Coffee extracts suppress tryptophan breakdown in mitogen-stimulated peripheral blood mononuclear cells.

OBJECTIVES: Coffee consumption is considered to exert an influence on mood, the immune system, cardiovascular disease, and cancer development, but the mechanisms of action of coffee and its compounds are only partly known and understood. METHODS: Immunomodulatory effects of filtered extracts of coffee and decaffeinated coffee as well as coffee compounds were investigated in human peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA). The activation of PBMCs was monitored by the breakdown of tryptophan to kynurenine via enzyme indoleamine 2,3-dioxygenase (IDO) and the production of the immune activation marker neopterin by GTP-cyclohydrolase I (GCH1). Both of these biochemical pathways are induced during cellular immune activation in response to the Th1-type cytokine interferon-γ (IFN-γ). RESULTS: Filtered extracts of coffee and decaffeinated coffee both suppressed tryptophan breakdown and neopterin formation in mitogen-stimulated PBMCs efficiently and in a dose-dependent manner. Of 4 coffee compounds tested individually, only gallic acid and less strong also caffeic acid had a consistent suppressive influence but also affected cell viability, whereas pure caffeine and chlorogenic acid exerted no relevant effect in the PBMC assay. CONCLUSION: The parallel influence of extracts on tryptophan breakdown and neopterin production shows an anti-inflammatory and immunosuppressive property of coffee extracts and some of its compounds. When extrapolating the in vitro results to in vivo, IFN-γ-mediated breakdown of tryptophan could be counteracted by the consumption of coffee or decaffeinated coffee. This may increase tryptophan availability for the biosynthesis of the neurotransmitter 5-hydroxytryptamine (serotonin) and thereby improve mood and quality of life.

Pathway-focused bioassays and transcriptome analysis contribute to a better activity monitoring of complex herbal remedies.

BACKGROUND: Transcriptome analysis in combination with pathway-focused bioassays is suggested to be a helpful approach for gaining deeper insights into the complex mechanisms of action of herbal multicomponent preparations in living cells. The polyherbalism based concept of Tibetan and Ayurvedic medicine considers therapeutic efficacy through multi-target effects. A polyherbal Indo-Tibetan preparation, Padma 28, approved by the Swiss drug authorities (Swissmedic Nr. 58436), was applied to a more detailed dissection of mechanism of action in human hepatoma HepG2 cells. Cell-free and cell-based assays were employed to evaluate the antioxidant capacity. Genome-wide expression profiling was done by applying Human Genome U133 Plus 2.0 Affymetrix arrays. Pathway- and network-oriented analysis elucidated the affected biological processes. The results were validated using reporter gene assays and quantitative real-time PCR. RESULTS: To reveal the direct radical scavenging effects of the ethanolic extract of the Indo-Tibetan polyherbal remedy Padma 28, an in vitro oxygen radical absorbance capacity assay (ORAC) was employed, which resulted in a peroxyl-radical scavenging activity of 2006 ± 235 µmol TE/g. Furthermore, the antioxidant capacity of Padma 28 was analysed in living HepG2 cells, by measuring its scavenging potential against radical induced ROS. This formulation showed a considerable antioxidant capacity by significantly reducing ROS levels in a dose-dependent manner.Integrated transcriptome analysis revealed a major influence on phase I and phase II detoxification and the oxidative stress response. Selected target genes, such as heme oxygenase 1, were validated in qPCR experiments. Network analysis showed 18 interrelated networks involved in important biological functions such as drug and bio-molecule metabolism, molecular transport and cellular communication. Some molecules are part of signaling cascades that are active during development and morphogenesis or are involved in pathological conditions and inflammatory response. CONCLUSIONS: The identified molecular targets and pathways suggest several mechanisms that underlie the biological activity of the preparation. Although extrapolation of these findings to the in vivo situation is not possible, the results obtained might be the basis for further investigations and new hypotheses to be tested. This study demonstrates the potential of the combination of focused and unbiased research strategies in the mode of action analysis of multicomponent herbal mixtures.

Prevention of lethal murine pancreas ischemia reperfusion injury is specific for tetrahydrobiopterin.

Tetrahydrobiopterin has been shown to efficiently abrogate ischemia reperfusion injury (IRI). However, it is unclear, whether its beneficial action relies on cofactor activity of one of the five known tetrahydrobiopterin-dependent reactions or on its antioxidative capacity. We therefore compared tetrahydrobiopterin with the pterin derivate tetrahydroneopterin (similar biochemical properties, but no nitric oxide synthase cofactor activity) and the antioxidants vitamin C and 5-methyltetrahydrofolate. Donor mice were pretreated with tetrahydrobiopterin, tetrahydroneopterin, vitamin C, or 5-methyltetrahydrofolate. Pancreatic grafts were subjected to 16-h cold ischemia time and implanted in syngeneic recipients. Untreated and nontransplanted animals served as controls. Following 2-h reperfusion, microcirculation was analyzed by intravital fluorescence microscopy. Graft damage was assessed by histology and nitrotyrosine immunostaining, and tetrahydrobiopterin levels were determined by HPLC. Recipient survival served as ultimate readout. Prolonged cold ischemia time resulted in microcirculatory breakdown. Only tetrahydrobiopterin pretreatment succeeded to preserve the capillary net, whereas all other compounds showed no beneficial effects. Along with increased intragraft tetrahydrobiopterin levels during recovery and implantation, only tetrahydrobiopterin pretreatment led to significant reduction of IRI-related parenchymal damage enabling recipient survival. These results show a striking superiority of tetrahydrobiopterin in preventing lethal IRI compared with related compounds and suggest nitric oxide synthases as treatment target.

An update on the strategies in multicomponent activity monitoring within the phytopharmaceutical field.

BACKGROUND: To-date modern drug research has focused on the discovery and synthesis of single active substances. However, multicomponent preparations are gaining increasing importance in the phytopharmaceutical field by demonstrating beneficial properties with respect to efficacy and toxicity. DISCUSSION: In contrast to single drug combinations, a botanical multicomponent therapeutic possesses a complex repertoire of chemicals that belong to a variety of substance classes. This may explain the frequently observed pleiotropic bioactivity spectra of these compounds, which may also suggest that they possess novel therapeutic opportunities. Interestingly, considerable bioactivity properties are exhibited not only by remedies that contain high doses of phytochemicals with prominent pharmaceutical efficacy, but also preparations that lack a sole active principle component. Despite that each individual substance within these multicomponents has a low molar fraction, the therapeutic activity of these substances is established via a potentialization of their effects through combined and simultaneous attacks on multiple molecular targets. Although beneficial properties may emerge from such a broad range of perturbations on cellular machinery, validation and/or prediction of their activity profiles is accompanied with a variety of difficulties in generic risk-benefit assessments. Thus, it is recommended that a comprehensive strategy is implemented to cover the entirety of multicomponent-multitarget effects, so as to address the limitations of conventional approaches. SUMMARY: An integration of standard toxicological methods with selected pathway-focused bioassays and unbiased data acquisition strategies (such as gene expression analysis) would be advantageous in building an interaction network model to consider all of the effects, whether they were intended or adverse reactions.

Activation of PKCzeta and PKMzeta in the nucleus accumbens core is necessary for the retrieval, consolidation and reconsolidation of drug memory.

One of the greatest challenges in the treatment of substance dependence is to reverse the control that drug-associated stimuli have gained over the addict's behavior, as these drug-associated memories increase the risk of relapse even after long periods of abstinence. We report here that inhibition of the atypical protein kinase C isoform PKCzeta and its constitutively active isoform PKMzeta with the pseudosubstrate inhibitor ZIP administered locally into the nucleus accumbens core reversibly inhibited the retrieval of drug-associated memory and drug (remifentanil) seeking, whereas a scrambled ZIP peptide or staurosporine, an effective inhibitor of c/nPKC-, CaMKII-, and PKA kinases that does not affect PKCzeta/PKMzeta activity, was without effect on these memory processes. Acquisition or extinction of drug-associated memory remained unaffected by PKCzeta- and PKMzeta inhibition.

Food additives such as sodium sulphite, sodium benzoate and curcumin inhibit leptin release in lipopolysaccharide-treated murine adipocytes in vitro.

Obesity leads to the activation of pro-inflammatory pathways, resulting in a state of low-grade inflammation. Recently, several studies have shown that the exposure to lipopolysaccharide (LPS) could initiate and maintain a chronic state of low-grade inflammation in obese people. As the daily intake of food additives has increased substantially, the aim of the present study was to investigate a potential influence of food additives on the release of leptin, IL-6 and nitrite in the presence of LPS in murine adipocytes. Leptin, IL-6 and nitrite concentrations were analysed in the supernatants of murine 3T3-L1 adipocytes after co-incubation with LPS and the food preservatives, sodium sulphite (SS), sodium benzoate (SB) and the spice and colourant, curcumin, for 24 h. In addition, the kinetics of leptin secretion was analysed. A significant and dose-dependent decrease in leptin was observed after incubating the cells with SB and curcumin for 12 and 24 h, whereas SS decreased leptin concentrations after 24 h of treatment. Moreover, SS increased, while curcumin decreased LPS-stimulated secretion of IL-6, whereas SB had no such effect. None of the compounds that were investigated influenced nitrite production. The food additives SS, SB and curcumin affect the leptin release after co-incubation with LPS from cultured adipocytes in a dose- and time-dependent manner. Decreased leptin release during the consumption of nutrition-derived food additives could decrease the amount of circulating leptin to which the central nervous system is exposed and may therefore contribute to an obesogenic environment.

Antimalarial drug chloroquine counteracts activation of indoleamine (2,3)-dioxygenase activity in human PBMC.

Antimalarial chloroquine is also used for the treatment of immune-mediated diseases. The interference of chloroquine with interferon-γ-induced tryptophan breakdown and neopterin production has been investigated in human peripheral blood mononuclear cells (PBMC) in vitro. Micromolar concentrations (2-50 µM) of chloroquine dose-dependently suppressed mitogen-induced tryptophan breakdown in PBMC but not in the myelomonocytic THP-1-Blue cell line, after 48 h of treatment. In stimulated PBMC, neopterin production was super-induced by 10 µM chloroquine, while it was significantly suppressed at a concentration of 50 µM. These anti-inflammatory effects may relate to the therapeutic benefit of chloroquine in inflammatory conditions and may widen the spectrum of its clinical applications.

Crinum latifolium leave extracts suppress immune activation cascades in peripheral blood mononuclear cells and proliferation of prostate tumor cells.

Plants of the genus Crinum (Amaryllidaceae) are widely used in folk medicine in different tropical and subtropical regions around the world. The Indian species Crinum latifolium (L.) was traditionally used to treat rheumatism, fistula, tumors, earaches, rubefacient, tubercle and whitlow. In Vietnamese and Chinese traditional medicine Crinum latifolium preparations are used until nowadays because of their antiviral and antitumor properties. In this study, we demonstrate potent in vitro antioxidant activity of an aqueous Crinum latifolium extract by an oxygen radical absorbance capacity (ORAC) value of 1610 ± 150 µmol Trolox equivalents/g. Furthermore, significant anti-inflammatory effects of this extract were shown by its potential to suppress indoleamine 2,3-dioxygenase (IDO) mediated tryptophan degradation in unstimulated- and mitogen-stimulated PBMC at IC(50) doses of 241 ± 57 µg/ml and 92 ± 20 µg/ml, respectively. Concentrations of the immune activation marker neopterin were slightly diminished in unstimulated PBMC, whereas a dose-dependent inhibition of neopterin formation was observed in mitogen-stimulated PBMC (IC(50) = 453 ± 86 µg/ml). Additionally, we measured also dose-dependent inhibitory effects of this aqueous Crinum latifolium extract on cell proliferation of highly metastatic human prostate carcinoma PC3 cells (IC(50) = 4.5 ± 0.8 mg/ml), androgen-sensitive prostate adenocarcinoma LNCaP cells (IC(50) =2.3 ± 0.1 mg/ml), and benign prostate hyperplasia BPH-1 cells (IC(50) = 2.1 ± 0.04 mg/ml). We conclude that both effects, inhibition of tumor cell growth and recovery of immune functions, are important for the antitumor properties of Crinum latifolium.

Testing for immunomodulatory properties of nanoparticles.

Immune activation and inflammation can precipitate a variety of diseases. A screening assay for immunomodulatory properties is described. It is based on freshly isolated human peripheral blood mononuclear cells and allows testing for effects of nanoparticles on the human immune system.

Myelomonocytic THP-1 cells for in vitro testing of immunomodulatory properties of nanoparticles.

The use of nanoparticles for new therapeutic and diagnostics options represents a new risk for individuals exposed to such compounds. The myelomonocytic cell line THP-1 could be a useful alternative to human peripheral blood mononuclear cells (PBMC) to test for effects of drugs and compounds. Stimulation degree of cells can be monitored by measurement of neopterin and/or the kynurenine to tryptophan ratio. The method is robust and reproducible in the range of 0.1-1.0 microg/ml of LPS. However, compared to the PBMC assay it will not reveal any effect on the T-cell interaction.

LPS-induced NF-kappaB expression in THP-1Blue cells correlates with neopterin production and activity of indoleamine 2,3-dioxygenase.

Neopterin production is induced in human monocyte-derived macrophages and dendritic cells upon stimulation with Th1-type cytokine interferon-gamma (IFN-gamma). In parallel, IFN-gamma induces the tryptophan-(trp)-degrading enzyme indoleamine 2,3-dioxygenase (IDO) and triggers the formation of reactive oxygen species (ROS). Translocation of the signal transduction element nuclear factor-kappaB (NF-kappaB) is induced by ROS and accelerates the pro-inflammatory response by activation of other pro-inflammatory pathways. Therefore, a close relationship between NF-kappaB expression, the production of neopterin and the degradation of trp can be assumed, although this has not been demonstrated so far. In the present in vitro study we compared the influence of lipopolysaccharide (LPS) on NF-kappaB activation, neopterin formation and the degradation of trp in THP-1Blue cells, which represent the human myelomonocytic cell line THP-1 stably transfected with an NF-kappaB inducible reporter system. In cells stimulated with LPS, a significant induction of NF-kappaB was observed, and this was paralleled by an increase of kynureunine (kyn) and neopterin concentrations and a decline of trp. The increase of the kyn to trp quotient indicates accelerated IDO activity. Higher LPS concentrations and longer incubation of cells were associated with higher activities of all three biochemical pathways and significant correlations existed between NF-kappaB activation, neopterin release and trp degradation (all p<0.001). We conclude that there is a parallel induction of NF-kappaB, neopterin formation and trp degradation in monocytic THP-1 cells, which is elicited by pro-inflammatory triggers like LPS during innate immune responses.

Bovine colostrum modulates immune activation cascades in human peripheral blood mononuclear cells in vitro.

Bovine colostrum (BC) is the thick yellow fluid a lactating cow gives to a suckling calf during its first days of life to support the growth of the calf and prevent gastrointestinal infections until the calf has synthesized its own active immune defense system. BC contains a complex system of immune factors and has a long history of use in traditional medicine. In an approach to evaluate the effects of bovine colostrum (BC) on the T-cell/macrophage interplay, we investigated and compared the capacity of BC containing low and high amounts of lactose and lactoferrin to modulate tryptophan degradation and neopterin formation in unstimulated and mitogen-stimulated human peripheral blood mononuclear cells (PBMC). The present study shows significant immunomodulatory effects of these BC preparations in human PBMC, either by enhancing or suppressing the occurrence of a Th-1 type immune response. The amount of lactose present in BC seems to diminish the activity of BC in our test system, since BC with higher amounts of lactose attenuated the stimulatory as well as the suppressive activity of BC.

Atypical protein kinase C zeta exhibits a proapoptotic function in ovarian cancer.

Intracellular signaling governed by serine/threonine kinases comprises the molecular interface between cell surface receptors and the nuclear transcriptional machinery. The protein kinase C (PKC) family members are involved in the control of many signaling processes directing cell proliferation, motility, and survival. Here, we examined a role of different PKC isoenzymes in protein phosphatase 2A (PP2A) and HRSL3 tumor suppressor-dependent cell death induction in the ovarian carcinoma cell line OVCAR-3. Phosphorylation and activity of PKC isoenzymes were measured in response to PP2A or phosphoinositide 3-kinase inhibition or HRSL3 overexpression. These experiments indicated a regulation of PKC, epsilon, zeta, and iota through PP2A and/or HRSL3, but not of PKCalpha and beta. Using isoform-specific peptide inhibitors and overexpression approaches, we verified a contribution to PP2A- and HRLS3-dependent apoptosis only for PKCzeta, suggesting a proapoptotic function of this kinase. We observed a significant proportion of human ovarian carcinomas expressing high levels of PKCzeta, which correlated with poor prognosis. Primary ovarian carcinoma cells isolated from patients also responded to okadaic acid treatment with increased phosphorylation of PKCzeta and apoptosis induction. Thus, our data indicate a contribution of PKCzeta in survival control in ovarian carcinoma cells and suggest that upregulation or activation of tyrosine kinase receptors in this tumor might impinge onto apoptosis control through the negative regulation of the atypical PKCzeta.

Food preservatives sodium benzoate and propionic acid and colorant curcumin suppress Th1-type immune response in vitro.

Food preservatives sodium benzoate and propionic acid and colorant curcumin are demonstrated to suppress in a dose-dependent manner Th1-type immune response in human peripheral blood mononuclear cells (PBMC) in vitro. Results show an anti-inflammatory property of compounds which however could shift the Th1-Th2-type immune balance towards Th2-type immunity.

The Potential Role of Cannabinoids in Modulating Serotonergic Signaling by Their Influence on Tryptophan Metabolism.

Phytocannabinoids present in Cannabis plants are well known to exert potent anti-inflammatory and immunomodulatory effects. Previously, we have demonstrated that the psychoactive D9-tetrahydrocannabinol (THC) and the non-psychotropic cannabidiol (CBD) modulate mitogen-induced Th1-type immune responses in peripheral blood mononuclear cells (PBMC). The suppressive effect of both cannabinoids on mitogen-induced tryptophan degradation mediated by indoleamine-2,3-dioxygenase (IDO), suggests an additional mechanism by which antidepressive effects of cannabinoids might be linked to the serotonergic system. Here, we will review the role of tryptophan metabolism in the course of cell mediated immune responses and the relevance of cannabinoids in serotonergic signaling. We conclude that in particular the non-psychotropic CBD might be useful for the treatment of mood disorders in patients with inflammatory diseases, since this cannabinoid seems to be safe and its effects on activation-induced tryptophan degradation by CBD were more potent as compared to THC.

Delta9-tetrahydrocannabinol and cannabidiol modulate mitogen-induced tryptophan degradation and neopterin formation in peripheral blood mononuclear cells in vitro.

Nanomolar concentrations of Delta9-tetrahydrocannabinol or cannabidiol are demonstrated to enhance mitogen-induced degradation of tryptophan in human peripheral blood mononuclear cells in dependence of CB1- or CB2-receptor activation. In contrast, suppression of this pathway by cannabinoids in the micromolar concentration range was achieved independent of cannabinoid receptor activation. Both cannabinoids also suppressed tryptophan degradation in myelomonocytic THP-1 cells stimulated with lipopolysaccharide. We conclude, that suppression of tryptophan degradation by cannabinoids via indoleamine-2,3-dioxygenase, which is independent of cannabinoid receptor activation, might enhance the availability of tryptophan for serotonin biosynthesis and consequently can be important in the action of cannabinoids to improve mood disturbances.

N-benzoxazol-2-yl-N'-1-(isoquinolin-3-yl-ethylidene)-hydrazine, a novel compound with antitumor activity, induces radicals and dissipation of mitochondrial membrane potential.

The novel compound N-benzoxazol-2-yl-N'-1-(isoquinolin-3-yl-ethylidene)-hydrazine (EPH136) has been shown to exhibit antitumor activity in vitro and in vivo. A COMPARE analysis showed that the patterns of cellular effects of EPH136 are not related to any of 175 standard antitumor agents with a known mechanism of action. In order to help identify the mechanism of action we employed a bioinformatics approach called partial least squares modelling in latent variables in which the expression levels of approximately 8,000 genes in each of 56 untreated NCI panel cell lines were correlated with the respective IC(50) values of each cell line following treatment with EPH136. The 60 genes found to be most important for the antiproliferative effect of EPH136 are involved in nucleoside, nucleotide, nucleic acid binding and metabolism, developmental processes, protein modification and metabolism. In addition, using a DNA microarray we measured the expression of approximately 5,000 known genes following treatment of HT-29 colon carcinoma cells with a two-fold IC(50) concentration of EPH136. The genes that were up-regulated more than two-fold compared to untreated controls belong to the same classes as found by the bioinformatic approach. Many of these proteins are regulated by oxidation/reduction and so we concluded that formation of radicals may be involved in the mechanism of action. We show here that EPH136 leads to generation of oxygen radicals, swelling of mitochondria and dissipation of the mitochondrial membrane potential. The antiproliferative activity of EPH136 was prevented by the radical scavenger N-acetylcysteine. Cells with elevated glutathione exhibited resistance to EPH136. In summary, the mechanism of the novel experimental anticancer drug EPH136 is generation of radicals and dissipation of the mitochondrial membrane potential.

Non-peptidic delta-opioid receptor antagonists suppress mitogen-induced tryptophan degradation in peripheral blood mononuclear cells in vitro.

Opioid receptors are expressed not only on neuroendocrine cells but also on immunocompetent cells such as lymphocytes, monocytes and macrophages. micro-Opioid receptor agonists were found to exert immunosuppressive effects, whereas delta-opioid receptor agonists have been shown to act as immunostimulants. delta-Opioid receptor agonists stimulate T and B cells and activate granulocytes and monocytes, conversely, immunostimulation can be blocked by the non-peptidic delta-opioid receptor antagonist (NTI). We investigated the impact of NTI and of the two structurally related compounds HS-378 and HS-459 on degradation of tryptophan and formation of neopterin in mitogen-stimulated human peripheral blood mononuclear cells (PBMC). Both these biochemical pathways were found to be suppressed by all three opioid receptor antagonists, HS-378 and HS-459 exhibiting slightly greater potency than NTI. The suppression of tryptophan degradation suggests that the tested delta-opioid antagonists are able to influence the serotonergic system via a non-opioid action.

PKCtheta cooperates with atypical PKCzeta and PKCiota in NF-kappaB transactivation of T lymphocytes.

Using yeast two-hybrid, we isolated atypical PKCzeta as a PKCtheta-interacting kinase and demonstrated that it selectively interacted with, and was phosphorylated by, PKCtheta. Importantly, however, both atypical PKCzeta and PKCiota were functionally required in TCR/CD28-mediated activation of NF-kappaB downstream of PKCtheta in Jurkat T cells albeit, activation responses of PKCzeta-deficient CD3+ T cells were comparable with wildtype controls. This normal activation thresholds of PKCzeta-/- T cells suggested that PKCiota, the closest structural relative, might play a compensatory role in TCR/CD28-induced signalling. Consistently, both PKCzeta and PKCiota resided in the plasma membrane lipid raft microdomains of Jurkat as well as primary mouse CD3+ T cells. Thus, PKCtheta, the established constituent of the immunological synapse, physically and functionally interacted with PKCzeta and PKCiota. Together, these data demonstrate that atypical PKCzeta/iota isotypes serve as direct downstream targets of PKCtheta in the signalling pathway leading to NF-kappaB activation in T lymphocytes.

Apoptosis induced by the Tibetan herbal remedy PADMA 28 in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2.

The Tibetan herbal remedy PADMA 28 revealed promising results to support treatment of atherosclerosis, Charot syndrome (intermittent claudication), chronic active hepatitis and infection of the respiratory tract. The remedy was confirmed to be closely linked with anti- and pro-oxidative properties in vitro. In this study, apoptogenic and survival effects of PADMA 28 were investigated in the T cell-derived lymphocytic leukaemia cell line CEM-C7H2. PADMA 28 led to a concentration-dependent inhibition of cell proliferation accompanied by the accumulation of CEM-C7H2 cells in subG1 phase, fragmentation of poly (ADP-ribose) polymerase (PARP) and nuclear body formation. Treatment with PADMA 28 rescued to some extent cells over-expressing Bcl-2 from apoptosis. This finding suggests that the mechanism of action of PADMA 28 may be via interference with Bcl-2 triggered survival pathways.