Inhibitory effects of the platelet-activating factor receptor antagonists, CV-3988 and Ginkgolide B, on alkali burn-induced corneal neovascularization.

PURPOSE: Platelet-activating factor (PAF) has been found in various ocular tissues; the activity of PAF depends on the binding to its specific receptor, PAF-receptor. We investigated the therapeutic effects of PAF-receptor antagonists (CV-3988 and Ginkgolide B) on alkali burn-induced corneal neovascularization (CNV). METHODS: CNV was induced by applying a 0.2 N sodium hydroxide (3 µl, NaOH) solution directly on mice corneas. CV-3988 (1 mM/10 µl) and Ginkgolide B (1 mM/10 µl) were administered topically on the corneas three times daily for three consecutive days. CNV was evaluated under a slit-lamp microscope. Corneas were processed for histological, immunohistochemical and reverse transcription polymerase chain reaction analysis. Human umbilical vein endothelial cells were used for the migration and tube formation assay. RESULTS: Application of CV-3988 and Ginkgolide B inhibited CNV caused by alkali burn. CV-3988 and Ginkgolide B attenuated the expression of PAF-receptor mRNA. Alkali injury induced a massively increased intraocular mRNA expression of an angiogenic factor in cornea tissues, whereas these increments were attenuated by the application of CV-3988 and Ginkgolide B. CONCLUSIONS: CV-3988 and Ginkgolide B reversed opacity and neovascularization in alkali burn-induced corneas. Our findings suggest that CV-3988 and Ginkgolide B may be therapeutically useful in the treatment of CNV and inflammation.

Selective inhibition of phosphoinositide 3-kinase p110α preserves lymphocyte function.

Class IA phosphoinositide 3-kinase (PI3K) is essential for clonal expansion, differentiation, and effector function of B and T lymphocytes. The p110δ catalytic isoform of PI3K is highly expressed in lymphocytes and plays a prominent role in B and T cell responses. Another class IA PI3K catalytic isoform, p110α, is a promising drug target in cancer but little is known about its function in lymphocytes. Here we used highly selective inhibitors to probe the function of p110α in lymphocyte responses in vitro and in vivo. p110α inhibition partially reduced B cell receptor (BCR)-dependent AKT activation and proliferation, and diminished survival supported by the cytokines BAFF and IL-4. Selective p110δ inhibition suppressed B cell responses much more strongly, yet maximal suppression was achieved by targeting multiple PI3K isoforms. In mouse and human T cells, inhibition of single class IA isoforms had little effect on proliferation, whereas pan-class I inhibition did suppress T cell expansion. In mice, selective p110α inhibition using the investigational agent MLN1117 (previously known as INK1117) did not disrupt the marginal zone B cell compartment and did not block T cell-dependent germinal center formation. In contrast, the selective p110δ inhibitor IC87114 strongly suppressed germinal center formation and reduced marginal zone B cell numbers, similar to a pan-class I inhibitor. These findings show that although acute p110α inhibition partially diminishes AKT activation, selective p110α inhibitors are likely to be less immunosuppressive in vivo compared with p110δ or pan-class I inhibitors.

Caffeic acid phenethyl ester inhibits the inflammatory effects of interleukin-1ß in human corneal fibroblasts.

CONTEXT: Expression of various inflammatory mediators in corneal fibroblasts contributes to corneal inflammation. OBJECTIVE: The purpose of this study was to assess the possible effects of caffeic acid phenethyl ester (CAPE) on the expression of inflammatory mediators during an inflammatory response in human corneal fibroblasts. MATERIALS AND METHODS: The levels of interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule-1 (ICAM-1) from IL-1ß-exposed human corneal fibroblasts were measured with enzyme-linked immunosorbent assays (ELISA). The regulatory mechanisms of CAPE on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays. A functional validation was carried out by evaluating the inhibitory effects of CAPE on neutrophil and monocyte migration in vitro. RESULTS: CAPE inhibited the expression of IL-6, MCP-1 and ICAM-1 induced by the pro-inflammatory cytokine IL-1ß in corneal fibroblasts. The activation of AKT and NF-κB by IL-1ß was markedly inhibited by CAPE, whereas the activity of mitogen-activated protein kinases (MAPKs) was not affected. CAPE significantly suppressed the IL-1ß-induced migration of differentiated (d)HL-60 and THP-1 cells. DISCUSSION: These anti-inflammatory effects of CAPE may be expected to inhibit the infiltration of leukocytes into the corneal stroma in vivo.

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 µl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 µl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

Expression of galectin-9 by IFN-γ stimulated human nasal polyp fibroblasts through MAPK, PI3K, and JAK/STAT signaling pathways.

Galectin-9 exhibited potent and selective eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo. Nasal polyposis is a chronic inflammatory disease of the upper airway characterized by the marked presence of inflammatory cells, particularly eosinophils. Thus, galectin-9 may be implicated in the pathogenesis of nasal polyposis. The study was designed to investigate whether interferon-gamma (IFN-γ) can induce the augmentation of galectin-9 expression and induce the expression of galectin-9 in nasal polyps. We examined the correlation between galectin-9 expression and eosinophil infiltration in nasal polyps. In addition, we identified the signaling pathways involved in the elevation of galectin-9 expression in response to IFN-γ. Our data demonstrate that the involvement of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3 phosphate kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) may play important roles in the selective recruitment of eosinophils in nasal polyp tissues through the production of galectin-9. These findings suggest that galectin-9 expression is associated with eosinophil infiltration in polyps of patients with nasal polyposis.

Correction: Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells.

[This corrects the article DOI: 10.1371/journal.pone.0220807.].

Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells.

Prostate cancer (PCa) is the most common cancer among men worldwide. Most PCa cases are not fatal; however, the outlook is poor when PCa spreads to another organ. Bone is the target organ in about 80% of patients who experience metastasis from a primary PCa tumor. In the present study, we characterized the secretome of PC3/nKR cells, which are a new subline of PC3 cells that were originally isolated from nude mice that were implanted with PC3 cells without anti-natural killer (NK) cell treatment. Wound healing and Transwell assays revealed that PC3/nKR cells had increased migratory and invasive activities in addition to a higher resistance to NK cells-induced cytotoxicity as compared to PC3 cells. We quantitatively profiled the secreted proteins of PC3/nKR and PC3 cells by liquid chromatography-tandem mass spectrometry analysis coupled with 2-plex tandem mass tag labeling. In total, 598 secretory proteins were identified, and 561 proteins were quantified, among which 45 proteins were secreted more and 40 proteins were secreted less by PC3/nKR cells than by PC3 cells. For validation, the adapter molecule crk, serpin B3, and cystatin-M were analyzed by western blotting. PC3/nKR cells showed the selective secretion of NKG2D ligand 2, HLA-A, and IL-6, which may contribute to their NK cell-mediated cytotoxicity resistance, and had a high secretion of crk protein, which may contribute to their high migration and invasion properties. Based on our secretome analysis, we propose that PC3/nKR cells represent a new cell system for studying the metastasis and progression of PCa.

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways.

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E2, and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-kappaB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-kappaB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor kappaB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-alpha and interleukin-1beta. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-kappaB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.

Amplification of an RNA ligase ribozyme under alternating temperature conditions.

Amplification of an RNA template molecule was examined using the ligase ribozyme and its corresponding RNA substrates under alternating temperature conditions. Alternating temperatures enhanced the rate of the thermodynamically unfavorable dissociation of the annealed products into the two separate RNA templates, reminiscent of the polymerase chain reaction. Under these conditions, the RNA ligase ribozyme system was observed to amplify through a mainly cross-catalytic process, generating additional copies of the starting RNA template molecules. Thus, template-directed RNA ligation using the ribozyme under thermally fluctuating conditions will be an intriguing point to consider when explaining the primordial event of chemical evolution.

Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular beta-catenin protein.

The Wnt/beta-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/beta-catenin signaling pathway. BIM increased beta-catenin responsive transcription (CRT) and up-regulated intracellular beta-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of beta-catenin protein in 3T3-L1 preadipocyte cells.

Identification and characterization of potent CYP2B6 inhibitors in Woohwangcheongsimwon suspension, an herbal preparation used in the treatment and prevention of apoplexy in Korea and China.

Woohwangcheongsimwon is a traditional medicine for treating hypertension, arteriosclerosis, coma, and stroke in China and Korea. To assess potential interactions of herb and drug metabolism, commercially available Woohwangcheongsimwon suspensions were examined for their potential to inhibit the activity of nine human cytochrome P450 enzymes. The Woohwangcheongsimwon suspensions showed strong inhibition of CYP2B6 activity. To identify individual constituents with inhibitory activity, the suspension was partitioned using hexane, ethyl acetate, and dichloromethane, and each fraction was tested for its inhibitory effect on CYP2B6-catalyzed bupropion hydroxylation. The hexane fraction possessed inhibitory activity, and gas chromatography/mass spectrometry analysis identified borneol and isoborneol as major constituents of the hexane fraction. These two terpenoids moderately inhibited CYP2B6-catalyzed bupropion hydroxylase activity in a competitive manner, with K(i) values of 9.5 and 5.9 microM, respectively, as well as efavirenz 8-hydroxylase activity, with K(i) values of 22 and 26 microM, respectively. Additionally, reconstituted mixtures of borneol and isoborneol, at the same concentrations as in the Woohwangcheongsimwon suspension, had comparable potency in inhibiting bupropion hydroxylation. These in vitro data indicate that Woohwangcheongsimwon preparations contain constituents that can potently inhibit the activity of CYP2B6 and suggest that these preparations should be examined for potential pharmacokinetic drug interactions in vivo.

Caffeic acid phenethyl ester attenuates allergic airway inflammation and hyperresponsiveness in murine model of ovalbumin-induced asthma.

Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of propolis, which has several interesting biological properties, including antioxidant and anti-inflammatory; however, its anti-allergic effects are poorly understood. The objective of this study was to determine whether treatment with CAPE results in significant inhibition of asthmatic reactions in a mouse model. Mice sensitized and challenged with ovalbumin (OVA) had the following typical asthmatic reactions: an increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways, and airway luminal narrowing; the development of airway hyperresponsiveness (AHR); the presence of tumor necrosis factor-alpha (TNF-alpha) and Th2 cytokines, including IL-4 and IL-5, in the BAL fluid; and the presence of allergen-specific IgE in the serum. Five successive intraperitoneal administrations of CAPE before the last airway OVA challenge resulted in significant inhibition of characteristic asthmatic reactions. We determined that increased generation of reactive oxygen species (ROS) by inhalation of OVA was diminished via the administration of CAPE in BAL fluid, as well as nuclear factor-kappaB (NF-kappaB) DNA binding activity. These findings indicate that oxidative stress may have a crucial function in the pathogenesis of bronchial asthma, and that CAPE may be useful as an adjuvant therapy for the treatment of bronchial asthma.

Effects of Ecklonia cava ethanolic extracts on airway hyperresponsiveness and inflammation in a murine asthma model: role of suppressor of cytokine signaling.

Ecklonia cava (EC) is a brown alga that evidences radical scavenging activity, bactericidal activity, tyrosinase inhibitory activity, and protease inhibitory activity. However, its anti-allergic effects remain poorly understood. In the current study, we attempted to determine whether pretreatment with EC induces a significant inhibition of asthmatic reactions in a mouse asthma model. Mice sensitized and challenged with ovalbumin (OVA) evidenced typical asthmatic reactions, as follows: an increase in the number of eosinophils in bronchoalveolar lavage fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways, and airway luminal narrowing; the development of airway hyperresponsiveness; the detection of tumor necrosis factor-alpha (TNF-alpha) and Th2 cytokines, including IL-4 and IL-5 in the bronchoalveolar lavage (BAL) fluid; and the detection of allergen-specific immunoglobulin E (IgE) in the serum. However, the administration of EC extract prior to the final airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. We also demonstrated that EC extracts treatment resulted in significant reductions on matrix metalloproteinase-9 (MMP-9) and Suppressor of cytokine signaling-3 (SOCS-3) expression and a reduction in the increased eosinophil peroxidase (EPO) activity. The treatment of animals with EC extracts resulted in a significant reduction in the concentrations of the Th2 cytokine (IL-4 and IL-5) in the airways, without any concomitant increase in the concentration of Th1 cytokines. These findings indicate that EC extracts may prove useful as an adjuvant therapy for allergic airway reactions via the inhibition of the Th2 response. Accordingly, this study may provide evidence that EC extract performs a critical function in the amelioration of the pathogenetic process of asthma in mice.

A PI3K p110α-selective inhibitor enhances the efficacy of anti-HER2/neu antibody therapy against breast cancer in mice.

Combination therapies with phosphoinositide 3-kinase (PI3K) inhibitors and trastuzumab (anti-human epidermal growth factor receptor [HER]2/neu antibody) are effective against HER2+ breast cancer. Isoform-selective PI3K inhibitors elicit anti-tumor immune responses that are distinct from those induced by inhibitors of class I PI3K isoforms (pan-PI3K inhibitors). The present study investigated the therapeutic effect and potential for stimulating anti-tumor immunity of combined therapy with an anti-HER2/neu antibody and pan-PI3K inhibitor (GDC-0941) or a PI3K p110α isoform-selective inhibitor (A66) in mouse models of breast cancer. The anti-neu antibody inhibited tumor growth and enhanced anti-tumor immunity in HER2/neu+ breast cancer TUBO models, whereas GDC-0941 or A66 alone did not. Anti-neu antibody and PI3K inhibitor synergistically promoted anti-tumor immunity by increasing functional T cell production. In the presence of the anti-neu antibody, A66 was more effective than GDC-0941 at increasing the fraction of CD4+, CD8+, and IFN-γ+CD8+ T cells in the tumor-infiltrating lymphocyte population. Detection of IFN-γ levels by enzyme-linked immunospot assay showed that the numbers of tumor-specific T cells against neu and non-neu tumor antigens were increased by combined PI3K inhibitor plus anti-neu antibody treatment, with A66 exhibiting more potent effects than GDC-0941. In a TUBO (neu+) and TUBO-P2J (neu-) mixed tumor model representing immunohistochemistry 2+ tumors, A66 suppressed tumor growth and prolonged survival to a greater extent than GDC-0941 when combined with anti-neu antibody. These results demonstrate that a PI3K p110α-isoform-selective inhibitor is an effective adjunct to trastuzumab in the treatment of HER2-positive breast cancer.

Replication of an RNA ligase ribozyme under alternating temperature condition.

Self-replication process of the RNA ligase ribozyme molecules was investigated by using the modified RNA ligase ribozyme under alternating temperature condition that enhances turnover rate of the RNA ligation reaction. In our experiment, the RNA ligase ribozyme system mainly undergoes a cross-catalytic replication process, in which two ribozymes catalyze each other's synthesis from a total of four RNA substrates under alternating temperature condition, resulting in time-dependent accumulation of additional copies of the starting ribozymes in a reaction mixture. The present study demonstrates that cross-catalytic replication in nucleic acids system can be efficiently devised under the alternating temperature condition.

Isoeugenol suppression of inducible nitric oxide synthase expression is mediated by down-regulation of NF-kappaB, ERK1/2, and p38 kinase.

Isoeugenol, which is a naturally occurring o-methoxyphenol in a variety of foods and essential oils, is known to have anti-inflammatory effects, although the mechanism is not clear. In the present study, we investigated the effect of isoeugenol on NF-kappaB signaling leading to inducible nitric oxide synthase (iNOS) expression in RAW 264.7 murine macrophages stimulated with lipopolysaccharide (LPS). Isoeugenol markedly inhibited nitric oxide (NO) production in dose- and time-dependent manners. The decrease in NO production was found to correlate with a decrease in iNOS expression, as determined by Western blot analysis and real-time RT-PCR. To characterize further the inhibitory mechanisms of isoeugenol at the transcriptional level, we examined the DNA-binding and transcriptional activities of NF-kappaB. Isoeugenol inhibited NF-kappaB-dependent transcriptional activity and DNA-binding activity by decreasing the nuclear translocation of p65, which is a component of NF-kappaB. In addition, isoeugenol blocked signaling upstream of NF-kappaB activation, such as degradation of I-kappaBalpha and the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK), in LPS-stimulated RAW 264.7 cells. The isoeugenol analogues eugenol and allylbenzene also inhibited LPS-induced NF-kappaB signaling and iNOS expression, albeit with less potency than isoeugenol. These results suggest that isoeugenol and its analogues inhibit NO production and iNOS expression in LPS-stimulated RAW 264.7 cells, and that these effects are mediated, at least in part, by blocking the phosphorylation of ERK1/2 and p38 kinase, degradation of I-kappaBalpha, and activation of NF-kappaB.

Suppression of interleukin-2 gene expression by isoeugenol is mediated through down-regulation of NF-AT and NF-kappaB.

Isoeugenol is a naturally occurring methoxyphenol found in a variety of foods and essential oils. We investigated the effect of isoeugenol on T-cell function and the regulatory mechanism underlying its effect. Isoeugenol and its structural analog eugenol suppressed the lymphoproliferative response to concanavalin A stimulation in B6C3F1 mouse splenocyte cultures. Isoeugenol inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced IL-2 mRNA expression and protein secretion in B6C3F1 mouse splenocytes, and in EL4.IL-2 mouse T-cells, as determined by real-time RT-PCR and ELISA, respectively. To further characterize the inhibitory mechanism of isoeugenol at the transcriptional level, we examined the DNA binding activity of the transcription factors for IL-2 using an electrophoretic mobility shift assay. Isoeugenol decreased the binding activity of NF-AT and NF-kappaB in PMA/Io-stimulated EL4.IL-2 cells, but no significant effect was observed for AP-1 or Oct binding activity. Western blot analysis showed that isoeugenol also decreased the nuclear translocation of cytoplasmic NF-AT and NF-kappaB. These results suggest that isoeugenol suppresses IL-2 production through a decrease of IL-2 mRNA expression and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT and NF-kappaB.

Gene expression analysis of primary normal human hepatocytes infected with human hepatitis B virus.

AIM: To find the relationship between hepatitis B virus (HBV) and hepatocytes during the initial state of infection by cDNA microarray. METHODS: Primary normal human hepatocytes (PNHHs) were isolated and infected with HBV. From the PNHHs, RNA was isolated and inverted into complement DNA (cDNA) with Cy3- or Cy5- labeled dUTP for microarray analysis. The labeled cDNA was hybridized with microarray chip, including 4224 cDNAs. From the image of the microarray, expression profiles were produced and some of them were confirmed by RT-PCR, immunoblot analysis, and NF-kappaB luciferase reporter assay. RESULTS: From the cDNA microarray, we obtained 98 differentially regulated genes. Of the 98 genes, 53 were up regulated and 45 down regulated. Interestingly, in the up regulated genes, we found the TNF signaling pathway-related genes: LT-alpha, TRAF2, and NIK. By using RT-PCR, we confirmed the up-regulation of these genes in HepG2, Huh7, and Chang liver cells, which were transfected with pHBV1.2x, a plasmid encoding all HBV messages. Moreover, these three genes participated in HBV-mediated NF-kappaB activation. CONCLUSION: During the initial state of HBV infection, hepatocytes facilitate the activation of NF-kappaB through up regulation of LT-alpha, TRAF2, and NIK.

Inhibitory effect of anethole on T-lymphocyte proliferation and interleukin-2 production through down-regulation of the NF-AT and AP-1.

Anethole is a naturally occurring alkenylbenzene found in a variety of foods and essential oils. In the present study, we investigated the effect of anethole on T-cell function and the regulatory mechanism of its effect. Direct addition of anethole to B6C3F1 mouse splenocyte cultures produced a concentration-dependent inhibition of the lymphoproliferative response to concanavalin A stimulation. Anethole inhibited phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io)-induced interleukin-2 (IL-2) mRNA expression and protein secretion in EL4 mouse T-cells as determined by quantitative/competitive RT-PCR and ELISA, respectively. To further characterize the mechanism for the transcriptional regulation of IL-2, an electrophoretic mobility shift assay was performed to evaluate the binding activity of the nuclear factor of activated T-cells (NF-AT), activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and octamer binding protein (Oct) in PMA/Io-stimulated EL4 cells. Anethole decreased the NF-AT and AP-1 binding activity, but no significant effect was observed on NF-kappaB or Oct binding activity. These results suggest that anethole suppress T-cell proliferation and IL-2 production and that the inhibition is mediated, at least in part, through the down-regulation of NF-AT and AP-1.

Expression of cyclooxygenase-2 and 5-lipoxygenase in nasal polyps associated with interleukin-4 promoter polymorphism -590.

OBJECTIVE: To evaluate expression of cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LO) associated with interleukin (IL)-4 promoter polymorphism -590 in nasal polyp tissues. STUDY DESIGN AND SETTING: A prospective controlled study. A venous blood sample was taken to determine the genotype in 61 nasal polyp subjects. The C-590T variant was determined by the polymerase chain reaction-restriction fragment length polymorphism method. The expression of 5-LO and COX-2 was determined with immunohistochemical staining in 37 nasal polyp tissues associated with genotype. RESULTS: The genotype frequencies at position -590 of the IL-4 gene in the patients with nasal polyp were C/C (8.20%), C/T (40.98%), and T/T (50.82%). There was no significantly increased expression of COX-2 among genotypes. The 5-LO expression was significantly increased in C/C compared with C/T and T/T. CONCLUSION: We suggested that the IL-4 promoter polymorphism -590 C/C is associated with the expression of 5-LO in the patients with nasal polyp.