IL-37 regulates allergic inflammation by counterbalancing pro-inflammatory IL-1 and IL-33.

BACKGROUND: Children with asthma have impaired production of interleukin (IL) 37; in mice, IL-37 reduces hallmarks of experimental allergic asthma (EAA). However, it remains unclear how IL-37 exerts its inhibitory properties in asthma. This study aimed to identify the mechanism(s) by which IL-37 controls allergic inflammation. METHODS: IL-37 target cells were identified by single-cell RNA-seq of IL-1R5 and IL-1R8. Airway tissues were isolated by laser-capture microdissection and examined by microarray-based gene expression analysis. Mononuclear cells (MNC) and airway epithelial cells (AECs) were isolated and stimulated with allergen, IL-1ß, or IL-33 together with recombinant human (rh) IL-37. Wild-type, IL-1R1- and IL-33-deficient mice with EAA were treated with rhIL-37. IL-1ß, IL-33, and IL-37 levels were determined in sputum and nasal secretions from adult asthma patients without glucocorticoid therapy. RESULTS: IL-37 target cells included AECs, T cells, and dendritic cells. In mice with EAA, rhIL-37 led to differential expression of >90 genes induced by IL-1ß and IL-33. rhIL-37 reduced production of Th2 cytokines in allergen-activated MNCs from wild-type but not from IL-1R1-deficient mice and inhibited IL-33-induced Th2 cytokine release. Furthermore, rhIL-37 attenuated IL-1ß- and IL-33-induced pro-inflammatory mediator expression in murine AEC cultures. In contrast to wild-type mice, hIL-37 had no effect on EAA in IL-1R1- or IL-33-deficient mice. We also observed that expression/production ratios of both IL-1ß and IL-33 to IL-37 were dramatically increased in asthma patients compared to healthy controls. CONCLUSION: IL-37 downregulates allergic airway inflammation by counterbalancing the disease-amplifying effects of IL-1ß and IL-33.

Constitutive immune activity promotes JNK- and FoxO-dependent remodeling of Drosophila airways.

Extensive remodeling of the airways is a major characteristic of chronic inflammatory lung diseases such as asthma or chronic obstructive pulmonary disease (COPD). To elucidate the importance of a deregulated immune response in the airways for remodeling processes, we established a matching Drosophila model. Here, triggering the Imd (immune deficiency) pathway in tracheal cells induced organ-wide remodeling. This structural remodeling comprises disorganization of epithelial structures and comprehensive epithelial thickening. We show that these structural changes do not depend on the Imd pathway's canonical branch terminating on nuclear factor κB (NF-κB) activation. Instead, activation of a different segment of the Imd pathway that branches off downstream of Tak1 and comprises activation of c-Jun N-terminal kinase (JNK) and forkhead transcription factor of the O subgroup (FoxO) signaling is necessary and sufficient to mediate the observed structural changes of the airways. Our findings imply that targeting JNK and FoxO signaling in the airways could be a promising strategy to interfere with disease-associated airway remodeling processes.

The alpha-melanocyte-stimulating hormone acts as a local immune homeostasis factor in experimental allergic asthma.

BACKGROUND: Originally, the neuropeptide α-melanocyte-stimulating hormone (α-MSH) has been described as a mediator of skin pigmentation. However, recent studies have shown that α-MSH is able to modulate inflammation in various tissues including the lung. So far, it is still not clear whether α-MSH also plays a role in allergic bronchial asthma. OBJECTIVE: This study aimed at investigating the role and regulatory mechanisms of α-MSH in asthma pathogenesis. METHODS: α-MSH levels were measured in bronchoalveolar lavage (BAL) fluid of asthmatic and non-asthmatic individuals as well as of healthy mice and mice with experimental asthma. Wild-type mice were sensitized to ovalbumin (OVA) and exposed to an OVA aerosol in order to induce experimental allergic asthma. α-MSH was administrated intratracheally, the α-MSH antibody intraperitoneally prior each OVA challenge. Airway inflammation, cytokine production, mucus production, airway hyperresponsiveness and receptor expression were assessed. RESULTS: α-MSH levels in BAL of asthmatic individuals and mice were significantly higher compared to healthy controls. In a mouse model of experimental asthma, α-MSH neutralization increased airway inflammation and mucus production, whereas local administration of α-MSH significantly reduced inflammation of the airways. The beneficial effects were further associated with decreased levels of eosinophilic chemoattractant factors that are released by MC5R-positive T helper 2 and airway epithelial cells. CONCLUSION AND CLINICAL RELEVANCE: α-MSH acts as a regulatory factor to maintain local immune homeostasis in allergic bronchial asthma.

Tumstatin fragment selectively inhibits neutrophil infiltration in experimental asthma exacerbation.

BACKGROUND: Asthma is a chronic inflammatory disease with structural changes present. Burgess and colleagues recently found tumstatin markedly reduced in adult asthmatic lung tissue compared with nonasthmatics. ECM fragments such as tumstatin are named matrikines and act independently of the parent molecule. The role of Col IV matrikines in neutrophil inflammation (eg. exacerbation in asthma) has not been investigated to date. Severe adult asthma phenotypes are dominated by neutrophilic inflammation and show a high frequency of severe exacerbations. OBJECTIVE: This study sought to investigate the role of a novel active region within tumstatin (CP17) and its implication in neutrophil inflammatory responses related to asthma exacerbation. METHODS: For reactive oxygen production, isolated neutrophils were preincubated with peptides or vehicle for 1 hour and stimulated (PMA). Luminescence signal was recorded (integration over 10 seconds) for 1.5 hours. Neutrophil migration was performed according to the SiMA protocol. Mice were sensitized to OVA/Alumn by intraperitoneal (i.p.) injections. Mice were then treated with CP17, vehicle (PBS) or scrambled peptide (SP17) after OVA exposure (days 27 and 28, polyI:C stimulation). All animals were killed on day 29 with lung function measurement, histology and lavage. RESULTS: CP17 decreased total ROS production rate to 52.44% (0.5 µmol/L, P < 0.05 vs SP17), reduced the in vitro directionality (vs SP17, P = 1 × 10-6 ) and migration speed (5 µmol/L, P = 1 × 10-3 ). In vivo application of CP17 decreased neutrophil inflammation ~1.8-fold (P < 0.001 vs SP17) and reduced numbers of mucus-producing cells (-29%, P < 0.05). CONCLUSION: CP17 reduced the ROS production rate, migrational speed and selectively inhibited neutrophil accumulation in the lung interstitium and lumen. CLINICAL RELEVANCE: CP17 may serve as a potential precursor for drug development to combat overwhelming neutrophil inflammation.

Novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ARDS model: a translational study.

The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit acute respiratory distress syndrome (ARDS) model (repeated airway lavage, overventilation, and LPS instillation into airways), we assessed whether the supplementation of surfactant (S; poractant alfa) with inositol derivatives [inositol 1,2,6-trisphosphate (IP3) or phosphatidylinositol 3,5-bisphosphate (PIP2)] or phosphatidylglycerol subfractions [16:0/18:1-palmitoyloleoyl-phosphatidylglycerol (POPG) or 18:1/18:1-dioleoyl-phosphatidylglycerol (DOPG)] would result in improved clinical parameters and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3, S+PIP2, and S+POPG), the ventilation efficiency index (S+IP3 and S+POPG), the compliance (S+IP3 and S+POPG) and resistance (S+POPG) of the respiratory system, and the extravascular lung water index (S+IP3 and S+POPG) significantly improved compared with S treatment alone. The inositol derivatives (mainly S+IP3) exerted their actions by suppressing acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome nucleotide-binding domain, leucine-rich repeat-containing protein-3 (NLRP3)-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-caspase-1 complex, and the profibrotic response represented by the cytokines transforming growth factor-ß1 and IFN-γ, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IκB kinase activity was significantly reduced. S+POPG and S+DOPG treatment inhibited polymorphonuclear leukocyte activity (MMP-8 and myeloperoxidase) and the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis, all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol derivatives and phosphatidylglycerol subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.

Nuclear Localization of Suppressor of Cytokine Signaling-1 Regulates Local Immunity in the Lung.

Suppressor of cytokine signaling 1 (SOCS1) is a negative feedback inhibitor of cytoplasmic Janus kinase and signal transducer and activator of transcription (STAT) signaling. SOCS1 also contains a nuclear localization sequence (NLS), yet, the in vivo importance of nuclear translocation is unknown. We generated transgenic mice containing mutated Socs1ΔNLS that fails to translocate in the cell nucleus (MGLtg mice). Whereas mice fully deficient for SOCS1 die within the first 3 weeks due to excessive interferon signaling and multiorgan inflammation, mice expressing only non-nuclear Socs1ΔNLS (Socs1-/-MGLtg mice) were rescued from early lethality. Canonical interferon gamma signaling was still functional in Socs1-/-MGLtg mice as shown by unaltered tyrosine phosphorylation of STAT1 and whole genome expression analysis. However, a subset of NFκB inducible genes was dysregulated. Socs1-/-MGLtg mice spontaneously developed low-grade inflammation in the lung and had elevated Th2-type cytokines. Upon ovalbumin sensitization and challenge, airway eosinophilia was increased in Socs1-/-MGLtg mice. Decreased transepithelial electrical resistance in trachea epithelial cells from Socs1-/-MGLtg mice suggests disrupted epithelial cell barrier. The results indicate that nuclear SOCS1 is a regulator of local immunity in the lung and unravel a so far unrecognized function for SOCS1 in the cell nucleus.

Effect of IL-37 on Allergic Airway Inflammation.

RATIONALE: The new cytokine IL-37 has been described as a negative regulator of innate immunity. It reduces activation of dendritic cells and the production of proinflammatory mediators in murine and human immune cells. Although recent results from the CLARA childhood asthma cohort suggested an impact of IL-37 on human asthma pathogenesis, the receptor for IL-37 and its implication in adaptive immune responses have not been determined. OBJECTIVES: This study aimed to clarify whether IL-37 also provides antiinflammatory effects on adaptive immune responses and through which receptor it exerts its effects. METHODS: IL-37 levels in supernatants of restimulated peripheral blood mononuclear cells isolated from children with asthma and healthy children were determined. Mice (wild-type, IL-18Rα(-/-), and SIGIRR/IL-1R8(-/-)) were sensitized to ovalbumin (OVA) and challenged with OVA aerosol to induce acute allergic experimental asthma, and IL-37 was applied intranasally during OVA challenge. Airway hyperresponsiveness was determined. A cytometric bead array was used to assess cytokine levels in bronchoalveolar lavage fluid. Epithelial mucus was quantified on the basis of lung sections stained with periodic acid-Schiff reagent, using the newCAST microscope system. MEASUREMENTS AND MAIN RESULTS: Human peripheral blood mononuclear cells of subjects with allergic asthma produce less IL-37 compared with healthy control subjects. In mice, intranasal administration of IL-37 dampened allergic airway inflammation as well as proinflammatory cytokine production, mucus hyperproduction, and airway hyperresponsiveness. However, the antiinflammatory effects of IL-37 were completely abolished in mice deficient for IL-18Rα or SIGIRR/IL-1R8. CONCLUSIONS: This study demonstrates that IL-37 reduces allergic airway inflammation directed by type 2 helper T cells and the hallmarks of experimental asthma in mice, suggesting that IL-37 may be critical for asthma pathogenesis in particular and may have an impact on adaptive immunity in general. Furthermore, these data suggest a mode of action of IL-37 that involves binding to IL-18Rα and subsequent heterodimerization with or activation of SIGIRR/IL-1R8. Therefore, IL-37 or its receptors could be potential targets for asthma intervention.

High dose CD11c-driven IL15 is sufficient to drive NK cell maturation and anti-tumor activity in a trans-presentation independent manner.

The common gamma (γc)-chain cytokine interleukin 15 (IL15) is a multifunctional immune-modulator which impacts the generation, maturation and activity of many cell types of the innate, as well as the adaptive immune system, including natural killer (NK) and CD8(+) T cells. Using a new series of transgenic mice, we analyzed the in vivo potential of IL15 as an immune-regulator when available at different concentrations or delivery modes, i.e. soluble monomer or complexed to its specific receptor α (Rα)-chain. We have identified distinct effects on selected IL15-responsive populations. While CD8(+) T cells required complexed forms of IL15/IL15Rα for full functionality, mature NK populations were rescued in an IL15/IL15Rα-deficient environment by high levels of CD11c-restricted IL15. These IL15-conditions were sufficient to limit tumor formation in a lung metastasis model indicating that the NK cell populations were fully functional. These data underline the potential of "free" IL15 in the absence of Rα-complex as a powerful and specific immuno-modulator, which may be beneficial where selective immune-activation is desired.

Poly(inosinic-cytidylic) acid-triggered exacerbation of experimental asthma depends on IL-17A produced by NK cells.

Viral infection of the respiratory tract represents the major cause of acute asthma exacerbations. dsRNA is produced as an intermediate during replication of respiratory viruses and triggers immune responses via TLR3. This study aimed at clarifying the mechanisms underlying TLR3 triggered exacerbation of experimental allergic asthma. The TLR3 ligand poly(inosinic-cytidylic) acid was applied intranasally to mice with already established experimental allergic asthma. Airway inflammation, cytokine expression, mucus production, and airway reactivity was assessed in wild-type, IL-17A, or IL-23p19-deficient, and in NK cell-depleted mice. Local application of poly(inosinic-cytidylic) acid exacerbated experimental allergic asthma in mice as characterized by enhanced release of proinflammatory cytokines, aggravated airway inflammation, and increased mucus production together with pronounced airway hyperresponsiveness. This was further associated with augmented production of IL-17 by Th17 cells and NK cells. Whereas experimental exacerbation could be induced in IL-23p19-deficient mice lacking mature, proinflammatory Th17 cells, this was not possible in mice lacking IL-17A or in NK cell-depleted animals. These experiments indicate a central role for IL-17 derived from NK cells but not from Th17 cells in the pathogenesis of virus-triggered exacerbation of experimental asthma.

HOPE-BAL: improved molecular diagnostics by application of a novel technique for fixation and paraffin embedding.

The bronchoalveolar lavage (BAL) and its cells have been widely used as a support for clinical diagnosis and as a versatile tool for research questions since many years. Because there are no sufficient possibilities of long-term storage, the authors explore in this study the utility of a new fixative for fixation and paraffin embedding of human lavage cells with the possibility of implementing standard molecular biology techniques. HOPE-fixed, paraffin-embedded BAL cells of patients with different lung diseases (asthma, chronic obstructive pulmonary diseases, tuberculosis, sarcoidosis, emphysema, and fibrosis) were subjected to immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, and transcription microarray analysis. Furthermore, two-dimensional gel electrophoresis was conducted to evaluate the range of possible applications for research, diagnostics, and further implementing in biobanks. The authors show, by targeting some exemplary molecules, the power of screening and validating HOPE-BAL for new biomarkers. The transforming growth factor ß signaling pathway may play a central role in immunomodulation upon infection as well as asthma. Furthermore, haptoglobin was overexpressed in asthma and sarcoidosis. Because of the excellent preservation of nucleic acids, protein, and morphologic structures, HOPE-BAL is a step forward into enhanced molecular diagnostics and biobanking in pulmonary medicine.

The other T helper cells in asthma pathogenesis.

The complex phenotype of allergic bronchial asthma involves a variable degree of bronchoobstruction, increased mucus production, and airway remodeling. So far it is suggested that it arises from multiple interactions of infiltrating and structural cells in the context of chronic airway inflammation that is orchestrated by T helper 2 (TH2) cells. By secreting a plethora of typical mediators such as interleukin (IL) 4, IL-5, and IL-13, these cells hold a key position in asthma pathogenesis. However, therapeutic approaches targeting these TH2-type mediators failed to improve asthma symptoms and impressively showed that asthma pathogenesis cannot be reduced by TH2 cell functions. Recently, other T helper cells, that is, TH9 and TH17 cells, have been identified and these cells also contribute to asthma pathogenesis, the processes leading to formation or aggravation of asthma. Furthermore, TH25 cells, TH3 cells, and regulatory T cells have also been implicated in asthma pathogenesis. This paper aims at summarizing recent insights about these new T helper cells in asthma pathogenesis.

ACBP knockdown leads to down-regulation of genes encoding rate-limiting enzymes in cholesterol and fatty acid metabolism.

The human Acyl-CoA binding protein (ACBP) is a structural and functional highly conserved protein. As an intracellular pool former and carrier of acyl-CoAs, ACBP influences overall lipid metabolism. Its nuclear abundance and physical interaction with hepatocyte nuclear factor 4alpha suggested a gene regulatory function of ACBP. To identify ACBP target genes we performed genome-wide transcript profiling under siRNA-mediated ACBP knockdown in human liver HepG2 cells. Based on a single sided permutation T-test (p<0.05) we identified 256 down-regulated and 198 up-regulated transcripts with a minimal fold change of 1.32 (log 0.5). Gene annotation enrichment analysis revealed ACBP-mediated down-regulation of 18 genes encoding key enzymes in glycerolipid (i.e. mitochondrial glycerol-3-phosphate acyltransferase), cholesterol (i.e. HMG-CoA synthase and HMG-CoA reductase) and fatty acid (i.e. fatty acid synthase) metabolism. Integration of these genes in common pathways suggested decreased lipid biosynthesis. Accordingly, saturated (16:0) and monosaturated (16:1, 18:1) fatty acids were significantly reduced to 75% in ACBP-depleted cells. Taken together, we obtained evidence that ACBP functions in lipid metabolism at the level of gene expression. This effect seems to be translated into certain metabolites. The identified 454 ACBP regulated genes present a first reference for further studies to define the ACBP regulon in mammalian cells.

Comparative analyses of disease risk genes belonging to the acyl-CoA synthetase medium-chain (ACSM) family in human liver and cell lines.

The human ACSM1, 2A and B, 3, and 5 genes, located on chromosome 16p12-13, encode for enzymes catalyzing the activation of medium-chain length fatty acids. Association studies have linked several polymorphisms of these genes to traits of insulin resistance syndrome. In our study, ACSM transcripts showed 3 to >400-fold higher expression levels in human liver when compared to cell lines by qRT-PCR. This difference was also evident at the protein level, as shown for ACSM2. In liver, ACSM2 was the most abundant transcript, showing sixfold (vs. ACSM3) to >300-fold higher expression levels (vs. ACSM1). Mitochondrial localization of the ACSM2 protein and the presence of an N-terminal targeting sequence were shown by GFP-tagging. We have shown ACSM2B to be the predominant transcript in human liver, and genetic variations of this gene could therefore play an important role in disease susceptibility.

Transcriptional regulation of HMG-CoA synthase and HMG-CoA reductase genes by human ACBP.

The acyl-CoA binding protein (ACBP) is an ubiquitary expressed multi-functional protein which regulates basic cellular functions such as fatty acid and steroid metabolism. Since ACBP is described to interact with the transcription factor hepatocyte nuclear factor 4 alpha (HNF-4alpha), we investigated the role of human ACBP on transcriptional regulation of the putative HNF-4alpha target gene HMG-CoA synthase 1 (HMGCS1). As shown by promoter-reporter assays ACBP represses the HNF-4alpha-induced activity of a 617bp HMGCS1 promoter fragment by approximately 80% in HepG2 cells as well as in non-endodermal HeLa cells devoid of HNF-4alpha. Interestingly, reporter assays without co-transfection of HNF-4alpha revealed that ACBP reduces the activity of the HMGCS1 promoter by about 60 to 80% in both cell lines. Activities of 417bp and 317bp HMGCS1 promoter fragments were 2.5 to 4 fold decreased by ACBP. Concordantly, the levels of HMGCS1-mRNA and -protein were diminished to 60% and 70% in ACBP-expressing HeLa cells, respectively. Additionally, ACBP reduces the promoter activity and the mRNA levels of the cholesterogenic HMG-CoA reductase (HMGCR). In conclusion, we provide evidence that ACBP is a transcriptional regulator of the HMGCS1 and HMGCR genes encoding rate-limiting enzymes of cholesterol synthesis pathway.

Oleate regulates genes controlled by signaling pathways of mitogen-activated protein kinase, insulin, and hypoxia.

Oleate (C18:1) is, besides palmitate (C16:0), the most abundant fatty acid in the human diet, and its involvement in the development of insulin resistance is broadly discussed. Because its influence on gene expression is poorly defined in mammalian cells, we performed whole genome expression profiling and quantitative real-time polymerase chain reaction in the human hepatocyte cell line HepG2 to identify oleate-regulated genes. In this respect, HepG2 cells were exposed for 24 hours to a physiologic concentration of oleate coupled to bovine serum albumin (BSA) (200 micromol/L) or BSA alone. Subsequent microarray analysis revealed 14 genes that were significantly (single-sided permutational t test, P < .05) regulated after oleate treatment. To decipher the functional and regulatory connections of these genes, a text mining approach combined with transcription factor binding site analysis was performed using Genomatix BiblioSphere (Munich, Germany) and MatInspector (Munich, Germany). The oleate-inducible genes encoding early growth response 1, c-fos, S-phase kinase-associated protein 2, and splicing factor 2 are mapped into a network, which is controlled by signaling pathways of mitogen-activated protein kinase, insulin, or hypoxia. Comparative in silico promoter analysis revealed putative regulation of oleate-sensitive genes through v-ets erythroblastosis virus E26 oncogene homolog 1 and retinoid X receptor family. In sum, a physiologic oleate concentration modulates genes expression in a very sensitive way as 14 genes were regulated.

Nutrition concepts for elite distance runners based on macronutrient and energy expenditure.

CONTEXT: Elite distance runners (EDR) must optimize their nutrition to maintain their demanding training schedules. OBJECTIVE: To develop a nutrition concept for EDR based on energy and macronutrient expenditures. DESIGN: This theoretical study provides calculations for macronutrient and energy expenditures of EDR. Anthropometric and metabolic characteristics of EDR were assumed based on average real EDR. SETTING: University of Kiel. PATIENTS OR OTHER PARTICIPANTS: Three prototypic types of male EDR described in the literature as type I (TI; body mass = 72 kg, respiratory quotient = 0.9 at rest, fast-twitch muscle fibers = 60% to 70%), type II (TII; body mass = 67 kg, respiratory quotient = 0.82 at rest, fast-twitch muscle fibers = 50%), and type III (TIII; body mass = 60 kg, respiratory quotient = 0.75 at rest, fast-twitch muscle fibers = 30% to 40%). MAIN OUTCOME MEASURE(S): We calculated the macronutrient and energy expenditures of the 3 types of EDR according to body mass, respiratory quotient, and percentage of fast-twitch muscle fibers. RESULTS: We found that the average energy expenditure was 3750 kcal . d(-1) for TI runners, 3463 kcal . d(-1) for TII runners, and 3079 kcal . d(-1) for TIII runners. The carbohydrate (CHO) expenditure reached an average value of 10.0 g . kg(-1) . d(-1) for TI runners, 8.0 g . kg(-1) . d(-1) for TII runners, and 4.7 g . kg(-1) . d(-1) for TIII runners. When the EDR accomplished running sessions at a pace >or=100% of maximum oxygen consumption, all types of runners had a CHO demand of about 10 g . kg(-1) . d(-1). The TI and TII runners need a CHO intake of 8 to 10 g . kg(-1) . d(-1). For the TIII runners, a CHO intake >6 g . kg(-1) . d(-1) is necessary during anaerobic training sessions. CONCLUSIONS: Nutrition concepts must be differentiated for EDR according to metabolic and anthropometric characteristics of the runners and their special training emphases.

Functional connections and pathways of coenzyme Q10-inducible genes: an in-silico study.

Coenzyme Q10 (CoQ10, ubiquinone) is an essential cofactor in the electron transport chain, serves as a potent antioxidant in mitochondria and lipid membranes, and is often used as a dietary supplement for a number of diseases including cardiovascular diseases. Recently, we obtained evidence that CoQ10 (Kaneka Q10) affects the expression of hundreds of human genes. To decipher the functional and regulatory connections of these genes, a literature search combined with transcription factor binding site analysis was performed using Genomatix BiblioSphere and MatInspector. This in-silico analysis revealed 17 CoQ10-inducible genes which are functionally connected by signalling pathways of G-protein coupled receptors, JAK/STAT, integrin, and beta-arrestin. Promoter analysis of these CoQ10-inducible genes showed one group of NF B-regulated genes, namely IL5, thrombin, vitronectin receptor and C-reactive protein (CRP). Furthermore, a common promoter framework containing binding sites of the transcription factor families EVI1, HOXF, HOXC, and CLOX was identified in the promoters of IL5, CRP, and vitronectin receptor. The identified CoQ10-inducible genes and pathways play an important role in inflammatory response. Since these effects are based on an in-vitro study, the effect of CoQ10 on vascular health in vivo needs to be addressed in further animal and/or human intervention studies.

Identification of palmitate-regulated genes in HepG2 cells by applying microarray analysis.

Palmitate is the most abundant saturated fatty acid in the human diet and the major one synthesized de novo. To identify palmitate-regulated genes we performed whole genome mRNA expression profiling by using human hepatoma HepG2 cells. We identified eleven genes which are significantly (single-sided permutational t-test, p<0.05) regulated by low concentration of palmitate (50 microM). We observed a decreased expression of five metallothioneins, and an increased expression of liver expressed plasminogen activator inhibitor-1 protein and insulin-like growth factor II, which play a prominent role in the development of the metabolic syndrome. Comparative promoter analysis in-silico revealed common transcriptional regulation of differentially expressed genes through erythroid kruppel-like factor and members of the zinc binding protein factor family. In conclusion, low physiological palmitate concentrations changed expression of very responsive genes.

Expression analysis of genes involved in fat assimilation in human monocytes.

The metabolic syndrome X is characterized by a group of risk factors such as obesity, atherogenic dyslipidemia, hypertension, and insulin resistance. To study the functional alterations resulting from genetic variations, ex vivo studies are one option to be carried out. Since it is not an easy procedure to obtain cells from the related tissues ex vivo, the aim of the present study was to investigate whether monocytes can serve as model cells. The purpose was to check if monocytes are insulin target cells or not and to elucidate the expression of genes involved in fat assimilation. Human monocytes were drawn from venous blood of healthy donors, aged 25 - 30, using density gradient separation and antibody-based magnetic cell sorting of CD14-positive cells. An expression analysis of genes was performed using RT-PCR and Western Blot. Transcripts of the three splice-variants of the Acyl-CoA binding protein (ACBP), the Medium-chain Acyl-CoA Synthetase 1 (MACS1), the Insulin Receptor (INSR) and the Peroxisome Proliferator-activated Receptor gamma (PPARgamma) are consistently expressed in monocytes of all donors. Differences in gene expression between donors are found for two other members of the MACS-family, the fatty acid transport protein 3 (FATP3) and the FATP4. On protein level, we tested for ACBP expression. The ACBP protein is consistently expressed in monocytes of all donors. Human monocytes are insulin target cells and capable of fatty acid metabolism to some extent. Ex vivo-derived monocytes could be used in additional studies for analyzing differences in genotype-dependent expression levels of genes involved in fat assimilation such as ACBP, MACS1 or PPARgamma.