Basidiomycete allergy: what is the best source of antigen?

Skin prick test activity and antigenicity of extracts of in vitro growth of the Basidiomycete Pleurotus ostreatus (PO) were compared to extracts of spores from PO growing in the wild. Patients demonstrated significant differences in skin test reactivity to the PO extracts. Some reacted only to in vitro growth extracts, others only to the spore extracts and 1 patient to all extracts. Further studies analyzed antigens present in all extracts with rabbit antisera to PO. Common as well as unique antigens were present in the spore extracts as compared to those from in vitro preparations. The fact that spores contain unique antigens suggests that basidiospores may be the best source of relevant allergens for clinical studies.     

Alternaria spore and mycelium sensitivity in allergic patients: in vivo and in vitro studies.

Extracts from Alternaria spores and mycelia were prepared to evaluate their allergenic potencies in allergic patients. Twelve Alternaria-sensitive patients, with histories of rhinitis or asthma, were submitted to skin prick tests and five of 12 patients received nasal challenges with spores and mycelia. In vitro, the allergenic activity of each extract was determined by RAST, basophil histamine-release and RAST-inhibition. All patients demonstrated skin reactivity to both extracts while skin reactivity to mycelia was higher than that induced by the spore extract (P < .005). Of the 12 patients, 11 had positive mycelia-RAST and 9/12 had positive spore-RAST. It was found that mycelium-IgE antibody levels were higher than spore-IgE antibody levels (P < .005). Nine RAST-positive patients had positive histamine release tests (> 50%) and basophils challenged with mycelia appeared 10-fold more sensitive compared to the spore challenge. In four of five patients subjected to nasal provocation tests, immediate-type rhinitis was elicited either after spore or mycelium challenge. The patients exhibited a higher nasal reactivity with the mycelium challenge than with spores. RAST-inhibition studies demonstrated that mycelial extracts shared common allergens with spore. These results indicated that Alternaria spore and mycelium were potent allergens in allergic patients and there was a variability in the pattern of in vivo and in vitro reactions between the patients for each allergen.     

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.     

Basidiospore allergens: determination of optimal extraction methods

Five methods were tested by RAST for allergen extraction from seven basidiospore species Armillaria tabescens, Chlorophyllum molybdites, Coprinus quadrifidus, Pleurotus ostreatus, Calvatia cyathiformis, Pisolithus tinctorius , and Scleroderma sp. With each basidiospore type, extracted allergen activity varied according to the method employed. In general, defatting of spores with ethyl ether, followed by homogenization in 0.125 m NH₄HCO₃ buffer, resulted in greatest allergen yield. In order to compare extracts from different isolates, batches of Pleurotus ostreatus spores obtained from different locations and over different time periods were analysed. Spores harvested from the same basidiomycete species in different areas varied in allergen (RAST) and protein (HPLC profile) content. The spores obtained from the same location over a 1–2 year period did not differ significantly. These results indicate that basidiospores can be stored for several years and that spore extracts from different locations can vary. These studies will help in the future to provide better characterized extracts for clinical studies.     

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.     

Ultrastructural demonstration of specific IgG and IgE antibodies binding to Aspergillus fumigatus from patients with aspergillosis.

Aspergillus-induced diseases usually demonstrate elevated circulating antibodies belonging to different isotypes. The antigens currently used to detect antibodies are crude culture filtrate and mycelial extracts of A. fumigatus (Af). Most Af-associated diseases result from the inhalation of the spores of the organisms present in the environment. However, it is not known whether specific circulating antibodies directed only against spore or mycelia of Af exist in the sera of patients with Af-induced diseases. With colloidal gold we have investigated thin sections of spores and hyphae of Af for their reactivity with Af-specific IgG and IgE antibodies. The results indicate that both spores and hyphae reacted identically with IgG and IgE antibodies from patients. None of the sera from normal control subjects reacted in this system, although low levels of antibodies were detected in the sera by ELISA. Sera from both patients with allergic bronchopulmonary aspergillosis or aspergilloma reacted with cell envelope antigens, whereas sera from patients with invasive aspergillosis also bound to cell sap. This method therefore demonstrates localization of antigens binding to different isotypes in the sera from different clinical forms of aspergillosis and may be useful in purifying specific antigens for immunodiagnosis.     

Respiratory and immunological reactions among Shiitake (Lentinus edodes) mushroom workers

Four workers, the total work force employed at a Shiitake farm, developed cough and sputum production following a variable period of exposure to Shiitake mushrooms. All four had abnormal diffusing capacity and three had abnormal spirometry values. Chest roentgenograms demonstrated an interstitial pattern in one worker. Pulmonary function tests performed before and during several days of work demonstrated a significant decrease (greater than 20%) in forced vital capacity (FVC) and/or maximal mid-expiratory flow (MMEF) in three workers. Although specific antibodies to an extract of Shiitake spores were detected in sera from three workers none were IgE. High levels of Shiitake spores were detected in growing rooms (greater than 10(6)/m3) as well as other locations at the farm. Shiitake spore airborne antigen, detected by an immunochemical assay, was present in dust collected with a volumetric sampler from different locations at the farm. Antigenic determinants of Shiitake spore antigens, in common with antigens from other cultivated mushrooms (Agaricus and Pleurotus) were demonstrated by ELISA inhibition assay. This study demonstrates that workers exposed to high levels of Shiitake spores develop symptoms and laboratory findings suggestive of hypersensitivity pneumonitis (HP). Strict environmental control and the wearing of a face mask is probably needed to reduce the high risk of sensitization and possible development of immunological lung disease. Shiitake spores must be considered as an aetiological agent of mushroom workers' lung.     

*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 cause 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.     

Identification of crustacea allergens by crossed radioimmunoelectrophoresis

Crossed immunoelectrophoresis (CIE) detected 18 precipitating antigens in extracts of shrimp. Of these antigens, crossed-line immunoelectrophoresis (CLIE) of shrimp extract demonstrated that 5 cross-reacted with crayfish, 3 with lobster and 1 with crab extract. Allergens present in the shrimp CIE plates were identified by crossed radioimmunoelectrophoresis (CRIE) using sera from 6 study subjects who were skin-test and RAST positive to shrimp extract. Of the 7 allergens detected, 3 (precipitins 1, 3 and 6) reacted with most of the 6 sera tested from shrimp-sensitive subjects. Precipitins 1 and 6 appear to be common crustacea allergens (present in shrimp, crayfish, lobster and crab) whereas precipitin 3 may be a specific allergen since it is present only in shrimp.     

The development of species-specific immunodiagnostics for Stachybotrys chartarum: the role of cross-reactivity

Mold contamination and exposure to fungi in indoor environments has been associated with various adverse health effects but little is known about the significance of individual fungal species in the initiation or exacerbation of such effects. Using Stachybotrys chartarum as a model fungus we sought to demonstrate that monoclonal antibodies (mAbs) can provide species-specific diagnostic reagents and also be used to investigate immunological cross-reactivity patterns among fungi. Mice were immunized with S. chartarum spore walls and monoclonal antibodies were screened against 60 fungal species and 24 different isolates of S. chartarum using an indirect ELISA. One species-specific mAb (IgG(1)) reacted only with spore preparations but not mycelium of S. chartarum or propagules of any other fungus. Five cross-reactive mAbs (IgM) documented extensive cross-reactivity among nine related Stachybotrys species and several non-related genera including several species of Cladosporium, Memnoniella, Myrothecium and Trichoderma. We also found that the ELISA reactivity for cross-reactive antigens and different isolates of S. chartarum differed considerably for normalized total amounts of mycelial antigen. We demonstrate that mAbs and immunoassays have the potential to detect S. chartarum species-specifically. The observed reactivity patterns with cross-reactive mAbs suggest that several fungi may share common antigens and that the majority of antigens are expressed by spores and mycelia. The observed cross-reactivity patterns need to be considered for accurate interpretations of environmental and serological analyses.     

Spore antigens of Clostridium sporogenes.

By means of spore-agglutination of fluorescent-antibody techniques, three serological types were identified among 84 strains of Clostridium sporogenes. Four spore antigens were identified, designated A, B, C and D. A, B and C were specific for the respective types whilst D was a group antigen shared by strains of the three types. The spore antigens had corresponding somatic antigens; the type-specific somatic antigens were designated I, II, III, and the shared somatic antigen IV. The flagellar antigens were found to be type specific and were designated 1, 2 and 3; no common flagellar antigen was detected. The results of precipitation tests with spore extracts depended on the method of testing. By a capillary-tube ring method there were cross reactions among the three types of C. sporogenes, whilst by immunodiffusion in agar layers the reactions were generally type specific. In a disintegrated spore extract, two non-protein antigenic components, possibly polysaccharide, were detected by means of immunoelectrophoresis. This extract showed cross reaction with antisera to strains of all types of all types of C. sporogenes by the ring test and by immunodiffusion.     

Basidiospore extracts: evidence for common antigenic/allergenic determinants

Spore extracts, prepared from Armillariella tabescens, Pleurotus ostreatus, Coprinus quadrifidus, Amanita muscaria, Ganoderma lucidum, Psilocybe cubensis, Pisolithus tinctorius, Scleroderma sp. and Calvatia cyathiformis, were examined for antigenic/allergenic relationships by Ouchterlony and radioallergosorbent testing (RAST) inhibition, respectively. Ouchterlony, using hyperimmunized rabbit sera, demonstrated a high degree of cross-antigenicity among the extracts tested; however, some unique antigens were also present. RAST inhibition, evaluated by comparing extract concentrations which inhibited the RAST by 50% (IC-50), varied with the allergen tested. P. cubensis was the most potent inhibitor (IC-50 ranged from 0.034 mg/ml for A. tabescens RAST to 0.29 mg/ml for G. lucidum RAST). P. tinctorius was the least potent inhibitor, failing to reach IC-50 at 10 mg/ml for any basidiospore extract. Evaluation of slopes and intercepts of the dose-response lines demonstrated qualitative and quantitative differences among allergens in these extracts. These results indicate the presence of shared allergenic epitopes, and suggest that representative extract panels could be developed for future use in diagnosis and treatment of basidiospore-sensitive individuals.     

The occurrence and nature of alveolitis-inducing substances in Aspergillus clavatus.

Five groups of antigens were identified in culture filtrates and extracts from spores and mycelia of Aspergillus clavatus fractionated by gel filtration, affinity chromatography, electrophoresis in polyacrylamide gels and chemical analysis. Some particulate and soluble fractions given by nasal inoculation provoked murine allergic alveolitis in non-sensitized and sensitized precipitin-negative, and sensitized, precipitin-positive, animals. Alveolitis-inducing substances appeared to be glycoprotein precipitinogens, which withstood proteolysis, were preferentially adsorbed by concanavalin A, but which were rendered almost inert by sodium periodate oxidation. Spore walls were particularly rich in allergenic substances extractable by alkaline hydrolysis. Delipidated dead spores provoked more severe disease in all immunological groups of mice than live spores. Polysacchraride extracts and acid hydrolysates of spore walls were unreactive.     

Antigenic mimicry of eel tissues by a myxosporidian parasite?

Parasite antigens were prepared fromMyxobolus spores removed from skin cysts in New Zealand eels and from eel muscle tissue. Rabbit antisera prepared against these antigens reacted with either eel muscle proteins orMyxobolus antigens, as demonstrated by immunodiffusion and immunoelectrophoresis in agarose. This indicated that an antigenic component of the parasite mimics eel tissues. The question of the origin of the spore antigen is discussed.     

Respiratory allergy to mushroom spores: not well recognized, but relevant.

BACKGROUND: Although basidiospores are a major component of the air spora in many parts of the world, their clinical significance as triggers of respiratory allergy has rarely been demonstrated. Therefore, the class of basidiomycetes as an aeroallergen is not well known. OBJECTIVE: To demonstrate a cause and effect relationship between respiratory allergy and basidiospores, we illustrate this case report of a 38-year-old housewife. METHODS: Skin prick test, immunoblot, and active anterior rhinomanometry were used as diagnostic tools to verify specific reactivity of a Pleurotus pulmonalis spore extract. Two atopic subjects served as controls. RESULTS: The skin prick test positive study subject reacted with subjective and objective signs including a significant drop of the FEV1 by nasal challenge at a concentration of 0.1 mg/mL of the Pleurotus spore extract while both controls were negative even at a higher test concentration. IgE-immunoblot revealed several distinct bands in the serum of the Pleurotus-sensitized subject. CONCLUSION: Spores of Pleurotus pulmonalis, a common mushroom of the fungal class of basidiomycetes, can cause specific, IgE-mediated acute rhinoconjuncivitis and asthma in sensitized individuals.     

Crawfish and lobster allergens: identification and structural similarities with other crustacea

Antigenic and allergenic components in crawfish and lobster extracts were studied using crossed immunoelectrophoretic techniques. Crossed immunoelectrophoresis with rabbit antisera revealed 23 antigens in crawfish and 17 antigens in lobster extracts. Both extracts exhibited structural similarities in antigens mutually and with other crustacea in cross-line immunoelectrophoresis. Crossed radioimmunoelectrophoresis (CRIE) demonstrated 6 crawfish and 4 lobster allergens when individual or pooled sera from radioallergosorbent test (RAST)-positive crustacea-sensitive subjects were used. Since radiostaining was also observed with sera from RAST-negative nonsensitive subjects, specificity of IgE binding was tested using CRIE-inhibition. Preincubation of RAST-positive sera with crawfish or lobster extract decreased radiostaining in CRIE, while no changes occurred when using control sera. These results confirmed the presence of IgE-mediated mechanisms in seafood allergy and demonstrated a number of shared antigenic determinants among crustacea allergens.     

Candida albicans group A-specific soluble antigens demonstrated by quantitative immunoelectrophoresis.

Soluble cytoplasmic extracts of Candida albicans groups A and B were prepared and compared by quantitative immunoelectrophoresis experiments performed with a commercial anti-C. albicans group A immune serum. Although crossed immunoelectrophoresis, tandem crossed immunoelectrophoresis, and line immunoelectrophoresis revealed many cross-reactions between the two groups, some components seemed to be specific to group A. However, the complexity of the extracts studied did not allow us to demonstrate specific constituents with these methods. Crossed-line immunoelectrophoresis with and without absorption of antibodies in situ was then used, and four specific antigens unique to group A cytoplasmic extract were demonstrated, one of which appeared to be quantitatively important. The value of various quantitative immunoelectrophoretic methods applied to complex antigenic preparations is discussed.     

Cross-reactivity between the house dust mite Dermatophagoides pteronyssinus and the mange mites Psoroptes cuniculi and P. ovis. I. Demonstration of antibodies to the house dust mite allergen Dpt 12 in sera from P. cuniculi-infested rabbits

The cross-reactivity of the mange mites Psoroptes cuniculi (PC) and Psoroptes ovis (PO) antigens with the house dust mite Dermatophagoides pteronyssinus (DP) antigens has been studied. Cross-reactivity between mange mite and house dust mite antigens was demonstrated by both ELISA and immunoelectrophoresis by use of a sheep anti-DP antiserum. Both PC and PO were demonstrated to contain eight cross-reacting antigens. Sera from rabbits infested with PC were demonstrated to produce antibodies to the homologous immunogen, to PO antigens, and to DP antigens. Of the seven sera from infested rabbits tested, four were demonstrated to produce a strong antibody response to a major DP antigen Dpt 12, and two were demonstrated to produce a weak response that was judged empirically by double-diffusion analysis. Two sera were judged to react with the DP lipoprotein, Dpt 4. Sera from control rabbits did not demonstrate reactivity with any extract tested. Despite the detection of anti-Dpt 12 antibodies, however, an antigen corresponding to Dpt 12 was not detected in either PC or PO extracts. The findings that mange mite-infested rabbits produce antibodies that recognize DP antigens probably explain previous observations in which it was demonstrated that commercially obtained rabbit anti-immunoglobulin antisera contain anti-DP antibodies, a finding that suggests caution in the use of such reagents in studies designed to measure antibody responses to DP allergens.     

Investigations of culture medium-free house dust mites. IV. Cross antigenicity and allergenicity between the house dust mites, Dermatophagoides farinae and D. pteronyssinus

Heterologous crossed immunoelectrophoresis (CIE) analysis demonstrated that Dermatophagoides pteronyssinus body and D. farinae body extracts contained up to 21 cross-reacting antigens (Ags). Incubation of CIE gels with two mite-sensitive individual sera and a serum pool of mite-sensitive patients indicated that up to nine cross-reacting Ags bound mite-specific IgE on crossed radioimmunoelectrophoresis and were common allergens. Likewise, heterologous CIE analysis of D. pteronyssinus feces and D. farinae feces extracts demonstrated approximately 13 cross-reacting Ags. Up to eight of these cross-reacting fecal Ags bound mite feces-specific human IgE. Additional cross antigenicity and allergenicity was evident between body extract of one species and feces extract of the other species. The amount of specific IgE binding to cross-reacting Ags in interspecific extracts varied both between and within individual sera. Different cross-reacting Ags contained potent allergens, and these varied from patient to patient. Also of major significance was the fact that body and fecal extracts of each species contained Ags and allergens that were unique and species specific.     

Production of polyclonal antibodies against spores of Clostridium tyrobutyricum, a contaminant affecting the quality of cheese: characterisation of the immunodominant protein

The effect of different treatments in the production of polyclonal antibodies against Clostridium tyrobutyricum spores and its immunodetection was studied. Moreover, the main protein that causes the immunological response has been characterised. Antisera from rabbits immunised with non-treated, heated or sonicated spores were tested by ELISA. Heated and sonicated spores gave a faster increase in antibody titre than non-treated spores a long time after immunisation. Heat treatment improved reactivity when it was applied to the spores prior to immunodetection. However, when the highest titre was reached, the treatment effect was not observed either in the immune response or in the immunodetection. The immunodominant protein was identified in a spore extract by transfer-blotting, analysed by on-line liquid chromatography tandem mass spectrometry, and characterised by comparison of its peptide fragmentation spectrum with the NCBI database. Five proteins from the family of chaperonins were shown related to the immunodominant protein of C. tyrobutyricum spore extract.