Modulation of T Regulatory and Dendritic Cell Phenotypes Following Ingestion of Bifidobacterium longum, AHCC® and Azithromycin in Healthy Individuals.

The probiotic Bifidus BB536 (BB536), which contains Bifidobacterium longum, has been shown to have enhanced probiotic effects when given together with a standardized extract of cultured Lentinula edodes mycelia (AHCC®, Amino Up Co. Ltd., Sapporo, Japan). BB536 and AHCC® may modulate T cell and dendritic cell (DC) phenotypes, and cytokine profiles to favour anti-inflammatory responses following antibiotic ingestion. We tested the hypothesis that orally administered BB536 and/or AHCC®, results in modulation of immune effector cells with polarisation towards anti-inflammatory responses following antibiotic usage. Forty healthy male volunteers divided into 4 equal groups were randomised to receive either placebo, BB536, AHCC® or a combination for 12 days in a double-blind manner. After 7 days volunteers also received 250 mg azithromycin for 5 days. Cytokine profiles from purified CD3+ T cells stimulated with PDB-ionomycin were assessed. CD4+ CD25+ forkhead box P3 (Foxp3) expression and peripheral blood DC subsets were assessed prior to treatment and subsequently at 7 and 13 days. There was no difference in cytokine secretion from stimulated CD3+ T cells between treatment groups. Compared with baseline, Foxp3 expression (0.45 ± 0.1 vs. 1.3 ± 0.4; p = 0.002) and interferon-gamma/interleukin-4 (IFN-γ/IL-4) ratios were increased post-treatment in volunteers receiving BB536 (p = 0.031), although differences between groups were not significant. For volunteers receiving combination BB536 and AHCC®, there was an increase in myeloid dendritic cells (mDC) compared with plasmacytoid DC (pDC) counts (80% vs. 61%; p = 0.006) at post treatment time points. mDC2 phenotypes were more prevalent, compared with baseline, following combination treatment (0.16% vs. 0.05%; p = 0.002). Oral intake of AHCC® and BB536 may modulate T regulatory and DC phenotypes to favour anti-inflammatory responses following antibiotic usage.

Histological Features of Flagellate Erythema.

Flagellate erythema is a distinctive eruption characterized by "whip-like" linear or curvilinear streaks and plaques, occurring mainly on the trunk. It has classically been described in 2 disparate clinical settings: chemotherapy with bleomycin and ingestion of mushrooms (most commonly Shiitake mushrooms). Most of the literature comprises single case reports, often with minimal histological description of rather nonspecific features. We describe in detail the histological features of 3 cases of flagellate erythema (2 related to bleomycin therapy and one related to ingestion of mushrooms) and review the findings described in the literature to define the spectrum of histological changes encountered in this eruption. Our 3 cases showed mild epidermal changes, with spongiosis and variable interface inflammation. All 3 showed a relatively prominent dermal lymphohistiocytic infiltrate, with features suggestive of a lymphocytic vasculopathy extending to at least the mid-reticular dermis. Eosinophils were a prominent component of the inflammatory infiltrate in 2 cases. Our review of the literature identified a total of 45 publications, representing reports of 46 patients, containing histological information. As well as bleomycin- and mushroom-related cases, similar eruptions have been reported in the context of connective tissue disease and other drugs. Although cases related to connective tissue disease show features of the underlying condition, cases secondary to drugs or mushrooms predominantly show features compatible with common patterns of exanthematous/morbilliform drug reaction. In particular, subtle spongiosis and/or interface dermatitis combined with a dermal lymphocytic infiltrate that includes increased numbers of eosinophils is a common finding. Features of a lymphocytic vasculopathy may be seen in a subset of these cases.

The Effects of AHCC®, a Standardized Extract of Cultured Lentinura edodes Mycelia, on Natural Killer and T Cells in Health and Disease: Reviews on Human and Animal Studies.

Mushrooms have been used for various health conditions for many years by traditional medicines practiced in different regions of the world although the exact effects of mushroom extracts on the immune system are not fully understood. AHCC® is a standardized extract of cultured shiitake or Lentinula edodes mycelia (ECLM) which contains a mixture of nutrients including oligosaccharides, amino acids, and minerals obtained through liquid culture. AHCC® is reported to modulate the numbers and functions of immune cells including natural killer (NK) and T cells which play important roles in host defense, suggesting the possible implication of its supplementation in defending the host against infections and malignancies via modulating the immune system. Here, we review in vivo and in vitro effects of AHCC® on NK and T cells of humans and animals in health and disease, providing a platform for the better understanding of immune-mediated mechanisms and clinical implications of AHCC®.

Active hexose correlated compound modulates LPS-induced hypotension and gut injury in rats.

We hypothesized that AHCC; (Amino UP Chemical Co., Ltd., Sapporo, Japan), a mushroom mycelium extract obtained from liquid culture of Lentinula edodes, restores immune function in LPS-induced inflammation in the gut, especially when the nitric oxide signaling pathway is impaired. This is the first inter-disciplinary proposal to identify molecular mechanisms involved in LPS-induced immune dysfunction in the gut in conscious animals treated or non-treated with AHCC, a promoter of immune support. Specifically, we have tested the effects of AHCC on LPS-induced deleterious effects on blood pressure and gut injury in conscious rats. The time course of biological markers of innate/acquired immune responses, and inflammation/oxidative stress is fully described in the present manuscript. Rats were randomly assigned into 3 groups (N=6 per group). Group 1 received 10% of AHCC in drinking water for 5days; Group 2 received lipopolysaccharide (LPS; Escherichia coli 0111:B4 purchased from Sigma) only at 20mg/kg IV; Group 3 received combined treatments (AHCC + LPS). LPS was administered at 20mg/kg IV, 5days following AHCC treatment. We have demonstrated that AHCC decreased the LPS-deleterious effects of blood pressure and also decreased inflammatory markers e.g., cytokines, nitric oxide and edema formation. Finally, AHCC diminished lymphocyte infiltration, restoring gut architecture. Because AHCC was administered prior to LPS, our results indicate the potential impact of AHCC's prophylactic effects on LPS inflammation. Consequently, additional experiments are warrant to assess its therapeutic effects in sepsis-induced inflammation.

Shiitake mushroom (Lentinus edodes): a poorly known allergen in Western countries responsible for severe work-related asthma.

OBJECTIVES: The aim of this study was to investigate the IgE-mediated pathogenesis of severe asthma presented by a patient only after handling shiitake (Lentinus edodes) mushrooms (SM). MATERIAL AND METHODS: Skin tests were performed using in-house extracts from mushrooms that the patient usually handled, i.e., shiitake, porcini, oyster and black fungus mushroom varieties. Specific IgE to champignons and various molds were determined. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting was performed to detect IgE-binding components. Four negative controls were included in the study. RESULTS: Skin prick tests performed with in-house mushroom extracts from varieties other than shiitake were completely negative, in contrast to the positive test obtained for shiitake mushrooms. Serum specific IgE levels for common molds and champignons were all negative. SDS-PAGE revealed many protein bands in the four mushroom extracts. Immunoblotting using the patient's serum showed allergenic bands at about 15 and 24 kDa exclusively for SM that were not shared with negative controls. Another faint band was detectable at approximately 37 kDa for SM and porcini varieties. CONCLUSIONS: Here, we present the first European case of SM-induced occupational asthma, a disease more frequently occurring in Asia. Asthma attacks stopped when the patient avoided contact with shiitake mushrooms. No skin reactions and no IgE-binding proteins by immunoblotting were detectable with the other mushrooms tested. The positive skin test with shiitake mushrooms and IgE-binding components in the shiitake extract confirmed the IgE-mediated etiology of the reaction.

Hypersensitivity pneumonitis induced by Shiitake mushroom spores.

Hypersensitivity pneumonitis (HP) is a pulmonary granulomatosis involving an immunoallergic mechanism caused by chronic inhalation of antigens, most frequently organic substances, as well as chemicals. We report the first European case of hypersensitivity pneumonitis due to the inhalation of Shiitake mushroom spores. A 37-year-old French Caucasian man with a one-month history of persistent dry cough, shortness of breath and loss of weight was admitted to our hospital on December 2010. Anamnesis showed he was involved in mushroom production beginning in the summer of 2010. His temperature on admission was 36.6°C and he had a normal blood pressure (135/90 mmHg). Bilateral fine crackles were audible in the base of both lungs. Pulmonary function tests showed a mild restrictive pattern with decreased DLco and a PaO(2) of 65 mmHg, Chest CT scan revealed reticulo-nodular shadows, slight ground glass opacities, liner atelectasis, and subpleural opacities in both lung fields. Bronchoscopy was normal but cytological examination of BAL revealed a predominant lymphocytosis (55%). Serum precipitins to the Shiitake mushroom spores were positive (3 precipitins arcs with high intensity) and as a result we advised the patient to cease his mushroom production activities. The diagnosis of hypersensitivity pneumonitis due to inhalation of Shiitake mushroom spores was established as a result of the improvement of all of his clinical symptoms, i.e., cough, weight loss, bilateral fine crackles, mild restrictive pattern of pulmonary function, and reticulo-nodular shadows on chest CT, once exposure was eliminated. Recent interest in exotic mushrooms varieties, e.g., Shiitake, in developed countries because of their possible medicinal properties might increase the potential risk of HP among mushrooms workers. Therefore, healthcare professionals have to take this new potential respiratory disease into account.

Immunoprophylactic effects of shiitake mushroom (Lentinula edodes) against Bordetella bronchiseptica in mice.

Antimicrobials are used as feed additives to improve growth performance and to prevent subclinical disease challenge in industrial animals. However, these drugs can lead to the development of resistant strains of bacteria. Shiitake mushrooms (SM) (Lentinula edodes) have long been popular as a health food in East Asia. Moreover, SM-derived polysaccharides are well-known as immunostimulants that possess antimicrobial properties. The aim of the present study was to evaluate the immunoprophylactic effects of SM against Bordetella bronchiseptica infection in mice as an initial step towards the development of eco-friendly feed additives to reduce the use of antimicrobials. Although SM had no effect on body weight gain under the un-infected conditions, SM alleviated progressive weight loss and helped in the recovery of body weight in B. bronchiseptica infected mice. Dietary supplementation with SM reinforced bacterial clearance in the infected mice. Of note, SM markedly increased the percentage of various T lymphocytes and the relative mRNA expression levels of tumor necrosis factor-α and interferon-γ in the bronchial lymph node early in the infection. Taken together, these findings suggest that SM could help in the improvement of body weight gain during B. bronchiseptica infection and may enhance the protective immune activity against a subclinical disease challenge, such as B. bronchiseptica infection in mice, probably by a strong stimulation of non-specific immune responses. Hence, SM may provide an alternative to reduce use of antimicrobials. Confirmation of the beneficial effects of SM as a feed additive is now required in industrial animals.

Immunomodulatory beta-glucan from Lentinus edodes activates mitogen-activated protein kinases and nuclear factor-kappaB in murine RAW 264.7 macrophages.

Lentinan, a cell wall ß-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.

Effects of two basidiomycete species on interleukin 1 and interleukin 2 production by macrophage and T cell lines.

Two basidiomycete species, Lentinus edodes mycelia (LEM) and Cordyceps sinensis (CS) were examined for induction of cytokines in murine macrophage cell line R309 (R309) and T cell line LBRM-33 1A5 (1A5). When lipopolysaccharide (LPS)-activated R309 were exposed to the extracts of basidiomycetes, R309 induced significant levels of interleukin 1 (IL-1). Interleukin 2 (IL-2) induction was recognized in 1A5 cultures in the presence of IL-1 and phytohemagglutinin (PHA). However, no enhancement of IL-2 production by these basidiomycetes was discerned in 1A5 cultures with IL-1 and PHA, i.e., direct action of basidiomycetes was not found on IL-2 production of 1A5. PHA-stimulated 1A5 exposed to basidiomycetes induced IL-2 without IL-1 when co-cultured with LPS-activated R309 as a source of IL-1. Effects of basidiomycetes on IL-2 production in 1A5 seemed to be caused through their action on macrophages. The induction of IL-2, Th1 type cytokine in T lymphocyte, is a significant finding since basidiomycetes, taken as a dietary supplement for immuno-suppressed patients, especially cancer patients, would be helpful in improving their immune activity against cancer.

Study on immunostimulating activity of macrophage treated with purified polysaccharides from liquid culture and fruiting body of Lentinus edodes.

Lentinus edodes is a well-known edible and medicinal mushroom used in Oriental cultures. Recently, L. edodes has attracted a lot of attention owing to its antifungal activity, antibacterial activity, antiviral activity, hepatoprotective effect, antitumor activities, and immunomodulatory and cytotoxic effects. In this study, the water-soluble crude polysaccharides, CPF and CPB, which were obtained from the fruiting body and culture cell-free broth of L. edodes by hot-water extraction and ethanol precipitation, were fractionated by DEAE cellulose and Sepharose CL-6B column chromatography, resulting in six polysaccharide fractions, CPFN-G-I, CPFN-G-II, CPFN-G-III, CPFA-G, CPBN-G, and CPBA-G. Among these fractions, CPFN-GI, CPBN-G, and CPBA-G were shown to stimulate the functional activation of macrophages including NO production cytokine expression and phagocytosis.

Structural characteristics of immunostimulating polysaccharides from lentinus edodes.

There is a significant amount of experimental evidence suggesting that polysaccharides from mushrooms enhance the host immune system by activating various mechanisms in immune cells, including macrophages. In this study, polysaccharides from Lentinus edodes were found to stimulate the functional activation of macrophages to secrete inflammatory mediators and cytokines and increase the phagocytotic uptake. The chemical properties of the stimulatory polysaccharides, CPFN-G-I, CPBN-G, and CPBA-G, were determined based on their monosaccharide composition, which mainly consisted of glucose and mannose. According to FT-IR and GC/MS, the structure of CPFN-G-I, purified from the fruiting body of L. edodes, was found to consist of a beta-1,6-branched-beta-1,4-glucan, whereas CPBN-G and CPBA-G, purified from the liquid.

[Chronic hypersensitivity pneumonitis induced by Shiitake mushroom cultivation: case report and review of literature].

A 72-year-old man, a Shiitake mushroom grower over fifty years, was admitted to our hospital because of bilateral chest interstitial shadow with chronic cough and breathlessness. Chest computed tomography showed traction bronchiectasis, subpleural micro-cystic changes and partial ground-glass opacities in both lungs, and mild mediastinal lymphadenopathy. A diagnosis of chronic hypersensitivity pneumonitis induced by Shiitake mushrooms was comprehensively confirmed by occupational history, radiological findings, and positive findings of an incidental environmental provocation test and lymphocyte stimulation test for Shiitake mushroom extracts. We reviewed the clinical features in five patients with chronic hypersensitivity pneumonitis induced by Shiitake mushrooms reported in Japan. There was a tendency toward increasing lymphocytes and high CD4/CD8 ratio in bronchoalveolar lavage fluids. Treatment with steroids seems to have a limited effect, while avoidance of the antigen is important.

Molecular mechanism of macrophage activation by Exopolysaccharides from liquid culture of Lentinus edodes.

Mushrooms are regarded as one of the well-known foods and biopharmaceutical materials with a great deal of interest. beta-Glucan is the major component of mushrooms that displays various biological activities such as antidiabetic, anticancer, and antihyperlipidemic effects. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using exopolysaccharides prepared from liquid culture of Lentinus edodes. We found that fraction II (F-II), with large molecular weight protein polysaccharides, is able to strongly upregulate the phenotypic functions of macrophages such as phagocytic uptake, ROS/NO production, cytokine expression, and morphological changes. F-II triggered the nuclear translocation of NF-kappaB and activated its upstream signaling cascades such as PI3K/ Akt and MAPK pathways, as assessed by their phosphorylation levels. The function-blocking antibodies to dectin-1 and TLR-2, but not CR3, markedly suppressed F-II-mediated NO production. Therefore, our data suggest that mushroom-derived beta-glucan may exert its immunostimulating potency via activation of multiple signaling pathways.

Occupational protein contact dermatitis from shiitake mushroom and demonstration of shiitake-specific immunoglobulin E.

Shiitake are popular edible mushrooms all over the world, and eating raw shiitake may lead to relatively common 'shiitake dermatitis' or toxicodermia. Workers involved in shiitake cultivation and marketing have distinct occupational respiratory and skin diseases unrelated to 'shiitake dermatitis'. There are no previous reports of protein contact dermatitis (PCD) from shiitake, and there is only 1 report of shiitake-specific immunoglobulin (Ig) E. We report 2 shiitake growers who developed work-related eczematous eruption on their hands. Both of the patients had small prick test reactions to fresh shiitake, and specific IgE to shiitake was detected in their sera by immunospot. One of the patients had a large prick test reaction to dry shiitake and also a positive wheal reaction to fresh shiitake in an open application test. Neither of the patients had noticed any symptoms of contact urticaria at work. Both of the patients had immediate IgE-mediated allergy to shiitake, and the diagnosis of occupational PCD was made. There are no commercial in vitro tests for shiitake-specific IgE. Tests for immediate allergy are important when shiitake contact dermatitis is investigated.

Chronic hypersensitivity pneumonitis induced by Shiitake mushroom spores associated with lung cancer.

A 61-year-old man was admitted to our hospital with a 6-month history of productive cough. He, along with his wife, had been involved with Shiitake mushroom cultures for a period of 12 years. On admission, chest radiography showed bilateral fine-nodular shadow and CT scans showed reticulonodular opacities and a ground-glass appearance predominantly in the subpleural area in both lungs, and a mass in the left S6. Resected pathological specimens obtained by left lower lobectomy revealed lung adenosquamous carcinoma (stage IB), interstitial changes accompanied with lymphocyte proliferation and fibrosis, and granuloma with giant cells. Serum precipitins for Shiitake mushroom antigens were positive. The productive cough improved after the hospital admission and occurred again when he returned to work with the Shiitake mushroom production. Therefore, chronic hypersensitivity pneumonitis (HP) caused by Shiitake mushroom spores was diagnosed. Moreover, his wife was found to have HP caused by mushrooms at this time. There are only two previous reports of chronic HP caused by Shiitake mushroom in Japan, and this is the first case of chronic HP associated with lung cancer.