Kanda Kasetsinsombat

Inhibition of UVA-mediated melanogenesis by ascorbic acid through modulation of antioxidant defense and nitric oxide system

Ascorbic acid (AA) has been well known as a skin whitening agent, although attempts have been made to evaluate its protective role against ultraviolet (UV)-induced skin hyperpigmentation or increased melanin production. While melanogenesis is a defense mechanism of the skin against UV irradiation, melanin overproduction may also contribute to melanoma initiation. UVA might play a role in melanogenesis through promoting oxidative stress, which occurs as the result of increased formation of oxidants and/or reactive nitrogen species (RNS) including nitric oxide (NO). Therefore, we investigated the antimelanogenic effect of AA (7.5–120 μM) in association with its inhibitory effect on UVA-induced oxidant formation, NO production through endothelial and inducible NO synthases (eNOS and iNOS) activation and impairment of antioxidant defense using G361 human melanoma cells. Our study demonstrated a comparable ability of AA with that of kojic acid, a well-known tyrosinase inhibitor in inhibiting mushroom tyrosinase. Melanin content was reduced by AA, but neither tyrosinase activity nor mRNA levels were reduced by AA at non-cytotoxic concentrations in UVA-irradiated G361 cells. AA was shown to inhibit UVA-mediated catalase (CAT) inactivation, glutathione (GSH) depletion, oxidant formation and NO production through suppression of eNOS and iNOS mRNA. We report herein that AA can protect against UVA-dependent melanogenesis possibly through the improvement of antioxidant defense capacity and inhibition of NO production through down-regulation of eNOS and iNOS mRNA.

Der p 1 suppresses indoleamine 2, 3-dioxygenase in dendritic cells from house dust mite–sensitive patients with asthma

BACKGROUND Indoleamine 2, 3-dioxygenase (IDO), a tryptophan-degrading enzyme in dendritic cells (DCs), mediates an immunosuppressive effect on activated T lymphocytes. However, little is known about the effect of Der p 1 on IDO in human DCs. OBJECTIVE The aim was to investigate the effect of Der p 1 on the expression and activity of IDO in monocyte-derived DCs from house dust mite (HDM)-sensitive patients with asthma. METHODS Using real-time RT-PCR and HPLC, the expression and activity of IDO were assessed in TNF-alpha-induced mature DCs from HDM-sensitive and nonatopic patients with asthma in response to Der p 1 exposure ex vivo. We also monitored the alteration of IDO activity in Der p 1-pulsed DCs after the coincubation with autologous T cells. RESULTS With a reliance on its protease activity, Der p 1 suppressed functional IDO in DCs from HDM-sensitive patients with asthma but enhanced IDO activity in DCs from nonatopic patients with asthma. This suppression was maintained by the reciprocally induced IL-4 from the coculturing autologous HDM-sensitive T cells. Conversely, the upregulation of IDO activity in Der p 1-pulsed DCs was maintained by IFN-gamma released from autologous nonatopic T cells and the regulatory T-cell subset. Der p 1 pulsation to sensitive DCs failed to raise regulatory T cells but raised progenitor fractions from cloned HDM-sensitive CD4(+) cells through direct contact and soluble mediators. CONCLUSION House dust mite-sensitive DCs exposed to Der p 1 downregulated IDO activity and tipped the T(H)1/T(H)2 cytokine balance toward IL-4, resulting in sustainable IDO suppression.

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.

Statins enhance the effects of corticosteroids on the balance between regulatory T cells and Th17 cells

Plasticity of CD4+ lymphocyte Th17/regulatory T cell (Treg) subsets is involved in the pathogenesis of chronic airway inflammatory diseases, such as asthma. Reversal of Th17/Treg cell balance towards Treg cells may be beneficial for the suppression of chronic Th2 cell‐mediated inflammatory diseases, such as asthma. However, the effect of the combination of corticosteroids and a statin on the ratio of Treg/Th17 cells is unknown.

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.

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

BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

Human Cytokine-Induced Killer Cells Specifically Infiltrated and Retarded the Growth of the Inoculated Human Cholangiocarcinoma Cells in SCID Mice

Cytokine-induced killer (CIK) cells were examined for safety and efficacy for cholangiocarcinoma treatment. Several conditions of human CIK cells were examined using ex vivo cytotoxic assay and SCID mice pre-inoculated with cholangiocarcinoma cells. We monitored the ex vivo cytotoxicity, tumor sizes and immunohistochemistry. Optimal tumor suppression was observed when CIK cells were pre-exposed to dendritic cells (DCs). Unexpectedly, pulsing of tumor RNA to DCs rendered the co-culturing CIK cells ineffective and raised the proportion of CD4+CD25+ subset. The use of CD3+CD56+ subset instead of the whole population of CIK cells for the co-culture with RNA-pulsed DCs restored the efficacy. Tumor-infiltrating human CD3+ cells were observed from day 2 – 14. The CD3+CD56+ cells are logical candidates for clinical trial while the DC-co-cultured CIK cells produced similar efficacy and more feasible for clinical application. The RNA pulsation of DCs up-regulated the regulatory subset of CIK cells and abrogated the anti-tumor efficacy.

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

BackgroundAyurved 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.MethodsCIK 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.ResultsAVS073 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).ConclusionsAVS073 upregulated Th1 and Th17, but downregulated Th2 and Treg phenotypes within CD3+CD56+ cells. The treatment of mDCs drove Th1 and Th17-polarizations.

Abstract A39: Cholangiocarcinoma-exposed cytokine-induced killer cells developed senescence and exhaustion

The existence of cellular exhaustion and senescence have been well-documented in T cells, but not yet in CIK cells. The exposure of CIK cells to HubCCA1 cholangiocarcinoma cells resulted in sustaining the proportion of CD3 + CD56 + subset at the expense of that of the pure T cell (CD3 + CD56 - ) subset. All major subsets (CD3 + CD56 + , T cell subsets, and NK cell (CD3 - CD56 + subset) of the survived CIK cells were inflicted with increasing senescence markers (KLRG1, CTLA4, and CD57) upon the exposure length. However, CD28, a T cell activation marker, was up-regulated in CD3 + CD56 + and NK cell subsets upon tumor exposure, but was suppressed in T cell subset. The co-culture of CIK cells with dendritic cells (DCs) could alleviate CD57, but not KLRG1 nor CTLA4. For exhaustion, the co-culture with DCs could lower the level of PD-1 in CD3 + CD56 + and T cell subset, but was unable to withstand the massive up-regulation of PD-1 and Tim-3 in all 3 major subsets upon the tumor exposure. The co-culture with DCs raised the proportion of Treg (CD4 + CD25 + Foxp3 + ) subset in CIK cells, but surprisingly reversed by the eventual exposure to tumor cells. In summary, the exposure of CIK cells to tumor cells rendered the development of senescence and exhaustion to the CIK cells. The co-culture of CIK cells with DCs had limited role in preventing these opposing effects. Citation Format: Adisak Wongkajornsilp, Nathawadee Sawatpiboon, Sunisa Duangsa-ard, Kanda Kasetsinsombat, Kittipong Maneechotesuwan, Suradej Hongeng. Cholangiocarcinoma-exposed cytokine-induced killer cells developed senescence and exhaustion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A39.