Basidiospore aeroallergens.
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Air Microbiology↗
Biomedical subjects
W E Horner
Publications and source records attributed to W E Horner.
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↗
Stability studies of Calvatia cyathiformis basidiospore allergens.
Calvatia cyathiformis allergens in unfractionated extract (crude), and in extract sequentially fractionated by gel filtration (GF) and hydrophobic interaction chromatography (HIC) were tested for stability. C. cyathiformis allergen sources (crude, GF, HIC) were sampled immediately (0 h), or incubated at 4, 24 or 37 degrees C and then sampled at 8, 24 or 96 h. Polyacrylamide gel-isoelectric focusing immunoprints revealed 3 allergen(s) groups, or bands (Bds) with respective pI of 3.6-4.6, 6.6 and 9.3. Only Bds 3.6-4.6 were stable at 37 degrees C. At 24 degrees C, Bds 3.6-4.6 persisted to 96 h, Bd 6.6 persisted 24 h, and Bd 9.3 waned in 8 h. At 4 degrees C all 3 allergens in HIC were stable for 8 and 24 h; Bd 9.3 was reduced at 96 h. All allergen activity was labile to low pH conditions except for Bds 3.6-4.6. Proteinase K degraded Bd 9.3 more rapidly than Bd 6.6. Immunoprint patterns corresponded to the stained gels and were consistent among different sources. Bd 9.3 is very labile, but reactive with 63% of sera tested. Since 10-15% of C. cyathiformis reactors bind IgE only to Bd 9.3, this is notable variability, and significant for diagnosis and treatment.
Allergens↗
Identification and analysis of basidiospore allergens from puffballs.
Spore extracts from four common puffballs (Calvatia cyathiformis, Geaster saccatum, Pisolithus tinctorius, and Scleroderma areolatum) were fractionated by Sephadex G-75 gel filtration. Pooled fractions, analyzed by skin test or direct RAST, contained allergenic components over a molecular weight range of 10,500 to 20,000 daltons for all puffball spores studied except for S. areolatum (greater than 70,000 daltons). The Sephadex G-75 allergenic pool of C. cyathiformis was further fractionated by hydrophobic interaction chromatography. Fractions were successively eluted with 4 and 1 mol/L buffered saline solutions and water. The 1 mol/L fraction contained maximal allergenic activity as determined by RAST-inhibition studies. At least 15 Coomassie blue staining bands were identified after isoelectrofocusing, of which 7 were shown by immunoprinting to bind IgE from a serum pool of five patients with allergy. Our results demonstrate that a combination of gel filtration, hydrophobic interaction chromatography, RAST-inhibition, and immunoprinting studies can be used to fractionate, characterize, and standardize spore allergen extracts of puffballs for diagnostic and therapeutic purpose.
Allergens↗
Characterization of allergens from spores of the oyster mushroom, Pleurotus ostreatus.
Crude extracts of Pleurotus ostreatus spores obtained from a single local source were fractionated by gel filtration to resolve the allergenic components. The fraction pool corresponding to 10.5 to 25 kd molecular weight contained allergenic activity as demonstrated by both RAST and skin testing. Similar results were obtained with extracts from spores that originated in four other areas and with extracts prepared from P. sajor-caju spores obtained from commercially produced caps. The RAST-active fraction was further separated by hydrophobic interaction chromatography (HIC). HIC fraction pools were assayed for allergen(s) by RAST inhibition and immunoblotting of isoelectric focused polyacrylamide gels. RAST-inhibition data indicated that the allergen(s) was reversibly bound to the HIC column, eluting with 2, 1, and 0.15 mol/L of buffered salt solutions. After electrofocusing, these fractions yielded 15, 12, and 11 Coomassie brilliant blue-staining bands, respectively. IgE binding occurred with 7, 8, and 6 of these bands, as revealed by radiostaining of the immunoblots. These procedures help identify P. ostreatus spore allergens and allow a greater degree of standardization in the preparation of allergen extracts from basidiospores for use in diagnosis and therapy of fungal allergy.
Allergens↗
Basidiospore allergens: analysis of Coprinus quadrifidus spore, cap, and stalk extracts.
Atopic individuals with symptoms of respiratory allergy have been shown to have IgE-mediated reactions to spores from the basidiomycete fungi. Because our earlier studies suggested that parts of the fungus other than spores may contain allergens, the current study was performed. Extracts of Coprinus quadrifidus spores, caps, and stalks were prepared and fractionated by gel filtration column chromatography on Sephadex G-75. Analysis of column fractions of each separation by ultraviolet absorption demonstrated at least three peaks of absorbency in spore, cap, and stalk extracts. Pooled column fractions were analysed by direct radio-allergosorbent test (RAST) using pooled sera from C. quadrifidus skin-test positive subjects. Enhanced allergenic activity was present in the same portion of the column eluate for cap, spore, and stalk fractionations, corresponding to a molecular weight of approximately 10.5-25 kD. Pools with allergenic activity were used to test volunteers by skin prick and RAST. Skin test and RAST activities were similar for each of the three Coprinus extracts, with stalk being the most potent. Evidence of common allergenic epitopes was demonstrated by inhibition of spore RAST by spore, cap, and stalk extracts. These results suggest that C. quadrifidus cap and stalk extracts contain allergens similar to those in spores extract and may provide useful sources of allergen for further study.
Agaricales↗