Improved methods for the differentiation of hypothalamic vasopressin neurons using mouse induced pluripotent stem cells.

High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies.

Arabinoxylan-Based Particles: In Vitro Antioxidant Capacity and Cytotoxicity on a Human Colon Cell Line.

Background and objectives: Arabinoxylans (AX) can gel and exhibit antioxidant capacity. Previous studies have demonstrated the potential application of AX microspheres as colon-targeted drug carriers. However, the cytotoxicity of AX gels has not been investigated so far. Therefore, the aim of the present study was to prepare AX-based particles (AXM) by coaxial electrospraying method and to investigate their antioxidant potential and cytotoxicity on human colon cells. Materials and Methods: The gelation of AX was studied by monitoring the storage (G') and loss (G'') moduli. The morphology of AXM was evaluated using optical and scanning electron microscopy (SEM). The in vitro antioxidant activity of AX before and after gelation was measured using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) methods. In addition, the effect of AX and AXM on the proliferation of human colon cells (CCD 841 CoN) was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: The final G' and G'' values for AX gels were 293 and 0.31 Pa, respectively. AXM presented spherical shape and rough surface with a three-dimensional and porous network. The swelling ratio and mesh size of AXM were 35 g water/g AX and 27 nm, respectively. Gelation decreased the antioxidant activity of AX by 61-64 %. AX and AXM did not affect proliferation or show any toxic effect on the normal human colon cell line CCD 841 CoN. Conclusion: The results indicate that AXM could be promising biocompatible materials with antioxidant activity.

Effect of copper variation in yeast hydrolysate on C-terminal lysine levels of a monoclonal antibody.

The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C-terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large-scale production of a murine cell line exhibited variation in the C-terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small-scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C-terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C-terminal lysine levels of the batches in manufacturing. Small-scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C-terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast-hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463-468, 2017.

Evidence of isotopic fractionation of natural uranium in cultured human cells.

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO22+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

Agrimonia eupatoria L. and Cynara cardunculus L. Water Infusions: Phenolic Profile and Comparison of Antioxidant Activities.

Reactive oxygen species (ROS) are highly considered in the ethiopathogenesis of different pathological conditions because they may cause significant damage to cells and tissues. In this paper, we focused on potential antioxidant properties of two medical plants such as the Agrimonia eupatoria L. and Cynara cardunculus L. Both plants have previously been studied for their pharmacological activities, especially as hepatoprotective and hypoglycemic activities. It has been suggested, that their effects are related to the antioxidant properties of polyphenols, which are dominant compounds of the plants' extracts. In the present study HPLC-MS analysis of water infusion was performed allowing the identification of several phenolic constituents. Furthermore, antioxidant effects of the two extracts were compared showing higher effects for agrimony extract compared to artichoke. Thus, agrimony was selected for the in vivo study using the skin flap viability model. In conclusion, our results provide evidence that the A. eupatoria extract may be a valuable source of polyphenols to be studied for the future development of supplements useful in the prevention of diseases linked to oxidative stress.

Novel Oxazolidinone Antibacterial Analogues with a Substituted Ligustrazine C-ring Unit.

A series of novel oxazolidinone compounds with a substituted ligustrazine C-ring unit and different substituted groups at the C-5 side chain were designed and synthesized using linezolid as a lead and based on a scaffold hopping strategy. Their antibacterial and anti-inflammatory activities were evaluated. The results of in vitro antibacterial assays showed that all fourteen target compounds displayed potent activity against Gram-positive pathogens, particularly 8b, 13b, 14a, 14b, 15a, and 15b. Moreover, 14a and 14b exhibited significant inhibitory activities on the production of inflammatory mediators, including nitric oxide, interleukin-6, and tumor necrosis factor-alpha. Thus, these derivatives could serve as valuable candidates to develop anti-infective agents for the treatment of chronic wounds.

The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function.

In this study we have investigated the effects of different cell culture conditions on the Calu-3 epithelial cell model. Calu-3 cells were cultured in media A-MEM at the air-liquid (A-L) or liquid-liquid (L-L) interface for one or three wks (weeks). Different cryomethods were tested and the cell line was characterized using histochemistry, immunofluorescence, transmission and scanning electron microscopy, transepithelial resistance (TEER) measurements, permeability studies, and gene profiling of 84 drug transporters. Cell culture was successful in A-MEM with only 2.5% FBS. Cell proliferation and viability depended on the cryopreservation method. All Calu-3 models expressed CK7, occludin, and E-cadherin. The A-L interface resulted in a more biomimetic native bronchial epithelium displaying pseudostratified columnar epithelium with more microvilli and secretory vesicles than at the L-L interface, where the epithelium was cuboidal, but exhibited higher TEER values and lower dextran permeabilities. Longer time in culture significantly decreased dextran permeability and increased the expression of specific drug transporters. Drug transporter expression was also notably influenced by the culture interface, where the A-L interface yielded a higher expression of drug transporter genes than the L-L interface. Since cell culture interface and time in culture affect Calu-3 cell differentiation, barrier integrity, permeability properties, and drug transporter expression, culture conditions need to be considered and standardized when using the Calu-3 cell line as an in vitro model for aerosol drug delivery and screening of bronchial drug candidates.

Pegylation of high-density lipoprotein decreases plasma clearance and enhances antiatherogenic activity.

RATIONALE: Infusions of apolipoprotein AI (apoAI), mimetic peptides, or high-density lipoprotein (HDL) remain a promising approach for the treatment of atherosclerotic coronary disease. However, rapid clearance leads to a requirement for repeated administration of large amounts of material and limits effective plasma concentrations. OBJECTIVE: Because pegylation of purified proteins is commonly used as a method to increase their half-life in the circulation, we determined whether pegylation of apoAI or HDL would increase its plasma half-life and in turn its antiatherogenic potential. METHODS AND RESULTS: Initial pegylation attempts using lipid-poor apoAI showed a marked tendency to form multi-pegylated (PEG) species with reduced ability to promote cholesterol efflux from macrophage foam cells. However, pegylation of human holo-HDL or reconstituted phospholipid/apoAI particles (rHDL) led to selective N-terminal monopegylation of apoAI with full preservation of cholesterol efflux activity. The plasma clearance of PEG-rHDL was estimated after injection into hypercholesterolemic Apoe-/- mice; the half-life of pegylated PEG-apoAI after injection of PEG-rHDL was increased ≈7-fold compared with apoAI in nonpegylated rHDL. In comparison with nonpegylated rHDL, infusion of PEG-rHDL (40 mg/kg) into hypercholesterolemic Apoe-/- mice led to more pronounced suppression of bone marrow myeloid progenitor cell proliferation and monocytosis, as well as reduced atherosclerosis and a stable plaque phenotype. CONCLUSIONS: We describe a novel method for effective monopegylation of apoAI in HDL particles, in which lipid binding seems to protect against pegylation of key functional residues. Pegylation of apoAI in rHDL markedly increases its plasma half-life and enhances antiatherogenic properties in vivo.

Mouse hypothalamic GT1-7 cells demonstrate AMPK-dependent intrinsic glucose-sensing behaviour.

AIMS/HYPOTHESIS: Hypothalamic glucose-excited (GE) neurons contribute to whole-body glucose homeostasis and participate in the detection of hypoglycaemia. This system appears defective in type 1 diabetes, in which hypoglycaemia commonly occurs. Unfortunately, it is at present unclear which molecular components required for glucose sensing are produced in individual neurons and how these are functionally linked. We used the GT1-7 mouse hypothalamic cell line to address these issues. METHODS: Electrophysiological recordings, coupled with measurements of gene expression and protein levels and activity, were made from unmodified GT1-7 cells and cells in which AMP-activated protein kinase (AMPK) catalytic subunit gene expression and activity were reduced. RESULTS: Hypothalamic GT1-7 neurons express the genes encoding glucokinase and ATP-sensitive K(+) channel (K(ATP)) subunits K ( ir ) 6.2 and Sur1 and exhibit GE-type glucose-sensing behaviour. Lowered extracellular glucose concentration hyperpolarised the cells in a concentration-dependent manner, an outcome that was reversed by tolbutamide. Inhibition of glucose uptake or metabolism hyperpolarised cells, showing that energy metabolism is required to maintain their resting membrane potential. Short hairpin (sh)RNA directed to Ampkα2 (also known as Prkaa2) reduced GT1-7 cell AMPKα2, but not AMPKα1, activity and lowered the threshold for hypoglycaemia-induced hyperpolarisation. shAmpkα1 (also known as Prkaa1) had no effect on glucose-sensing or AMPKα2 activity. Decreased uncoupling protein 2 (Ucp2) mRNA was detected in AMPKα2-reduced cells, suggesting that AMPKα2 regulates UCP2 levels. CONCLUSIONS/INTERPRETATION: We have demonstrated that GT1-7 cells closely mimic GE neuron glucose-sensing behaviour, and reducing AMPKα2 blunts their responsiveness to hypoglycaemic challenge, possibly by altering UCP2 activity. These results show that suppression of AMPKα2 activity inhibits normal glucose-sensing behaviour and may contribute to defective detection of hypoglycaemia.

Functional characterization of lipopolysaccharide derived from symbiotic bacteria in rice as a macrophage-activating substance.

BACKGROUND AND AIM: Lipopolysaccharide derived from a symbiotic bacterium in wheat (Pantoea agglomerans, LPSp) has shown multiple positive effects, such as prophylactic, antiallergic and antitumour effects, without serious side-effects. LPSp has differential biological activities in comparison to other LPS, such as those from Escherichia coli (LPSe). The only difference between LPSp and LPSe is in the O-antigen polysaccharide structure (O-PS). This led us to the hypothesis that the O-PS structure would seem to participate in biological activities. Thus, the characterization of properties of O-PS in LPS is of the utmost importance for understanding cell activation in the maintenance of homeostasis. However, little is known about the correlation between the O-PS structure of LPS and its biological activities. In this study, we extracted LPS derived from a symbiotic bacterium in rice (strain A46, related species of Pantoea), which has a long history of use in foods, and investigated its putative structures and functions. MATERIALS AND METHODS: LPS derived from strain A46 was prepared using a hot phenol extraction method. The properties of LPS-A46 were analysed by thin-layer chromatography, Tricine SDS-PAGE and Western blotting. The function of LPS-A46 was analyzed by quantative real-time PCR and flow cytometry using THP-1 cells and Peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: In Tricine SDS-PAGE, high molecular mass LPS-A46 had a molecular mass lower than that of LPSp. In Western blotting, LPS-A46 reacted with lipid A antibody but did not react with an O-PS antibody of LPSp. In comparison to other LPS, LPS-A46 induced a differential cytokine gene expression profile in THP-1 cells and PBMC-derived macrophages. CONCLUSION: The present study suggests that LPS derived from symbiotic bacterium in rice is a bioactive functional LPS which may have different functional activities compared to other types of LPS.

ω-3 fatty acids suppress inflammatory cytokine production by macrophages and hepatocytes.

OBJECTIVE: Long-term total parenteral nutrition (TPN) in children is often complicated by parental nutrition-associated liver disease and may even lead to liver failure. Recently, the addition of ω-3 fatty acids to TPN has been shown to reduce the risk of parental nutrition-associated liver disease. The purpose of this study was to explore the anti-inflammatory effects of ω-3 fatty acids (eicosapentaenoic acid [EPA]) to demonstrate the protection of the liver against hepatic steatosis and damage. MATERIALS AND METHODS: Lipopolysaccharide (LPS) and prostaglandin E(2) (PGE(2)) were used to stimulate human macrophages and hepatocytes (THLE-3) to induce in vitro inflammatory condition. The cells were then incubated with either ω-3 (EPA) or ω-6 (arachidonic acid) fatty acids. Supernatants were collected at different time points for the measurement of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10) using enzyme-linked immunosorbent assay. Furthermore, pretreated macrophages by LPS stimulation and after incubation with EPA were added to prestimulated hepatocytes for the subsequent measurement of cytokine response. RESULTS: Eicosapentaenoic acid effectively reduced LPS-induced or PGE(2)-induced TNF-α and IL-6 expression, and increased IL-10 expression significantly when compared with arachidonic acid. Furthermore, supernatant collected after co-culturing EPA with macrophages also suppressed the levels of TNF-α and IL-6 in hepatocytes. This would suggest that EPA not only had an anti-inflammatory effect on macrophages and hepatocytes directly, it could indirectly reduce hepatocyte inflammation through activated macrophages. CONCLUSIONS: The addition of ω-3 fatty acids in TPN suppresses the inflammatory response via direct and indirect routes. The findings may help explain the clinical benefits of EPA in pediatric patients receiving long-term TPN.

Paclitaxel-dependent cell lines reveal a novel drug activity.

We previously described the isolation of Tax 18 and Tax 11-6, two paclitaxel-dependent cell lines that assemble low amounts of microtubule polymer and require the drug for cell division. In the present studies, fluorescence time-lapse microscopy was used to measure microtubule dynamic instability behavior in these cells. The mutations were found to cause small decreases in microtubule growth and shortening, but the changes seemed unable to explain the defects in microtubule polymer levels or cell division. Moreover, paclitaxel further suppressed microtubule dynamics at low drug concentrations that were insufficient to rescue the mutant phenotype. Wild-type (WT) cells treated with similar low drug concentrations also had highly suppressed microtubules, yet experienced no problems with cell division. Thus, the effects of paclitaxel on microtubule dynamics seemed to be unrelated to cell division in both WT and mutant cell lines. The higher drug concentrations needed to rescue the mutant phenotype instead inhibited the formation of unstable microtubule fragments that appeared at high frequency in the drug-dependent, but not WT, cell lines. Live cell imaging revealed that the fragments were generated by microtubule detachment from centrosomes, a process that was reversed by paclitaxel. We conclude that paclitaxel rescues mutant cell division by inhibiting the detachment of microtubule minus ends from centrosomes rather than by altering plus-end microtubule dynamics.

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells.

AIM: The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD: Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS: The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION: CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders.

Selective apoptosis induction by the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide is achieved by modulating mitochondrial bioenergetics in premalignant and malignant human prostate epithelial cells.

Prostate tumorigenesis is coupled with an early metabolic switch in transformed prostate epithelial cells that effectively increases their mitochondrial bioenergetic capacity. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) inhibits prostate cancer development in vivo, and triggers reactive oxygen species (ROS)-dependent prostate cancer cell apoptosis in vitro. The possibility that 4HPR-induced ROS production is associated with mitochondrial bioenergetics and required for apoptosis induction in transformed prostate epithelial cells in vitro would advocate a prospective mechanistic basis for 4HPR-mediated prostate cancer chemoprevention in vivo. We investigated this tenet by comparing and contrasting 4HPR's effects on premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. 4HPR promoted a dose- and/or time-dependent apoptosis induction in PWR-1E and DU-145 cells, which was preceded by and dependent on an increase in mitochondrial ROS production. In this regard, the PWR-1E cells were more sensitive than the DU-145 cells, and they consumed roughly twice as much oxygen as the DU-145 cells suggesting oxidative phosphorylation was higher in the premalignant cells. Interestingly, increasing the [Ca(2+)] in the culture medium of the PWR-1E cells attenuated their proliferation as well as their mitochondrial bioenergetic capacity and 4HPR's cytotoxic effects. Correspondingly, the respiration-deficient derivatives (i.e., rho(0) cells lacking mitochondrial DNA) of DU-145 cells were markedly resistant to 4HPR-induced ROS production and apoptosis. Together, these observations implied that the reduction of mitochondrial bioenergetics protected PWR-1E and DU-145 cells against the cytotoxic effects of 4HPR, and support the concept that oxidative phosphorylation is an essential determinant in 4HPR's apoptogenic signaling in transformed human prostate epithelial cells.

Influence of culture conditions on recombinant Drosophila melanogaster S2 cells producing rabies virus glycoprotein cultivated in serum-free medium.

The recombinant G glycoprotein from the surface of the rabies virus (RVGP) is a promising candidate as a rabies vaccine component and also for diagnostic purposes. In this study, RVGP production by transfected Drosophila melanogaster S2 cells cultivated in a serum-free medium (supplemented IPL-41 medium) was carried out. The effects of pH and pO(2) were evaluated in batch culture in parallel spinner flasks. The use of a pH equal to 6.3 and a pO(2) of 40% air saturation resulted in the highest RVGP content. These conditions were also used in fed-batch mode, yielding a RVGP content level of 98g/10(7) cells. The main nutrients consumed were glucose, glutamine, asparagine, serine and proline and the major metabolites produced were alanine and ammonia, according to the metabolism studies performed. Since RVGP is a transmembrane protein, two different methods for protein recovery were assessed and compared. Detergent-based cell disruption showed to be more effective than mechanical disruption with glass beads for glycoprotein recovery.

Oleanolic acid and its derivatives: new inhibitor of protein tyrosine phosphatase 1B with cellular activities.

Protein tyrosine phosphatase 1B is a key factor in the negative regulation of insulin pathway and a promising target for treatment of diabetes and obesity. Herein, a series of competitive inhibitors were optimized from oleanolic acid, a natural triterpenoid identified against PTP1B by screening libraries of traditional Chinese medicinal herbs. Modifying at 3 and 28 positions, we obtained compound 13 with a K(i) of 130 nM, which exhibited good selectivity between other phosphatases involved in insulin pathway except T-cell protein tyrosine phosphatase. Further evaluation in cell models illustrated that the derivatives enhanced insulin receptor phosphorylation in CHO/hIR cells and also stimulated glucose uptake in L6 myotubes with or addition of without insulin.

Simultaneous effect of ursolic acid and oleanolic acid on epidermal permeability barrier function and epidermal keratinocyte differentiation via peroxisome proliferator-activated receptor-alpha.

Ursolic acid (UA) and oleanolic acid (ONA) are pentacyclic triterpenoids, which naturally occur in many medicinal herbs and plants. Recent research revealed that several pharmacological effects could be attributed to UA and ONA, such as anti-tumor, anti-inflammatory and anti-microbial activities. To evaluate the effects of UA and ONA on epidermal permeability barrier recovery and normal skin, both flanks of hairless mice were topically treated with either 0.01-0.1 mg/mL UA or 0.1-1.0 mg/mL ONA after tape stripping and transepidermal water loss (TEWL) were assessed, and then hydration and TEWL were measured for 3 weeks with application of UA and ONA (2 mg/mL). We also investigated the morphological changes using light (LM) and electron microscopic (EM) examination. Finally, we observed that UA and ONA stimulated epidermal keratinocyte differentiation via peroxisome proliferator-activated receptor (PPAR)-alpha using Western immunoblotting. The recovery rate of epidermal permeability barrier after tape stripping increased in the UA- and ONA-treated groups (0.1 mg/mL UA and 0.5 mg/mL ONA) at 6 h to more than 20% when compared to the vehicle-treated group (P < 0.05). In both groups, hydration was increased compared to the vehicle group from 1 week without TEWL alteration (P < 0.05). An LM finding showed that epidermal thickening was frequently observed (UA > ONA > vehicle). EM examination revealed an increase in secretion and in the number of lamellar bodies in treated groups and that complete formation of lipid bilayers was also prominent (ONA > UA > vehicle). Protein expression of PPAR-alpha, involucrin, loricrin and filaggrin increased twofold and threefold in HaCaT cells treated for 24 h with either ONA (10 micromol/L) or UA (10 micromol/L), respectively, reflecting that the UA and ONA can improve the recovery of skin barrier function and induce epidermal keratinocyte differentiation via PPAR-alpha. Taken together, these results suggest that UA and ONA will be pertinent candidates for the improvement of epidermal permeability barrier function.

Inhibitory effects of Ligusticum chuanxiong on the proliferation of rat hepatic stellate cells.

BACKGROUND AND AIMS: Platelet-derived growth factor (PDGF) is a very potent mitogen for hepatic stellate cells (HSC) in hepatic fibrogenesis. Ligusticum chuanxiong Hort. (LC), a traditional Chinese herb used for cerebrovascular diseases, has been shown to exert anti-inflammatory and free radical scavenging effects. The aims of the present study were to investigate the effects of LC extract on the proliferation-related biomarkers in a rat HSC cell line (HSC-T6) stimulated with PDGF. METHODS: DNA synthesis via bromodeoxyuridine (BrdU) incorporation, cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of LC. RESULTS: The results revealed that LC extract (25-100 microg/mL) concentration-dependently decreased the PDGF-induced cell proliferation as well as alpha-smooth muscle actin expression in HSC. The inhibitory activity of LC on HSC was associated with: (i) inhibition of BrdU incorporation; (ii) induction of apoptosis with the activation of caspase-3, up-regulation of cell cycle inhibitory proteins p21 and p27, and down-regulation of cell cycle stimulatory proteins cyclins D1 and D2; and (iii) increased phosphorylation of mitogen-activated protein kinases (JNK). LC at the studied concentrations showed no direct cytotoxicity on primary hepatocytes. CONCLUSION: The results suggest that LC significantly inhibited PDGF-activated HSC proliferation, possibly through apoptotic mechanisms and the potential of LC as an antifibrotic agent warrants further investigation.

Inhibition of glycinamide ribonucleotide formyltransferase results in selective inhibition of macrophage cytokine secretion in vitro and in vivo efficacy in rat adjuvant arthritis.

OBJECTIVE: To determine the effects of a glycinamide ribonucleotide formyltransferase (GARFT) inhibitor on macrophage inflammatory processes and in vivo in rat adjuvant arthritis. METHODS: GARFT inhibitors, LY309886 (6S-2',5'-thienyl-5, 10-dideazatetrahydrofolic acid) and LY329201 (R)-N-[[5-[2-(2-amino-1,4,5,6,7,8-hexahydro-4-oxopyrido[2,3-d]pyrimidin-6-yl)ethyl]-2-thienyl]carbonyl]-L-glutamatic acid disodium salt, were investigated in vitro and ex vivo on primary murine peritoneal macrophages and in the RAW macrophage cell line for both purine depletion and inhibition of LPS induced monokine secretion. In vivo efficacy following GARFT inhibition was evaluated in modified rat adjuvant arthritis. RESULTS: LY309886 inhibited purine biosynthesis in the RAW cell line with an EC50 of 90 nM, an effect readily reversible with exogenous hypoxanthine. LY309886 and LY329201 also inhibited LPS induced TNF alpha and MIP1 alpha in these cells and in primary macrophages. A similar effect could be demonstrated ex vivo with mice dosed for two days with 3 mg/kg of LY329201. LY329201 as well as methotrexate demonstrated a dose dependent reduction in both paw and spleen weight and improved joint histology following 2 weeks of dosing in a rat adjuvant arthritis study. CONCLUSION: These results suggest that GARFT inhibitors should be tested in the treatment of rheumatoid arthritis by considering their mechanism of action, here successfully tested on activated macrophages.

Functional interplay between the macrophage scavenger receptor class B type I and pitavastatin (NK-104).

BACKGROUND: Scavenger receptor class B type I (SR-BI), a receptor for high-density lipoprotein (HDL), plays an important role in the bidirectional cholesterol exchange between cells and HDL particles and the atherosclerotic lesion development. Enhancement of SR-BI expression significantly reduces, whereas lack of SR-BI expression accelerates, the atherosclerotic lesion development in proatherogenic mice. Statins, a class of inhibitors for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, significantly suppress de novo cholesterol synthesis and reduce the incidence of coronary heart disease. Statins also display multiple pleiotropic effects independently of cholesterol synthesis in the vascular cells. Here, we investigated the effects of pitavastatin (NK-104), a newly synthesized statin, on macrophage SR-BI expression. METHODS AND RESULTS: We found that pitavastatin significantly increased SR-BI mRNA and protein expression in a macrophage cell line in a concentration- and time-dependent manner. It also increased SR-BI expression in both mouse peritoneal and human monocyte-derived macrophages. Associated with increased SR-BI expression, pitavastatin enhanced macrophage HDL binding, uptake of [14C]cholesteryl oleate/HDL, and efflux of [3H]cholesterol to HDL. Pitavastatin abolished the inhibition of macrophage SR-BI expression by cholesterol biosynthetic intermediates. It also restored SR-BI expression inhibited by lipopolysaccharide and tumor necrosis factor-alpha through its inactivation of the transcription factor nuclear factor-kappaB. CONCLUSIONS: Our data demonstrate that pitavastatin can stimulate macrophage SR-BI expression by reduction of cholesterol biosynthetic intermediates and antiinflammatory action and suggest additional pleiotropic effects of statins by which they may reduce the incidence of coronary heart disease.