Development and Performance of a CD8 Gene Signature for Characterizing Inflammation in the Tumor Microenvironment across Multiple Tumor Types.

Across multiple tumor types, immune checkpoint inhibitors (ICIs) have demonstrated clinical benefit to patients with cancer, yet there is a need to identify predictive biomarkers of response to these therapies. A multiparameter gene expression profiling-based tumor inflammation assay may offer robust characterization of the tumor microenvironment, thereby extending the utility of single-gene analysis or immunohistochemistry (IHC) in predicting response to ICIs. The authors interrogated 1778 commercially procured, formalin-fixed, paraffin-embedded samples using gene expression profiling and pathology-assisted digital CD8 IHC. A machine-learning approach was used to develop gene expression signatures that predicted CD8+ immune cell abundance as surrogates for tumor inflammation in melanoma and squamous cell carcinoma of the head and neck samples. An assay for a 16-gene CD8 signature was developed and analytically validated across 12 tumor types. CD8 signature scores correlated with CD8 IHC in a platform-independent manner, and inflammation prevalence was similar between assay methods for all tumor types except prostate cancer and small cell lung cancer. In retrospective analyses, CD8 signature scores were associated with progression-free survival and overall survival with nivolumab in patients with urothelial carcinoma from CheckMate 275. This study demonstrated that the CD8 signature assay can be used to accurately quantify CD8+ immune cell abundance in the tumor microenvironment and has potential clinical utility for determining patients with cancer likely to respond to ICIs.

Genome-wide transcriptome analysis identifies novel dysregulated genes implicated in Alzheimer's pathology.

INTRODUCTION: Abnormal gene expression patterns may contribute to the onset and progression of late-onset Alzheimer's disease (LOAD). METHODS: We performed transcriptome-wide meta-analysis (N = 1440) of blood-based microarray gene expression profiles as well as neuroimaging and cerebrospinal fluid (CSF) endophenotype analysis. RESULTS: We identified and replicated five genes (CREB5, CD46, TMBIM6, IRAK3, and RPAIN) as significantly dysregulated in LOAD. The most significantly altered gene, CREB5, was also associated with brain atrophy and increased amyloid beta (Aß) accumulation, especially in the entorhinal cortex region. cis-expression quantitative trait loci mapping analysis of CREB5 detected five significant associations (P < 5 × 10-8 ), where rs56388170 (most significant) was also significantly associated with global cortical Aß deposition measured by [18 F]Florbetapir positron emission tomography and CSF Aß1-42 . DISCUSSION: RNA from peripheral blood indicated a differential gene expression pattern in LOAD. Genes identified have been implicated in biological processes relevant to Alzheimer's disease. CREB, in particular, plays a key role in nervous system development, cell survival, plasticity, and learning and memory.

Reliable Gene Expression Profiling from Small and Hematoxylin and Eosin-Stained Clinical Formalin-Fixed, Paraffin-Embedded Specimens Using the HTG EdgeSeq Platform.

Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (H&E)-stained, and immunohistochemistry-stained slides from patients with small-cell and non-small-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with H&E staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-γ signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with H&E-stained slides and a significant Spearman correlation (P = 0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm2 of a section (5 µm thick); sample equivalents ≥0.31 mm2 showed alignment rates >69% and pairwise Pearson correlation coefficients ≥0.87. EdgeSeq can, thus, be used to profile small and H&E-stained FFPE tumor specimens to obtain biomarker data from limited tissue in oncology clinical trials and enable research into tumor microenvironment and immune cell engagement with tumors at the locoregional level.

Evaluation and selection of a non-PCR based technology for improved gene expression profiling from clinical formalin-fixed, paraffin-embedded samples.

AIM: Formalin-fixed, paraffin-embedded (FFPE) clinical tissue samples have the potential to provide valuable gene-expression data for the development of cancer biomarkers. However, FFPE RNA is extensively fragmented, presenting a significant challenge for reliably detecting gene expression using traditional qPCR methods. RESULTS: We evaluated three novel methodologies along with the traditional qPCR method for their ability to detect Notch pathway gene expression in colorectal cancer FFPE tissue RNAs. We found that quantitative nuclease protection assay-detected gene expression in high-quality RNAs as sensitively as qPCR, and consistently detected mRNAs in highly fragmented FFPE tissue RNAs. CONCLUSION: Quantitative nuclease protection assay represents an improved methodology for detecting gene expression in FFPE tissue and has the potential to advance the development of cancer biomarkers.

Genetic basis for clinical response to CTLA-4 blockade in melanoma.

BACKGROUND: Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. METHODS: We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. RESULTS: Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab. CONCLUSIONS: These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).

Anti-inflammatory effect and mechanism of the green fruit extract of Solanum integrifolium Poir.

The green fruit of Solanum integrifolium Poir. has been used traditionally as an anti-inflammatory and analgesic remedy in Taiwanese aboriginal medicine. The goal of this study is to evaluate the anti-inflammatory activity and mechanism of the green fruit extract of S. integrifolium. A bioactivity-guided fractionation procedure was developed to identify the active partition fraction. The methanol fraction (ME), with the highest phenolic content, exhibited the strongest inhibitory effect against LPS-mediated nitric oxide (NO) release and cytotoxicity in RAW264.7 macrophages. ME also significantly downregulated the expression of LPS-induced proinflammatory genes, such as iNOS, COX-2, IL-1ß, IL-6, CCL2/MCP-1, and CCL3/MIP1α. Moreover, ME significantly upregulated HO-1 expression and stimulated the activation of extracellular-signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with the HO-1 inhibitor zinc protoporphyrin and MEK/ERK inhibitor U0126 attenuated ME's inhibitory activity against LPS-induced NO production. Taken together, this is the first study to demonstrate the anti-inflammatory activity of green fruit extract of S. integrifolium and its activity may be mediated by the upregulation of HO-1 expression and activation of ERK1/2 pathway.

Lipid peroxidation end product 4-hydroxy-trans-2-nonenal triggers unfolded protein response and heme oxygenase-1 expression in PC12 cells: Roles of ROS and MAPK pathways.

This study investigates the roles of ROS overproduction and MAPK signaling pathways in the induction of unfolded protein response (UPR) and the expression of Phase II enzymes in response to 4-hydroxy-trans-2-nonenal (4-HNE) in a neuronal-like catecholaminergic PC12 cells. Our results showed that 4-HNE triggered three canonical pathways of UPR, namely IRE1-XBP1, PERK-eIF2α-ATF4 and ATF6, and induced the expression of UPR-targeted genes, GRP78, CHOP, TRB3, PUMA, and GADD34, as well as Phase II enzymes, HO-1 and GCLC. 4-HNE also induced apoptosis, intracellular calcium accumulation, caspase-3 activation, and G0/G1 cell cycle arrest, which was correlated with the increased expression of GADD45α. The addition of tiron, a cellular permeable superoxide scavenger, scavenged 4-HNE-mediated ROS formation, but did not alleviate cytotoxicity, or the expression of UPR-targeted genes or Phase II enzymes, indicating that ROS overproduction per se did not play a major role in 4-HNE-caused deleterious effects. HO-1 expression was attenuated by Nrf2 siRNA and chemical chaperone 4-phenylbutyrate (4-PBA), suggesting HO-1 expression was regulated by Nrf2-ARE, which may work downstream of ER stress. 4-HNE treatment promptly induced ERK, JNK and p38 MAPK activation. Addition of p38 MAPK specific inhibitor SB203580 attenuated HO-1 upregulation, but enhanced expression of CHOP, PUMA and TRB3, and cytotoxicity. These results indicate that 4-HNE-induced transient p38 MAPK activation may serve as an upstream negative regulator of ER stress and confer adaptive cytoprotection against 4-HNE-mediated cell injury.

Arsenic trioxide induces unfolded protein response in vascular endothelial cells.

Chronic arsenic exposure has been linked to endothelial dysfunction and apoptosis. We investigate the involvement of unfolded protein response (UPR) signaling in the arsenic-mediated cytotoxicity of the SVEC4-10 mouse endothelial cells. The SVEC4-10 cells underwent apoptosis in response to As2O3 dose- and time-dependently, accompanied by increased accumulation of calcium, and activation of caspase-3. These phenomena were completely inhibited by α-lipoic acid (LA), which did not scavenge ROS over-production, but were only partially or not ameliorated by tiron, a potent superoxide scavenger. Moreover, arsenic activated UPR, leading to phosphorylation of eukaryotic translation initiation factor 2 subunit α (eIF2α), induction of ATF4, and processing of ATF6. Treatment with arsenic also triggered the expression of endoplasmic reticulum (ER) stress markers, GRP78 (glucose-regulated protein), and CHOP (C/EBP homologous protein). The activation of eIF2α, ATF4 and ATF6 and expression of GRP78 and CHOP are repressed by both LA and tiron, indicating arsenic-induced UPR is mediated through ROS-dependent and ROS-independent pathways. Arsenic also induced ER stress-inducible genes, BAX, PUMA (p53 upregulated modulator of apoptosis), TRB3 (tribbles-related protein 3), and SNAT2 (sodium-dependent neutral amino acid transporter 2). Consistent with intracellular calcium and cell viability data, ROS may not be important in arsenic-induced death, because tiron did not affect the expression of these pro-apoptotic genes. In addition, pretreatment with salubrinal, a selective inhibitor of eIF2α dephosphorylation, enhanced arsenic-induced GRP78 and CHOP expression and partially prevented arsenic cytotoxicity in SVEC4-10 cells. Taken together, these results suggest that arsenic-induced endothelial cytotoxicity is associated with ER stress, which is mediated by ROS-dependent and ROS-independent signaling.

Gene expression profiling of whole blood in ipilimumab-treated patients for identification of potential biomarkers of immune-related gastrointestinal adverse events.

BACKGROUND: Treatment with ipilimumab, a fully human anti-CTLA-4 antibody approved for the treatment of advanced melanoma, is associated with some immune-related adverse events (irAEs) such as colitis (gastrointestinal irAE, or GI irAE) and skin rash, which are managed by treatment guidelines. Nevertheless, predictive biomarkers that can help identify patients more likely to develop these irAEs could enhance the management of these toxicities. METHODS: To identify candidate predictive biomarkers associated with GI irAEs, gene expression profiling was performed on whole blood samples from 162 advanced melanoma patients at baseline, 3 and 11 weeks after the start of ipilimumab treatment in two phase II clinical trials (CA184004 and CA184007). Overall, 49 patients developed Grade 2 or higher (grade 2+) GI irAEs during the course of treatment. A repeated measures analysis of variance (ANOVA) was used to evaluate the differences in mean expression levels between the GI irAE and No-GI irAE groups of patients at the three time points. RESULTS: In baseline samples, 27 probe sets showed differential mean expression (≥ 1.5 fold, P ≤ 0.05) between the GI irAE and No-GI irAE groups. Most of these probe sets belonged to three functional categories: immune system, cell cycle, and intracellular trafficking. Changes in gene expression over time were also characterized. In the GI irAE group, 58 and 247 probe sets had a ≥ 1.5 fold change in expression from baseline to 3 and 11 weeks after first ipilimumab dose, respectively. In particular, on-treatment expression increases of CD177 and CEACAM1, two neutrophil-activation markers, were closely associated with GI irAEs, suggesting a possible role of neutrophils in ipilimumab-associated GI irAEs. In addition, the expression of several immunoglobulin genes increased over time, with greater increases in patients with grade 2+ GI irAEs. CONCLUSIONS: Gene expression profiling of peripheral blood, sampled before or early in the course of treatment with ipilimumab, resulted in the identification of a set of potential biomarkers that were associated with occurrence of GI irAEs. However, because of the low sensitivity of these biomarkers, they cannot be used alone to predict which patients will develop GI irAEs. Further investigation of these biomarkers in a larger patient cohort is warranted.

Curcuminoids distinctly exhibit antioxidant activities and regulate expression of scavenger receptors and heme oxygenase-1.

SCOPE: Curcumin (CUR), demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC) have been demonstrated as having antioxidant, anticarcinogenic, and hypocholesterolemic activities. We report the diverse antiatherogenic effects and mechanisms of curcuminoids. METHODS AND RESULTS: We found that CUR was the most potent antioxidant against copper-mediated LDL oxidation as measured by thiobarbituric acid-reactive substances assay, oxidized LDL (oxLDL) ELISA, and electrophoretic mobility. CUR upregulated heme oxygenase-1, modifier subunit of glutamate-cysteine ligase (GCLM), and CD36 expression in undifferentiated THP-1 cells, supporting the possible involvement of Nrf2 pathway in CD36 expression. Monocyte-to-macrophage differentiation plays a vital role in early atherogenesis. BDMC reduced oxLDL uptake most effectively, while CUR was the best inhibitor for CD36, scavenger receptor A, and lectin-like oxidized LDL receptor-1 expression during phorbol 12-myristate 13-acetate (PMA)-induced THP-1 differentiation. In PMA-differentiated THP-1 macrophages, CUR and DMC effectively induced heme oxygenase-1 expression, but attenuated oxLDL-induced CD36 expression, leading to decreased oxLDL uptake. CONCLUSION: This result indicates curcuminoids, despite structural similarities, exert different atheroprotective effects. Curcuminoids, especially CUR and DMC, are hormetic compounds, which induce Phase II enzyme expression and confer resistance to PMA- and oxLDL-induced scavenger receptor expression and activity.

Arsenic modulates heme oxygenase-1, interleukin-6, and vascular endothelial growth factor expression in endothelial cells: roles of ROS, NF-κB, and MAPK pathways.

Chronic arsenic exposure has been linked to an increased risk of vascular diseases. To clarify the molecular mechanisms through which arsenic causes injuries to blood vessels, we analyzed the effects of arsenic trioxide on the cytotoxicity, intracellular reactive oxygen species (ROS), the expression of related genes, and signaling pathways involved in the SVEC4-10 mouse endothelial cells. Arsenic dose-dependently caused SVEC4-10 cell death, which is completely inhibited by α-lipoic acid (LA), a thioreductant, but partially ameliorated by Tiron, a potent superoxide scavenger. The mRNA levels of heme oxygenase-1 (HO-1), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF) were significantly increased by arsenic. The up-regulation of these can be blocked by LA instead of Tiron, suggesting ROS is not important in their increase. HO-1 competitive inhibitor zinc protoporphyrin improved the cytotoxicity of arsenic in an inverted-U dose-response curve, indicating the biphasic hormetic effect of HO-1. HO-1 siRNA decreased VEGF expression in response to arsenic. Arsenic exposure also enhanced NF-E2-related factor 2 (Nrf2) expression and increased activation of nuclear factor-κB (NF-κB). NF-κB inhibitor Bay 11-7082 reduced arsenic-mediated expression of HO-1 and IL-6. Selective blocking of the MAPK pathways with p38 inhibitor SB203580 significantly decreased arsenic-induced HO-1 and VEGF expression, while JNKs inhibitor SP600125 increased IL-6 expression. These results suggest that in arsenic-treated SVEC4-10 cells, HO-1 expression is mediated through Nrf2-, NF-κB-, and p38 MAPK-dependent signaling pathways and serves as an upstream regulator of VEGF. IL-6 expression is regulated by NF-κB and JNKs. In conclusion, oxidative stress may be associated with arsenic-induced cytotoxicity and endothelial gene up-regulation, but signaling transduction dominates the direct effects of ROS.

An immune-active tumor microenvironment favors clinical response to ipilimumab.

PURPOSE: Ipilimumab, a fully human monoclonal antibody specific to CTLA-4, has been shown to improve overall survival in metastatic melanoma patients. As a consequence of CTLA-4 blockade, ipilimumab treatment is associated with proliferation and activation of peripheral T cells. To better understand various tumor-associated components that may influence the clinical outcome of ipilimumab treatment, gene expression profiles of tumors from patients treated with ipilimumab were characterized. EXPERIMENTAL DESIGN: Gene expression profiling was performed on tumor biopsies collected from 45 melanoma patients before and 3 weeks after the start of treatment in a phase II clinical trial. RESULTS: Analysis of pre-treatment tumors indicated that patients with high baseline expression levels of immune-related genes were more likely to respond favorably to ipilimumab. Furthermore, ipilimumab appeared to induce two major changes in tumors from patients who exhibited clinical activity: genes involved in immune response showed increased expression, whereas expression of genes for melanoma-specific antigens and genes involved in cell proliferation decreased. These changes were associated with the total lymphocyte infiltrate in tumors, and there was a suggestion of association with prolonged overall survival in these patients. Many IFN-γ-inducible genes and Th1-associated markers showed increased expression after ipilimumab treatment, suggesting an accumulation of this particular type of T cell at the tumor sites, which might play an important role in mediating the antitumor activity of ipilimumab. CONCLUSIONS: These results support the proposed mechanism of action of ipilimumab, suggesting that cell-mediated immune responses play an important role in the antitumor activity of ipilimumab.

Lipoic acid ameliorates arsenic trioxide-induced HO-1 expression and oxidative stress in THP-1 monocytes and macrophages.

Inorganic arsenic is a common environmental contaminant; chronic exposure to arsenic can alter the physiology of various key immune cells, particularly macrophages. The aim of this research is to elucidate the key parameters associated with arsenic-induced toxicity and investigate the potential and mechanism of α-lipoic acid (LA), a potent thioreducant, for reducing the toxicity in human promonocytic THP-1 cells. We found that a non-lethal concentration of arsenic trioxide (1 µM) significantly induced the expression of heme oxygenase-1 (HO-1), a response biomarker to arsenic, without stimulating measurable superoxide production. Co-treatment of cells with the HO-1 competitive inhibitor zinc protoporphyrin (Znpp) potentiated arsenic-induced cytotoxicity, indicating that HO-1 confers a cytoprotective effect against arsenic toxicity. In addition, low concentrations of arsenic trioxide (1 and 2.5 µM) markedly inhibited monocyte-to-macrophage differentiation and expression of macrophage markers. Treatment of cells with LA attenuated arsenic trioxide-induced cytotoxicity and HO-1 over-expression and restored the redox state. In addition, LA neutralized arsenic trioxide-inhibition of monocyte maturation into macrophages and reversed the expression and activity of scavenger receptors. In conclusion, the cytotoxicity of arsenic trioxide is associated with an imbalance of the cellular redox state, and LA can protect cells from arsenic-induced malfunctions either through its reducing activity, direct interacting with arsenic or stimulating other unidentified signaling pathways.

Regulation of heme oxygenase-1 expression and MAPK pathways in response to kaempferol and rhamnocitrin in PC12 cells.

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. Our aim of research is to investigate the protective effects and mechanisms of kaempferol and rhamnocitrin (kaempferol-7-methyl ether) on oxidative damage in rat pheochromocytoma PC12 cells induced by a limited supply of serum and hydrogen peroxide (H2O2). The current result demonstrated that kaempferol protected PC12 cells from serum deprivation-induced apoptosis. Pretreatment of cells with kaempferol also diminished intracellular generation of reactive oxygen species (ROS) in response to H2O2 and strongly elevated cell viability. RT-Q-PCR and Western blotting revealed that kaempferol and rhamnocitrin significantly induced heme oxygenase (HO)-1 gene expression. Addition of zinc protoporphyrin (Znpp), a HO-1 competitive inhibitor, significantly attenuated their protective effects in H2O2-treated cells, indicating the vital role of HO-1 in cell resistance to oxidative injury. While investigating the signaling pathways responsible for HO-1 induction, we observed that kaempferol induced sustained extracellular signal-regulated protein kinase 1/2 (ERK1/2) in PC12 cells grown in low serum medium; while rhamnocitrin only stimulated transient ERK cascade. Addition of U0126, a highly selective inhibitor of MEK1/2, which is upstream of ERK1/2, had no effect on kaempferol- or rhamnocitrin-induced HO-1 mRNA expression, indicating no direct cross-talk between these two pathways. Furthermore, both kaempferol and rhamnocitrin were able to persistently attenuate p38 phosphorylation. Taking together, the above findings suggest that kaempferol and rhamnocitrin can augment cellular antioxidant defense capacity, at least in part, through regulation of HO-1 expression and MAPK signal transduction.

Fisetin, morin and myricetin attenuate CD36 expression and oxLDL uptake in U937-derived macrophages.

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds, including fisetin, morin and myricetin, on the susceptibility of low-density lipoprotein (LDL) to oxidative modification and on oxLDL uptake in macrophages. The results demonstrated that fisetin had stronger inhibitory activity than the other two on inhibiting Cu(2+)-mediated LDL oxidation measured by thiobarbituric acid-reactive substances assay (TBARS), conjugated diene formation and electrophoretic mobility. The class B scavenger receptor, CD36, to which oxLDL binds, is present in atherosclerotic lesions. Treatment of U937-derived macrophages with myricetin (20 microM) significantly inhibited CD36 cell surface protein and mRNA expression (p<0.01). Fisetin, morin and myricetin (20 microM) also reduced the feed-forward induction of CD36 mRNA and surface protein expression by PPARgamma. The inhibition of CD36 by flavonols was mediated by interference with PPARgamma activation thus counteracting the deleterious autoamplification loop of CD36 expression stimulated by PPARgamma ligand. All three flavonols (10 and 20 microM) markedly decreased the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled oxLDL uptake in U937-derived macrophages dose-dependently. Current evidences indicate that fisetin, morin and myricetin not only prevent LDL from oxidation but also block oxLDL uptake by macrophages at least in part through reducing CD36 gene expression on macrophages. In conclusion, flavonols may play a role in ameliorating atherosclerosis.

Oral administration of submerged cultivated Grifola frondosa enhances phagocytic activity in normal mice.

Grifola frondosa fruiting body (Maitake) has been used as a dietary supplement due to its antitumour and immunomodulatory properties. The aim of this study was to evaluate the immunomodulatory effects of orally administered submerged cultivated G. frondosa mixture, including both mycelium and culture broth, in a healthy murine model. Composition analyses showed that submerged cultivated G. frondosa mixture contained only 32.48% carbohydrate, which was less than half of fruiting bodies. The content of adenosine, a potential immunomodulatory agent in medicinal mushrooms, was 2.8 mg g(-1). After feeding 8-week-old female BALB/cByJ mice with AIN-93G diet containing 0% (C), 1% (G1), 3% (G3) or 5% (G5) (wt/wt) G. frondosa mixture for 31 days, neither body weight nor the outward appearance of organs showed any significant difference among different diet groups. Splenocyte subpopulation, mitogen-activated cytokine release and splenic NK activity were not affected by G. frondosa administration, either. On the other hand, the phagocytic activity was enhanced in leucocytes of groups G3 and G5, without exerting detectable levels of serum proinflammatory cytokines. These results suggested that oral administration of submerged cultivated G. frondosa mixture may enhance host innate immunity against foreign pathogens without eliciting adverse inflammatory response.

Distinctive antioxidant and antiinflammatory effects of flavonols.

The antioxidant and antiinflammatory effects of flavonols have been suggested to be structure-related. Results revealed that selected flavonols, including fisetin (F), kaempferol (K), morin (MO), myricetin (MY), and quercetin (Q), exhibited distinctive free radical scavenging properties against different kinds of free radicals. The H donation (DPPH bleaching) potential was Q > F approximately equals MY > MO > K, indicating that the presence of a 3',4'-catechol moiety in the B ring correlated with high activity. The 4'-OH in the B ring was suggested to be important for reducing xanthing/xanthine oxidase-generated superoxide; while an additional OH moiety on the ortho sites (3' or 5') attenuated the effect as the observed inhibitory potency was K approximately equals MO > Q > F > MY. The relative inhibitory effect for Fenton-mediated hydroxyl radical was K approximately equals MO approximately equals Q > F > MY. This result implies the involvement of 4-keto, 5-OH region in Fe++ chelating and the negative effect of pyrogallol moiety in the B ring. Similar to the inhibitory activity against a N-formyl-methionyl-leucyl-phenylalanine (f-MLP)-stimulated oxidative burst in human polymorphonuclear neutrophils (PMN), our result showed that the structural peculiarity of the di-OH in the B ring obviously rendered F, Q, and MO more potent as ROS inhibitors than MY and K, which have tri- and mono-OH in the B ring, respectively. All of the previous data indicated that the structure prerequisite to reinforce the free radical scavenging activity varies with the type of free radical. We further analyzed the effects of flavonols on nitric oxide (NO) production in endotoxin-stimulated murine macrophages, RAW264.7 cells. Results showed that all flavonols (up to 10 microM) inhibited NO production without exerting detectable cytotoxicity. F, K, and Q dose-dependently repressed iNOS mRNA expression and prostaglandin E2 (PGE2) production, in part through an attenuating NF-kappaB signaling pathway. This result indicates that flavonols, despite structural similarity, have different antioxidant and antiinflammatory effects.

Immunomodulatory properties of Grifola frondosa in submerged culture.

Maitake (Grifola frondosa) is a popular mushroom in Asia for its tasty flavor and immune-stimulating property. The aim of the study is to investigate the innate immunity augmentation effects of different extracts of mycelia and culture filtrate from G. frondosa in submerged cultures. The hot water extract of mycelia showed the strongest cytokine induction effect as a function of its concentration in human whole blood culture. The most potent fractions of hot water extract, Fr. I and II, were mainly composed of polysaccharides with molecular masses of 43-140 and 13-38 kDa, respectively. These fractions (0.025 mg/mL) showed marked activity in enhancing phagocytosis of human polymorphonuclear neutrophils (PMN). In parallel, the expression of CD11b, an early marker of PMN activation, was also up-regulated dose dependently. This result suggested that complement receptor 3 was primed by these fractions. In addition to activation of phagocytes, these bioactive fractions also increased human peripheral blood natural killer cell cytotoxicity. These results imply that the relatively low molecular mass polysaccharides isolated from mycelia of G. frondosa can enhance innate immunity in vitro and therefore may serve as biological response modifiers.

Immunomodulatory effect of Glossogyne tenuifolia in murine peritoneal macrophages and splenocytes.

Glossogyne tenuifolia Cass., a medicinal plant native to Taiwan, is traditionally used as an anti-inflammatory remedy. Oleanolic acid and luteolin-7-glucoside have been previously identified as active components of Glossogyne tenuifolia in the murine macrophage-like cell line, RAW264.7. Current study investigates the effect and mechanism of the ethanol extract of Glossogyne tenuifolia (GT) and its major constituents on the release of inflammatory mediators in activated elicited murine peritoneal macrophages and splenocytes. Our results showed that GT (up to 0.15 mg/ml) inhibited the production of proinflammatory mediators, TNF-alpha, IL-1beta, IL-6, nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in LPS-activated macrophages, and IFN-gamma in PHA-activated splenocytes. GT also inhibited LPS-activated murine iNOS and COX-2 promoter activities in transiently transfected RAW264.7 cells. The major constituents, oleanolic acid and luteolin-7-glucoside, as well as its aglycone, luteolin, inhibited the release of NO, PGE(2), TNF-alpha and IL-1beta in activated peritoneal macrophages. However, only luteolin-7-glucoside and luteolin were able to reduce IFN-gamma release in PHA-stimulated splenocytes. To further investigate the possible mechanisms that interfere with LPS- and PHA-signaling, this study focused on nuclear factor-kappaB activation signaling pathways. Our results demonstrate that GT (0.075-0.15 mg/ml) treatment reduces nuclear factor-kappaB (NF-kappaB) DNA binding activity, as demonstrated by electrophoretic mobility shift assay (EMSA). Collectively, the results suggest that GT inhibits proinflammatory mediator synthesis in activated murine peritoneal macrophages and splenocytes, in part through NF-kappaB-dependent pathways.

Antioxidant activity of Glossogyne tenuifolia.

Glossogyne tenuifolia is a native traditional anti-inflammatory herb in Taiwan. It has previously been shown that the ethanol extract of G. tenuifolia (GT) inhibited the LPS-induced inflammatory mediator release from murine macrophage cell line and human whole blood. In the present work, the ethanol extracts of G. tenuifolia and its major constituent, luteolin-7-glucoside, were shown to be scavengers of 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radicals. Moreover, copper-induced low-density lipoprotein oxidation was suppressed by GT and luteolin-7-glucoside as measured by decreased formation of malondialdehyde and conjugated diene as well as reduced electrophoretic mobility. GT and luteolin-7-glucoside were also against N-formyl-methionyl-leucyl-phenylalanine-induced reactive oxygen species (ROS) production in human polymorphonuclear neutrophils and peripheral blood mononuclear cells. In summary, these data indicated that GT is a potential ROS scavenger and may prevent atherosclerosis via inhibiting LDL oxidation or ROS production in human leukocytes. Moreover, luteolin-7-glucoside may serve as the active principal of GT.