Fusarium solani: Evidence for common antigenic/allergenic determinants with other Fungi Imperfecti

Aqueous extracts of Fusarium solani and other members of the Fungi Imperfecti were evaluated for the presence of common antigenic/allergenic determinants using skin-prick testing, radio-allergo-sorbent test (RAST) inhibition, and immunoelectrophoretic methods. Prevalence of skin reactivity in forty-four atopic individuals, tested with commercially available fungal extracts, ranged from 27.3% for Alternaria tenuis to 6.8% for Penicillium notatum. No specific patterns of reactivity emerged from statistical analyses of skin test data. In contrast, RAST inhibition demonstrated common allergenic determinants. P. notatum and Aspergillus glaucus inhibited F. solani RAST by 79% and 84%, respectively. This was supported by crossed line immunoelectrophoresis; both P. notatum and A. glaucus had antigenic determinants in common with F. solani. Collectively, these studies suggest that F. solani, P. notatum , and A. glaucus have several common antigenic/allergenic determinants.     

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.     

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.     

Isolation and characterization of cat allergens

A crude (saline soluble) extract of cat skins capable of eliciting a strong positive prick skin test in cat sensitive individuals was fractionated on Sephadex G200. Active fractions were pooled and successively fractionated on isoelectric focusing gradients of pH 3–5 and pH 4–5. Allergenic activity was localized in two peaks with mean isoelectric points of 4.1 and 4.35 respectively. On immunoelectrophoresis the allergen with pI = 4.35 was associated with a protein which was subsequently found to be immunologically indistinguishable from serum albumin and to have a molecular weight of 69,000 daltons. The allergen with pI = 4.1 migrated in the α₂-region on immunoelectrophoresis and had a mean molecular weight of 55,000 daltons. This allergen was isolated and analysed for amino acid and carbohydrate content. A combined extract of both allergens coupled to microcrystalline cellulose and used in a RAST procedure readily distinguished between two groups of individuals classified as skin test positive and skin test negative to cat allergen.     

High-performance liquid chromatography of Cladosporium herbarum. Identification of allergens with immunological techniques

High-performance liquid chromatography (HPLC) using a gel permeation column was applied to separate components in a crude extract of the mould Cladosporium herbarum. Allergenic activity as well as individual allergens in collected fractions were analyzed by direct radioallergosorbent test (RAST) and fused rocked radioimmunoelectrophoresis (FRRIE), respectively. Thus, information of the molecular weight as well as the importance of the individual allergens were obtained simultaneously in a system showing a high degree of resolution. The study demonstrated that the crude extract of C. herbarum contained allergens mainly in the molecular weight range between 10,000 and 300,000 daltons. The results demonstrated the importance of carrying out both RAST and FRRIE to detect all allergens.     

Characterization of water-soluble shrimp allergens released during boiling

Water-soluble shrimp allergens released during boiling (shrimp water) were characterized and compared to allergen extracts from boiled shrimp (shrimp meat). Both shrimp extracts contained acidic proteins (isoelectrofocusing) and demonstrated similar allergenic activity (RAST and RAST inhibition). Shrimp-water extract was analyzed further by immunoprinting with sera from 14 shrimp-sensitive, RAST-positive subjects, and six nonsensitive, RAST-negative individuals. Although none of the sera from shrimp-tolerant individuals reacted, 12/14 sera (85.7%) from shrimp-sensitive subjects reacted with shrimp-water proteins with acid isoelectric points. Shrimp-water extract was fractionated by chromatofocusing with pH and NaCl gradients. A number of eluted ultraviolet-absorbing peaks contained allergens as determined by RAST inhibition. Isoelectrofocusing demonstrated many protein bands present in these peaks, some of which bound IgE from a RAST-positive sera pool. These studies indicate that shrimp water is an excellent source of shrimp allergens, that chromatofocusing is a useful method for fractionation of shrimp allergens, and that shrimp allergens are generally protein molecules with acid isoelectric points.     

Identification of allergens in dog dander extract. I. Clinical and immunological aspects of allergenicity activity

Allergenic activity of mixed dog dander extract was compared with dog serum albumin by skin testing, RAST testing and RAST inhibition. Whereas albumin is a potent allergen, additional lower molecular weight allergens determined by sucrose density gradient centrifugation and immunoelectrophoresis were found. Identification of the most potent allergens and standardization of extract used for testing and treatment is a necessary prerequisite in understanding dog dander hypersensitivity.     

Comparative studies on tree-pollen allergens. V. Immunochemical mapping of the antigens and allergens of birch pollen extract (Betula verrucosa)

A comparative electrophoretic and autoradiographic analysis of the different fractions of birch pollen crude extract was performed. This extract included a minimum of 17 reproducible and distinct antigens located in the gel filtration fractions BV2-BV4. Only one of these precipitates had the ability to bind IgE, as demonstrated by immunoelectrophoretic and autoradiographic techniques using several poly- and monospecific rabbit antibodies. The allergenicity of the different fractions was examined in vitro by RAST and RAST inhibition, and in vivo by passive cutaneous transfer and skin prick tests. The data suggested that Betula verrucosa pollen extract contains a group of isoallergens with related antigenicity but with variable molecular sizes. The findings have also confirmed the presence of identical antigenic properties of the previously isolated pI 5.18 and BV45 as deduced from their immunoelectrophoretic and autoradiographic studies.     

Monoclonal antibodies against selected peanut allergens: Production and use as affinity agents

An extract of dry‐roasted commercial peanut mix (CPE) was examined for allergenic activity in peanut‐sensitive individuals, using skin tests and radioallergosorbent (RAST) assays. Proteins in the extract were characterized by sodium dodecyl sulfate polyacry‐lamide gel electrophoresis (SDS‐PAGE) and immunoblotting. The proteins were electro‐eluted in three fractions in the ranges 15–25, 26–58 and 65 kDa. The 15–25 kDa molecular weight fraction produced the most reactive skin tests in peanut‐sensitive subjects and was chosen for monoclonal antibody production. Six hybridoma cell lines secreting peanut‐specific antibodies of the IgM isotype (kappa light chain) were produced. Immunopurified CPE proteins were then subjected to SDS‐PAGE, resulting in five major bands with approximate molecular weights of 14, 25, 38, 40 and 44 kDa. Immunoblotting of these separated proteins revealed: (1) three bands with approximate molecular weights of 38, 44 and 65 kDa, which bound IgE from peanut‐sensitive patients; and (2) that the monoclonal antibodies recognized epitopes in bands at approximate molecular weights of 12, 14, 23 and 25 kDa. RAST inhibition assays showed that the affinity‐purified proteins were able to inhibit the binding of serum IgE from peanut‐allergic individuals to solid‐phase CPE.     

Studies on allergens of Dermatophagoides pteronyssinus by direct and indirect RAST.

Dermatophagoides pteronyssinus (DP) extract was fractionated by Sephadex G-75 and liquid isoelectric focusing (IEF) and the allergenic activity of different fractions was monitored by direct and indirect RAST. The fractionation on Sephadex G-75 showed that the allergenic activity of DP extract was related to wide molecular weight spectrum components, even though the maximum amount was recovered in effluent that contained protein with a molecular weight ranging between 25,000 and 12,500 daltons. By fractionation of the mite extract on IEF, three main peaks of allergenic activity (pI less than 3.0; pI = 4.3 +/- 0.25; pI = 6.4 +/- 0.25) were found. Cross-inhibition experiments showed a high degree of cross-reactivity between allergenic material eluted in very distant regions of molecular weight or isoelectric point. The allergenic activity of unfractionated mite extract and of its IEF fractions was destroyed by pronase - but not by neuraminidase - treatment. These results suggest that DP extract probably contains one main allergen existing in multiple molecular forms rather than several distinct allergens and that a protein moiety of the allergen is necessary for the combination with IgE.     

Antigenic/allergenic characterization of American and German cockroach extracts

Cockroach allergens have been implicated as clinically significant sensitizing agents in the induction/exacerbation of "urban asthma." In the present study, approximately 50% of atopic, predominantly inner-city residents had immediate wheal-and-flare cutaneous reactivity to a commercial American cockroach whole body extract. Crude whole body extracts were prepared in our laboratory from both American (Periplaneta americana) and German (Blatella germanica) cockroach species. Crossed immunoelectrophoresis of American cockroach whole body extract (AWBE) and German cockroach whole body extract (GWBE) detected a total of 50 and 56 precipitin peaks, respectively, when extracts were reacted with hyperimmunized rabbit antisera. Crossed radioimmunoelectrophoresis identified at least five electrophoretically distinct allergens each in AWBE and GWBE. Cockroach whole body extracts produced two major protein peaks when extracts were fractionated on Sephadex G-75. RAST-inhibition studies demonstrated allergens in both peak 1 and the immediate trailing fractions of the column. Direct RAST and end point prick skin testing confirmed the presence of significant/important allergens in column "fraction 2" of AWBE. Skin testing and RAST analysis suggested the occurrence of shared and species-specific allergens between AWBE and GWBE. Collectively, these studies confirm the important sensitizing potential of cockroach allergens, characterize their number and size distribution by crossed radioimmunoelectrophoresis and column chromatography, support the occurrence of significant allergens in column "fraction 2," and suggest the occurrence of both species-specific and shared interspecies allergens.     

IgE sensitization in snow crab-processing workers

Occupational asthma is a highly prevalent disease among snow crab-processing workers, but its immunologic mechanism has not been identified. Prick skin tests with snow crab-meat extract, commercial extracts from other crab genera, and snow crab cooking water collected in 1984 were performed on 119 workers. Crab-specific IgE was assessed by RAST in sera from 115 workers with meat and water extracts. Both skin and RAST tests were performed in 58 individuals. Diagnosis of occupational asthma had previously been confirmed in 54 individuals. A highly significant relationship was demonstrated between the presence of immediate skin reactivity or increased serum levels of specific IgE to crab extracts and the occurrence of occupational asthma. There was good agreement between the results of skin and RAST tests with extracts of either meat or snow crab cooking water. Cooking water and snow crab-meat extracts were more sensitive than commercial preparations. Water extract was more potent and more sensitive than meat extract. We conclude that there is evidence that occupational asthma in snow crab-processing workers is mediated through an IgE mechanism.     

Antigens and Allergens in Birch Pollen Extract

More than 70% of the total allergenic activity of a birch pollen (BP) extract was detected within the first 30 min of extraction. Fractionation of the BP extract by gel filtration and analysis of the eluted antigens by a fused rocket immunoelectrophoresis revealed at least three antigens with molecular weights of about 29 000, and 17 000-10 000, corresponding to antigens Nos. 7-8 and No. 2, respectively, in crossed-immunoelectrophoresis (CIE) and in crossed-radioimmuno-electrophoresis (CRIE). Gel isoelectrofocusing of the pooled allergenic fractions revealed two major protein bands with pI's around 5.6 and 5.7, probably corresponding to antigens Nos.7-8 and No. 2, respectively. Antigens Nos. 7-8 were thermoresistant, while antigen No. 2 was thermolabile. The allergenic activity was determined by prick skin testing and by the RAST inhibition method. More than 90% of the allergenic activity in the fractions was located in the protein peak C (mol. wt. 10 000-17 000) containing antigens 7-8. About 30% of the total allergenic activity of the extract (1:10 w/v) was recovered in the peak C fractions, and only less than 0.5% outside these fractions. Higher allergenic activity was obtained for the peak B fractions (mol. wt. 29 000) by skin prick testing than by the RAST. Peak B contained allergens (antigen 2) distinct from those of peak C by the CRIE and by the RAST. The allergenic material in the low molecular weight fractions of peak D (mol. wt. 2000-5000) was allergenically similar to that of peak C in the RAST. Only weak and even negative skin reactions were observed with the peak D fractions in allergic subjects.     

Basidiomycete allergy: Measurement of spore-specific IgE antibodies

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.     

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.     

Influence of IgG antibody and glycopeptide allergens on the correlation between the radioallergosorbent test (RAST) and skin testing or bronchial challenge with alternaria.

The radioallergosorbent test (RAST) for alternaria was compared to skin tests and bronchial challenges in children suffering from chronic intractable asthma. In contrast to when such children were tested with a timothy grass pollen extract, the bronchial challenge and skin test results against alternaria did not correlate significantly. When alternaria allergens were coupled to cyanogen bromide-activated microcrystalline cellulose, the RAST correlated with the results of skin testing but not bronchial challenge. It was demonstrated by column immunabsorption that some allergic sera contained sufficient IgG antibody against alternaria to competitively inhibit the RAST. When Sepharose 2B was substituted for cellulose as the insoluble support, the inhibition by IgG antibody was largely overcome and then the RAST correlated with both skin test and bronchial challenge results. Glycopeptides contribute significantly to the allergenicity of alternaria, and when these materials were coupled to a Sepharose 2B conjugate by mild oxidation, the RAST correlated with bronchial challenge, but not skin test, results. It was concluded that in this group of steroid-dependent asthmatic children, the correlation of the RAST with the in vivo challenges was strongly influenced by the presence of IgG antibody in the allergic sera and the chemical nature of the mould allergens investigated.     

Studies on Alternaria allergens. I. Isolation of allergens from Alternaria tenuis and Alternaria solani.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.     

Allergenic cross-reactivity between Callistemon citrinis and Melaleuca quinquenervia pollens

Aqueous extracts of both Callistemon citrinis (bottlebrush) and Melaleuca quinquenervia (melaleuca) were analyzed for allergenic cross-reactivity. Inhibition analysis using the radioallergosorbent test (RAST) was performed on the ammonium bicarbonate extracts of bottlebrush (NH4B) and melaleuca (NH4M) pollens. RAST inhibition analysis demonstrated that the extracts contained allergenically cross-reactive components. Sephadex G-100 column chromatography of NH4B and NH4M extracts resulted in at least 4 distinct peaks for each extract analyzed. These fractions were designated NH4B1-NH4B4 and NH4M1-NH4M4. A modified dot-blot assay for detection of allergenic components was utilized to show that the first elution peaks of bottlebrush and melaleuca, NH4B1 and NH4M1, respectively, contained allergenic components. These allergenic components, NH4B1 and NH4M1, had estimated molecular weights of 50,000 and 35,000 daltons, respectively.     

Allergenic activity of fractions of cocksfoot (Dactylis glomerata) pollen

A cocksfoot (Dactylis glomerata) pollen extract has been fractionated by isoelectric focusing in polyacrylamide gel, the separated components extracted from the gel and assayed by three methods: prick tests in human skin, RAST and PCA tests in monkey skin. Prick testing in human skin showed that subjects responded differently to the separated components, indicating the presence of more than one antigenic determinant, and a prolile of antibody activity against each fraction could be constructed. In general, good agreement was observed between RAST profiles and skin test profiles, although some patients who gave positive skin tests lacked circulating IgE and consequently did not give a RAST profile or a monkey PCA. These studies emphasize the current difficulty in attempting to isolate a single pure allergen from cocksfoot which can be used either diagnostically for the detection of pollen allergy, or for the standardization of diagnostic extracts.     

Comparison of the antigenic and allergenic properties of three types of bovine epithelial material.

The antigenic and allergenic characteristics of three bovine epithelial extracts, dander, skin scrapings and whole skin, were compared using IgE-ELISA inhibition and SDS-PAGE with immunoblotting. Cow dander extract was shown to contain more allergenic activity than skin scrapings or whole skin extracts which were needed in about three times higher amounts than cow dander extract to induce the same degree of inhibition in ELISA. Skin scrapings and whole skin extracts contained more high-molecular weight components than dander extract. These components were at least partly serum-derived and reacted often with the IgG but not with the IgE of both the cow-asthmatics and their control subjects. The antigenic characteristics of the low-molecular weight components as well as the allergenic qualities of these three epithelial preparations were generally similar. Using the sera of 49 cow-asthmatic farmers, two major allergens were detected at 20 and 22 kD in all three extracts. Our results show that the highest amount of allergenic material and all the essential allergens are present in cow dander extract. In addition, the normally non-allergenic high molecular weight components are detected in low concentrations in dander extract. Therefore it is concluded that cow dander extract is the best alternative for allergen purification and allergen extract preparation.