Cytokine Levels After Consumption of a Medicinal Agaricus blazei Murill-Based Mushroom Extract, AndoSan™ , in Patients with Crohn's Disease and Ulcerative Colitis in a Randomized Single-Blinded Placebo-Controlled Study.

Ingestion of the Agaricus blazei Murill-based mushroom extract AndoSan™ has been shown in randomized placebo-controlled studies to improve symptoms in Crohn's disease (CD) and ulcerative colitis (UC) and also fatigue and quality of life in the latter patients. The aim was to examine whether this clinical impact of AndoSan™ intake could be explained by influence on foremost pro-inflammatory cytokines in the patients. Fifty patients with symptomatic UC and CD were randomized and blinded for oral daily intake of AndoSan™ or placebo. Blood samples taken before (visit 1) and after 21 days' (visit 3) consumption were analysed for cytokines IL-1ß, IL-2, IL-4-8, IL-10, IL-12-13, IL-17, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1ß and TNF-α. Baseline cytokine levels were similar in CD and UC. In CD, cytokine levels at visit 1 versus visit 3 were unaltered within the AndoSan™ and the placebo groups. Only IL-2 was significantly reduced at visit 3 in the Andosan™ compared with the placebo group. However, when combining IL-1ß, IL-6 and G-CSF in the patients with CD, the cytokine levels were significantly lower in the AndoSanTM - versus the placebo group, visit 3. In UC, levels of IL-2, IL-5 and MIP-1ß were reduced within the AndoSan™ group. IL-5 was also reduced at visit 3 compared with placebo. Generally, the effect on reduction in systemic cytokine levels by consumption of AndoSan™ was limited and supported only marginally anti-inflammatory effects in these patients. Therefore, other explanations behind the clinical anti-inflammatory effects than the contribution of cytokines seem more pertinent, including anti-allergic and antioxidant activities.

Effect of an extract based on the medicinal mushroom Agaricus blazei Murill on expression of cytokines and calprotectin in patients with ulcerative colitis and Crohn's disease.

An immunomodulatory extract (AndoSan™) based on the medicinal mushroom Agaricus blazei Murill (AbM) has shown to reduce blood cytokine levels in healthy volunteers after 12 days' ingestion, pointing to an anti-inflammatory effect. The aim was to study whether AndoSan™ had similar effects on cytokines in patients with ulcerative colitis (UC) and Crohn's disease (CD). Calprotectin, a marker for inflammatory bowel disease (IBD), was also measured. Patients with CD (n = 11) and with UC (n = 10) consumed 60 ml/day of AndoSan™. Patient blood plasma was harvested before and after 6 h LPS (1 ng/ml) stimulation ex vivo. Plasma and faecal calprotectin levels were analysed using ELISA and 17 cytokines [IL-2, IFN-γ, IL-12 (Th1), IL-4, IL-5, IL-13 (Th2), IL-7, IL-17, IL-1ß, IL-6, TNF-α, IL-8, MIP-1ß, MCP-1, G-CSF, GM-CSF and IL-10] by multiplex assay. After 12 days' ingestion of AndoSan™, baseline plasma cytokine levels in UC was reduced for MCP-1 (40%) and in LPS-stimulated blood for MIP-1ß (78%), IL-6 (44%), IL-1ß (41%), IL-8 (30%), G-CSF (29%), MCP-1 (18%) and GM-CSF (17%). There were corresponding reductions in CD: IL-2 (100%), IL-17 (55%) and IL-8 (29%) and for IL-1ß (35%), MIP-1ß (30%), MCP-1 (22%), IL-8 (18%), IL-17 (17%) and G-CSF (14%), respectively. Baseline concentrations for the 17 cytokines in the UC and CD patient groups were largely similar. Faecal calprotectin was reduced in the UC group. Ingestion of an AbM-based medicinal mushroom by patients with IBD resulted in interesting anti-inflammatory effects as demonstrated by declined levels of pathogenic cytokines in blood and calprotectin in faeces.

Gender differences in indoor allergen exposure and association with current rhinitis.

BACKGROUND: Differences between boys and girls in allergic manifestations are well known, and this difference is possibly not attributed to physiological differences alone. OBJECTIVE: We, therefore, investigated whether boys and girls could be exposed to different allergen levels at home and whether indoor allergen levels could be differently associated with rhinitis in boys and girls at 10 years of age. METHODS: Cat, dog and house dust mite (HDM) allergen levels in mattress dust and interview data regarding current allergic disease were available for 797 10-year-old children (360 girls) in The Environment and Childhood Asthma Study in Oslo. RESULTS: Girls had higher concentrations of cat and dog allergens in their mattresses compared with boys, also in homes without cats [geometric mean 95% confidence intervals (95% CI): 0.37 (0.31, 0.44) for girls and 0.26 (0.23, 0.30) microg cat allergen/g dust for boys, P=0.002], and without dogs [girls: 0.74 (0.63, 0.86) and boys: 0.55 (0.48, 0.62) microg dog allergen/g dust, P=0.003]. No difference was observed for HDM allergen (Der p 1) levels. Of the 190 (23.8%) children reporting current rhinitis, 144 (75.8%) were sensitized to at least one allergen. The adjusted odds ratio for current rhinitis increased with 1.20 (95% CI: 1.01, 1.42) per 1 microg/g dust increase in Der p 1 for girls (P=0.037), but not for boys (P=0.91). CONCLUSION: Girls had higher levels of cat and dog allergens in mattress dust compared with boys, whereas no difference was observed for Der p 1 allergen. Nevertheless, only increasing levels of Der p 1 and not cat and dog allergens significantly increased the risk of current rhinitis in girls, whereas no significant association was observed for boys.

An extract based on the medicinal mushroom Agaricus blazei Murill stimulates monocyte-derived dendritic cells to cytokine and chemokine production in vitro.

The edible mushroom Agaricus blazei Murill (AbM), which has been used in traditional medicine against a range of diseases and possess immunomodulating properties, probably due to its high content of beta-glucans. Others and we have demonstrated stimulatory effects of extracts of this mushroom on different immune cells. Dendritic cells are major directors of immune function. We wanted to examine the effect of AbM stimulation on signal substance release from monocyte-derived dendritic cells (MDDC). After 6d incubation with IL-4 and GM-CSF, the cells were true MDDC. Then the cells were further incubated with up to 10% of the AbM-based extract, AndoSan, LPS (0.5 microg/ml) or PBS control. We found that the AbM extract promoted dose-dependent increased levels of IL-8, G-CSF, TNFalpha, IL-1beta, IL-6 and MIP-1beta, in that order. The synthesis of IL-2, IL-8 and IFNgamma were similar for the AbM extract and LPS. However, AndoSan induced a 10- to 2-fold higher production than did LPS of G-CSF, TNFalpha and IL-1beta, respectively. AbM did not induce increased synthesis of Th2 or anti-inflammatory cytokines or the Th1 cytokine IL-12. We conclude that stimulation of MDDC with an AbM-based extract resulted in increased production of proinflammatory, chemotactic and some Th1-type cytokines in vitro.

Effect of an extract based on the medicinal mushroom Agaricus blazei murill on release of cytokines, chemokines and leukocyte growth factors in human blood ex vivo and in vivo.

An immunostimulatory extract based on the medicinal mushroom Agaricus blazei Murill (AbM) has been shown to stimulate mononuclear phagocytes in vitro to produce pro-inflammatory cytokines, and to protect against lethal peritonitis in mice. The present aim was to study the effect of AbM on release of several cytokines in human whole blood both after stimulation ex vivo and in vivo after oral intake over several days in healthy volunteers. The 17 signal substances examined were; T helper 1 (Th1) cytokines [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], T helper 2 cytokines (IL-4, IL-5 and IL-13), pleiotropic (IL-7, IL-17), pro-inflammatory [IL-1beta, IL-6, tumour necrosis factor (TNF)-alpha (mainly produced by Th1 cells)]--and anti-inflammatory (IL-10) cytokines, chemokines [IL-8, macrophage inhibitory protein (MIP)-1beta and monocyte chemoattractant protein (MCP)-1] and leukocyte growth factors [granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony stimulating factor]. After stimulation of whole blood ex vivo with 0.5-5.0% of a mushroom extract, AndoSan mainly containing AbM, there was a dose-dependent increase in all the cytokines studied, ranging from two to 399-fold (TNF-alpha). However, in vivo in the eight volunteers who completed the daily intake (60 ml) of this AbM extract for 12 days, a significant reduction was observed in levels of IL-1beta (97%), TNF-alpha (84%), IL-17 (50%) and IL-2 (46%). Although not significant, there was a trend towards reduced levels for IL-8, IFN-gamma and G-CSF, whilst those of the remaining nine cytokines tested, were unaltered. The discrepant results on cytokine release ex vivo and in vivo may partly be explained by the antioxidant activity of AbM in vivo and limited absorption of its large, complex and bioactive beta-glucans across the intestinal mucosa to the reticuloendothelial system and blood.

Mould-specific immunoglobulin antibodies quantified by flow cytometry reflect mould exposure in Norwegian children.

BACKGROUND: Studies from many countries have shown an association between dampness in buildings and airway symptoms. Little is known about the role of mould-specific IgG antibodies in this context. Objective To examine the IgG antibody response to mould applying a new flow cytometric assay, compare the results with the standardized ImmunoCap method, and evaluate the association of IgG to IgE antibodies, dampness in buildings, and airway symptoms like wheeze and asthma. METHODS: A population of 3713 children 9-11 years of age living in Northern Norway was investigated for airway symptoms and dampness at homes by a parental questionnaire, using protocols of the International study of Asthma and Allergy in Childhood (ISAAC). Among these, a case-control study of 100 wheezers and 100 non-wheezers was established that included home inspection, a parental structured interview, and serum samples analysed for mould-specific IgG and IgE antibodies, total IgE, and specific IgE to an allergen panel (Phadiatop). RESULTS: Self-reported visible signs of mould or moisture at home during the child's first year of life were a significant risk factor for both wheeze and asthma. The levels of mould-specific IgG antibodies were associated with mould and moisture findings, but only when IgG antibodies were measured by flow cytometry. CONCLUSIONS: The results support that dampness at home can increase the risk of airway symptoms. IgG antibodies determined by flow cytometry reflect mould exposure better than antibodies measured by the conventional method. IgG antibodies measured by flow cytometry may be used as an indicator of mould exposure.

Specific IgE to respiratory allergens and IgG antibodies to Toxoplasma gondii and Streptococcus pneumoniae in Norwegian military recruits.

Declining exposure to infections has been implicated as a cause for the rising trend of allergy. Gastrointestinal infections, in particular, have been suggested to have a protective effect against allergy development. In contrast, there are only limited data available regarding the effect of respiratory tract infections with encapsulated bacteria on allergy development. We investigated the association between IgG antibodies against the gastrointestinal parasite Toxoplasma gondii and the bacterium Streptococcus pneumoniae and specific IgE to common allergens in Norwegian military recruits. IgG antibodies to T. gondii and to a 23-valent pneumococcal polysaccharide vaccine (Pneumovax) were determined by ELISA. Specific IgE to common airborne allergens was determined by Phadiatop. Individuals with Phadiatop values from class 0-2 were classified as non-atopic (n = 419), while those with class 3-6 were classified as atopic (n = 201). No significant difference was observed in IgG antibody levels to pneumococcal polysaccharides between atopic and non-atopic recruits, whereas seropositivity to T. gondii was found to be less frequent among the atopic recruits (odds ratio, 0.37; 95% CI, 0.17-0.81; P = 0.01). Hence, in Norwegian recruits, the serological response to a gastrointestinal pathogen, but not to the respiratory encapsulated bacteria, was found to be associated with a lower probability for being sensitized according to the criteria used. The present study conforms to the hypothesis that reduced rates of infection with certain microorganisms may be associated with an increased risk of allergic sensitization.

Effects of the medicinal mushroom Agaricus blazei Murill on immunity, infection and cancer.

Agaricus blazei Murill (AbM) is an edible, medicinal mushroom of Brazilian origin. It is used traditionally against a range of diseases, including cancer and chronic hepatitis, and has been cultivated commercially for the health food market. AbM has recently been shown to have strong immunomodulating properties, which has led to increasing scientific interest. In this article, we review current knowledge as to the immunological properties of AbM, and its possible clinical use in connection with infections and cancer. We also present some novel findings, which point to highly different biological potency between AbM extracts of different source and manufacturing.

IL-1beta as a determinant in silica-induced cytokine responses in monocyte-endothelial cell co-cultures.

Alveolar macrophages and endothelial cells are both involved in lung inflammation and remodeling of lung alveolar structures. In the present study, monocytes (precursors for macrophages) were exposed to crystalline silica and examined for pro- and anti-inflammatory cytokine responses in non-contact co-cultures with endothelial cells. The time courses for silica-induced release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-8 both from co-cultures and monocyte mono-cultures showed an early peak at 5-10 h, almost no response at 20 h, and a strong increase at 43 h. At 43 h, co-cultures also showed strongly increased IL-6 levels. Steady-state levels of mRNA roughly exhibited the same pattern of early up-regulation and reduced levels at 20 h. Compared with monocyte mono-cultures, silica induced a strong release of IL-1beta, IL-6, and IL-8, but not of TNF-alpha, after 43 h in co-cultures, whereas at 5 and 10 h a significant difference was only observed for the silica-induced IL-8 response. An antagonist to the IL-1 receptor strongly reduced IL-6 and IL-8 levels, whereas antibodies to TNF-alpha increased the levels of IL-1beta and IL-8. Thus, IL-1beta is suggested to be an important triggering factor that determines the silica-induced release of several of the other cytokines in this co-culture system.

Effects on gene expression and viral load of a medicinal extract from Agaricus blazei in patients with chronic hepatitis C infection.

Extracts from the mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer and infections, including hepatitis. We previously demonstrated a potent immunomodulating effect of a particular preparation on monocytes in vitro, and a protective effect on bacterial infections in mice. Here we report the effect on gene expression in peripheral blood cells from four chronic hepatitis C patients, using global (29 k) oligo-based, single channel microarrays. The viral load was slightly, but not significantly, decreased after 1 week of AbM treatment. The cytokine genes most strongly induced in vitro were not induced in vivo. The more notable changes in mRNA levels were related to genes involved in the G-protein coupled receptor signalling pathway, in cell cycling, and in transcriptional regulation. The results suggest that the beta-glucans of the extract, which presumably are responsible for cytokine induction, did not readily enter the blood, while other components, such as substances proposed to have anticancer effects, were active in the blood.

Effect of a medicinal extract from Agaricus blazei Murill on gene expression in a human monocyte cell line as examined by microarrays and immuno assays.

Extracts from the edible mushroom Agaricus blazei Murill (AbM) are used extensively as a non-prescription remedy against cancer, infections, and immune related diseases. The presumed effect is to activate certain parts of the immune system. In order to investigate the effect, we examined the changes of gene expression caused by the extract on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine gene products by immuno assays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had drastic effects on gene expression. Genes related to immune function were selectively up-regulated, particularly proinflammatoric genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2).

Processing of C5a by human polymorphonuclear leukocytes.

Uptake of human C5a by neutrophils was monitored in vitro using both 125I-labeled and unlabeled C5a. The ligand was internalized by the cells in a dose-dependent manner and maximal binding/uptake was observed after 5 min of incubation. Neutrophils were incubated with labeled C5a and the cytosol and supernatant were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. C5a degradation products were primarily observed in the supernatant, whereas most of the protein remained intact in the cytosol even after 60 min of incubation. Cytosol from neutrophils incubated for 20 min with unlabeled C5a was examined by radioimmunoassay and found to contain antigenically intact C5a and retained the ability to induce a neutrophil (shape change) response. The functional activity of C5a recovered from the cytosol was inhibited by antibodies to either C5a or the C5a receptor (CD88). This data supports our hypothesis that although C5a is internalized it remains antigenically intact and functionally active inside the cell and is primarily degraded extracellularly.

The use of flow cytometry to detect the biosynthesis of complement components.

Agarose beads, an activator of complement, were incubated with MRC-5 or He 9 fibroblast cell lines under serum-free conditions. The beads were tested for binding of anti-complement antibodies by flow cytometry with a FACS 440 using FITC-labelled anti-Ig detection antibodies. Controls consisted of co-cultured beads incubated with irrelevant antibody or albumin, beads maintained in cell cultures containing cycloheximide, and beads which were not exposed to cells. The histograms demonstrated positive staining with anti-C3c, -C5, -C7 and -C9, but not with anti-C6 and -C8. Flow cytometry with multiple histogram analysis confirmed that the differences between the positive curves and the controls were statistically significant. The results show that cell-derived complement components (C3, C5, C7 and C9) were deposited on the beads and could be detected by flow cytometry.

Cytotoxic effect of radiolabeled C5a on U937 cells in vitro.

We studied whether the receptor (R) for C5a could be exploited to deliver the radiolabeled ligand into U937 cells. A dose-response for uptake of 125I-C5a was demonstrated. Incorporation of [3H]leucine by unstimulated or gamma-INF-stimulated U937 cells treated with 125I-C5a, was significantly lower compared with cells treated with 125I alone. Trypan blue exclusion experiments indicated that gamma-INF stimulated cells incubated with 125I-C5a were less viable than cells exposed to 125I or C5a alone. The results suggest that 125I-C5a is internalized into myeloid cells via C5a-R and is more cytotoxic in vitro than the radiolabel alone, but only at/above a specific activity of 4 microCi/microg.

The B chain, but not the A chain, of Ricinus communis activates human complement.

Complement activation properties of ricin holotoxin, its A or B chain were assessed in two enzyme immunoassays (EIA). One was specific for C3 activation products and the other detected the terminal SC5b-9 complement complex (TCC) and thus determined activation of the initial and terminal part of the complement pathways, respectively. Ricin and its A and B chains were incubated with normal human serum or EDTA-serum. It was found that ricin B chain activated both the initial and terminal complement pathways in a time- and concentration-dependent fashion, whereas the A chain and the holotoxin did not.

Human peritoneal macrophages. Production in vitro of the active terminal complement components C5 to C9 and a functional alternative pathway of complement. Brief report.

Endotoxin-stimulated human peritoneal macrophages were cultured in serum-free medium with agarose beads. Monospecific antibodies to human C3c, C3g, C5, C6, C7, C8, C9 and to C9-neoantigen bound to the beads. This shows that activated C3 and the terminal complement complex (TCC), made from complement components C5 to C9, were generated on the beads. De novo synthesis was confirmed by agarose binding of tritium-labelled protein. Moreover, C3-derivatives and C9-neoantigen were detected on normal serum-treated agarose beads but not on beads kept in factor B-depleted or heat-inactivated sera, implying that an intact alternative complement pathway was required for our findings. The macrophages thus synthesize the active complement components of the alternative and terminal pathways in vitro.

Detection of mRNA for the terminal complement components C5, C6, C8 and C9 in human umbilical vein endothelial cells in vitro.

Human umbilical vein endothelial cells (HUVEC) have previously been shown to synthesize the functional terminal pathway of complement based on the detection by radioimmunoassay of the terminal complement complex (TCC) on coincubated agarose beads. In addition, C7 secretion by these cells in amounts comparable to C3, as well as C7 mRNA, has recently been demonstrated. However, it has not been possible to detect C5-6 and C8 in the fluid phase, and only trace amounts of soluble C9. Against this background we examined whether mRNA for the remaining terminal complement factors was present in HUVEC. By the use of reverse transcription (RT)-polymerase chain reaction (PCR) and Northern blot the presence of mRNA for complement factors C5, C6, C8 and C9 was demonstrated.

Both plasma- and leukocyte-associated C5a are essential for assessment of C5a generation in vivo.

BACKGROUND: Measurement of C5a in plasma is hampered by the rapid clearance of C5a as a result of cell binding. Therefore, an assessment of whether cell-bound C5a might better reflect C5a generation in vivo is essential. METHODS: We quantified plasma and leukocyte-bound C5a in samples from patients undergoing cardiopulmonary bypass, which is known to be associated with complement activation. C3 activation products and the terminal complement complex were measured as well. RESULTS: Plasma levels of C3 activation products and the terminal complement complex increased rapidly and significantly after the onset of cardiopulmonary bypass until they reached a plateau after 30 minutes. The concentration of plasma C5a increased steadily to twice baseline at the end of bypass. The concentration of leukocyte-associated C5a increased threefold after 10 minutes of cardiopulmonary bypass, when a plateau was reached. A positive correlation was found between levels of plasma C3 activation products or terminal complement complex and plasma C5a plus cell-associated C5a but not between C3 activation products or terminal complement complex and either one of the C5a variables. CONCLUSIONS: We conclude that both plasma C5a and leukocyte-associated C5a are needed for monitoring in vivo C5a generation.

Protective effect of beta-glucan against systemic Streptococcus pneumoniae infection in mice.

The antimicrobial effect of soluble beta-1,3-D-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3-48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.

Effects of C5a and FMLP on interleukin-8 production and proliferation of human umbilical vein endothelial cells.

Interleukin-8 (IL-8), C5a and N-formyl-methionyl-leucyl-phenylalanine (fMLP) are chemotactic peptides with predominant effects on leukocytes during inflammation. With emphasis on C5a we studied the regulation of the production of IL-8 by human umbilical vein endothelial cells (HUVEC) in vitro. Primary HUVEC cultures were incubated with IL-1alpha, TNFalpha, C5a and fMLP for 24 h and 48 h prior to measurement of IL-8 in supernatants of the cells by an enzyme immunoassay. Whereas IL-1alpha and TNFalpha significantly increased the levels of IL-8, C5a decreased the IL-8 production after 48 h. In addition, the ability of IL-1alpha, TNFalpha, C5a, fMLP and IL-8 to induce cell proliferation was compared by means of a 3H-thymidine incorporation assay. In contrast with IL-1alpha and TNFalpha, both C5a and fMLP increased cell proliferation of HUVEC. This increase occurred with increasing concentrations of C5a contrary to IL-8, which showed increased cell proliferation at low, but not high IL-8 concentrations.