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BACKGROUND: Shiitake mushrooms are a dietary staple in Asia and are increasingly popular worldwide. A cholesterol-lowering study with shiitake showed that 17 of 49 participants withdrew because of rash or abdominal discomfort, and two had marked eosinophilia. One of these latter participants was subsequently challenged for 14 days with shiitake powder and again had eosinophilia. OBJECTIVE: We investigated whether ingestion of shiitake mushroom powder induces eosinophilia or symptoms. METHODS: We studied 10 normal persons. Each participant ingested 4 gm shiitake powder (open label) daily for 10 weeks (trial 1), and the protocol was repeated in these same subjects after 3 to 6 months (trial 2). Blood counts and serum samples were obtained biweekly (trial 1) or weekly along with stool specimens (trial 2). Eosinophil major basic protein and IL-5, IgE, and IgG antishiitake antibodies were measured in sera. Eosinophil-derived neurotoxin was measured in stool extracts. We defined responders as subjects having peak eosinophil counts four or more times their average baseline counts. RESULTS: Each trial had four responders, and trial 2 had one new and three repeat responders. Eosinophilia ranged from 400 to 3900/mm3. Responders had increased blood eosinophils, serum major basic protein, stool eosinophil-derived neurotoxin, and factors that enhanced eosinophil viability. Antishiitake IgE was not detected, and antishiitake IgG increased in two responders. Gastrointestinal symptoms coincided with eosinophilia in two subjects. Symptoms and eosinophilia resolved after discontinuing shiitake ingestion. CONCLUSIONS: Daily ingestion of shiitake mushroom powder in five of 10 healthy persons provoked blood eosinophilia, increased eosinophil granule proteins in serum and stool, and increased gastrointestinal symptoms. Shiitake ingestion suggests a model to study the eosinophil's role in the blood and gastrointestinal tract. Finally, our report raises concerns of possible adverse systemic reactions to this increasingly popular food.

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Many immune modulating compounds have been isolated from fungal extracts, but the molecular mechanisms of their action have rarely been elucidated. In this study we isolated a proteoglycan from cultured mycelia of Lentinus lepideus and tested its effects on murine spleen cells. The acidic-polysaccharide fraction was obtained by extraction with hot water followed by purification using DEAE-cellulose anion exchange. The molecular mass of the compound was determined by Sepharose CL-4B gel filtration to be approximately 47 kDa. When cultured in the presence of the compound, spleen cells from C3H mice underwent rapid cell proliferation and cell aggregation. Treatment with the compound also caused a 10-fold increase in [3H]-thymidine incorporation compared to a control, confirming cell proliferation. Flow cytometry analysis indicated that the affected cell population was mainly B cells. As one approach to understanding the molecular mechanism of this action, we investigated the effects of the compound on cellular transcription factors which are known to control the proliferation of immune cells. Using gel retardation assays, we found that the compound significantly activated NF-kappa B but not AP-1 in spleen cells. Taken together, the data suggest that the proteoglycan compound is a biological response modifier that stimulates B cell proliferation, probably by regulating cellular transcription factors such as NF-kappa B.

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The effect of fungal and yeast glucan on different immune functions in mice was examined and compared. The simultaneous administration of glucan and a sensitizing dose of DNFB on the different sites significantly stimulated delayed-type hypersensitivity (DTH) response only when using fungal glucan. Both glucans tested, when administered before sensitization, significantly increased DTH response, but with a significantly higher level at the beginning of the investigation (on day 7) when using fungal glucan. The increase in phagocytic activity by the blood leucocytes started in the 1st week after fungal-glucan treatment, and in the 2nd week after yeast-glucan treatment, and took longer after administration of fungal glucan. The values of the phagocytic-activity index were significantly influenced only after fungal-glucan injection. The results of the study indicate that fungal glucan isolated from Pleurotus ostreatus could be a prospective immunomodulating substance.

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Two different bands with laccase activity were obtained after nondenaturing PAGE of the culture filtrate of Pleurotus ostreatus. Immunoblot analysis revealed that antisera raised against laccase I were not reactive to laccase II. Laccase I, which exhibited faster mobility on nondenaturing polyacrylamide gel, was purified 42.9-fold with an overall yield of 10.8%. Gel filtration and SDS-PAGE revealed that laccase I is a single polypeptide with a molecular mass of approximately 64 kDa. Laccase I contained 12.5% carbohydrate by weight and 3.9 mol copper (mol protein)-1. The absorption spectrum of laccase I showed a type 1 signal at 605 nm and EPR spectra showed that the parameters of the type 1 and type 2 Cu signals were g parallel = 2.197 and A parallel = 0.009 cm-1, and g parallel = 2.263 and A parallel = 0.0176 cm-1, respectively. The data obtained from the pH profiles suggested that two ionization groups, whose pKa values were 5.60-5.70 and 6.70-6.85, may play an important role in the active site of laccase I as the ligand of copper metal. The optimal pH and temperature for the activity of laccase I were 6.0-6.5 and 30-35 degrees C, respectively. The enzyme had affinity for various lignin-related phenolic compounds: the Km values for ferulic acid and syringic acid were 48 and 89 microM, respectively. EPR spectroscopic study of the action of laccase I on 3,5-dimethoxy-5-hydroxyacetophenone indicated that this enzyme catalyses single electron transfer with the formation of the phenoxy radical as an intermediate.

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Inherent proteolytic activity was estimated in cockroach and basidiomycete extracts by quantifying acid soluble peptides that were released by incubating extracts with 1% bovine serum albumin as measured by Lowry (Sigma). Reference proteases released 740 (Proteinase K, 0.1 U), 248 (Trypsin, 1.0 U), and 533 micrograms/ml (Pronase, 0.5 U) of soluble peptides. American whole body cockroach extract (0.1 mg dry weight) released 330 micrograms/ml of soluble peptides, representing 13 trypsin equivalent units (TEU)/mg. Extracts from spores of the mushroom Pleurotus ostreatus released 230 micrograms/ml (0.9 TEU/mg) and Pleurotus cap extract released 112 micrograms/ml (0.5 TEU/mg). Mycelium of Pleurotus and the mushroom Psilocybe cubensis and spores of Psilocybe and the puffball Calvatia cyathiformis showed negligible amounts of proteolytic activity. The protease inhibitor phenylmethylsulfonyl flouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@1 mM) and the inhibitor ethylene diaminetetraacetic acid inhibited the proteolytic activity of Pleurotus spores by 95% (@1 mM). Loss of allergen activity as determined by RAST inhibition and immunoprinting correlated with protease activity. Thus, in the preparation and handling of allergen extracts, one should employ conditions that minimize proteolysis.

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Indoor cultivation of the edible mushroom Shii-take (Lentinus edodes) regularly leads to symptoms of mushroom worker's lung (MWL) in workers. An immunologic test is described allowing detection of IgG type antibodies against Shii-take spore antigens. It was found that MWL patients employed in Shii-take picking (n = 5) have significantly increased antibody titres against Shii-take spore antigens. Different control groups, viz, MWL patients employed in the cultivation of the white button mushroom Agaricus bisporus (n = 14) and of the oyster mushroom Pleurotus spp (n = 3), patients with Bechterew's syndrome (n = 7), sarcoidosis (n = 7), rheumatoid arthritis (n = 9), and healthy controls were found in the same range of low titres. The use of protective masks during picking reduced complaints of the workers (n = 14). However, their antibody titres increased with duration of employment despite the protection.

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We report the case of a young man who developed severe asthma a few months after starting work in a factory producing a single type of mushroom: Pleurotus cornucopiae (a basidiomycete). Immunological investigations, performed with material recovered from the filtering devices of the mushroom's bed, led to demonstration of specific IgE and IgG against spore extracts and to isolation of one discriminant antigen (molecular weight: 10.5 kd). Current data concerning the underestimated role of the basidiomycetes in allergic asthma are reviewed.

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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.

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The biological and immunomodulating activities of polysaccharide fraction (GF-1), an antitumor poysaccharide fraction from cultured fruiting bodies of Grifola frondosa, was examined in mice. GF-1 showed no cytocidal effect on culturing tumor cells. However, GF-1 induced resistance against Sarcoma 180 in ICR mice which had completely regressed from the tumor by the effect of GF-1. The administration of GF-1 into mice increased the weights or cell numbers of spleen, and peritoneal cavity. GF-1 enhanced the antigen specific antibody response and carbon clearance activity, whereas GF-1 did not show polyclonal B cell activation and mitogenic activities, and the effect on delayed type hypersensitivity.

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Immunological techniques are useful to clarify the sistematic scheme utilized for mushrooms. By comparing the various fruiting portions, a different composition in protein was observed in the cell wall of spores, pileus and stipe. The differences were present both in Agaricus bisporus and in Pleurotus ostreatus. The results indicate that a well definite choice of the utilized portions is necessary for comparative studies. In the Basidiomycetes the meiospores represent the more useful development stage to carry out immunological comparisons.

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In Pleurotus ostreatus protocatechuic acid is degraded by protocatechuate 3,4-dioxygenase (protocatechuate: oxygen 3,4-oxidoreductase, EC 1.13.11.3) via "intradiol" cleavage of aromatic ring to form beta-carboxy-cis,cis-muconic acid. The enzyme was isolated from the mycelium induced with p-hydroxybenzoic acid. An about 460-fold purification of the enzyme was achieved by ammonium sulphate fractionation, and DEAE-cellulose and Sephadex G-200 chromatography. The enzyme was homogeneous on analytical electrophoresis under non-denaturing conditions, whereas in the presence of sodium dodecyl sulphate several polypeptides of low molecular weight appeared additionally in trace amounts. Molecular weight of the enzyme, determined by gel filtration and electrophoresis was 200 000 and 205 000, respectively. The enzyme showed low substrate specificity, its pH optimum was 8.0 and Michaelis constant for protocatechuic acid was 14.2 microM.

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Continuous air sampling with a Hirst volumetric spore trap over 3 yr has identified basidiospores of Ganoderma applanatum, a bracket fungus, as the most numerous fungal spores in two southern Ontario locations. The particle size is small and the calculated total spore mass approximates that of the spores of Cladosporium and Alternaria. Extracts of Ganoderma applanatum bracket fungus and spores in w/v, 1:10 concentration were prepared after collection of samples of the fungus from local woods. Skin prick tests with the extracts were performed in 294 consecutive children and adults attending two chest/allergy clinics. Of these patients, 182 (61.9%) reacted to 1 or more of the common inhalant allergen extracts and 24 (8.2%) reacted to Ganoderma antigen. There was no consistent relationship between reactivity to Ganoderma antigen and any of the common inhaled allergens. IgE-dependent sensitization to Ganoderma was confirmed by the radioallergosorbent test (RAST). Rabbit antisera to Ganoderma antigen preparations did not appear to cross-react with preparations of the various clinically important allergens. The findings indicate that Ganoderma antigen is commonly encountered, can induce human sensitization, and has unique antigenicity among common allergens of clinical importance.

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Gloeophyllum trabeum (Fr.) Murr. produced eight precipitin lines in homologous serological reactions in double-diffusion experiments. Five of these lines were similar to the precipitin lines developed with G. saepiarium (Fr.) Karst. antigens, indicating a close serological relationship between the two organisms.

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