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Research supports the theory that the microbiome of plants and mushrooms produce potent activators of pathogen recognition receptors which are principal contributors to the stimulation of macrophages. We have previously reported that the in vitro macrophage stimulatory activity of water-soluble extracts from 13 different types of edible mushrooms is predominantly due to bacterial components originating from the naturally occurring bacterial communities within these materials. The purpose of the current study was to further investigate the bacterial-dependent activity of the water-soluble extracts and assess whether these 13 types of mushrooms contain water-insoluble beta glucans that activate the dectin-1b signaling pathway. Activity of the water-soluble extracts was predominantly due to Toll-like receptor 2 (TLR2) and TLR4 agonists. For dectin-1b-dependent activity (indicative of water-insoluble beta glucans), culinary mushrooms (Agaricus bisporus varieties) were essentially inactive, whereas most of the medicinal mushrooms (Lentinula edodes, Grifola frondosa, Hypsizygus marmoreus varieties, Flammulina velutipes) exhibited potent activation. A. bisporus samples with no detectable dectin-1b-dependent activity had yeast colony forming units that were 687 times lower than L. edodes exhibiting high activity, indicating that the active insoluble beta glucans are derived from colonizing yeast. In addition, co-stimulation of macrophages with the TLR agonists and insoluble beta glucan was found to result in a synergistic enhancement of in vitro cytokine production. Taken together, these findings indicate that the in vitro macrophage activating potential of edible mushrooms is due to the collaborative interaction of water-soluble TLR agonists (derived from colonizing bacteria) and water-insoluble beta glucans (derived from colonizing yeast).

The purpose of this study was to determine if a methanolic extract of the Pulsatilla patens (L.) Mill. can inhibit the progression of cancer through the modulation of cancer-related metabolic signaling pathways. We analyzed a panel of 13 inducible luciferase reporter gene vectors which expression is driven by enhancer elements that bind to specific transcription factors for the evaluation of the activity of cancer signaling pathways. The root extract of P. patens exhibited strong inhibition of several signaling pathways in HeLa cells, a cervical cancer cell line, and was found to be the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt and Hdghog, at a concentration of 40 µg/mL. The methanolic extracts of P. patens enhanced apoptotic death, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of Pulsatilla species on cancer signaling pathways. Further, our detailed phytochemical analysis of the methanolic extracts of the P. patens allowed to deduce that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.

Enthusiasm for the use of dietary bioactive compounds as chemopreventive agents and adjuvants for current therapies has increased laboratory research conducted on several types of cancers including Head and Neck Squamous Cell Carcinoma (HNSCC). The green chemoprevention movement is a modern approach to highlight healthy lifestyle changes that aim to decrease the incidence of HNSCC. A healthy diet can be an effective way to prevent the development of oral cancers. Discovery of the naturally occurring plant based compounds called phytochemicals has facilitated the development of new treatment strategies for patients that are at risk for, or have developed HNSCC. Many of these compounds have been shown to elicit very potent anti-carcinogenic properties. While there are many compounds that have been studied, the compounds from two specific categories of phytochemicals, phenolics (resveratrol, EGCG, curcumin, quercetin, and honokiol) and glucosinolates (sulforaphane, PEITC and BITC), are emerging as potent and effective inhibitors of oral carcinogenesis. These compounds have been shown to inhibit HNSCC growth through a variety of mechanisms. Research has demonstrated that these compounds can regulate cancer cell proliferation through the regulation of multiple cell signaling pathways. They can impede cell cycle progression, induce differentiation and apoptosis, prevent angiogenesis, and inhibit cancer cell invasive and metastatic properties. They can protect normal cells during treatment and reduce the damage caused by chemotherapy and radiotherapy. This review aims to provide an overview of some of the most effective phytochemicals that have the potential to successfully prevent and treat head and neck squamous cell carcinoma.

The anticancer activities of Rubia cordifolia and its constituents have been reported earlier, but their influence on the crosstalk of complex cancer-related signaling metabolic pathways (i.e., transcription factors, TF) has not yet been fully investigated. In this study, R. cordifolia root extract was subjected to the cancer signaling assay based bioactivity-guided fractionation, which yielded the following compounds viz., three anthraquinones, namely alizarin (1), purpurin (2), and emodin (3), two lignans, namely eudesmin (4) and compound 5, and two cyclic hexapeptides, namely deoxybouvardin RA-V (6), and a mixture of 6+9 (RA-XXI). The structures of the isolated compounds were determined by NMR spectroscopy and HRESIMS. The isolated compounds 1, 2, 3, 6, and a mixture of 6+9 were tested against a panel of luciferase reporter genes that assesses the activity of a wide-range of cancer-related signaling pathways. In addition, reference anthraquinones viz., chrysophanol (11), danthron (12), quinizarin (13), aloe-emodin (14), and &alpha, lapachone (15) were also tested. Among the tested compounds, the cyclic hexapeptide 6 was found to be very active against several signaling pathways, notably Wnt, Myc, and Notch with IC50 values of 50, 75, and 93 ng/mL, respectively. Whereas, the anthraquinones exhibited very mild or no inhibition against these signaling pathways. Compound 6 being the most active, we tested it for stability in simulated intestinal (SIF) and gastric fluids (SGF), since the stability in biological fluid is a key short-coming of cyclic hexapeptides. The anticancer activity of 6 was found to remain unchanged before and after the treatment of simulated gastric/intestinal fluids, indicating that RA-V was stable. As a result, it could be bioavailable when orally used in therapeutics and possibly a drug candidate for cancer treatment. The mechanism for the preferential inhibition of these pathways and the possible crosstalk effect with other previously reported signaling pathways has been discussed.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

We previously demonstrated that extracts from Echinacea purpurea material varied substantially in their ability to activate macrophages in vitro and that this variation was due to differences in their content of bacterial components. The purpose of the current study was to identify soil conditions (organic matter, nitrogen, and moisture content) that alter the macrophage activation potential of E. purpurea and determine whether these changes in activity correspond to shifts in the plant-associated microbiome. Increased levels of soil organic matter significantly enhanced macrophage activation exhibited by the root extracts of E. purpurea (p E. purpurea. Bacterial communities also differed significantly between root materials cultivated in soils with different levels of organic matter (p E. purpurea roots. Since ingestion of bacterial preparation (e.g., probiotics) is reported to impact human health, it is likely that the medicinal value of Echinacea is influenced by cultivation conditions that alter its associated bacterial community.

Through genetic analysis of the Dahl salt-sensitive (SS) rat, a model of hypertension and chronic kidney disease (CKD), Arhgef11, a Rho guanine nucleotide exchange factor was implicated in kidney injury. Arhgef11, via exchange of GDP for GTP, plays a role in the activation of RhoA signaling cascades through a number of cell stimuli that impact cytoskeletal structure and influence cell-cell contacts and promotes cell transformation. Previously, we demonstrated that reduced Arhgef11 expression/protein function in an SS- Arhgef11 SHR -minimal congenic strain resulted in significantly decreased proteinuria, fibrosis, and improved renal hemodynamics compared to SS-WT, without impacting BP. More recently, an SS-Arhgef11 -/- knockout rat model was studied. On low-salt (0.3% NaCl), SS-Arhgef11 -/- animals demonstrated reduced proteinuria and renal injury, with no impact on BP versus SS-WT. In contrast, SS-Arhgef11 -/- animals on an elevated-salt diet (2% NaCl) demonstrated a significant (pArhgef11 (on low-salt) similarly leads to renoprotection, whereas the loss of Arhgef11 (vs reduced expression) leads to blunting of salt-induced elevations in BP vs SS-WT. These animal studies, in combination with in vitro work, suggest that inhibition of Arhgef11-RhoA could be an effective therapeutic for CKD. Using an in vitro cell screen, several hundred natural product compounds were tested for ability to inhibit Arhgef11/RhoA activity and identified 3 compounds. A pilot study done using one compound (sufficient for a one-week study) was very encouraging as animals treated with XTL-019-G7 (20mg/kg/day) exhibited an ~25% reduction in proteinuria compared to vehicle (VEH) treated animals (70±8.5 versus 95±9.6 mg/24hours, p=0.07), demonstrating that in vivo testing is feasible and that the compound warrants further testing. In summary, genetic analysis of a model of CKD identified a gene/pathway involved in kidney injury that served as a target to screen natural product derived small molecules, and may ultimately lead to a new treatment for CKD.

Our previous studies indicate that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea depends on bacterial components. In the present study, total bacterial load was determined within E. purpurea samples and ranged from 6.4 × 10(6) to 3.3 × 10(8) bacteria/g of dry plant material. To estimate total bacterial load, we developed a PCR-based quantification method that circumvents the problems associated with nonviable/nonculturable cells (which precludes using plate counts) or the coamplification of mitochondrial or chloroplast DNA with the use of universal bacterial primers (which precludes the use of qPCR). Differences in total bacterial load within Echinacea samples were strongly correlated with the activity (NF-κB activation in THP-1 cells) and content of bacterial lipopolysaccharides within extracts of this plant material. These results add to the growing body of evidence that bacteria within Echinacea are the main source of components responsible for enhancing innate immune function.

Recent studies have indicated that a major contributor to the innate immune enhancing properties of some medicinal plants is derived from the cell wall components of bacteria colonizing these plants. The purpose of the current study was to assess if the bacteria present within edible and medicinal mushrooms substantially contribute to the innate immune stimulating potential of these mushrooms. Whole mushrooms from thirteen types of edible fungi and individual parts from Agaricus bisporus were analyzed for in vitro macrophage activation as well as bacterial lipopolysaccharides (LPS) content, cell load, and community composition. Substantial variation between samples was observed in macrophage activation (over 500-fold), total bacterial load (over 200-fold), and LPS content (over 10 million-fold). Both LPS content (ρ = 0.832, p < 0.0001) and total bacterial load (ρ = 0.701, p < 0.0001) correlated significantly with macrophage activation in the whole mushroom extracts. Extract activity was negated by treatment with NaOH, conditions that inactivate LPS and other bacterial components. Significant correlations between macrophage activation and total bacterial load (ρ = 0.723, p = 0.0001) and LPS content (ρ = 0.951, p < 0.0001) were also observed between different tissues of Agaricus bisporus. Pseudomonas and Flavobacterium were the most prevalent genera identified in the different tissue parts and these taxa were significantly correlated with in vitro macrophage activation (ρ = 0.697, p < 0.0001 and ρ = 0.659, p = 0.0001, respectively). These results indicate that components derived from mushroom associated bacteria contribute substantially to the innate immune enhancing activity exhibited by mushrooms and may result in similar therapeutic actions as reported for ingestion of bacterial preparations such as probiotics.

Tissue culture techniques have been used to establish and maintain a repository of medicinal Echinacea. In vitro clones obtained from hypocotyls of germinated seeds, varied macroscopically, microscopically and exhibited variation in immune enhancing activity. Two in vitro produced clones of Echinacea tennesseensis (Beadle) Small (ETN 03 and ETN 11) were identified as high and low activity based on the activation of human monocytes. Phenotypic analyses of ETN 03 and ETN 11 clones were done using AFLP (Amplified Fragment Length Polymorphism) assay. Results of the AFLP assay revealed that no mutation has occurred during in vitro multiplication, storage, and acclimatization into soil. Plants of ETN 03, ETN 11 clones were cultivated for two growing seasons. Extracts of their dry leaves and roots exhibited immune enhancing activity; however, the variation in activity noticed between clones during micropropagation diminished and was no longer statistically relevant.

WOS: 000264114300012, PubMed ID: 18597088, Purpose Breast cancer is one of the most prevalent woman cancers. Genomic instability, accumulative mutations, and subsequent changes in intracellular signaling cascades play key roles in the development of human breast cancers. Activation of nuclear factor-kappa B(NF-kappa B) has been implicated in oncogenesis of breast cancers and is known to be associated with resistance to anticancer agents and apoptosis. Blocking NF-kappa B signaling may represent a therapeutic strategy in breast cancer therapy. The objective of this study is to investigate the in vitro effects of epoxypseudoisoeugenol-2-methyl butyrate (EPB), a phenylpropranoid isolated from Pimpinella corymbosa, on the activation of NF-kappa B, cell growth, cell cycle progression and apoptosis in MCF-7 (estrogen-dependent) and BT-549 (estrogen-independent) breast cancer cells. Methods Transcriptional activity of NF-kappa B was measured by cell based reporter gene assay. Cell proliferation was determined by MTT assay. Cell cycle analysis was carried out by flow cytometry and apoptosis was observed by DAPI staining assy. Results EPB inhibited the NF-kappa B-mediated transcription activity induced by tumor necrosis factor-alpha (TNF-alpha) and phorbol myristate acetate (PMA) in MCF-7 cells. EPB also inhibited constitutive NF-kappa B transcriptional activity in BT-549 cells. EPB inhibited the proliferation of both MCF-7 and BT-549 cells in a concentration- and time-dependent manner. EPB induced cell cycle arrest in G(1)/G(0) phase and apoptosis in both MCF-7 and BT 549 cells. Conclusions These in vitro results indicated that EPB has a potential for use against both hormone-dependent and hormone-independent breast cancers and its effects seem to be mediated by inhibiting the NF-kappa B activity., USDA Agricultural Research Service SpeciWc Cooperative Agreement [58-6408-2-0009], This study was supported in part by USDA Agricultural Research Service SpeciWc Cooperative Agreement NO. 58-6408-2-0009.

WOS: 000248556900006, PubMed ID: 17450505, Pimpinella essential oils and isolated compounds were screened for their inhibitory activity against NF-kappa B mediated transcription in SW1353 cells. Twelve oils were effective in inhibiting NF-kappa B mediated transcription. Especially the roots of P. corymbosa, P. tragium and P. rhodanta showed potent activities with IC50 values of 2, 3 and 6 mu g/mL, respectively. Five pure compounds, 7 (4-(2-propenyl)phenylangelate), 12 (4-(3methyloxiranyl)phenyltiglate), 17 (4-methoxy-2-(3-methyloxiranyl)phenyl isobutyrate), 18 (4-methoxy-2-(3methyloxiranyl)phenylangelate) and 21 (epoxy pseudoisoeugenol-2-methylbutyrate) inhibited NF-kappa B mediated transcription with IC50 values of 5.5, 1.2, 0.01, 3.6 and 11 mu g/mL, respectively. None of the compounds were cytotoxic to mammalian cells. These findings add significant information to the pharmacological activity of Pimpinella species and their beneficial effects and use in disease prevention especially those related to inflammation. Copyright (c) 2007 John Wiley & Sons, Ltd.

Spirulina (Spirulina platensis) is a dietary supplement valued for its immune-enhancing properties. We previously reported that the immunostimulatory effect of spirulina can be traced to a high-molecular- weight polysaccharide fraction. This fraction, labeled Immolina, activates nuclear factor kappa-B in human monocytic THP-1 cells and increases expression of proinflammatory cytokines.To characterize further the immunostimulatory effects of Immolina on THP-1 cells, we evaluated its effect on genes encoding the chemokines interleukin (IL)-8, MCP-1, MIP-1alpha, MIP-1beta, IP-10, the cytokines tumor necrosis factor (TNF)-alpha, IL-1beta, and the enzyme cyclo-oxygenase-2 (COX-2).THP-1 cells were exposed to concentrations of Immolina ranging from 1 ng/mL to 100 microg/mL and changes in gene expression were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). For comparison, THP-1 cells were activated with 1 ng/mL of TNF-alpha, 10 ng/mL of IL-1beta, or 10 ng/mL of lipopolysaccharide using the same assay conditions. To assess the response of THP-1 cells to Immolina at the protein level, we probed culture supernatants using a cytokine array immunoblot assay.RT-PCR analysis revealed that Immolina dose-dependently increased the expression of all 5 chemokines tested as well as the expression of TNF-alpha, IL-1beta, and COX-2. The cytokine array immunoblot assay revealed an increase in the chemokines IL-8 and MIP-1beta. Thymidine uptake experiments verified that Immolina did not affect the viability and growth rate of THP-1 cells.The results of the experiments demonstrate that Immolina activates THP-1 cells in a manner that is consistent with the recruitment of diverse populations of leukocytes in response to inflammatory and infectious signals.

Structure activity relationships of aristolochic acid analogues: Toxicity in cultured renal epithelial cells. Background Aristolochia species are nephrotoxic and carcinogenic. Recent studies showed that aristolochic acid (AA) could induce acute renal failure and tubular lesions in several species and available evidences demonstrate the unequivocal role of AA in so called Chinese herbs nephropathy. Methods A series of AA derivatives isolated from Aristolochia spp. were analyzed for their nephrotoxic potential using the neutral red dye exclusion assay in cultures of LLC-PK 1 cells. The structural relationships between AA I and its analogues were compared with their cytotoxic effects to predict structural determinants for AA toxicity. Further, caspase-3 assay was performed on toxic compounds to determine if caspases, the enzymes that play a critical role in apoptosis are involved in AA-induced cytotoxicity. Results AA I was found to be most toxic followed by AA II, AA VIIIa, and AA Ia in decreasing levels of toxicity. The other compounds, nitrophenanthrene carboxylic acid analogues of AA I, aristolactams, and other derivatives did not exhibit considerable toxicity. The results showed significant relationships between cytotoxicity of AA compounds and the localization of functional groups in their structure. Analogues containing hydroxyl groups diminished cytotoxicity. The demethylated analogues of AA I are markedly less active. The negative impact on cytotoxicity was found on nitroreduction of AA I. AA induced caspase activation was also observed. Conclusion These cytotoxic data suggest that the nitro and methoxy groups are critical determinants of nephrotoxicologic potency of AA.

The present study was undertaken to determine the effects of catecholamines, agonists, and antagonists of β-adrenergic receptors (AR) in the LNCaP cell line. Changes in cellular cyclic adenosine-3′,5′-monophosphate (cAMP) levels were quantified by the use of a 6 cAMP response element (CRE)-luciferase reporter gene assay. LNCaP cells were transiently transfected with this gene construct, incubated in 96-well microtiter plates for 24 hr, and then treated with β-AR agonists and/or antagonists for 4 hr. The rank order of potency for catecholamines and known β-AR agonists was terbutaline (3.31 nM )> isoproterenol (8.31 nM )≥ fenoterol (15 nM )= epinephrine (16.2 nM )> norepinephrine (77.5 nM )> BRL -37344 [( R ∗ , R ∗ )-(±)4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxy acetic acid, sodium salt] (1000 nM )> dobutamine (1770 nM )> CGP 12177 (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2 H -benzimidazole-2-one hydrochloride) (inactive). The non-selective β 1 -/-β 2 -AR antagonists; propranolol and CGP 12177, at 10 −7 M, inhibited luciferase activity induced by these agonists by 80–96%. Propranolol blocked isoproterenol-induced luciferase responses in a competitive manner ( K B =1.4 nM). In addition, isoproterenol-activated luciferase expression was blocked more potently by ICI 118,551 [(±)-1-[2,3-(dihydro-7-methyl-1 H -inden-4-yl)oxy]-3-[(1-methylethy) amino]-2-butanol], a β 2 -AR antagonist than by ICI 89,406 [(±)- N -[2-[3-(2-cyanophenoxy-)]-2-hydroxypropylamino]ethyl- N -phenylurea], a β 1 -AR antagonist, giving K B values of 1.07 and 161 nM, respectively. These results suggest that the β 2 -AR is the major subtype mediating catecholamine-induced cAMP changes in LNCaP cells.

A phytochemical investigation of the CHCl(3) fraction of an ethanol extract of the root of Guatteria multivenia furnished nine compounds, of which four are sesquiterpenes (1-4) and five are alkaloids (5-9). Of the four sesquiterpenes, two are new (1, 3), named guatterin A (1) and dihydromadolin-K (3), and two are known (2, 4), identified as madolin-K (2) and madolin-W (4). The five known alkaloids were identified as liriodenine (5), lysicamine (6), lanuginosine (7), guadiscine (8), and O-methylpallidine (9). All the known compounds were isolated from this species for the first time. Structures of the new compounds were determined by extensive NMR studies, including DEPT, COSY, HMQC, HMBC, and NOESY. Compound 7 showed weak inhibitory effect against Candida albicans secreted aspartic proteases (SAP) with IC(50) of 45 microg/mL. Compound 5 was found to have antimicrobial activity against C. albicans, Cryptococcusneoformans, Staphylococcus aureus, and methicillin-resistant S. aureus (MRS) with IC(50)/MIC values of 3.5/6.25, 2.0/12.5, 2.0/3.13, and 2.0/3.13 microg/mL, respectively.

A growing body of research indicates that oral administration of bacteria (such as probiotics) can exhibit a protective effect against influenza A (H1N1) viral infection in mice. In the present study, we used a mouse model to examine whether oral administration of Immulina(®), a commercial extract from the cyanobacteria Arthrospira (Spirulina) platensis, can reduce the severity of illness resulting from influenza A (H1N1) viral infection. The main active compounds within Immulina(®) are bacterial Braun-type lipoproteins that activate innate immune cells through a toll-like receptor (TLR) 2-dependent pathway. Mice that were fed Immulina(®) for 30 days before and 21 days after infection with influenza A (H1N1) virus exhibited a statistically significant reduction in the severity of infection. Compared to the control group, Immulina(®)-fed mice exhibited less weight loss, increased appetite, decreased clinical signs of disease, and lower lung histopathology scores. The results from the present study adds to the increasing evidence that oral administration of bacterial components that activate innate immune cells, whether derived from a bacterial preparation (probiotics or cyanobacteria) or from plant material containing endophytic bacteria, can exhibit a protective effect against influenza A (H1N1) viral infection.

Previous studies indicate that Immulina®, a commercial extract of Arthrospira (Spirulina) platensis, is a potent activator of innate immune cells and that bacterial Braun-type lipoproteins (a principal toll-like receptor 2 ligand) are the main active components within this product. In the present study, mice fed Immulina® for 30 days prior to infection with influenza A (H1N1) virus exhibited a statistically significant reduction in the severity of infection. Compared to the control group, Immulina®-fed mice exhibited more appetite, reduced weight loss, diminished clinical signs of disease and lower lung histopathology scores. This study adds to the growing body of evidence that oral administration of bacterial components that activate immune cells, whether derived from a bacterial preparation (probiotics or cyanobacterial extracts) or from bacteria associated with plants as endophytes, can exhibit a protective effect against influenza A (H1N1) viral infection. Acknowledgements: This research was funded by Scandinavian Clinical Nutrition AB (Stockholm, Sweden). NDP and DSP were partially supported by the USDA, Agricultural Research Service Specific Cooperative Agreement Nos. 58 – 6408 – 6-067 and 58 – 6408 – 1-603 (NCNPR/University of Mississippi).

This research describes the identification of three new high molecular weight polysaccharide preparations isolated from food-grade microalgae that are potent activators of human monocytes/macrophages: "Immulina" from Spirulina platensis, "Immunon" from Aphanizomenon flos-aquae, and "Immurella" from Chlorella pyrenoidosa. These polysaccharides are structurally complex and have estimated molecular weights above ten million daltons. All three polysaccharides are highly water soluble and comprise between 0.5 % and 2.0 % of microalgal dry weight. Immunostimulatory activity was measured using a transcription factor-based bioassay for nuclear factor kappa B (NF-kappa B) activation in THP-1 human monocytes/macrophages. Using this system the EC(50) values for these microalgal polysaccharides are between 20 and 110 ng/ml (about 10pM). THP-1 activation was confirmed by measuring immune cytokine mRNA induction using reverse transcriptase-polymerase chain reaction (RT-PCR). Each polysaccharide substantially increased mRNA levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). These polysaccharides are between one hundred and one thousand times more active for in vitro monocyte activation than polysaccharide preparations that are currently used clinically for cancer immunotherapy.

There exists a growing body of research which indicates that antimitotics such as taxol and colchicine influence cytokine gene expression. In the present study we examined the effect of podophyllotoxin and six analogs on nuclear factor kappa B (NF-kappa B) activation, and on interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) mRNA expression in human THP-1 monocytes. All compounds were inactive between 0.001microM and 10microM when tested alone. However, podophyllotoxin (0.1 microM) enhanced LPS-induced NF-kappa B activation and IL-1beta mRNA expression between 2 and 3-fold. In contrast, LPS-induced TNF-alpha mRNA expression was decreased between 3 and 6-fold. Comparable results were also observed with the three analogs acetylpodophyllotoxin, 4'-demethylpodophyllotoxin and alpha-peltatin. The remaining three analogs (podophyllotoxin-4-O-glucoside, beta-peltatin-beta-D-glucopyransoide and 1,2,3,4-dehydrodesoxypodophyllotoxin) were inactive. Clearly certain structural features such as the presence of a glycosidic group or ring aromatization results in loss of biological activity. Interestingly, the analogs that were inactive in our assays have also been previously shown to lack affinity for tubulin binding. These results suggest that during the initial hours of exposure to podophyllotoxin or specific analogs these compounds do not act as independent stimulants of human monocyte activation, but can selectively enhance or suppress LPS-induced cytokine gene expression.

Inflammasome activation has been shown to regulate both innate and adaptive immune responses. It is important to investigate whether immune-enhancing natural products can also activate inflammasome. The current study examined the potential of protein-bound polysaccharide-K (PSK), a hot water extract from Trametes versicolor, to activate inflammasome. Using THP-1 cells, we have demonstrated that PSK induces both pro-IL-1β and mature IL-1β in THP-1 cells in a caspase 1- and NLRP3-dependent manner. PSK also induces IL-1β and IL-18 in human PBMC. Cathepsin B is required for PSK-induced inflammasome activation as CA-074-Me, a cathepsin B inhibitor, significantly decreased PSK-induced IL-1β. PSK induces NLRP3 at both mRNA and protein level. Comparison of PSK-induced IL-1β in bone marrow-derived macrophages from wild type C57BL/6 mice, TLR2-/-, P2X7R-/- and NLRP3-/- mice demonstrated that PSK-induced IL-1β is dependent on both TLR2 and NLRP3. P2X7R is not required for PSK-induced inflammasome activation, but enhances PSK-induced caspase-1 activation and IL-1β induction. Altogether, these results demonstrated that PSK induces inflammasome activation and production of IL-1β in a TLR2- and NLRP3-dependent mechanism. These results provide novel insights into the mechanisms of the immune modulatory effects of PSK.

In the course of our research on the oligoglycosidic constituents of Turkish Astragalus species, we have isolated a number of cycloartane-type triterpene glycosides. The current study examines the immunostimulatory effects of nineteen of these cycloartane-type compounds using a transcription-based bioassay for Nuclear Factor kappa B (NF-kappaB) activation in a human macrophage/monocyte cell line, THP-1. All compounds were inactive at 100 microg/ml except astragaloside I which increased NF-kappaB directed luciferase expression to levels about 65% as compared with maximal stimulation by E. coli lipopolysaccharide (LPS) at 10 microg/ml. None of the compounds were active at low dosage levels (0.1 microg/ml) in combination with 50 ng/ml LPS. Astragaloside I also increased mRNA expression of the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) as measured using reverse transcriptase-polymerase chain reaction (RT)-PCR. Based on these results it is clear that certain structural features are required for immunostimulation of cycloartane-type triterpene glycosides.

The potential involvement of vicinal dithiols in the transformation of the aryl hydrocarbon (Ah) receptor from its ligand binding to DNA binding form in Hepa-1 cells was explored through the use of diamide and phenylarsine oxide (PAO), which have been shown to specifically form a stable ring complex with vicinal sulfhydryl groups in selected proteins. Pretreatment with diamide and PAO rapidly prevented the inducer-dependent formation of the Ah receptor/xenobiotic response element complex detected by electrophoretic mobility shift assays and suppressed Ah receptor-mediated transcription. Diamide and PAO also inhibited DNA binding activity of the nuclear Ah receptor subsequent to its translocation to the nucleus but to a lesser extent than that observed with pretreatment conditions. The Ah receptor exhibited much higher sensitivity to cellular redox changes than Sp1, a transcription factor previously shown to be very sensitive to redox regulation. Diamide added to nuclear extracts inhibited Ah receptor DNA binding more than when it was added in intact cells. In contrast, Ah receptor DNA binding activity was more sensitive to PAO when it was added to intact cells than when it was added to nuclear extracts. Finally, dithiol 2,3-dimercaptopropanol was over 100 times more effective than monothiol 2-mercaptoethanol in reversing the PAO-dependent inhibition of Ah receptor DNA binding activity. This suggests that vicinal sulhydryl residues may be involved in DNA binding of the Ah receptor.

Bioassay-guided fractionation of the leaves of Eugenia rigida yielded three stilbenes, (Z)-3,4,3',5'-tetramethoxystilbene (1), (E)-3,4,3',5'-tetramethoxystilbene (2), and (E)-3,5,4'-trimethoxystilbene (3). Their structures were determined using 1D- and 2D-NMR spectroscopy and HRESIMS. The sterically hindered Z-stereoisomer 1, a new natural product, was prepared by time-dependent photoisomerization of the E-isomer (2) under UV irradiation at λ254 nm, while 2,3,5,7-tetramethoxyphenanthrene (5) was identified at λ365 nm by UHPLC/APCI-MS and NMR spectroscopy. Compounds 1-3 were tested against a panel of luciferase reporter gene assays that assess the activity of many cancer-related signaling pathways, and the Z-isomer (1) was found to be more potent than the E-isomer (2) in inhibiting the activation of Stat3, Smad3/4, myc, Ets, Notch, and Wnt signaling, with IC50 values between 40 and 80 μM. However, both compounds showed similar inhibition against Ap-1 and NF-κB signaling. In addition, 1 demonstrated cytotoxic activity toward human leukemia cells, solid tumor cells of epidermal, breast, and cervical carcinomas, and skin melanoma, with IC50 values between 3.6 and 4.3 μM, while 2 was weakly active against leukemia, cervical carcinoma, and skin melanoma cells. Interestingly, 2 showed antioxidant activity by inhibition of ROS generation to 50% at 33.3 μM in PMA-induced HL-60 cells, while 1 was inactive at 100 μM (vs Trolox 1.4 μM).

We have found that the bacterial components lipopolysaccharides (LPS) and Braun-type lipoproteins are responsible for the majority of the in vitro macrophage activation exhibited by extracts of Echinacea and other immune enhancing botanicals. Furthermore, we show that endophytic bacteria are the principal source of these components. We also show that total bacterial load within Echinacea samples are strongly correlated with the innate immune stimulatory activity (NF-κB activation in THP-1 cells) and content of bacterial LPS within extracts of this plant material. Differences in the levels of these two bacterial components are responsible for the substantial variation observed (˜200-fold) in innate immune cell activation from E. purpurea material obtained from six major growers/commercial suppliers in North America. This substantial variability in bacterial component content/activity could be a factor contributing to the inconsistent outcomes exhibited in Echinacea clinical trials. We also demonstrate that the oral consumption of extracts rich in these bacterial components is therapeutically relevant with respect to enhancing immune parameters and for impacting disease outcomes in both animal models and in humans. Acknowledgements: This research was partially funded by grants from the National Institutes of Health R21AT01207, R01AT002360 and R01AT007042 (NCAAM) to DSP and by the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 02 – 1-612.

Immulina®, a commercial extract of Arthrospira (Spirulina) platensis is a potent activator of THP-1 monocytes and CD4+ T cells IN VITRO and enhances several immunological functions in mice. We further characterized Immulina® by determining that Braun-type lipoproteins are responsible for a major portion of the IN VITRO monocyte activation exhibited by this material. In order to understand the effect of Immulina® on NK cell activity, a pilot study was conducted on ten healthy North American individuals who supplemented their diet with Immulina® (400 mg/day) for seven days. We observed a 40% average increase in the killing of K562 tumor cells by NK cells (p 0.01) after Immulina® supplementation. In a separate placebo-controlled, crossover study involving 11 healthy Danish subjects, we observed increased mRNA expression of the NK cell marker NKG2D by 37% (p = 0.02) and by 55% (p = 0.0003) after administration of Immulina® (200 mg and 400 mg per day, respectively) for seven days. The mRNA expression of the NK- and T-cell marker perforin increased by 75% (p = 0.008) after administration of 400 mg Immulina® per day. Both markers displayed significant dose-dependent effects (p = 0.0003 and p = 0.02, respectively). The ratio between CD56 (bright) and CD56 (dim) NK cells was not affected by Immulina® administration. In summary, two independent studies showed enhancement of NK cell activity following administration of Immulina® for seven days.

Immulina is a commercial extract of Spirulina (Arthrospira) platensis that is standardized by biological activity. We previously reported that this extract is a potent activator of THP-1 monocytes in vitro and that oral consumption enhanced several immunological functions in mice [1]. In this study we further characterized Immulina by determining that Braun type lipoproteins are responsible for a major portion of the in vitro monocyte activation exhibited by this material. In order to understand the effect of Immulina on the human immune system, a pilot study was conducted on ten healthy individuals who supplemented their diet with Immulina (400 mg/day) for seven days. Blood was drawn from the participating individuals at two time points: before and after seven days of Immulina supplementation. Changes in mononuclear and polymorphonuclear phagocytosis were determined in heparinized whole blood as well as the cytotoxicity exhibited by natural killer (NK) and lymphokine activated killer cells. We observed statistically significant increases both in tumor cell killing by NK cells (p = 0.0019) and in phagocytosis by blood mononuclear cells (p = 0.0124) after Immulina supplementation. Acknowledgements: This research was partly funded by a USDA, Agricultural Research Service Specific Cooperative Agreement No. 58-6408-012. Immulina capsules were supplied by Scandinavian Clinical Nutrition Denmark A/S, Greve, Denmark. References: [1] Balachandran P, et al. (2006), Inteternational Immunopharmacology, 6: 1808–1814.

We previously reported that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea and other botanicals depends on bacterial lipopolysaccharides and Braun type bacterial lipoproteins. We determined the contribution made by these bacterial components to the overall immune enhancing activity detected in E. purpurea and E. angustifolia from bulk root and aerial material obtained from six major growers/suppliers in North America. Substantial variation in activity (up to 200-fold) was observed in extracts of these materials when tested in two monocyte/macrophage cell lines. The majority of activity was negated by treatment with agents that target bacterial lipoproteins (lipoprotein lipase) and lipopolysaccharides (polymyxin B). Experiments comparing the activity of freeze dried, freshly harvested Echinacea plants with those harvested and dried using various commercially relevant conditions, suggest that post-harvesting procedures do not substantially contribute to the variation observed in the commercial material.

We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacterially derived components when tested in in vitro monocyte/macrophage activation systems. In experiments using RAW 264.7 and mouse peritoneal macrophages the majority (85–98%) of the activity within extracts from eight immune enhancing botanicals was eradicated by treatment with agents (lipoprotein lipase and polymyxin B) known to target these two bacterial components. Alfalfa sprouts exhibited the highest activity of those botanicals tested but the appearance of this activity during the germination of surface sterilized seeds was abolished by the presence of antibiotics. These studies indicate that the majority of the in vitro macrophage activating properties in extracts from these botanicals can be attributed to the presence of lipoproteins and lipopolysaccharides derived from bacteria and that bacterial endophytes may be a significant source of these components.

Three nuclear factors, the Ah receptor, XF1, and XF2, bind sequence specifically to the Ah response elements or xenobiotic response elements (XREs) of the cytochrome P450IA1 (P450c) gene. The interactions of these factors with the Ah response element XRE1 were compared by three independent methods, methylation interference footprinting, orthophenanthroline-Cu+ footprinting, and mobility shift competition experiments, using a series of synthetic oligonucleotides with systematic alterations in the XRE core sequence. These studies established the following (i) all three factors interact sequence specifically with the core sequence of XRE1; (ii) the pattern of contacts made with this sequence by the Ah receptor are different from those made by XF1 and XF2; and (iii) although XF1 and XF2 can be distinguished by the mobility shift assay, the sequence specificities of their interactions with XRE1 are indistinguishable. Further characterization revealed the following additional differences among these three factors: (i) XF1 and XF2 could be extracted from nuclei under conditions quite different from those required for extraction of the Ah receptor; (ii) XF1 and XF2 were present in the nuclei of untreated cells and did not respond to polycyclic compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-napthoflavone, while nuclear Ah receptor was undetectable in untreated cells and rapidly increased in response to TCDD; (iii) inhibition of protein synthesis did not affect the TCDD-induced appearance of the Ah receptor but substantially decreased the constitutive activities of XF1 and XF2, suggesting that the Ah receptor must be present in untreated cells in an inactive form that can be rapidly activated by polycyclic compounds, while the constitutive expression of XF1 and XF2 depends on the continued synthesis of a relatively unstable protein; (iv) the receptor-deficient and nuclear translocation-defective mutants of the hepatoma cell line Hepa1, which are known to lack nuclear Ah receptor, expressed normal levels of XF1 and XF2, suggesting that the former factor is genetically distinct from the latter two; and (v) a divalent metal ion, probably Zn2+, is known to be an essential cofactor for the Ah receptor but was not required for the DNA-binding activities of XF1 and XF2. Together, these findings indicate that the Ah receptor is distinct from XF1 and XF2, while the latter two activities may be related. Because the DNA-binding domains of these three factors overlap substantially, their binding to XREs is probably mutually exclusive, which suggests that the interplay of these factors at Ah response elements may be important to the regulation of CYP1A1 gene transcription. The results of preliminary transfection experiments with constructs harboring XREs upstream of the chloramphenicol acetyltransferase gene driven by a minimal simian virus 40 promoter are presented that are consistent with this hypothesis.