The first synthesis and immunomodulatory properties of p-hydroxyphenyl caffeate derived from Wedelia trilobata.

Herein, we report a concise and efficient method for the synthesis of p-hydroxyphenyl caffeate, a novel natural caffeic acid ester derivative isolated from Wedelia trilobata. The key feature of this synthesis is the Verley-Doebner modification of the Knoevenagel condensation of the p-hydroxyphenyl malonate intermediate. The synthesized p-hydroxyphenyl caffeate enhanced interleukin 2 production by murine lymph node T cells, and suppressed interleukin 13 production by murine epidermal T cells. This implies that p-hydroxyphenyl caffeate might be a novel immunomodulatory drug candidate, specifically targeting T helper 2 cell type responses in skin diseases such as atopic dermatitis and vitiligo. [Formula: see text].

A novel sphingosylphosphorylcholine and sphingosine-1-phosphate receptor 1 antagonist, KRO-105714, for alleviating atopic dermatitis.

BACKGROUND: Atopic dermatitis (eczema) is a type of inflammation of the skin, which presents with itchy, red, swollen, and cracked skin. The high global incidence of atopic dermatitis makes it one of the major skin diseases threatening public health. Sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) act as pro-inflammatory mediators, as an angiogenesis factor and a mitogen in skin fibroblasts, respectively, both of which are important biological responses to atopic dermatitis. The SPC level is known to be elevated in atopic dermatitis, resulting from abnormal expression of sphingomyelin (SM) deacylase, accompanied by a deficiency in ceramide. Also, S1P and its receptor, sphingosine-1-phosphate receptor 1 (S1P1) are important targets in treating atopic dermatitis. RESULTS: In this study, we found a novel antagonist of SPC and S1P1, KRO-105714, by screening 10,000 compounds. To screen the compounds, we used an SPC-induced cell proliferation assay based on a high-throughput screening (HTS) system and a human S1P1 protein-based [35S]-GTPγS binding assay. In addition, we confirmed the inhibitory effects of KRO-105714 on atopic dermatitis through related cell-based assays, including a tube formation assay, a cell migration assay, and an ELISA assay on inflammatory cytokines. Finally, we confirmed that KRO-105714 alleviates atopic dermatitis symptoms in a series of mouse models. CONCLUSIONS: Taken together, our data suggest that SPC and S1P1 antagonist KRO-105714 has the potential to alleviate atopic dermatitis.

Biodegradable and injectable hydrogels as an immunosuppressive drug delivery system.

Cyclosporine A (CsA) is an extremely hydrophobic immunosuppressive drug, whose systemic administration to suppress the activity of T cells and T cell-based immune responses is frequently associated with a number of adverse drug reactions. Local delivery of CsA focused on a specific target organ has been proposed as a possible solution to this problem. In this study, we developed biodegradable sol-gel drug delivery systems, consisting of HA-Ca-Alg hydrogels combining hyaluronic acid calcium complex (HA-Ca) and sodium alginate (Alg-Na) components, for the local sustained delivery of CsA. A HA-Ca complex with very high degree of substitution was prepared by the acid-base reaction of hyaluronic acid and calcium acetate. The gelation was completed within about 2-45 min without external addition of calcium salts such as CaSO4 and CaCl2, indicating the high potential of the present hydrogel systems for drug delivery by injection in vivo. The HA-Ca system was characterized by high-resolution inductively coupled plasma-optical emission spectroscopy, 1H NMR, FT-IR, and thermogravimetric analysis methods. Moreover, the scanning electron microscopy analysis of the HA-Ca-Alg hydrogels showed an irregular porous morphology, with interconnected pores of 50-300 µm width. The sol-gel transition and the maximum viscosity (about 10,000 cP) of the HA-Ca-Alg hydrogels were characterized by examining the time evolution of the viscosity at 37 °C. The hydrolytic degradation of the HA-Ca-Alg hydrogel was also examined at 37 °C. CsA-encapsulated HA-Ca-Alg hydrogels exhibited sustained in vitro release of CsA over 14 days, which was confirmed through in vitro measurements of the activity of murine T cells over 2 weeks. These results show that the present injectable HA-Ca-Alg hydrogels can be used effectively for the sustained delivery of extremely hydrophobic immunosuppressive drugs, including CsA.

Potentiation of T Cell Stimulatory Activity by Chemical Fixation of a Weak Peptide-MHC Complex.

The stability of peptide-MHC complex (pMHC) is an important factor to shape the fate of peptide-specific T cell immune response, but how it influences on T cell activation process is poorly understood. To better understand that, we investigated various T cell activation events driven by Ld MHCI loaded with graded concentrations of P2Ca and QL9 peptides, respectively, with 2C TCR Tg T cells; the binding strength of P2Ca for Ld is measurably weaker than that of QL9, but either peptides in the context of Ld interact with 2C TCR with a similar strength. When their concentrations required for early T cell activation events, which occur within several minutes to an hour, were concerned, EC50s of QL9 were about 100 folds lower than those of P2Ca, which was expected from their association constants for Ld. When EC50s for late activation events, which takes over several hours to occur, were concerned, the differences grew even larger (> 300 folds), suggesting that, due to weak binding, Ld/P2Ca dissociate from each other more easily to lose its antigenicity in a short time. Accordingly, fixation of Ld/P2Ca with paraformaldehyde resulted in a significant improvement in its immunogenicity. These results imply that binding strength of a peptide for a MHC is a critical factor to determine the duration of pMHC-mediated T cell activation and thus the attainment of productive T cell activation. It is also suggested that paraformaldehyde fixation should be an effective tool to ameliorate the immunogenicity of pMHC with a poor stability.

Influences of Environmental Chemicals on Atopic Dermatitis.

Atopic dermatitis is a chronic inflammatory skin condition including severe pruritus, xerosis, visible eczematous skin lesions that mainly begin early in life. Atopic dermatitis exerts a profound impact on the quality of life of patients and their families. The estimated lifetime prevalence of atopic dermatitis has increased 2~3 fold during over the past 30 years, especially in urban areas in industrialized countries, emphasizing the importance of life-style and environment in the pathogenesis of atopic diseases. While the interplay of individual genetic predisposition and environmental factors contribute to the development of atopic dermatitis, the recent increase in the prevalence of atopic dermatitis might be attributed to increased exposure to various environmental factors rather than alterations in human genome. In recent decades, there has been an increasing exposure to chemicals from a variety of sources. In this study, the effects of various environmental chemicals we face in everyday life - air pollutants, contact allergens and skin irritants, ingredients in cosmetics and personal care products, and food additives - on the prevalence and severity of atopic dermatitis are reviewed.

Effect of ginseng and ginsenosides on melanogenesis and their mechanism of action.

Abnormal changes in skin color induce significant cosmetic problems and affect quality of life. There are two groups of abnormal change in skin color; hyperpigmentation and hypopigmentation. Hyperpigmentation, darkening skin color by excessive pigmentation, is a major concern for Asian people with yellow-brown skin. A variety of hypopigmenting agents have been used, but treating the hyperpigmented condition is still challenging and the results are often discouraging. Panax ginseng has been used traditionally in eastern Asia to treat various diseases, due to its immunomodulatory, neuroprotective, antioxidative, and antitumor activities. Recently, several reports have shown that extract, powder, or some constituents of ginseng could inhibit melanogenesis in vivo or in vitro. The underlying mechanisms of antimelanogenic properties in ginseng or its components include the direct inhibition of key enzymes of melanogenesis, inhibition of transcription factors or signaling pathways involved in melanogenesis, decreasing production of inducers of melanogenesis, and enhancing production of antimelanogenic factor. Although there still remain some controversial issues surrounding the antimelanogenic activity of ginseng, especially in its effect on production of proinflammatory cytokines and nitric oxide, these recent findings suggest that ginseng and its constituents might be potential candidates for novel skin whitening agents.

Erratum to "Microfluidic System Based High Throughput Drug Screening System for Curcumin/TRAIL Combinational Chemotherapy in Human Prostate Cancer PC3 Cells" [Biomol. Ther. 22 (2014) 355-362].

[This corrects the article on p. 355 in vol. 22, PMID: 25143816.].

Role of epidermal γδ T-cell-derived interleukin 13 in the skin-whitening effect of Ginsenoside F1.

Ginsenoside F1 (GF1) is a metabolite of ginsenoside Rg1. Although GF1 has several benefits for skin physiology, the effect of GF1 on skin pigmentation has not been reported. We found that a cream containing 0.1% GF1 showed a significant whitening effect on artificially tanned human skin after 8 weeks of application. However, GF1 did not inhibit mRNA expression of tyrosinase or dopachrome tautomerase (DCT) in normal human epidermal melanocytes (NHEMs) or cocultured NHEMs/normal human epidermal keratinocytes. Interestingly, GF1 enhanced production of interleukin 13 (IL-13) from human epidermal γδ T cells. IL-13 significantly reduced the mRNA expression and protein amount of both tyrosinase and DCT and reduced melanin synthesis activities in NHEMs, resulting in visible brightening of NHEM pellet. These results suggest that enhancement of IL-13 production by GF1 from epidermal γδ T cells might play a role in the skin-whitening effect of GF1 via the suppression of tyrosinase and DCT.

Microfluidic System Based High Throughput Drug Screening System for Curcumin/TRAIL Combinational Chemotherapy in Human Prostate Cancer PC3 Cells.

We have developed a fully automated high throughput drug screening (HTDS) system based on the microfluidic cell culture array to perform combinational chemotherapy. This system has 64 individually addressable cell culture chambers where the sequential combinatorial concentrations of two different drugs can be generated by two microfluidic diffusive mixers. Each diffusive mixer has two integrated micropumps connected to the media and the drug reservoirs respectively for generating the desired combination without the need for any extra equipment to perfuse the solution such as syringe pumps. The cell array is periodically exposed to the drug combination with the programmed LabVIEW system during a couple of days without extra handling after seeding the cells into the microfluidic device and also, this device does not require the continuous generation of solutions compared to the previous systems. Therefore, the total amount of drug being consumed per experiment is less than a few hundred micro liters in each reservoir. The utility of this system is demonstrated through investigating the viability of the prostate cancer PC3 cell line with the combinational treatments of curcumin and tumor necrosis factor-alpha related apoptosis inducing ligand (TRAIL). Our results suggest that the system can be used for screening and optimizing drug combination with a small amount of reagent for combinatorial chemotherapy against cancer cells.

Comparative Analysis of Human Epidermal and Peripheral Blood γδ T Cell Cytokine Profiles.

BACKGROUND: Human epidermal γδ T cells are known to play crucial roles in the defense and homeostasis of the skin. However, their precise mechanism of action in skin inflammation remains less clear. OBJECTIVE: In this study, we analyzed the cytokine expression profile of human epidermal γδ T cells and compared it to that of peripheral blood γδ T cells to investigate the specific activity of epidermal γδ T cells in modulating skin inflammation. METHODS: We isolated γδ T cells from epidermal tissue or peripheral blood obtained from healthy volunteers. Isolated γδ T cells were stimulated using immobilized anti-CD3 antibody and interleukin-2 plus phytohaemagglutinin, and were then analyzed using a cytokine array kit. RESULTS: Both epidermal and peripheral blood γδ T cells produced comparable levels of granulocyte-macrophage colony-stimulating factor, I-309, interferon-γ, macrophage migration inhibitory factor, macrophage inflammatory protein-1α, and chemokine (C-C) ligand 5. The epidermal γδ T cells produced significantly higher levels of interleukin-4, -8, -13, and macrophage inflammatory protein-1ß than the peripheral blood γδ T cells did. Notably, the epidermal γδ T cells produced several hundred-fold higher levels of interleukin-13 than interleukin-4. CONCLUSION: These results suggest that the epidermal γδ T cells have a stronger potential to participate in the Th2-type response than the peripheral blood γδ T cells do. Furthermore, epidermal γδ T cells might play an important role in the pathogenesis of Th2-dominant skin diseases because of their active production of interleukin-13.

IL-4 inhibits the melanogenesis of normal human melanocytes through the JAK2-STAT6 signaling pathway.

Skin diseases can be characterized by their predominant CD4-positive T-helper (Th) cell profiles. Chronic dermatological conditions often give rise to abnormal skin pigmentation. To understand the role of Th cells in pigmentation, the effects of the major Th cell cytokines, IFNγ, IL-4, and IL-17A, on melanogenesis were evaluated using cultured normal human melanocytes (NHMs) instead of relying on transformed melanoma cell lines. IL-4 directly inhibited melanogenesis in NHMs and downregulated both transcription and translation of melanogenesis-associated genes, such as microphthalmia-associated transcription factor (MITF) and dopachrome tautomerase. Despite the lack of a direct inhibition of melanin pigment synthesis, IFNγ and IL-17A increased the synthesis of an antimelanogenic cytokine IL-6 in NHMs. IFNγ activated signal transducers and activators of transcription 1 (STAT1) and STAT3 phosphorylation in NHMs, and IL-4 increased the STAT3 and STAT6 phosphorylation. The differential phosphorylation profile of STAT proteins between IFNγ and IL-4 may explain the difference in their effect on melanogenesis in NHMs. The IL-4-induced downregulation of melanogenesis was inhibited by treating NHMs with a JAK2 inhibitor AG490 or STAT6 siRNA. In conclusion, the involvement of the IL-4-induced JAK2-STAT6 signaling and the IFNγ- or IL-17A-dependent antimelanogenic IL-6 production should be considered as one of the mechanisms explaining the association with hypopigmention in skin diseases.

CXCR7 mediates SDF1-induced melanocyte migration.

Melanoblasts are derived from the neural crest and migrate to the dermal/epidermal border of skin and hair bulges. Although melanoblast migration during embryogenesis has been well investigated, there are only a few reports regarding the migration of mature melanocytes. Here, we demonstrate that a chemokine, stromal-derived factor-1 (SDF1, also known as CXCL12), and one of its receptor CXCR7 regulate normal human epidermal melanocyte (NHEM) migration. We found that SDF1 induces the directional migration of NHEMs. Interestingly, although both CXCR4 and CXCR7 are expressed in NHEMs, blockade of CXCR4 using a CXCR4-specific neutralizing antibody did not exert any influence on the SDF1-induced migration of NHEMs, whereas blockade of CXCR7 using a CXCR7-specific neutralizing antibody did influence migration. Furthermore, SDF1-induced NHEMs migration exhibited the early hallmark events of CXCR7 signaling associated with MAP kinase activation. It is known that the phosphorylation of ERK through CXCR7 signaling is mediated by ß-arrestins. The treatment of NHEMs with SDF1 resulted in the phosphorylation of ERK in a ß-arrestin 2-dependent manner. These results suggest that melanocytes may have a unique mechanism of migration via SDF1/CXCR7 signaling that is different from that of other cell types.

Kojic acid-induced IL-6 production in human keratinocytes plays a role in its anti-melanogenic activity in skin.

BACKGROUND: Kojic acid is a fungal metabolite widely used in medicinal and cosmetic formulations as a skin-lightening agent based on its de-pigmenting activity. Although in human clinical studies kojic acid has been shown to be effective in the treatment of hyper-pigmentation disorders such as melasma, the reasons for its apparent lack of anti-melanogenic activity in cultured mammalian melanocytes are unclear. OBJECTIVES: This study was aimed to elucidate pharmacological mechanisms of the in vivo anti-melanogenic activity of kojic acid in human skin. METHODS: A primary human melanocyte and keratinocyte co-culture system was used to evaluate whether kojic-acid-induced changes in keratinocytes were associated with anti-melanogenic activities in melanocytes. The cytokine secretion profiles in response to kojic acid were analyzed. RESULTS: Kojic acid increased interleukin (IL)-6 and IL-8 production in melanocyte/keratinocyte co-cultures; however, IL-6 directly inhibited melanogenesis whereas IL-8 did not. In melanocyte monocultures, kojic acid did not increase IL-6 production whereas in keratinocyte monocultures it significantly up-regulated IL-6 gene and protein expression. Therefore, the up-regulation of IL-6 in melanocyte/keratinocyte co-cultures seems to be originated from kojic acid-induced changes in keratinocytes. Anti-IL-6 antibody treatment antagonized the anti-melanogenic effect of kojic acid on the co-cultures. CONCLUSIONS: The pharmacological mechanism of kojic acid to explain clinically effective anti-melanogenic activity on hyper-pigmented skin is associated with the kojic acid-induced IL-6 production in keratinocytes. The cross-talk between melanocytes and keratinocytes should be determined in future studies on the pharmacological mechanisms of clinically effective dermatological drugs acting on the epidermis.

Neuroimmunological mechanism of pruritus in atopic dermatitis focused on the role of serotonin.

Although pruritus is the critical symptom of atopic dermatitis that profoundly affect the patients' quality of life, controlling and management of prurirtus still remains as unmet needs mainly due to the distinctive multifactorial pathogenesis of pruritus in atopic dermatitis. Based on the distinct feature of atopic dermatitis that psychological state of patients substantially influence on the intensity of pruritus, various psychotropic drugs have been used in clinic to relieve pruritus of atopic dermatitis patients. Only several psychotropic drugs were reported to show real antipruritic effects in atopic dermatitis patients including naltrexone, doxepin, trimipramine, bupropion, tandospirone, paroxetine and fluvoxamine. However, the precise mechanisms of antipruritic effect of these psychotropic drugs are still unclear. In human skin, serotonin receptors and serotonin transporter protein are expressed on skin cells such as keratinocytes, melanocytes, dermal fibroblasts, mast cells, T cells, natural killer cells, langerhans cells, and sensory nerve endings. It is noteworthy that serotonergic drugs, as well as serotonin itself, showed immune-modulating effect. Fenfluramine, fluoxetine and 2, 5-dimethoxy-4-iodoamphetamine significantly decreased lymphocyte proliferation. It is still questionable whether these serotonergic drugs exert the immunosuppressive effects via serotonin receptor or serotonin transporter. All these clinical and experimental reports suggest the possibility that antipruritic effects of selective serotonin reuptake inhibitors in atopic dermatitis patients might be at least partly due to their suppressive effect on T cells. Further studies should be conducted to elucidate the precise mechanism of neuroimmunological interaction in pruritus of atopic dermatitis.

Percutaneously placed covered retrievable stents for the treatment of biliary anastomotic strictures following living donor liver transplantation.

This study evaluated the feasibility and efficacy of covered retrievable stent placement compared with drainage catheter placement for treating biliary anastomotic strictures following living donor liver transplantation (LDLT). In 39 of 59 patients who underwent LDLT, balloon dilation of strictures and subsequent interposition of 14-French indwelling catheters were performed (group 1), whereas in 20 patients, 29 covered retrievable stents were percutaneously placed (group 2). The drainage catheters were positioned above the stricture after at least 12 weeks of internal drainage or following stent removal after at least 8 weeks of stent placement. The drainage catheters were removed when the follow-up cholangiogram revealed free passage of contrast media. In the presence of residual stricture, either of the following was performed: (1) balloon dilation of the stricture and replacement of the indwelling catheter or (2) placement of another covered retrievable stent for at least 12 weeks. Technical success was achieved in all recipients. Clinical success was higher in group 1 (95%) than in group 2 (70%) (P = 0.005), whereas the duration of treatment was significantly shorter in group 2 (197 ± 89 days) than in group 1 (278 ± 115 days) (P = 0.018). All stents were successfully removed at a median of 14 weeks after placement, except for 1 patient in whom early stent removal was inevitable and a second patient in whom the stent migrated completely. Stent migration rate was 24% (7 of 29 stents). In conclusion, treatment of biliary anastomotic strictures following LDLT is feasible using covered retrievable stents and has an acceptable clinical success rate with shorter treatment duration.

Sphingosylphosphorylcholine down-regulates filaggrin gene transcription through NOX5-based NADPH oxidase and cyclooxygenase-2 in human keratinocytes.

Sphingosylphosphorylcholine (SPC) mediates various inflammatory and behavioral responses in atopic dermatitis. Recent studies have shown that dysfunction of the epidermal permeability barrier itself plays a primary role in the etiology of atopic dermatitis. However, the effects of SPC on major proteins essential to the development of the epidermal permeability barrier such as filaggrin, loricrin, involucrin, keratin 1, keratin 10 and small proline-rich proteins are still unclear. In this study, we demonstrated that SPC significantly reduces filaggrin gene transcription, implying that SPC plays a pivotal role in impairment of the epidermal permeability barrier in atopic dermatitis lesional skin. In cultured normal human keratinocytes (NHKs), SPC increases the intracellular level of reactive oxygen species (ROS) and up-regulates NADPH oxidase 5 (NOX5) gene transcription. SPC also stimulates prostaglandin (PG) E(2) production by increasing cyclooxygenase (COX)-2 expression in NHK. The effects of the prostanoid EP receptor agonists, limaprost, butaprost, and sulprostone on filaggrin gene expression in NHK suggest that the prostanoid EP2 receptor plays a significant role in the PGE(2)-mediated filaggrin down-regulation. In contrast, limaprost and butaprost do not affect NOX5 expression in NHK, implying that the NOX5-regulated ROS pathway stimulated by SPC may be upstream of the COX-2 pathway. We propose that the increase in SPC levels further aggravates dermatological symptoms of atopic dermatitis through SPC-induced down-regulation of filaggrin in NHK.

Acute inhibition of selected membrane-proximal mouse T cell receptor signaling by mitochondrial antagonists.

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-gamma1 and entry of extracellular Ca(2+) following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

LFA-1-dependent Ca2+ entry following suboptimal T cell receptor triggering proceeds without mobilization of intracellular Ca2+.

A surge in cytosolic calcium ion concentration by entry of extracellular Ca2+ is a hallmark of T cell activation. According to store-operated Ca2+ entry mechanism, the Ca2+ entry is preceded by activation of phospholipase C-gamma1 (PLC-gamma1) and the consequent mobilization of intracellular Ca2+. Using membrane vesicles expressing the mouse class I major histocompatibility complex, i.e. Ld plus costimulatory ligands, i.e. B7-1 and intercellular adhesion molecule-1 along with 2C T cell receptor transgenic T cells, we investigated the roles of CD28 and LFA-1 (lymphocyte function-associated antigen-1) in the activation of PLC-gamma1 and Ca2+ signaling. Both CD28 and LFA-1 made significant and comparable contributions to the activation of PLC-gamma1 as gauged by the level of its phosphorylation at tyrosine 783. In contrast, their roles in Ca2+ signaling were quite distinct so that LFA-1/intercellular adhesion molecule-1 interaction exerted a determining role, whereas CD28/B7-1 interaction played only a minimal role. In particular, when the T cells were activated by suboptimal T cell receptor stimulation, LFA-1 played an indispensable role in the Ca2+ signaling. Further experiments using Ca2+-free medium demonstrated that the entry of extracellular Ca2+ was not always accompanied by mobilization of intracellular Ca2+. Thus, intracellular Ca2+ mobilization was hardly detected under the condition that LFA-1 played the indispensable role in the entry of extracellular Ca2+, while a distinct level of intracellular Ca2+ mobilization was readily detected under the condition that LFA-1 played only the supporting role. These results ensure the unique role of LFA-1 in T cell Ca2+ signaling and reveal that LFA-1-dependent Ca2+ entry proceeds via a mechanism separate from store-operated Ca2+ entry.