bd9.3, appeared to cross-react with spore extracts from 3 of 5 other species tested. To isolate this component for additional analysis, a protocol was used with two sequential stages of preparative isoelectric focusing (IEF) followed by hydrophobic interaction chromatography. This yielded purified Cal c Bd9.3 that retained IgE-binding activity by IP, was a single band by IEF and SDS-PAGE (16 kD) analysis when stained with Coomassie blue. Double diffusion in gel with rabbit antiserum to Cal c BD9.3 demonstrated a single precipitin band in crude extract that was identical to that obtained with purified Cal c Bd9.3. By IP this antiserum recognized pure Cal c Bd9.3 and the 9.3 band in crude extracts from C. cyathiformis and 3 other species, corroborating the cross-reactivity. However, other bands were also recognized. These studies indicate that basidiospores are major fungal aeroallergens, many of which contain a common basic allergen to which most sensitized subjects react.
Allergens↗
Evidence for cross-reactive allergens among basidiomycetes: immunoprint-inhibition studies.
Allergenic cross-reactivity among six basidiomycete species (Calvatia cyathiformis, Coprinus quadrifidus, Psilocybe cubensis, Pleurotus ostreatus, Ganoderma meredithae, and Pisolithus tinctorius) was determined by immunoprint inhibition. Extensive cross-reactivity was demonstrated among Coprinus quadrifidus, Psilocybe cubensis, and Pleurotus ostreatus of the order Agaricales, and Calvatia cyathiformis of the order Lycoperdales. However, G. meredithae (order Aphyllophorales) and Pisolithus tinctorius (order Sclerodermatales) did not demonstrate significant cross-reactivity with the other basidiomycete species. Generally, the two most potent inhibitors were Psilocybe cubensis and Pleurotus ostreatus. Inhibitory dose-response curves of a major allergenic band (isoelectric point, 9.3) were obtained by densitometry. Significant cross-reactivity was demonstrated for the 9.3 band among the species of the order Agaricales and with Calvatia cyathiformis. The most potent inhibitors were again Psilocybe cubensis and Pleurotus ostreatus. Thus, there is substantial allergenic cross-reactivity among the species of the order Agaricales tested and with Calvatia cyathiformis but not between these four species and G. meredithae or Pisolithus tinctorius. These studies support earlier RAST-inhibition observations of shared allergenic epitopes among basidiomycetes, especially epitopes within the Agaricales. The presence of shared epitopes suggests the possibility of devising a panel of skin test reagents representative of a large group of basidiomycetes.
Allergens↗
Identification and characterization of important cockroach allergens.
Allergens extracted from American and German cockroach species have been identified as significant sensitizing agents in the induction/exacerbation of asthma. In the present study, gel-filtration fractions of saline extracts of American cockroach (Periplaneta americana) whole bodies (AWBE fraction 2) and German cockroach (Blattella germanica) whole bodies (GWBE fraction 2) were used to identify and characterize important cockroach allergens by immunoprinting. In addition, allergens from AWBE and GWBE fractions 2 were additionally fractionated by chromatofocusing on polybuffer exchanger. Immunoprinting studies demonstrated several important acidic allergens in cockroach whole body extracts. All but one allergen had an acid isoelectric point (pH 2.80 to 5.20). Two allergens, one that focused at pH 3.50 and another allergen (or group of isoallergens) that focused between pH 4.15 to 4.55 were reactive with most sera obtained from cockroach-sensitive subjects. Chromatofocusing and subsequent skin test and RAST studies of AWBE and GWBE confirmed the presence of significant cockroach allergens with isoelectric points within the zone of pH 3.75 to 4.50. RAST-inhibition studies demonstrated the similarity of these allergens between AWBE and GWBE. Collectively, these observations identify the presence of several acidic cockroach allergens presumably shared between AWBE and GWBE.
Allergens↗
Evaluation of Basidiomycete and Deuteromycete (Fungi Imperfecti) extracts for shared allergenic determinants.
Aqueous extracts of select members of the Basidiomycetes and Deuteromycetes (Fungi Imperfecti) were evaluated for the presence of shared allergenic determinants using skin prick and radio-allergosorbent test (RAST) inhibition. Twenty adults with perennial symptoms of rhinitis, with or without asthma, were skin-prick tested with six species of Deuteromycetes and seven species of Basidomycetes. Positive weal-and-flare reactivity to Pleurotus ostreatus was associated with Alternaria alternata, Fusarium solani and Epicoccum purpurescens. Positive skin reactivity to Calvatia cyathiformis was also associated with A. alternata and F. solani. Coprinus quadrifidus was associated only with F. solani, and Psilocybe cubensis was only associated with Aspergillus fumigatus. No other skin test associations were demonstrated. For every allergen tested by RAST inhibition, significant dose-dependent homologous inhibition was demonstrated. Although the ability of an individual heterologous extract to inhibit the direct RAST varied, inhibition was generally minimal. In the most extreme example, no heterologous allergen inhibited the A. alternata RAST. However, the Armillaria tabescens RAST was inhibited 52.6%, 38.1% and 25.1% by A. fumigatus, E. purpurescens, and Penicillium notatum, respectively, suggesting significant cross-reactivity. These results suggest that, although shared allergenic determinants exist between select species of Basidiomycetes and Deuteromycetes, crossreactivity is minimal and its clinical significance is not clear. These data confirm that for reliable diagnosis of fungal allergy, representatives of both major groups must be used.
Adult↗
Common German cockroach whole body and fecal allergens: immunoprint inhibition studies.
Previous studies have established that cockroach allergens are important sensitizing agents in the induction/exacerbation of urban asthma. The present investigation compared saline extracts of German cockroach (Blattella germanica) whole bodies (GWBE) and feces (GFE) as important sources of allergens. Both extracts were tested prior to or following gel filtration on Sephadex G-75 (Fr2). Immunoprinting of unfractionated fecal extract using 10 RAST-positive sera detected a series of allergens with pI values between 4.15-4.55 and a single allergen that focused near the cathode. Multiple allergens were detected in both GWBE-Fr2 and GFE-Fr2. Generally, most RAST-positive sera reacted similarly, although GWBE-Fr2 had more allergenic proteins than GFE-Fr2 with pI less than 4.7 and fewer allergens with pI greater than 4.7. Immunoprint inhibition of GWBE and GFE by GWBE and GFE also demonstrated allergenic similarity, although homologous extracts were more effective inhibitors than heterologous preparations. Collectively, these studies demonstrate the allergenic similarities of cockroach whole body and fecal extracts. Thus fecal material may provide an important source of sensitizing antigen.
Allergens↗
Immunoprint analysis of Calvatia cyathiformis allergens. I. Reactivity with individual sera.
Basidiomycete allergens have received scant attention to date, compared to allergens from deuteromycetes. This is true even though in previous studies, 32% of atopic patients with respiratory allergies were skin test reactive to basidiospore extracts. Since sufficient quantities of Calvatia cyathiformis spores were available, their allergens were sequentially fractionated by gel filtration (GF) and hydrophobic interaction chromatography (HIC). Unfractionated extract (crude), GF, and HIC fractions were electrofocused in polyacrylamide gel (pH 3.5 to 9.5) and immunoprinted onto CNBr-activated nitrocellulose (0.2 microns) filters. Sera from 19 skin prick test positive and 10 negative subjects, with comparable total IgE levels, were used to screen individual strips of each blot. Blots were reacted with 125I-labeled anti-IgE and analyzed by autoradiography. IgE from 79%, 89%, and 89% of the positive sera bound to crude extract, GF, and HIC, respectively; IgE from none of the negative sera bound to crude extract. A series of bands (pH 3.6 to 4.6) reacted with 63%, one band (pH 6.6) reacted with 68%, and one band (pH 9.3) reacted with 63% of the sera tested. These studies demonstrate at least three important groups of allergens in C. cyathiformis spores. This characterization allows assignment of initial C. cyathiformis allergen designations and development of a purification protocol.
Allergens↗