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Background: Alternaria alternata is associated with allergic respiratory diseases, which can be managed with allergen extract-based diagnostics and immunotherapy. It is not known how spores and hyphae contribute to allergen content. Commercial allergen extracts are manufactured by extracting proteins without separating the different forms of the fungus., Objective: We sought to determine differences between spore and hyphae proteomes and how allergens are distributed in Aalternata., Methods: Data-independent acquisition mass spectrometry was used to quantitatively compare the proteomes of asexual spores (nongerminating and germinating) with vegetative hyphae., Results: We identified 4515 proteins in nongerminating spores, germinating spores, and hyphae; most known allergens are more abundant in nongerminating spores. On comparing significant protein fold-change differences between nongerminating spores and hyphae, we found that 174 proteins were upregulated in nongerminating spores and 80 proteins in hyphae. Among the spore proteins are ones functionally involved in cell wall synthesis, responding to cellular stress, and maintaining redox balance and homeostasis. On comparing nongerminating and germinating spores, 25 proteins were found to be upregulated in nongerminating spores and 54 in germinating spores. Among the proteins specific to germinating spores were proteases known to be virulence factors. One of the most abundant proteins in the spore proteome is sialidase, which has not been identified as an allergen but may be important in the pathogenicity of this fungus. Major allergen Alt a 1 is present at low levels in spores and hyphae and appears to be largely secreted into growth media., Conclusions: Spores and hyphae express overlapping but distinct proteomes. Most known allergens are found more abundantly in nongerminating spores., (Published by Elsevier Inc.)

Background: Childhood asthma is a significant public health problem and severe exacerbations can result in diminished quality of life and hospitalization., Objective: We sought to examine the contribution of outdoor fungi to childhood and adolescent asthma hospitalizations., Methods: The Melbourne Air Pollen Children and Adolescent study is a case-crossover study of 644 children and adolescents (aged 2-17 years) hospitalized for asthma. The Melbourne Air Pollen Children and Adolescent study collected individual data on human rhinovirus infection and sensitization to Alternaria and Cladosporium and daily counts of ambient concentrations of fungal spores, pollen, and air pollutants. Conditional logistic regression models were used to assess associations with increases in spore counts while controlling for potential confounding and testing interactions., Results: Exposure to Alternaria (adjusted odds ratio [aOR], 1.07; 95% CI, 1.03-1.11), Leptosphaeria (aOR, 1.05; 95% CI, 1.02-1.07), Coprinus (aOR, 1.04; 95% CI, 1.01-1.07), Drechslera (aOR, 1.03; 95% CI, 1.00-1.05), and total spores (aOR, 1.05; 95% CI, 1.01-1.09) was significantly associated with child asthma hospitalizations independent of human rhinovirus infection. There were significant lagged effects up to 3 days with Alternaria, Leptosphaeria, Cladosporium, Sporormiella, Coprinus, and Drechslera. Some of these associations were significantly greater in participants with Cladosporium sensitization., Conclusions: Exposures to several outdoor fungal spore taxa, including some not reported in previous research, are associated with the risk of child and adolescent asthma hospitalization, particularly in individuals sensitized to Cladosporium. We need further studies to examine cross-reactivity causing asthma exacerbations. Identifying sensitization to multiple fungal allergens in children with asthma could support the design and implementation of more effective strategies to prevent asthma exacerbations., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: It is well accepted that mold exposure is a major contributor to the development of asthma, and beta-glucans are often used as a surrogate for mold exposure in the environment. Beta-glucans are an important component of mold spores and are recognized by the immune system by their receptor, Dectin-1. Cladosporium cladosporioides spores have a high beta-glucan content, but the beta-glucans are not available on the surface of live spores., Objective: We sought to determine whether altering the exposure of beta-glucans in C cladosporioides through heat killing could alter the immune response through binding to Dectin-1., Methods: In a murine model of mold-induced asthma, mice were repeatedly exposed to either live or heat-killed C cladosporioides and the phenotype was determined by the measurement of airway hyperresponsiveness, airway inflammation, and cytokine production. Pro-inflammatory cytokines from dendritic cells were measured by using quantitative PCR and ELISA., Results: Live C cladosporioides induced robust airway hyperresponsiveness, eosinophilia, and a predominately TH2 response, while heat-killed C cladosporioides induced a strong TH17 response and neutrophilic inflammation, but very mild airway hyperresponsiveness. Heat killing of C cladosporioides spores effectively exposed beta-glucans on the surface of the spores and increased binding to Dectin-1. In the absence of Dectin-1, heat-killed spores induced a predominantly TH2 response analogous to live spores. Furthermore, the production of TH17-skewing IL-6, IL-23, and TNF-α by dendritic cells in response to heat-killed C cladosporioides was dependent on Dectin-1., Conclusions: The host immune response to C cladosporioides is dependent on the surface availability of beta-glucans rather than the total beta-glucan content., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Aspergillus fumigatus conidia aggravate asthmatic responses. Lung macrophages normally kill fungal conidia, but the presence of type 2 cytokines during asthma contributes to the alternative (or M2) activation of these cells, which secrete proallergic factors and exhibit impaired innate immunity., Objective: Considering that pentraxins modulate macrophage function, we examined the effect of C-reactive protein (CRP) and serum amyloid P (SAP) in an experimental model of A fumigatus-induced allergic airway disease., Methods: The effects of SAP and CRP on M2 macrophage differentiation were examined in vitro, and the in vivo effects of these pentraxins were analyzed in the asthma model., Results: SAP inhibited the generation of M2 markers, such as arginase and the chitinase Ym-1, through an FcγR-dependent mechanism in cultured macrophages. This effect correlated with a decrease in signal transducer and activator of transcription 6 (STAT6) phosphorylation in SAP-treated M2 macrophages. In vivo treatment with SAP significantly decreased methacholine-induced bronchial resistance, mucus cell metaplasia, the number of "found in inflammatory zone 1" (FIZZ1)-positive cells in the lungs, and collagen deposition compared with the control group. CRP had a modest effect on M2 differentiation, and in vivo treatment with CRP had a minor effect or exacerbated A fumigatus-induced lung disease. Finally, the adoptive transfer of SAP-pretreated M2 macrophages into allergic mice significantly attenuated disease when compared with nontransferred or M2-transferred control groups., Conclusions: These findings demonstrate that SAP is a potent inhibitor of M2 macrophage differentiation and represents a novel therapy in A fumigatus-induced allergic disease., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Although sensitization to fungal allergens is prevalent in inner-city children with asthma, the relationship between fungal exposure and morbidity is poorly understood., Objective: We examined relationships between fungal sensitization, exposure, and asthma morbidity in inner-city children., Methods: Participants were 5 to 11 years old and enrolled in the Inner-City Asthma Study. This report includes the subset of children with at least 1 positive skin test (PST) response to a fungal allergen extract; for these children, indoor and outdoor airborne culturable fungi levels were measured at baseline and throughout the 2-year study. Asthma morbidity measures were collected prospectively. The primary outcome was symptom days per 2 weeks., Results: At baseline, children with a PST response to a fungal allergen extract had significantly more symptom days compared with those without a PST response to any fungal allergen extract (6.3 vs 5.7 days per 2 weeks, P = .04). During the study, increases in total fungal exposure and indoor Penicillium species exposure were associated with increases in symptom days and asthma-related unscheduled visits. Indoor exposures to total fungi and to Penicillium species were associated with significant increases in unscheduled visits, even after controlling for outdoor fungal levels. Adverse effects associated with exposure to a specific fungus were stronger among children with PST responses to that fungal allergen extract compared with those seen in children with negative skin test responses., Conclusion: Outdoor fungal exposure is primarily associated with increased asthma symptoms and increased risk of exacerbations in this population.

Background: After July 29, 2002, an epidemic of asthma admissions was associated with a thunderstorm in the United Kingdom., Objective: We sought to study the cause of epidemics of asthma associated with thunderstorms., Methods: We performed a case-control study of 26 patients presenting to Cambridge University Hospital with asthma after the thunderstorm. Control subjects were 31 patients with summer seasonal asthma. Subjects underwent skin tests and specific IgE serology to inhaled aeroallergens. Meteorologic and aerobiologic data correlated with asthma admissions were analyzed., Results: Twenty-three of 26 cases had IgE sensitization to Alternaria species. Eleven of 31 control subjects gave a history of asthma exacerbation during thunderstorms. Ten of these 11 control subjects were sensitive to Alternaria species on skin testing, but Alternaria species sensitivity was only identified in 4 of the 20 remaining control subjects who did not report thunderstorm-related asthma symptoms. The odds ratio of having epidemic thunderstorm-related asthma if sensitive to Alternaria species was 9.31 (95% CI, 2.305-37.601; P = .0008) and 63.966 (95% CI, 3.577-1143.9; P < .0001) if sensitive to Alternaria species, Cladosporium species, or both. Poisson regression analysis showed that counts of broken Alternaria species and Didymella and Cladosporium species were significantly correlated with each other and with asthma admissions. The thunderstorm was associated with increased levels of Alternaria, Cladosporium, and Didymella species., Conclusions: Alternaria alternata sensitivity is a compelling predictor of epidemic asthma in patients with seasonal asthma and grass pollen allergy and is likely to be the important factor in thunderstorm-related asthma., Clinical Implications: Alternaria species sensitization in asthmatic subjects with grass pollen sensitivity predicts susceptibility to thunderstorm-associated asthma.

Background: Experimental studies have generated conflicting data regarding the role of CCR8 in antigen-driven allergic airway disease models, thereby dampening enthusiasm for further exploration of the targeting of CCR8 in asthma., Objective: Recent data show that the absence of CCR8 leads to a marked amplification of the innate immune response, and these data provided impetus for the current study, which addressed the role of this chemokine receptor in a model of fungal asthma., Methods: Wild-type (CCR8(+/+)) and CCR8-deficient (CCR8(-/-)) mice were sensitized to Aspergillus fumigatus antigens and challenged via intra-tracheal injection with live fungal conidia, and parameters of airway hyperresponsiveness, inflammation, and remodeling were examined., Results: At day 7 after conidia challenge in wild-type (CCR8(+/+)) and CCR8-deficient (CCR8(-/-)) mice sensitized to A. fumigatus antigens, markedly less fungal material was present in the lungs of the CCR8(-/-) group compared with the CCR8(+/+) group. At day 14 after conidia challenge, all characteristic airway physiology, inflammatory, and remodeling parameters of fungal asthma were significantly decreased or abolished in the CCR8(-/-) group relative to the CCR8(+/+) group., Conclusion: Together these data show that an enhanced innate immune response in the absence of CCR8 promotes the rapid clearance of fungal material from the lung, thereby facilitating the remission of fungal asthma., Clinical Implications: This study shows that the clearance of fungal material from the lung was enhanced in the absence of CCR8, which suggests that this receptor may be an attractive target in fungal-allergic asthma and other fungal-associated pulmonary diseases.

Background: Allergens dispersed by airborne fungal spores play an important but poorly understood role in the underlying cause and exacerbation of asthma. Previous studies suggest that spores of Alternaria and Aspergillus release greater quantities of allergen after germination than before germination. It is unknown whether this is true of other allergenic fungi., Objective: Our purpose was to investigate the release of allergen from a range of individual fungal spores before and after germination., Methods: Allergen expression from spores of Alternaria alternata, Cladosporium herbarum, Aspergillus fumigatus, Botrytis cinerea, Epicoccum nigrum, Exserohilum rostratum, Penicillium chrysogenum, Stemphylium botryosum, Curvularia lunata, Trichoderma viride, and Bipolaris spicifera was examined by halogen immunoassays through the use of pooled serum IgE from patients allergic to fungus. Spores were deposited onto protein-binding membranes direct from culture. To germinate spores, samples were incubated in high humidity at room temperature for 48 hours. Ungerminated and germinated samples were then laminated with an adhesive film and immunostained by the halogen assay. The samples were examined by light microscopy, and positive counts (haloed particles) were expressed as percentages of total spores., Results: For 9 of 11 species, between 5.7% and 92% of spores released allergen before germination. Spores of Penicillium and Trichoderma did not release detectable allergen. After germination, all spores that germinated had allergen elution from their hyphae. Eight of 11 species showed a significant increase (P <.05) in the percentage of spores eluting detectable allergen. Localization of allergen along the hyphae varied with species, such that some eluted allergen mainly from hyphal tips and septal junctions whereas others eluted allergen along the entire length., Conclusions: Increased elution of allergen after germination might be a common feature of many species of allergenic fungi. Although allergens from both spores and hyphae were recognized by human IgE, the extent to which human exposure occurs to allergens eluted from inhaled spores or from hyphae that germinate after deposition in the respiratory tract remains to be explored. The patterns of allergen expression might affect the clinical response to such exposure.

Background: Exposure to outdoor allergens has commonly been estimated by collecting airborne particles with a Hirst-type spore trap and then using morphologic criteria to identify the intact pollen grains and fungal spores that are recognized as allergen sources. Several antibody-based blotting or fixation methods have also been developed that enable the counting of amorphous airborne particles carrying allergen, but none of these methods allow the ready association of the released allergen with the morphologically identifiable particle of origin. A method has been developed that uses pressure-sensitive adhesive tape to sample the airborne particles and then allows the immunoidentification of the specific particles that are the allergen sources., Objective: Our purpose was to visualize and immunostain the particles carrying pollen allergen that are collected with a volumetric spore trap., Methods: A Burkard sampler was used to collect airborne particles onto pressure-sensitive adhesive tapes. The particles were permanently fixed between the tape and a protein-binding membrane when the tape was laminated with the membrane. Allergens that elute from the particles onto the membrane were detected with a range of antibodies. Both the particle and associated immunostained allergen were viewed through the transparent tape for final microscopic identification., Results: Polyclonal and monoclonal antibodies and IgE from allergic patients stained allergens in the periphery of particles collected on the tapes. Individual pollen grains and paucimicronic particles were seen with halos of immunostained allergen surrounding them. When IgE was used, the density of immunostaining in the halo surrounding Lolium perenne pollen grains was found to be proportional to the level of Lolium-specific IgE. The method is highly sensitive and can be used to detect different airborne particles that carry allergen. Both the particle and the immunostaining can be subjected to a range of simple measurement techniques., Conclusion: Individual particles carrying allergens and antigens were visualized. These particles included intact pollen grains, paucimicronic particles, and fungal spores.

Background: Basidiospores are universal components in the air and established as important causes of respiratory allergies. Recent reports indicate that aeroallergens may aggravate eczematous skin lesions in subjects with atopic dermatitis (AD)., Objective: The aim of the study was to investigate whether spores of Coprinus comatus, a species of basidiomycetes, may elicit delayed-type skin reactions in subjects with an atopic predilection, especially dermatitis., Methods: Sixty-six study subjects were categorized in groups having AD or respiratory allergy with regard to the skin prick test (SPT) reactivity to C comatus extract. Twenty nonatopic individuals served as control subjects. Atopy patch tests (APTs) were performed with extract of C comatus spore containing tissue at a concentration of 1. 35 mg of protein per gram of petrolatum (Vaseline) and C comatus cap at a concentration of approximately 5 mg of protein per gram of petroleum jelly. APT reactions were evaluated after 48 and 72 hours., Results: Of the subjects with AD completing the study, 12 (32%) of 38 showed a positive APT reaction, with 8 (57%) also having a positive SPT response to C comatus. Only 1 (9%) of 11 subjects with asthma had a positive SPT and APT response to C comatus. No positive test reaction was observed in the nonatopic control subjects or in subjects with respiratory allergy and negative SPT responses to C comatus., Conclusion: Our results demonstrate that C comatus can induce delayed-type reactions in atopic individuals, particularly in those with AD. Because spores of Coprinus species are ubiquitous, basidiomycetes have to be considered as possible aeroallergens when investigating causes of eczematous skin lesions in AD.

Background: Previous literature has indicated that environmental exposures in childhood influence development of atopic sensitization., Objective: We sought to study the association between childhood environment and adult atopy., Methods: Thirteen thousand nine hundred thirty-two subjects aged 20 to 44 years from 36 areas in Europe, New Zealand, the United States, and Australia took part in the European Community Respiratory Health Survey, answering interviewer-led questionnaires and providing blood tests for measurement of specific IgE to grass, house dust mite, cat, and Cladosporium allergens., Results: Atopy was negatively associated with family size (OR = 0. 93; 95% CI = 0.90-0.96 per 1 sib), partly attributable to an independent protective effect of a greater number of brothers (OR = 0.92; 95% CI = 0.89-0.95 per 1 brother). Accounting for total number of siblings, no further influence was detected for number of older or younger siblings. Bedroom sharing was associated with a lower prevalence of atopy, particularly to cat allergen. A protective effect of family size and bedroom sharing could only be detected in subjects reporting no parental allergy (family size, test for interaction P =.012). The presence of a dog in the home in childhood was negatively associated with adult atopy (OR = 0.85, 95% CI = 0. 78-0.92), an effect that remained after adjustment for parental allergy, sibling allergy, and adult pet ownership., Conclusion: Subjects from large families with brothers, shared bedrooms, and dogs in childhood were less often atopic as adults. Our findings are consistent with the hypothesis that infectious agents could inhibit development of atopy during childhood. However, in subjects with a strong genetic predisposition, environmental factors in childhood are possibly of less importance.

Objective: Many studies have noted an association of ambient aeroallergen levels with exacerbation of asthma. This study was undertaken to examine the relationship of aeroallergen levels with asthma-related mortality in Chicago., Methods: The association of environmental aeroallergen levels with death caused by asthma among 5- to 34-year-olds in Chicago was examined for the period of 1985 through 1989. Logistic regression analysis was used to compare the probability of a death caused by asthma occurring on the basis of environmental tree, grass, or ragweed pollen and mold spore levels., Results: Mean mold spore levels but not tree, grass, or ragweed pollen levels were significantly higher for days on which asthma-related death occurred than for days on which no deaths occurred (z = 2.80, p < 0.005). The odds of a death caused by asthma occurring on days with mold spore counts of 1000 spores per cubic meter or greater was 2.16 times higher (95% confidence interval = 1.31, 3.56, p = 0.003) than on days on which mold spore counts were less than 1000 spores per cubic meter. The association with mold spore levels remained significant on multivariate logistic regression with mold spore counts measured as a continuous variable and controlling for pollens, with the odds of an asthma-related death occurring being 1.2 times higher (95% confidence interval = 1.07-1.34) for every increase of 1000 spores per cubic meter in daily mold spore levels., Conclusion: Although death caused by asthma also involves personal, social, and medical access factors, these data suggest that exposure to environmental molds may play a role in asthma-related mortality and should be considered in prevention strategies.

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.

Two atmospheric isolates of Epicoccum nigrum (EN) were grown under sporulation conditions. Dialyzed extracts of spores, (greater than 95% pure) and pure mycelia were used for skin testing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. By skin testing, 49 of the 126 atopic patients were found to be sensitive to EN in St. Louis, Mo., and Corpus Christi, Texas, combined. On immunoblotting, which was performed on 17 sera, 44 bands (12.3 to 119.0 kd) were detected; six were unique to spore, four were unique to mycelium, and 34 were common to both. No single band bound IgE from all sera. The most frequent band corresponding to 42 kd occurred in 11 sera. Five other bands were recognized by more than one half, whereas the remainder bound fewer sera. All skin test-positive patients had positive immunoblots; the number of bands recognized varied from three to 25. Spore or mycelium-specific, as well as common bands were recognized by 13 of 17 sera. Two sera recognized only spore and mycelium-specific bands. Only spore-specific bands were bound by two sera. No strain differences were detected. The binding patterns were comparable in the sera from both St. Louis, Mo., and Corpus Christi, Texas. These data suggest that EN is a significant allergen in urban communities. Allergenic proteins occur in both spore and mycelium, suggesting that both must be included in the reagents for skin testing and immunotherapy.

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.

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.

Because particulate aeroallergens may exist in amorphous form as well as in pollen grains and fungal spores and because symptoms of allergic diseases presumably correlate with the total amount of allergen exposure, an immunochemical method of assay of aeroallergens would be useful. We report such a method based on (1) capture of airborne pollen, fungal spores, and amorphous particles 0.3 micrometer in diameter on fiberglass sheets with a high-volume air sampler; (2) elution of the sheets with buffered saline; and (3) analysis of eluate allergen content by radioallergosorbent test (RAST) inhibition assays. In preliminary indoor experiments we applied various quantities of short ragweed (SRW) pollen or dry Alternaria powder to the sheets while airflow was maintained at 1.19 m3/min. We compared techniques for extraction of allergen from the sheets, including homogenization, cutting and soaking, and descending elution of sheets. Although all three methods successfully extracted allergen from the sheets, an 8-hr descending elution procedure was optimal from the standpoint of yield and convenience. Eluates from filters exposed to as little as 4 mg of SRW pollen or Alternaria powder produced satisfactory RAST inhibition curves. When the sampler was operated outdoors continuously we could measure the atmospheric allergenic activity for both Alternaria and SRW from July to September. This allergenic activity was highly correlated with the traditional morphologic counts of airborne ragweed pollen and Alternaria spores.

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.

The ability to harvest spore-rich isolates of molds permitted quantitative studies of their role in the pathogenesis of asthma. Alternaria and Penicillium were selected as examples of ubiquitous molds that readily induce IgE antibodies and are of contrasting sizes. Extracts from those spores were prepared for skin tests and aerosol bronchial challenges. Intact spores were used in the same subjects in bronchial challenges delivered by a Spinhaler. Seven patients with a history of mild asthma received a total of 16 bronchial challenges with the mold to which they had been sensitized. Provocative doses in spore equivalents for a 35% drop in SGaw, 20% drop in FEV1, or 25% drop in PEFR were sought for each challenge. Density dependence-flow rates were also determined. Environmental spore survey data were obtained and compared with the challenge doses for these spores. It was found that immediate-type asthma was readily provoked by both whole spores and by their extracts, in some subjects fewer intact than extracted spores were required, delayed-type asthma occurred only after whole spore challenges, SGaw was the most sensitive and equally specific of the pulmonary function tests, and provocative doses of spore equivalents were within natural exposure ranges. The study confirmed that Alternaria and Penicillium spores in relatively natural states and numbers were potent immunopathogens for asthma.

RAST with 12 basidiospore extracts demonstrated that a significant percent of 42 individuals with symptoms of perennial rhinitis and/or asthma and skin reactivity to at least one spore extract had spore-specific IgE antibodies. These antibodies were not demonstrable in 14 atopic, symptomatic, skin test negative control subjects nor in five nonatopic, asymptomatic control subjects. There was a statistically significant association between RAST and skin test results for all but three of the spore extracts. A statistically significant association was also observed between RAST results obtained with most of the different spore extracts. A similar association was present for skin test results with different spore extracts. These results provide evidence that basidiospore extracts are suitable allergens for use in diagnostic studies of respiratory disease associated with exposure to basidiomycetes.

An experimental model of allergic lung disease has been described that is monitored by analysis of arterial oxygen tension following aerosol challenge with antigen. Rabbits immunized to a classical soluble antigen, human serum albumin (HSA), to the point where severe Arthus skin reactivity was demonstrable, were aerosol-challenged with antigen. Arterial oxygen tension measurements made on pre- and post-challenge samples yielded early, late, and continuous response patterns, reminiscent of those obtained in humans following provocation testing. Aerosol challenge of unimmunized animals with HSA resulted in no change from baseline conditions. Unimmunized rabbits exposed to small and massive (10X) aerosols of Aspergillus spores also demonstrated various postchallenge depressions in arterial oxygen tension as well as decreased levels in hemolytic complement activity, depending on the species of fungus and dose of spores used. Unimmunized animals pretreated with cobra venom factor in a manner known to achieve complement depletion failed to respond with altered arterial oxygen tensions following similar aerosol challenge. It is postulated that although precipitins may play a role in artificial disease initiated by soluble antigens, nonspecific complement activation may be more important in understanding the etiology of spontaneous disease in humans brought about by inhalation of moldy particulate matter.

Mold spore-induced respiratory allergy has been incompletely studied, and only a limited number of Fungi Imperfecti are well established as aeroallergens. Basidiomycetes, a complex and common group of fungi, which include mushrooms, rusts, smuts, brackets, and puffballs, have not been well studied. Although basidiospores can be present in high atmospheric concentrations, little is known of their aeroallergen potential. To examine this question, we performed skin prick and RASTs in 33 adult residents of Washington State using a panel of 15 common inhalant allergents that included four Fungi Imperfecti and 15 basidiospore extracts. Thirty-one of 33 (94%) subjects had positive immediate reactions to two or more common inhalants. Nine of 33 (27%) subjects responded to at least one Fungi Imperfecti; reactions were most common to Aspergillus sp. (21%), and least common to Penicillium sp., which were positive in 6%. Positive responses to basidiospore extracts were observed in 10 of 33 (30%) subjects. The prevalence of basidiospore reactivity was similar to that of Fungi Imperfecti, ranging from 18% for Scleroderma sp. to 6% for four different spore extracts. These results demonstrate that a significant number of subjects with respiratory allergies have skin test reactivity to basidiospore extracts, suggesting that these spores could be important aeroallergens in the Pacific Northwest.

Bronchoprovocation challenge with basidiospore extracts was performed in eight subjects with asthma and with positive wheal-and-flare skin reactivity and RAST to basidiospore allergens. Five of the eight patients demonstrated a significant decrease in FEV1 ranging from 20% to 47% after basidiospore-extract challenge. Both immediate and late-phase reactivity was observed. Six atopic control subjects with asthma and with negative skin reactivity to basidiospore extracts did not exhibit significant bronchospasm after basidiospore challenge. These results demonstrate that basidiospore extracts can induce bronchospasm in subjects with asthma who demonstrate IgE antibodies to basidiospore allergens.

One hundred fifty adults, with respiratory-allergic disease, and 14 control subjects, without symptoms of respiratory allergy, were skin prick tested with 16 common inhalant allergens, 12 extracts of mycelia from Basidiomycetes grown in vitro, and/or 10 to 15 basidiospore extracts. Eighty-three subjects (58%) had positive skin tests to two or more of the common inhalant allergens. Twenty-seven percent of the study subjects had positive skin reactions to one or more of the Basidiomycete mycelia extracts, and 32% demonstrated positive skin reactions to one or more basidiospore extracts. None of the 14 control subjects had positive skin reactivity to basidiospore extracts. Skin prick reactivity of study subjects to 15 different basidiospore extracts ranged from 5% for Cantharellus cibarius to 17% for Scleroderma sp. The prevalence of skin test reactivity to basidiospores did not differ significantly from the reactivity to commercial mold extracts of several common species of the Fungi Imperfecti (6% for Cladosporium herbarum or Penicillium notatum to 13% for Alternaria tenuis). These results demonstrate that a significant number of individuals reporting symptoms of respiratory allergy have skin test reactivity to basidiospores and suggest that these spores are important fungal aeroallergens in the New Orleans environment.