Immortalized hepatocyte-like cells: A competent hepatocyte model for studying clinical HCV isolate infection.

More than 58 million individuals worldwide are inflicted with chronic HCV. The disease carries a high risk of end stage liver disease, i.e., cirrhosis and hepatocellular carcinoma. Although direct-acting antiviral agents (DAAs) have revolutionized therapy, the emergence of drug-resistant strains has become a growing concern. Conventional cellular models, Huh7 and its derivatives were very permissive to only HCVcc (JFH-1), but not HCV clinical isolates. The lack of suitable host cells had hindered comprehensive research on patient-derived HCV. Here, we established a novel hepatocyte model for HCV culture to host clinically pan-genotype HCV strains. The immortalized hepatocyte-like cell line (imHC) derived from human mesenchymal stem cell carries HCV receptors and essential host factors. The imHC outperformed Huh7 as a host for HCV (JFH-1) and sustained the entire HCV life cycle of pan-genotypic clinical isolates. We analyzed the alteration of host markers (i.e., hepatic markers, cellular innate immune response, and cell apoptosis) in response to HCV infection. The imHC model uncovered the underlying mechanisms governing the action of IFN-α and the activation of sofosbuvir. The insights from HCV-cell culture model hold promise for understanding disease pathogenesis and novel anti-HCV development.

Transplantation of autologous cultivated oral mucosal epithelial sheets for limbal stem cell deficiency at Siriraj Hospital: a case series.

BACKGROUND: The loss of limbal stem cells owing to either corneal burn or inflammation leads to the repopulation of opaque skin over the raw surface of the cornea. It has been proposed that reconstitution of oral mucosal stem cells over this raw surface will mimic the limbal stem cells and restore vision. The efficacy and safety of applying a sheet of cultivated oral mucosal cells as an autologous graft for corneal replacement were evaluated. CASE PRESENTATION: The study was conducted during 2014-2015 and involved a total of six patients, of whom three had suffered a chemical burn and three had Stevens-Johnson Syndrome (SJS). Oral mucosal tissue was dissected from each patient, seeded onto irradiated J2 fibroblast feeder cells for 14 days, and analyzed for quality and safety 1 day before being transplanted onto the cornea of the affected eyes. After transplantation, topical antibiotic and anti-inflammatory eye drops were instilled four times daily, and the patients wore contact lenses. Subjects were clinically followed for visual acuities and adverse effects at 2, 4, and 6 weeks, 3 and 6 months, and 1 year post-transplantation. Data were presented descriptively. Visual acuities in patients improved at 2 weeks post-surgery. However, two patients with SJS had corneal ulcer at 2 weeks postoperatively. At the 1-year postoperative examination, the eyes of two patients were in good condition with decreased vascularization and epithelial defect. CONCLUSIONS: Cultivated oral mucosal epithelial sheet transplantation in limbal stem cell deficiency had a favorable efficacy. In this study, patients with chemical burn had more clinical benefit than those with SJS. Trial registration ClinicalTrials.gov: NCT02415218. Registered retrospectively 4 Apr 2015 ( https://clinicaltrials.gov/ct2/show/NCT02415218 ).

A Competent Hepatocyte Model Examining Hepatitis B Virus Entry through Sodium Taurocholate Cotransporting Polypeptide as a Therapeutic Target.

Hepatitis B virus (HBV) infection has been considered a crucial risk factor for hepatocellular carcinoma. Current treatment can only lessen the viral load but not result in complete remission. An efficient hepatocyte model for HBV infection would offer a true-to-life viral life cycle that would be crucial for the screening of therapeutic agents. Most available anti-HBV agents target lifecycle stages post viral entry but not before viral entry. This protocol details the generation of a competent hepatocyte model capable of screening for therapeutic agents targeting pre-viral entry and post viral entry lifecycle stages. This includes the targeting of sodium taurocholate cotransporting polypeptide (NTCP) binding, cccDNA formation, transcription, and viral assembly based on imHC or HepaRG as host cells. Here, the HBV entry inhibition assay used curcumin to inhibit HBV binding and transporting functions via NTCP. The inhibitors were evaluated for binding affinity (KD) with NTCP using isothermal titration calorimetry (ITC)-a universal tool for HBV drug screening based on thermodynamic parameters.

Curcumin inhibited hepatitis B viral entry through NTCP binding.

Hepatitis B virus (HBV) has been implicated in hepatitis and hepatocellular carcinoma. Current agents (nucleos(t)ide analogs and interferons) could only attenuate HBV infection. A combination of agents targeting different stages of viral life cycle (e.g., entry, replication, and cccDNA stability) was expected to eradicate the infection. Curcumin (CCM) was investigated for inhibitory action toward HBV attachment and internalization. Immortalized hepatocyte-like cells (imHCs), HepaRG and non-hepatic cells served as host cells for binding study with CCM. CCM decreased viral load, HBeAg, HBcAg (infectivity), intracellular HBV DNA, and cccDNA levels. The CCM-induced suppression of HBV entry was directly correlated with the density of sodium-taurocholate co-transporting polypeptide (NTCP), a known host receptor for HBV entry. The site of action of CCM was confirmed using TCA uptake assay. The affinity between CCM and NTCP was measured using isothermal titration calorimetry (ITC). These results demonstrated that CCM interrupted HBV entry and would therefore suppress HBV re-infection.

An Immortalized Hepatocyte-like Cell Line (imHC) Accommodated Complete Viral Lifecycle, Viral Persistence Form, cccDNA and Eventual Spreading of a Clinically-Isolated HBV.

More than 350 million people worldwide have been persistently infected with the hepatitis B virus (HBV). Chronic HBV infection could advance toward liver cirrhosis and hepatocellular carcinoma. The intervention with prophylactic vaccine and conventional treatment could suppress HBV, but could not completely eradicate it. The major obstacle for investigating curative antiviral drugs are the incompetence of hepatocyte models that should have closely imitated natural human infection. Here, we demonstrated that an immortalized hepatocyte-like cell line (imHC) could accommodate for over 30 days the entire life cycle of HBV prepared from either established cultured cells or clinically-derived fresh isolates. Normally, imHCs had intact interferon signaling with anti-viral action. Infected imHCs responded to treatments with direct-acting antiviral drugs (DAAs) and interferons (IFNs) by diminishing HBV DNA, the covalently closed circular DNA (cccDNA) surface antigen of HBV (HBsAg, aka the Australia antigen) and the hepatitis B viral protein (HBeAg). Notably, we could observe and quantify HBV spreading from infected cells to naïve cells using an imHC co-culture model. In summary, this study constructed a convenient HBV culture model that allows the screening for novel anti-HBV agents with versatile targets, either HBV entry, replication or cccDNA formation. Combinations of agents aiming at different targets should achieve a complete HBV eradication.

Cytokine-induced killer cells: A novel treatment for allergic airway inflammation.

The effectiveness of cytokine-induced killer (CIK) cells for treatment of cancers has long been appreciated. Here, we report for the first time that CIK cells can be applied to treat allergic airway inflammation. Adopting from an established protocol with some modifications, we generated CIK cells ex vivo from mouse T cells, and examined their effectiveness in treatment of allergic airway inflammation using the ovalbumin-induced model of allergic airway inflammation. Based upon evaluation of bronchoalveolar lavage cellularity, T helper type2 cytokine levels and lung histology, all of which are important parameters for determining the severity of allergic airway inflammation, diseased mice treated with CIK cells showed significant reductions in all the parameters without any obvious adverse effects. Interestingly, the observed effects were comparable to those treated with dexamethasone. Thus, our study provides a novel application of CIK cells in treatment of allergic airway inflammation.

Development of Hepatocyte-like Cell Derived from Human Induced Pluripotent Stem cell as a Host for Clinically Isolated Hepatitis C Virus.

This unit describes protocols to develop hepatocyte-like cells (HLCs) starting from mesenchymal stem cells (MSCs) as a natural host for hepatitis C virus (HCV). These include the preparation of MSCs from bone marrow, the reprogramming of MSCs into induced pluripotent stem cells (iPSCs), and the differentiation of iPSCs into HLCs. This unit also incorporates the characterization of the resulting cells at each stage. Another section entails the preparations of HCV. The sources of HCV are either the clinically isolated HCV (HCVser) and the conventional JFH-1 genotype. The last section is the infection protocol coupled with the measurement of viral titer. © 2017 by John Wiley & Sons, Inc.

An additional CD28 costimulatory signal enhances proliferation and cytotoxicity of murine T cell-derived CIK cell.

OBJECTIVE: Cytokine induced killer (CIK) cells are ex-vivo expanded T cells endowed with both T and Natural Killer cell properties. The standard protocol for generation of CIK cells is to culture peripheral blood mononuclear cells (PBMC) in the presence of interferon- gamma (IFN-γ), monoclonal antibody (mAb) against CD3 and interleukin-2 (IL-2). However, this protocol lacks costimulatory signal (CD28), crucial for T cell activation. Herein, the proliferation and functional properties of murine thymocytes derived CIK cells generated with or without costimulatory activation provided by anti-CD28 mAb were examined. METHOD: The proportion of CIK (Thy1.2+NK1.1+ and CD8+NK1.1+) cells in culture and the expression of cytotoxic granules (granzyme B and perforin) and proinflammatory cytokines (IFN-γ and tumor necrosis factor-alpha (TNF-α)) were determined by flow cytometry. Additionally, CIK cell cytotoxicity against YAC-1 murine lymphoma cells was measured by a propidium iodide-based assay. RESULTS: The addition of anti-CD28 to standard CIK culture conditions increased the number of Thy1.2+ NK1.1+ and CD8+ NK1.1+ (the major effector population) cells by almost 40% and 32%, respectively. Furthermore, the cytotoxic potential of CIK cells cultured with the addition of anti-CD28 mAb was also enhanced, with a corresponding increase in CIK cells expressing granzyme B, perforin, IFN-γ and TNF-α. CONCLUSIONS: The addition of anti-CD28 mAb generated more effective murine T cell-derived CIK cells.

Effects of the Ayurved Siriraj Wattana recipe on functional and phenotypic characterization of cytokine-induced killer cells and dendritic cells in vitro.

BACKGROUND: Ayurved Siriraj Wattana recipe (AVS073), has been prescribed as tonic, to increase appetite, and for pain relief. It also exhibits antioxidant, anti-inflammatory, immunomodulating and anti-cancer activities. However, the immunomodulatory effects on antigen-presenting cells and effector T cells remained elusive. We thus aimed to study the effects of AVS073 on differentiation, maturation, functions and proportions of CIK cells and monocyte-derived DCs. METHODS: CIK cells and monocyte-derived DCs were treated with AVS073, followed by the assessment of T-helper (Th) phenotypes using real-time RT-PCR and flow cytometry. RESULTS: AVS073 promoted Th1 phenotype in CD3+CD56+ subset of CIK cells through increasing STAT4, T-bet, and interferon-γ. AVS073 inhibited Th2 phenotype through decreasing STAT6. AVS073 inhibited Treg phenotype through decreasing STAT5A, STAT5B and IDO. AVS073 promoted Th17 phenotype through increasing STAT3, RORC and IL-17. AVS073 treatment of mDCs resulted in increasing Th1-prone cytokine (IL-12) and Th17-prone cytokines (IL-6 and IL-23). CONCLUSIONS: AVS073 upregulated Th1 and Th17, but downregulated Th2 and Treg phenotypes within CD3+CD56+ cells. The treatment of mDCs drove Th1 and Th17-polarizations.

Simvastatin up-regulates adenosine deaminase and suppresses osteopontin expression in COPD patients through an IL-13-dependent mechanism.

BACKGROUND: Adenosine deaminase (ADA) and osteopontin (OPN) may play opposing roles in the pathogenesis of COPD. Deficiency of ADA results in enhanced adenosine signaling which up-regulates OPN expression. Although statins suppress OPN in cancer cells, little is known about their effects on ADA and OPN in COPD patients. METHODS: We extended a previous randomized double-blind placebo crossover study to investigate the effects of simvastatin (20 mg/day) on sputum ADA and OPN expression and explored the underlying signaling pathways involved by conducting in vitro experiments with cigarette smoke extract (CSE)-treated monocyte-derived macrophages (MDM) from COPD patients and healthy subjects. RESULTS: Simvastatin decreased sputum IL-13, OPN and CD73, while increasing ADA expression, irrespective of inhaled corticosteroid treatment and smoking status in parallel to increased inosine levels. The degree of simvastatin-restored ADA activity was significantly correlated with the magnitude of changes in pre-bronchodilator FEV1. Mechanistic exploration showed that CSE enhanced the expression of IL-13, which induced an increase in OPN and inhibited ADA mRNA accumulation in MDM from COPD patients but not healthy subjects through a STAT6-dependent mechanism. Simvastatin treatment inhibited IL-13 transcription in a dose-dependent manner, and therefore diminished the IL-13-induced increase in OPN and restored IL-13-suppressed ADA. There was no effect of simvastatin on adenosine receptors in CSE-stimulated MDM, indicating that its effects were on the adenosine pathway. CONCLUSION: Simvastatin reversed IL-13-suppressed ADA activity that leads to the down-regulation of adenosine signaling and therefore inhibits OPN expression through the direct inhibition of IL-13-activated STAT6 pathway. Inhibition of IL-13 may reverse the imbalance between ADA and OPN in COPD and therefore may prevent COPD progression.

A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host.

BACKGROUND: Hepatitis C virus (HCV) could induce chronic liver diseases and hepatocellular carcinoma in human. The use of primary human hepatocyte as a viral host is restrained with the scarcity of tissue supply. A culture model restricted to HCV genotype 2a (JFH-1) has been established using Huh7-derived hepatocyte. Other genotypes including the wild-type virus could not propagate in Huh7, Huh7.5 and Huh7.5.1 cells. METHODS: Functional hepatocyte-like cells (HLCs) were developed from normal human iPS cells as a host for HCV infection. Mature HLCs were identified for selective hepatocyte markers, CYP450s, HCV associated receptors and HCV essential host factors. HLCs were either transfected with JFH-1 HCV RNA or infected with HCV particles derived from patient serum. The enhancing effect of α-tocopherol and the inhibitory effects of INF-α, ribavirin and sofosbuvir to HCV infection were studied. The HCV viral load and HCV RNA were assayed for the infection efficiency. RESULTS: The fully-developed HLCs expressed phase I, II, and III drug-metabolizing enzymes, HCV associated receptors (claudin-1, occludin, CD81, ApoE, ApoB, LDL-R) and HCV essential host factors (miR-122 and SEC14L2) comparable to the primary human hepatocyte. SEC14L2, an α-tocopherol transfer protein, was expressed in HLCs, but not in Huh7 cell, had been implicated in effective HCVser infection. The HLCs permitted not only the replication of HCV RNA, but also the production of HCV particles (HCVcc) released to the culture media. HLCs drove higher propagation of HCVcc derived from JFH-1 than did the classical host Huh7 cells. HLCs infected with either JFH-1 or wild-type HCV expressed HCV core antigen, NS5A, NS5B, NS3 and HCV negative-stand RNA. HLCs allowed entire HCV life cycle derived from either JFH-1, HCVcc or wild-type HCV (genotype 1a, 1b, 3a, 3b, 6f and 6n). Further increasing the HCVser infection in HLCs was achieved by incubating cell with α-tocopherol. The supernatant from infected HLCs could infect both naïve HLC and Huh7 cell. Treating infected HLC with INF-α and ribavirin decreased HCV RNA in both the cellular fraction and the culture medium. The HLCs reacted to HCVcc or wild-type HCV infection by upregulating TNF-α, IL-28B and IL-29. CONCLUSIONS: This robust cell culture model for serum-derived HCV using HLCs as host cells provides a remarkable system for investigating HCV life cycle, HCV-associated hepatocellular carcinoma development and the screening for new anti HCV drugs.

Autologous Fibrin-Base Scaffold for Chondrocytes and Bone Marrow Mesenchymal Stem Cells Implantation: The Development and Comparison to Conventional Fibrin Glue.

Objective: The authors developed the autologous fibrin-base scaffold for chondrocytes and bone marrow mesenchymal stem cells (BM-MSCs) implantation and evaluated cells viability in autologous fibrin-base scaffold comparing to commercial fibrin glue. Material and Method: The chondrocytes and BM-MSCs were seeded into autologous fibrin-base scaffold and commercial fibrin glue. The cell viability and proliferation were evaluated at 1 and 7 days. The histology were evaluated with hematoxylineosin (H&E) staining and cartilaginous matrices formation with Alcian blue, Saffanin-0, Toluidine blue, and Collagen type II staining at 6 weeks. The fixation of the scaffolds was observed. Results: The chondrocytes and BM-MSCs could not survive in commercial fibrin glue. The chondrocytes and BM-MSCs in autologous fibrin-base scaffold could proliferate and synthesize the cartilaginous matrices on Alcian blue, Saffanin-0, Toluidine blue, and Collagen type II staining at 6 weeks. The fixation strength is excellent. Conclusion: The developed autologous fibrin-base scaffold can be used as the scaffold for chondrocytes and BM-MSCs implantation with potential to implant chondrocytes and BM-MSCs arthroscopically.

A Prospective Randomized Controlled Trial Comparing Posterolateral Lumbar Fusion With and Without Bone Marrow Concentrate Augmentation in Single-Level Lumbar Spondylolisthesis.

Background: Bone marrow (BM), which is a good source of stem cells and biological factors, has the potential to enhance bone fusion. Simple centrifugation technique is one of the procedures used to concentrate BM aspirate for increasing number of cells. However, there are limited clinical study for using BM concentrate augmentation in spinal fusion. Objective: This study was designed to examine the spinal fusion enhancement effects of bone marrow (BM) concentrate augmentation on poster lateral lumbar fusion (PLF) with autologous local bone graft in terms of both quality and quantity, as compared with a control procedure without BM concentrate augmentation. Material and Method: Twelve patients with L4-L5 spondylolisthesis scheduled for PLF after decompressive laminectomy and pedicle screw instrumentation were included in this study. This prospective randomized controlled trial was conducted at Siriraj Hospital during the 2009 to 2012 study period. Patients were randomly assigned to two groups. One group underwent PLF with local bone graft with BM concentrate augmentation (BM group) and the other group underwent PLF with local bone graft only (non-BM group). Clinical outcomes were evaluated by the Oswestry Disability Index (ODI) preoperatively and at 3 and 6 months after PLF. Bone fusion quality was evaluated by bony bridging on 3D-CT imaging. Fusion mass volumes were measured on quantitative 3D-CT scans at 1 week and 6 months, postoperatively. Results: Clinical outcome scores did not differ between groups. Six-month postoperative 3D-CT imaging showed complete PLF bridging in 58.3% and 100% of patients in the BM and non-BM groups, respectively. PLF mass volumes were decreased at 6 months by 51.1% in the BM group and by 48.5% in the non-BM group. One patient in the BM group had local inflammation at the BM aspiration site. Conclusion: Bone marrow concentrate augmentation in this small randomized controlled trial failed to demonstrate positive effects on autologous local bone graft in posterolateral lumbar fusion relative to both quality and quantity. The high percentage of incomplete bridging should also be noted and further investigated.

Chronic spinal cord injury treated with transplanted autologous bone marrow-derived mesenchymal stem cells tracked by magnetic resonance imaging: a case report.

INTRODUCTION: Intrathecal transplantation is a minimally invasive method for the delivery of stem cells, however, whether the cells migrate from the lumbar to the injured cervical spinal cord has not been proved in humans. We describe an attempt to track bone marrow-derived mesenchymal stem cells in a patient with a chronic cervical spinal cord injury. CASE PRESENTATION: A 33-year-old Thai man who sustained an incomplete spinal cord injury from the atlanto-axial subluxation was enrolled into a pilot study aiming to track bone marrow-derived mesenchymal stem cells, labeled with superparamagnetic iron oxide nanoparticles, from intrathecal transplantation in chronic cervical spinal cord injury. He had been dependent on respiratory support since 2005. There had been no improvement in his neurological function for the past 54 months. Bone marrow-derived mesenchymal stem cells were retrieved from his iliac crest and repopulated to the target number. One half of the total cells were labeled with superparamagnetic iron oxide nanoparticles before transplantation to the intrathecal space between L4 and L5. Magnetic resonance imaging studies were performed immediately after the transplantation and at 48 hours, two weeks, one month and seven months after the transplantation. His magnetic resonance imaging scan performed immediately after the transplantation showed hyposignal intensity of paramagnetic substance tagged stem cells in the subarachnoid space at the lumbar spine area. This phenomenon was observed at the surface around his cervical spinal cord at 48 hours. A focal hyposignal intensity of tagged bone marrow-derived stem cells was detected at his cervical spinal cord with magnetic resonance imaging at 48 hours, which faded after two weeks, and then disappeared after one month. No clinical improvement of the neurological function had occurred at the end of this study. However, at 48 hours after the transplantation, he presented with a fever, headache, myalgia and worsening of his motor function (by one grade of all key muscles by the American Spinal Injury Association impairment scale), which lasted for 48 hours. CONCLUSION: Intrathecal injection of bone marrow-derived stem cells at the lumbar spine level could deliver the cells to the injured cervical spinal cord. Transient complications should be observed closely in the first 48 hours after transplantation. Further study should be carried out to evaluate the result of the treatment.

Sunitinib indirectly enhanced anti-tumor cytotoxicity of cytokine-induced killer cells and CD3⁺CD56⁺ subset through the co-culturing dendritic cells.

Cytokine-induced killer (CIK) cells have reached clinical trials for leukemia and solid tumors. Their anti-tumor cytotoxicity had earlier been shown to be intensified after the co-culture with dendritic cells (DCs). We observed markedly enhanced anti-tumor cytotoxicity activity of CIK cells after the co-culture with sunitinib-pretreated DCs over that of untreated DCs. This cytotoxicity was reliant upon DC modulation by sunitinib because the direct exposure of CIK cells to sunitinib had no significant effect. Sunitinib promoted Th1-inducing and pro-inflammatory phenotypes (IL-12, IFN-γ and IL-6) in DCs at the expense of Th2 inducing phenotype (IL-13) and regulatory phenotype (PD-L1, IDO). Sunitinib-treated DCs subsequently induced the upregulation of Th1 phenotypic markers (IFN-γ and T-bet) and the downregulation of the Th2 signature (GATA-3) and the Th17 marker (RORC) on the CD3⁺CD56⁺ subset of CIK cells. It concluded that sunitinib-pretreated DCs drove the CD3⁺CD56⁺ subset toward Th1 phenotype with increased anti-tumor cytotoxicity.

Decreased indoleamine 2,3-dioxygenase activity and IL-10/IL-17A ratio in patients with COPD.

RATIONALE: Indoleamine 2,3-dioxygenase (IDO) induces generation of regulatory T cells but suppresses Th17 cells and therefore might attenuate neutrophilic inflammation. The role of IDO in neutrophilic airway diseases such as chronic obstructive pulmonary disease (COPD) remains unknown. We evaluated IDO activity and expression and interleukin (IL)-10 and IL-17A levels in sputum from patients with COPD. METHODS: IDO activity and cytokine concentrations in sputum supernatants from patients with COPD of varying severity and in smoking and non-smoking control subjects were determined by high-performance liquid chromatography and ELISA, respectively. RESULTS: Patients with COPD had reduced sputum IDO activity and expression and IL-10 levels, with increased IL-17A, IL-6 and CXCL8 concentrations and sputum neutrophils. These changes were significantly correlated with disease severity. IDO activity was decreased, but to a lesser extent, in normal smokers compared with non-smoking controls. CONCLUSIONS: Patients with COPD have a progressive reduction in IDO activity with reversal of the balance between IL-10 and IL-17A, resulting in chronic airway neutrophilic inflammation.

The Phyllanthus emblica L. infusion carries immunostimulatory activity in a mouse model.

BACKGROUND: Phyllanthus emblica L. (Indian gooseberry, Ma khaam pom) has been an herbal component of Thai traditional recipes proposed to slow down the aging process. A number of methodologies have been employed to investigate the immunological aspects of the so called "anti-aging effects" of P. emblica in a BALB/c mice model. OBJECTIVE: 1) To investigate the immunological efficacy of the anti-aging effects of P. emblica infusion in a BALB/c mice model. 2) To verify the safety for the consumption of P. emblica infusion in BALB/c mice. MATERIAL AND METHOD: For in vitro studies, splenocytes were isolated from mice and examined in comparison with the human umbilical endothelial cells, fibroblasts and YAC-1 (mouse lymphoma) cells for proliferative activity upon the exposure to P. emblica infusion. For in vivo studies, mice were orally administered with P. emblica infusion at a dose range of 0, 50, 100, 200 mg/kg BW for 14 days. After the treatments, splenocytes isolated from these mice examined for proliferative and NK cell activities. RESULTS: For in vitro studies, the infusion of P. emblica could directly drive the proliferation of mouse splenocytes in a dose-dependent manner. The P. emblica infusion itself was already cytotoxic to YAC-1 in the studied dose, while sparing the human umbilical endothelial cells and fibroblasts. For in vivo studies, splenocytes isolated from these mice exhibited dose-dependent proliferative activities. Only the isolated splenocytes from mice ingesting 100 mg/kg BW exhibited an enhancement in NK cell activity. CONCLUSION: P. emblica infusion could drive proliferative activity of splenocyte in vitro and in vivo, with an enhancement in the NK cell-induced cytotoxic activity. The infusion in the aforementioned dose was safe throughout the study.

UVA-induced melanogenesis and modulation of glutathione redox system in different melanoma cell lines: the protective effect of gallic acid.

Oxidative stress has been suggested to play a role in ultraviolet A (UVA)-mediated melanogenesis. Glutathione (GSH) and GSH-related enzymes including γ-glutamate cysteine ligase (γ-GCL) and glutathione S-transferase (GST) are important antioxidant defenses responsible for maintaining cellular redox balance. Hence, improving GSH redox system to cope with oxidative insults may be essential for attenuation of abnormal melanin production. Gallic acid (GA), a dietary phenolic, has been shown to provide beneficial effects against hyperpigmentation possibly through its antioxidant properties. This study thus aimed to assess the antimelanogenic action of GA with regard to modulation of GSH-GCL system and GST in two melanoma cell lines, lightly pigmented G361 human melanoma and more pigmented B16F10 mouse melanoma cells, irradiated with UVA. G361 cells were shown to have lower basal GSH content and GST activity than B16F10 cells. Moreover, GA provided antimelanogenic effects in correlation with promotion of GSH levels, GST activity as well as γ-GCL and GST mRNA in both G361 and B16F10 cells at 2-h post-irradiation. In summary, GA exhibits protective effects on UVA-mediated melanogenesis possibly through improvement of GSH-related antioxidant defenses. Furthermore, different redox state in G361 and B16F10 cells may affect the responses of melanoma cells to GA.

Development of immortalized hepatocyte-like cells from hMSCs.

Clones of hepatocyte-like cells were reproducibly generated from human mesenchymal stem cells immortalized with a combined transduction of both Bmi-1 and TERT genes. These hepatocyte-like cells contained selective markers and several functional properties of hepatocytes, yet still carried proliferative potential. These cells had cuboidal morphology and arranged themselves as cord-like structure in culture. The cloned cells deposited glycogen and actively synthesized albumin. The basal expressions of CYP450 isozymes was observed, albeit only 10-20% that of primary hepatocytes. These expressions were promptly increased upon the addition of rifampicin, a known enzyme inducer. These hepatocyte-like cells may serve as a close alternative to the use of primary hepatocytes for in vitro studies.

Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers.

BACKGROUND: The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. RESULTS: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells. CONCLUSION: The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.