Resveratrol and ω-3 PUFAs Promote Human Macrophage Differentiation and Function.

Monocytes differentiate into M1 and M2 macrophages, which are classically activated by microbial products such as LPS or IFN-γ and interleukins (e.g., the anti-inflammatory and Th2 promoting IL-4), respectively. The contribution of nutrients or nutrient-based substances such as ω-3 polyunsaturated fatty acids (ω-3 PUFAs) and resveratrol (Res) on the differentiation and function of M1 and M2 macrophages was evaluated. THP-1 cells and peripheral blood mononuclear cells (PBMCs) were differentiated into M1 and M2 cells and activated with LPS/IFN-γ or IL-4/IL-13. Macrophage lineage specific surface determinants (e.g., CD11b, CD11c, CD14, CD206, CD209, CD274, HLA-DR, CCR7, CCR2) were analysed by cytofluorometry. Res and ω-3 PUFAs altered CD14, CD206, CD274 and HL-DR surface expression patterns in M1 and M2 macrophages differentiated from PBMC. LPS/IFN-γ or IL-14/IL-13 activated macrophages subpopulations, which secreted cytokines and chemokines as measured by multiplex ELISA. Res and ω-3 PUFA reduced IL-1ß, IL-6, TNF-α, CXCL10/IP-10, CCL13/MCP-4 and CCL20/MIP-3α in LPS/IFN-γ activated human leukaemia THP-1 cells, which is indicative of a dampening effect on M1 macrophages. However, Res increased M1 prototypic cytokines such as IL-1ß or IL-6 in macrophages derived from PBMCs and also modified the expression of IL-12p70. Collectively, Res and ω-3 PUFAs distinctly promoted the differentiation and function of M1 and M2 macrophages. We conclude that these substances strengthen the macrophage-mediated effects on the innate and adaptive immune response.

Resveratrol, EGCG and Vitamins Modulate Activated T Lymphocytes.

Vitamins and bioactives, which are constituents of the food chain, modulate T lymphocyte proliferation and differentiation, antibody production, and prevent inflammation and autoimmunity. We investigated the effects of vitamins (vitamin A (VA), D (VD), E (VE)) and bioactives (i.e., resveratrol (Res), epigallocatechin-3-gallate (EGCG)) on the adaptive immune response, as well as their synergistic or antagonistic interactions. Freshly isolated T lymphocytes from healthy individuals were activated with anti-CD3/CD28 antibodies for 4-5 days in the presence of bioactives and were analyzed by cytofluorometry. Interleukins, cytokines, and chemokines were measured by multiple ELISA. Gene expression was measured by quantitative RT-PCR. Res and EGCG increased CD4 surface intensity. EGCG led to an increased proportion of CD8+ lymphocytes. Anti-CD3/CD28 activation induced exuberant secretion of interleukins and cytokines by T lymphocyte subsets. VD strongly enhanced Th2 cytokines (e.g., IL-5, IL-13), whereas Res and EGCG favored secretion of Th1 cytokines (e.g., IL-2, INF-γ). Res and VD mutually influenced cytokine production, but VD dominated the cytokine secretion pattern. The substances changed gene expression of interleukins and cytokines in a similar way as they did secretion. Collectively, VD strongly modulated cytokine and interleukin production and favored Th2 functions. Resveratrol and EGCG promoted the Th1 response. VA and VE had only a marginal effect, but they altered both Th1 and Th2 response. In vivo, bioactives might therefore interact with vitamins and support the outcome and extent of the adaptive immune response.

Resveratrol distinctively modulates the inflammatory profiles of immune and endothelial cells.

BACKGROUND: The phenolic substance resveratrol (RES) is a plant metabolite known to modulate numerous physiological functions and to exert beneficial effects as a cancer-chemopreventing agent and on neurological, hepatic, and cardiovascular systems. Since the compound affects the lifespan of yeast and flies it might be an anti-aging substance. Mechanistically, RES is involved in down regulating the inflammatory response. The pleiotropic effects of RES in cells of the immune and endothelial system were examined in this study. RESULTS: Murine macrophages (RAW264.7 cells), human monocytic/leukemia cells (THP-1), PBLs and HUVECs were incubated with RES and activated with inflammatory stimuli such as LPS or TNF-α. Inflammatory mediators and adhesion molecules were measured by multiplex analysis and gene expression was quantified by RT-PCR. In PBLs, which were activated with LPS, RES blunted the production of TNF-α, CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, whereas it increased the production of IL-1ß, IL-6, CCL4/MIP-1ß and CXCL10/IP-10. Thus, in the blood compartment chemokines attracting mainly monocytes were up-regulated by RES, while those attracting T lymphocytes or neutrophils were diminished. At conditions of endothelial dysfunction (ED), RES reduced the expression of cytokines, chemokines, ICAM and GM-CSF in TNF-α activated HUVECs, whereas eNOS expression was corrected to pre-ED homeostasis. In macrophages nitric oxide, PGE2, cytokines (TNF-α, IL-1ß, IL-6) and chemokines (CCL2/MCP-1, CCL4/MIP-1ß, CCL5/RANTES, CXCL10/IP-10) were reduced by the phenolic substance. CONCLUSIONS: RES had cell-specific and context-dependent effects, in particular on the expression of IL-1ß, IL-6, CCL4/MIP-1ß and CXCL10/IP-10. It enhanced cellular features that mirror increased alertness to disturbed immune homeostasis in the vascular-endothelial compartment (e.g. increased production of IL-1ß or IL-6), whereas it blunted inflammatory mediators in macrophages and consequently chronic inflammation. We infer from the present in vitro study, that RES has unique properties in the regulation of inflammatory and immune responses, which are controlled in a complex hierarchical and temporal order.

Combinations of bio-active dietary constituents affect human white adipocyte function in-vitro.

BACKGROUND: Specific bio-active dietary compounds modulate numerous metabolic processes in adipose tissue (AT), including pre-adipocyte proliferation and differentiation. AT dysfunction, rather than an increased fat mass per se, is strongly associated with the development of insulin resistance and is characterized by impaired adipogenesis, hypertrophic adipocytes, inflammation, and impairments in substrate metabolism. A better understanding of mechanisms underlying AT dysfunction may provide new strategies for the treatment of obesity-associated metabolic diseases. Here we evaluated the role of (all-E)-lycopene (Lyc), eicosapentaenoic acid (EPA) or trans-resveratrol (Res) and combinations thereof on human white adipocyte function. METHODS: In-vitro differentiating human pre-adipocytes were treated with EPA, Lyc and Res or their combinations for 14 days. The effects on intracellular lipid droplet (LD) accumulation, secreted anti- and pro-inflammatory cyto-/adipokines (e.g. adiponectin, IL-6, IL-8/CXCL-8 and MCP-1/CCL2) and on gene expression of markers of adipocyte differentiation and substrate metabolism (e.g. PPAR-gamma, C/EBP-alpha, GLUT-4, FAS, ATGL, HSL, and PLIN-1) were measured by fluorescent microscopy (Cellomics™), multi-parametric LiquiChip® technology and quantitative RT-PCR, respectively. RESULTS: Treatment of differentiating adipocytes for 14 days with the combination of Lyc/Res and EPA/Res resulted in significantly inhibited LD formation (~ -25 and -20%, respectively) compared to the effects of the single compounds. These morphological changes were accompanied by increased mRNA levels of the adipogenic marker PPAR-gamma and the lipase ATGL and by decreased expression levels of lipogenic markers (LPL, FAS, GLUT-4) and the LD-covering protein PLIN-1. In addition, a blunted adipocyte secretion of pro-inflammatory cytokines (IL-6 and MCP-1) and adiponectin was observed following treatment with these compounds. CONCLUSION: The combination of the dietary bio-actives Lyc and EPA with Res might influence adipocyte function by affecting the balance between adipogenic, lipogenic and lipolytic gene expression, resulting in a reduced LD storage and a less inflammatory secretion profile. Taken together, our results indicate that combinations of dietary compounds may be beneficial for the prevention and treatment of metabolic disorders via effects on human white adipocyte function.

Mitochondrial and glycolytic activity of UV-irradiated human keratinocytes and its stimulation by a Saccharomyces cerevisiae autolysate.

Cutaneous aging is correlated with mitochondrial dysfunction and a concomitant decline in energy metabolism that can be accelerated by extrinsic factors such as UV radiation (UVR). In this study we compared cellular bioenergetics of normal and UV-irradiated primary human epidermal keratinocytes. Moreover, we investigated the influence of a Saccharomyces cerevisiae autolysate (SCA) on stressed keratinocytes to regain cellular homeostasis. Cellular metabolism was assessed by extracellular flux analysis which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as well as by ATP quantification. The expression level of ten mitochondria related genes in normal and UVR-stimulated (60mJ/cm(2) UVB) keratinocytes was quantified by real-time PCR and the impact of SCA addition was determined. Sublethal UV stress increased mitochondrial dysfunction in keratinocytes which resulted in reduced viability, uncoupled oxidative phosphorylation, and down-regulated mitochondrial gene expression. Particularly, gene expression of SHDA, UPC2, BID, and ATP5A1 was reduced about twofold within 4h. Treatment of keratinocytes with SCA shifted cellular metabolism towards a more energetic status by increasing the respiratory rate and glycolysis. SCA also stimulated cellular ATP production after short (4h) and prolonged (22h) incubations and induced the expression of genes related to mitochondrial function towards normal expression levels upon UV irradiation. The decreased respiratory capacity of UV-irradiated keratinocytes was partially compensated by the addition of SCA which enhanced glycolytic activity and thereby increased cellular resistance to environmental stress.

Carnosol and Related Substances Modulate Chemokine and Cytokine Production in Macrophages and Chondrocytes.

Phenolic diterpenes present in Rosmarinus officinalis and Salvia officinalis have anti-inflammatory and chemoprotective effects. We investigated the in vitro effects of carnosol (CL), carnosic acid (CA), carnosic acid-12-methylether (CAME), 20-deoxocarnosol and abieta-8,11,13-triene-11,12,20-triol (ABTT) in murine macrophages (RAW264.7 cells) and human chondrocytes. The substances concentration-dependently reduced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-stimulated macrophages (i.e., acute inflammation). They significantly blunted gene expression levels of iNOS, cytokines/interleukins (IL-1α, IL-6) and chemokines including CCL5/RANTES, CXCL10/IP-10. The substances modulated the expression of catabolic and anabolic genes in chondrosarcoma cell line SW1353 and in primary human chondrocytes that were stimulated by IL-1ß (i.e., chronic inflammation In SW1353, catabolic genes like MMP-13 and ADAMTS-4 that contribute to cartilage erosion were down-regulated, while expression of anabolic genes including Col2A1 and aggrecan were shifted towards pre-pathophysiological homeostasis. CL had the strongest overall effect on inflammatory mediators, as well as on macrophage and chondrocyte gene expression. Conversely, CAME mainly affected catabolic gene expression, whereas ABTT had a more selectively altered interleukin and chemokine gene exprssion. CL inhibited the IL-1ß induced nuclear translocation of NF-κBp65, suggesting that it primarily regulated via the NF-κB signalling pathway. Collectively, CL had the strongest effects on inflammatory mediators and chondrocyte gene expression. The data show that the phenolic diterpenes altered activity pattern of genes that regulate acute and chronic inflammatory processes. Since the substances affected catabolic and anabolic gene expression in cartilage cells in vitro, they may beneficially act on the aetiology of osteoarthritis.

Tomato Aqueous Extract Modulates the Inflammatory Profile of Immune Cells and Endothelial Cells.

Nutrients transiently or chronically modulate functional and biochemical characteristics of cells and tissues both in vivo and in vitro. The influence of tomato aqueous extract (TAE) on the in vitro inflammatory response of activated human peripheral blood leukocytes (PBLs) and macrophages was investigated. Its effect on endothelial dysfunction (ED) was analyzed in human umbilical vein endothelial cells (HUVECs). Murine macrophages (RAW264.7 cells), PBLs and HUVECs were incubated with TAE. They were activated with LPS or TNF-α in order to induce inflammatory processes and ED, respectively. Inflammatory mediators and adhesion molecules were measured by immune assay-based multiplex analysis. Gene expression was quantified by RT-PCR. TAE altered the production of interleukins (IL-1ß, IL-6, IL-10, IL-12) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, CXCL8/IL-8, CXCL10/IP-10) in PBLs. TAE reduced ED-associated expression of adhesion molecules (ICAM-1, VCAM-1) in endothelial cell. In macrophages, the production of nitric oxide, PGE2, cytokines and ILs (TNF-α, IL-1ß, IL-6, IL-12), which reflects chronic inflammatory processes, was reduced. Adenosine was identified as the main bioactive of TAE. Thus, TAE had cell-specific and context-dependent effects. We infer from these in vitro data, that during acute inflammation TAE enhances cellular alertness and therefore the sensing of disturbed immune homeostasis in the vascular-endothelial compartment. Conversely, it blunts inflammatory mediators in macrophages during chronic inflammation. A novel concept of immune regulation by this extract is proposed.

ω-3 PUFAs and Resveratrol Differently Modulate Acute and Chronic Inflammatory Processes.

ω-3 PUFAs and polyphenols have multiple effects on inflammation in vivo and in vitro. The effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and resveratrol (RV) were investigated in LPS-stimulated peripheral blood leukocytes (PBLs) (i.e., acute inflammation) and IL-1ß activated human chondrocytes (i.e., chronic inflammation). Inflammatory mediators including chemokines, cytokines, interleukins, and PGE2 were measured by multiplex analysis and gene expression was quantified by RT-PCR. In PBLs, RV decreased the secretion of PGE2, CCL5/RANTES, and CXCL8/IL-8 but increased IL-1ß, IL-6, and IL-10. In contrast to RV, ω-3 PUFAs augmented the production of PGE2 and CXCL8/IL-8. EPA and DHA similarly affected the pattern of inflammatory mediators. Combination of RV and ω-3 PUFAs exerted synergistic effects on CCL5/RANTES and had additive effects on IL-6 or CXCL8/IL-8. Both ω-3 PUFAs and RV reduced catabolic gene expression (e.g., MMPs, ADAMTS-4, IL-1ß, and IL-6) in activated chondrocytes. The data suggest that ω-3 PUFAs and RV differ in the regulation of acute inflammation of peripheral blood leukocytes but have common properties in modulating features related to chronic inflammation of chondrocytes.

Ascorbic acid modulates cell migration in differentiated HL-60 cells and peripheral blood leukocytes.

SCOPE: The impact of L-ascorbic acid (L-AA) on the chemokinesis (CK) and chemotaxis (CT) of HL-60 cells and polymorphonuclear cells (PMN) was investigated. METHODS AND RESULTS: HL-60 cells were differentiated with DMSO, retinoic acid (RA), vitamin D, or L-AA. Chemokinesis and chemotaxis of differentiated HL-cells were assayed. Vitamin D3-treated HL-60 cells (dHL-60vitD3 cells) and RA-treated cells (dHL-60RA cells) acquired monocyte/macrophage-like and neutrophil-like phenotypes, respectively. DMSO induced the differentiation of an intermediate phenotype (dHL-60DMSO cells), whereas L-AA downregulated neutrophil markers (dHL-60L-AA cells). dHL-60DMSO cells had increased CK and potent CT in gradients of IL-8 and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). dHL-60RA cells and dHL-60L-AA cells migrated less toward IL-8 and fMLP; dHL-60vitD3 cells preferably responded to fMLP. L-AA enhanced CK of dHL-60DMSO cells and was a weak chemo-attractant. In human leukocytes, IL-8 and fMLP triggered receptor-mediated chemotaxis. CXCR2 and fMLPR were downregulated by IL-8 and fMLP, respectively. L-AA stimulated chemotaxis although significantly less than IL-8 and fMLP. IL-8 targeted chemotaxis was enhanced both in HL-60 cells and leukocytes when cells were incubated with L-AA. CONCLUSION: L-AA modulated chemokinesis and had significant chemo-attractant properties, which were independent on fMLP or IL-8 receptors. The results suggest that L-AA improves leukocyte function in innate immune responses.

A novel rose hip preparation with enhanced anti-inflammatory and chondroprotective effects.

Rose hip powder (RHP) alleviates osteoarthritis (OA) due to its anti-inflammatory and cartilage-protective properties. Substances contained in RHP might contribute to its clinical efficacy. The activity of two RHP (i.e., RH-A, from the whole fruit, RH-B, from fruits without seeds) was investigated in human peripheral blood leukocytes (PBL) and primary chondrocytes (NHAC-kn). RH-A and RH-B diminished the secretion of chemokines and cytokines in LPS/IFN-γ-activated PBL, including CCL5/RANTES, CXCL10/IP-10, interleukin- (IL-) 6, and IL-12. Most effects were transcriptional, since gene expression levels were significantly influenced by RH-A and RH-B. In IL-1ß treated normal chondrocytes (NHAC-kn), both RH preparations reduced the expression of matrix metalloproteinase- (MMP-) 1, MMP-3, and MMP-13 and ADAMTS-4. These changes are associated with diminished inflammatory damage or cartilage erosion. Principal component analysis revealed that (1) RH-A and RH-B modified a large pattern of biomarkers, and (2) RH-B outperformed RH-A. Furthermore, RH-B contained more chondroprotective and anti-inflammatory constituents than RH-A. Thus, RHP contributed to restore cellular homeostasis in PBL and chondrocytes. RH preparations from fruits without seeds are thus expected to have an improved OA-preventive or OA-therapeutic profile, as subsequently shown in a related clinical trial.

Resveratrol, from experimental data to nutritional evidence: the emergence of a new food ingredient.

The polyphenol resveratrol is found notably in grapes and in a variety of medicinal plants. Recently, resveratrol has been suggested to have cardioprotective effects and to improve metabolic health by mimicking the effects of calorie restriction. Numerous animal and in vitro studies suggest that resveratrol could improve cardiovascular and metabolic health in humans. In view of this compelling preclinical evidence, several human studies investigating the effects of resveratrol on vascular and metabolic health have been initiated. Collectively, the animal, human epidemiological, and first human intervention studies support a role of resveratrol in vascular and metabolic health. This has led to the introduction of the first supplement and food products containing resveratrol and its emergence as a promising new health ingredient. Thus, supplementation with resveratrol may be included in nutritional and lifestyle programs aiming to reduce the risk of vascular and obesity-related problems.

Oral supplementation with 25(OH)D3 versus vitamin D3: effects on 25(OH)D levels, lower extremity function, blood pressure, and markers of innate immunity.

To test the effect of 25(OH)D(3) (HyD) compared to vitamin D(3) on serum 25-hydroxyvitamin D levels (25(OH)D), lower extremity function, blood pressure, and markers of innate immunity. Twenty healthy postmenopausal women with an average 25(OH)D level of 13.2 ± 3.9 ng/mL (mean ± SD) and a mean age of 61.5 ± 7.2 years were randomized to either 20 µg of HyD or 20 µg (800 IU) of vitamin D(3) per day in a double-blind manner. We measured on 14 visits over 4 months, 25(OH)D serum levels, blood pressure, and seven markers of innate immunity (eotaxin, interleukin [IL]-8, IL-12, interferon gamma-induced protein 10 kDa [IP-10], monocyte chemotactic protein-1 [MCP-1], macrophage inflammatory protein beta [MIP-1ß], and "Regulated upon Activation, Normal T-cell Expressed, and Secreted" [RANTES]). At baseline and at 4 months, a test battery for lower extremity function (knee extensor and flexor strength, timed up and go, repeated sit-to-stand) was assessed. All analyses were adjusted for baseline measurement, age, and body mass index. Mean 25(OH)D levels increased to 69.5 ng/mL in the HyD group. This rise was immediate and sustained. Mean 25(OH)D levels increased to 31.0 ng/mL with a slow increase in the vitamin D(3) group. Women on HyD compared with vitamin D(3) had a 2.8-fold increased odds of maintained or improved lower extremity function (odds ratio [OR] = 2.79; 95% confidence interval [CI], 1.18-6.58), and a 5.7-mmHg decrease in systolic blood pressure (p = 0.0002). Both types of vitamin D contributed to a decrease in five out of seven markers of innate immunity, significantly more pronounced with HyD for eotaxin, IL-12, MCP-1, and MIP-1 ß. There were no cases of hypercalcemia at any time point. Twenty micrograms (20 µg) of HyD per day resulted in a safe, immediate, and sustained increase in 25(OH)D serum levels in all participants, which may explain its significant benefit on lower extremity function, systolic blood pressure, and innate immune response compared with vitamin D(3).

Rose hip and its constituent galactolipids confer cartilage protection by modulating cytokine, and chemokine expression.

BACKGROUND: Clinical studies have shown that rose hip powder (RHP) alleviates osteoarthritis (OA). This might be due to anti-inflammatory and cartilage-protective properties of the complete RHP or specific constituents of RHP. Cellular systems (macrophages, peripheral blood leukocytes and chondrocytes), which respond to inflammatory and OA-inducing stimuli, are used as in vitro surrogates to evaluate the possible pain-relief and disease-modifying effects of RHP. METHODS: (1) Inflammatory processes were induced in RAW264.7 cells or human peripheral blood leukocytes (PBL) with LPS. Inflammatory mediators (nitric oxide (NO), prostaglandin E(2) (PGE(2)) and cytokines/chemokines) were determined by the Griess reaction, EIA and multiplex ELISA, respectively. Gene expression was quantified by RT-PCR. RHP or its constituent galactolipid, GLGPG (galactolipid (2S)-1, 2-di-O-[(9Z, 12Z, 15Z)-octadeca-9, 12, 15-trienoyl]-3-O-ß-D-galactopyranosyl glycerol), were added at various concentrations and the effects on biochemical and molecular parameters were evaluated. (2) SW1353 chondrosarcoma cells and primary human knee articular chondrocytes (NHAC-kn) were treated with interleukin (IL)-1ß to induce in vitro processes similar to those occurring during in vivo degradation of cartilage. Biomarkers related to OA (NO, PGE(2), cytokines, chemokines, metalloproteinases) were measured by multiplex ELISA and gene expression analysis in chondrocytes. We investigated the modulation of these events by RHP and GLGPG. RESULTS: In macrophages and PBL, RHP and GLGPG inhibited NO and PGE(2) production and reduced the secretion of cytokines (TNF-α, IFN-γ, IL-1ß, IL-6, IL-12) and chemokines (CCL5/RANTES, CXCL10/IP-10). In SW1353 cells and primary chondrocytes, RHP and GLGPG diminished catabolic gene expression and inflammatory protein secretion as shown by lower mRNA levels of matrix metalloproteinases (MMP-1, MMP-3, MMP-13), aggrecanase (ADAMTS-4), macrophage inflammatory protein (MIP-2, MIP-3α), CCL5/RANTES, CXCL10/IP-10, IL-8, IL-1α and IL-6. The effects of GLGPG were weaker than those of RHP, which presumably contains other chondro-protective substances besides GLGPG. CONCLUSIONS: RHP and GLGPG attenuate inflammatory responses in different cellular systems (macrophages, PBLs and chondrocytes). The effects on cytokine production and MMP expression indicate that RHP and its constituent GLGPG down-regulate catabolic processes associated with osteoarthritis (OA) or rheumatoid arthritis (RA). These data provide a molecular and biochemical basis for cartilage protection provided by RHP.

Hydroxytyrosol is the major anti-inflammatory compound in aqueous olive extracts and impairs cytokine and chemokine production in macrophages.

Substances in olive products contribute to improved health as suggested by epidemiological data. In this study we assessed the effects of hydroxytyrosol (HT) on inflammatory mediators, cytokines and chemokines, and identified anti-inflammatory constituents of aqueous olive extracts, I.E., olive vegetation water (OVW). Murine macrophages (RAW264.7 cells) were stimulated with lipopolysaccharide (LPS) in the absence or presence of substances; inflammatory mediators [nitric oxide (NO), prostaglandin E2 (PGE2), cytokines, interleukins, chemokines] were determined by the Griess reaction, EIA, or multiplex ELISA (Luminex technology). Expression of inflammatory genes was determined by RT-PCR. Aqueous olive extracts were fractionated by preparative HPLC and the fractions investigated for their effects on NO and PGE2 production. Results were further analyzed by principal component analysis. HT inhibited production of NO and PGE2 with an IC50 of 11.4 and 19.5 µM, respectively, reflecting strong anti-inflammatory activity. HT and OVW diminished secretion of cytokines (IL-1 α, IL-1 ß, IL-6, IL-12, TNF- α), and chemokines (CXCL10/IP-10, CCL2/MCP-1). HT and OVW concentration-dependently reduced the expression of genes of inducible nitric oxide synthase (iNOS), IL-1 α, CXCL10/IP-10, MIP-1 ß, matrix metalloproteinase-9, and prostaglandin E2 synthase (PGES). The effects of HT were partly mediated VIA the NF- κB pathway, as shown by RT-PCR analysis. HT was identified as the main bioactive compound of OVW. The data provide a molecular basis for elucidating the effects of HT on inflammatory processes. The effects of HT on NO and chemokine production point to their impact on chronic inflammatory processes in endothelium or arthritis.

Dietary constituents reduce lipid accumulation in murine C3H10 T1/2 adipocytes: A novel fluorescent method to quantify fat droplets.

BACKGROUND: Adipocyte volume (fat accumulation) and cell number (adipogenesis) is increased in obese individuals. Our objective was the identification of dietary constituents with inhibitory effects on triglyceride formation during adipogenesis. Therefore an in vitro adipose cell assay in murine C3H10 T1/2 cells was developed, which enabled rapid quantification of intracellular fat droplet accumulation during adipocyte differentiation. Results were corroborated by expression levels of several specific adipogenic and lipogenic genes which are known to regulate triglyceride accumulation. METHODS: C3H10 T1/2 adipocyte differentiation was conducted with rosiglitazone in the presence of test compounds for 7 days. Accumulation of intracellular lipid droplets was measured using the Cellomics® ArrayScan® VTI HCS reader and SpotDetector® BioApplication from ThermoFisher. Fluorescent images were automatically acquired and analysed employing the fluorescent dyes BODIPY® 493/503 and Hoechst 33342, for staining neutral lipids and localisation of nuclei, respectively. The expression levels of adipogenic and lipogenic genes, such as PPARα and PPARγ, C/EBPα, aP2, adiponectin, LPL and HSL, CPT-1ß, ACC1, Glut4 and FAS, were determined by quantitative RT-PCR. Dietary ingredients including PUFAs, carotenoids, polyphenols and catechins were tested for their effect on lipid accumulation. RESULTS: The ω-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the carotenoid ß-carotene and hydroxytyrosol exhibited the strongest inhibitory effects on the rosiglitazone-stimulated lipid formation. (all-E)-lycopene and epigallocatechin gallate (EGCG) showed a moderate inhibition, whereas resveratrol did not reduce fat droplet formation. Additionally, it was demonstrated that adipogenic and lipogenic gene expression was attenuated. DHA, ß-carotene and hydroxytyrosol inhibited the gene expression of PPARγ, C/EBPα, aP2 and CPT-1ß. CONCLUSION: This in vitro assay in differentiating adipocytes enables automated detection and quantification of changes in lipid droplet number, size and intensity. The observed inhibitory effects of identified dietary constituents such as ω-3 PUFAs and ß-carotene correlate with the modulation of genes involved in adipocyte differentiation.

Challenges in discovering bioactives for the food industry.

Nutritional interventions are associated with transient deviations of homeostasis within the human body. The role of a balanced nutrition is, firstly, to ensure an adequate intake of nutrients to efficiently enable all metabolic processes and, secondly, to contribute to sustained human health. This is a new challenge for the food industry because consumers demand taste and convenience in addition to healthy food. The attempts of the food industry to omit health-adverse compounds, and incorporate constituents with identified beneficial health effects has coined the term 'functional food', encompassing fresh or processed food with health-promoting and/or disease-preventing properties. The active ingredients of these products are known as 'nutraceuticals'. This paper outlines scientific concepts applied to the identification of novel bioactive food ingredients.

Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis.

Glycogen synthase kinase-3 (GSK-3) is known to modulate cell survival and apoptosis through multiple intracellular signaling pathways. However, its hepatoprotective function and its role in activation of NF-kappaB and anti-apoptotic factors are poorly understood and remain controversial. Here we investigated whether inhibition of GSK-3 could induce apoptosis in the presence of TNF-alpha in primary mouse hepatocytes. We show that pharmacological inhibition of GSK-3 in primary mouse hepatocytes does not lead to TNF-alpha-induced apoptosis despite reduced NF-kappaB activity. Enhanced stability of IkappaB-alpha appears to be responsible for lower levels of nuclear NF-kappaB and hence reduced transactivation. Additionally, inhibition of GSK-3 was accompanied by marked upregulation of beta-catenin, AP-1, and CREB transcription factors. Stimulation of canonical Wnt signaling and CREB activity led to elevated levels of anti-apoptotic factors. Hence, survival of primary mouse hepatocytes may be caused by the activation and/or upregulation of other key regulators of liver homeostasis and regeneration. These signaling molecules may compensate for the compromised anti-apoptotic function of NF-kappaB and allow survival of hepatocytes in the presence of TNF-alpha and GSK-3 inhibition.

Plant phenolics inhibit neutrophil elastase.

Human neutrophil elastase (HNE) is a serine protease, which is present in its active form in inflamed tissue as well as in psoriatic lesions. In extension of our research on natural compounds as inhibitors of HNE or of its release, several phenolics of different size were tested. The ellagitannins agrimoniin and pedunculagin were the most potent direct HNE inhibitors (IC (50) = 0.9 and 2.8 microM, respectively). Ligand docking calculations provided evidence that inhibition may occur in an unspecific manner. Agrimoniin also showed anti-proliferative effects in the ATP assay (IC (50) = 3.2 microM), suggesting that this type of tannin could have beneficial effects in the treatment of diseases such as psoriasis. Tests with other phenolics combined with ligand docking experiments revealed that, besides the presence of ORTHO-dihydroxy groups, a specific lipophilic shape is necessary for an inhibitory activity. The phenolic genistein deserves special interest as an inhibitor of elastase release because its effect was remarkably potent (IC (50) = 0.6 microM).

Analysis with respect to instrumental variables for the exploration of microarray data structures.

BACKGROUND: Evaluating the importance of the different sources of variations is essential in microarray data experiments. Complex experimental designs generally include various factors structuring the data which should be taken into account. The objective of these experiments is the exploration of some given factors while controlling other factors. RESULTS: We present here a family of methods, the analyses with respect to instrumental variables, which can be easily applied to the particular case of microarray data. An illustrative example of analysis with instrumental variables is given in the case of microarray data investigating the effect of beverage intake on peripheral blood gene expression. This approach is compared to an ANOVA-based gene-by-gene statistical method. CONCLUSION: Instrumental variables analyses provide a simple way to control several sources of variation in a multivariate analysis of microarray data. Due to their flexibility, these methods can be associated with a large range of ordination techniques combined with one or several qualitative and/or quantitative descriptive variables.