Search

Human induced pluripotent stem (hiPS) cells are attracting attention as a tool for regenerative medicine. However, several problems need to be overcome for their widespread and safe use, for example, the high cost of maintaining hiPS cells and the possibility of xenogeneic cell contamination in hiPS cell cultures. One of the main contributors to the high cost of maintaining hiPS cells is basic fibroblast growth factor (bFGF), which is essential for such cultures. Xenogeneic contamination can occur because of the use of mouse-derived feeder cells to culture hiPS cells. To overcome the problems of cell culture cost and xenogeneic contamination, we have developed a novel culture method in which the undifferentiated state and pluripotency of hiPS cells can be maintained under feeder-free and bFGF-free conditions. Our new approach involves the addition to the culture medium of highly sulfated hyaluronic acid (HA-HS), in which the hydroxyl groups of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) are chemically sulfated. HA-HS promotes bFGF signaling and maintains the undifferentiated state and pluripotency of hiPS cells under feeder-free and bFGF-free conditions. By contrast, non-sulfated hyaluronic acid and low sulfated hyaluronic acid do not maintain the undifferentiated state and pluripotency of hiPS cells. These results indicate that the maintenance of hiPS cells under feeder-free and bFGF-free conditions is an HA-HS specific effect. This study is the first to demonstrate the effects of sulfated hyaluronic acid on mammalian pluripotent stem cells, and provides a novel method for maintaining hiPS cells using HA-HS.

CD30+ lymphoproliferative disorders represent a spectrum of diseases from lymphomatoid papulosis (LyP) to primary cutaneous anaplastic large-cell lymphoma (PCALCL) (1, 2). LyP is characterized by recurrent papulonodular lesions, which undergo spontaneous regression (1, 2). Survival is unaffected by LyP, but patients are at risk of a second cutaneous or lymphoid malignancy (1). PCALCL has a favourable prognosis with a 5-year survival rate between 76% and 96%. In this report, we used immunohistochemical (IHC) staining of LyP type A and PCALCL specimens to examine for macrophage markers as well as dendritic cell (DC)-specific markers and their functional markers.

Purpose Telogen (resting phase) hair follicles (HFs) are more radioresistant than their anagen (growth phase) counterparts. Fibroblast growth factor (FGF) 18 is strongly expressed in telogen HFs to maintain the telogen phase, while several other FGFs exert radioprotective effects. However, the role of FGF18 in the radioresistance of HFs remains unknown. Therefore, this study focused on clarifying the role of FGF18 in the radioresistance of telogen HFs and its potential as a radioprotector. \nMethods and Materials BALB/c mice with telogen or plucking-induced anagen HFs were exposed to total body irradiation with γ-rays at 4 to 12 Gy after intraperitoneal treatment with a FGF18 or FGFR inhibitor. A time course analysis was performed histologically and hair growth was observed 14 or 15 days after depilation. Skin specimens were analyzed by DNA microarrays and western blotting. \nResults Telogen irradiation at 6 Gy resulted in transient cell growth arrest, leading to successful hair growth, whereas anagen irradiation failed to promote hair growth. Telogen irradiation did not induce apoptosis in HFs or reduce hair follicle stem cells, whereas anagen irradiation induced apoptosis and reduced stem cell numbers. The Inhibition of FGF receptor signaling during the telogen phase promoted HF cell proliferation; however, hair failed to grow after irradiation. In contrast, recombinant FGF18 induced transient cell growth arrest after anagen irradiation with enhanced DNA repair, leading to the inhibition of apoptosis, maintenance of hair follicle stem cells, and successful hair growth. Moreover, FGF18 reduced the expression levels of genes promoting G2/M transition as well as the protein expression levels of cyclin B1 and cdc2 in skin, and induced G2/M arrest in the keratinocyte cell line HaCaT. \nConclusion These results suggest that FGF18 signaling mediates radioresistance in telogen HFs by arresting the cell cycle, and that FGF18 has potential as a radioprotector for radiation-induced alopecia.

A variety of polypeptide growth factors are involved in the dynamic maintenance of the skin and hair. Here, we demonstrate the presence of high levels of fibroblast growth factor (FGF)-13 in the bulge region of hair follicles. Using real-time PCR, we found that expression of FGF-13 mRNA is comparable to, or higher than, that of other FGF known to regulate hair growth and wound healing. To gain additional insight into the function of FGF-13, we evaluated its distribution using in situ hybridization and immunohistochemical staining. Unlike other FGF, the distribution of FGF-13 mRNA and protein in adult mice was mainly restricted to cells in the bulge region of hair follicles, although lower levels were detected with less frequency in keratinocytes in the basal layer of the epidermis. FGF-13 protein was detectable in the bulge region throughout the hair growth cycle, but its distribution was especially wide during telogen and early anagen. During hair follicle morphogenesis in newborn mice, FGF-13 protein was first detected in the bulge region and basal layer keratinocytes 3 d after birth. These findings suggest that FGF-13 may play a role in regulating the function of cells in the bulge region and basal layer of the epidermis.

Metal allergy is thought to be caused by the release of ions from metallic materials. Extensive use of metal in jewelry, coins, surgical instruments, and dental restorations may be responsible for recent increases in allergy incidence. Metal allergic disease is categorized as a delayed-type hypersensitivity, which is developed more than 24 h after exposure to the causal metal. The hallmark of delayed-type hypersensitivity is the recruitment of lymphocytes and inflammatory cells, including T cells and granulocytes, to the site of allergic inflammation. During the development of metal allergy, T cells are known to play a role, and since metal ions are thought to function as haptens, T cell-mediated responses likely contribute to allergic disease. While the involvement of pathogenic T cells in the development of metal allergy has not been explored using animal models, studies utilizing human patient samples have been conducted. T cell clones, both CD4+ and CD8+, have been established from peripheral blood mononuclear cells of patients with metal allergy, and their responsiveness to the causal metal has been analyzed. It was found that metal ions induced proliferation of these T cells in vitro and some of the T cell clones produced IFN-γ or IL-4 after metal stimulation, while others produced both T helper 1- and 2-type cytokines. However, the subset of pathogenic T cells involved in the development of metal allergy and their cytokine profiles remain controversial. Recently, a novel animal model that reproduces human metal allergy has been established. Here we show the pathogenesis of metal allergy in the view of immunological responses using this animal model.

T helper 17 cells, characterized by interleukin-17 (IL-17) production, play a critical role in the pathogenesis of autoimmune disease, including alopecia areata (AA). In this report, we employed immunohistochemical staining for IL-17-producing cells, as well as interferon-γ-producing cells, granulysin-bearing cells and Foxp3+ regulatory T cells, and performed a quantitative analysis of IL-17-producing cells in the lesional skin of several clinical forms of AA by TissueFAXS analysis. Among them, interestingly, the ratio of IL-17-producing cells in acute, diffuse and total alopecia was significantly lower than those of multiple types of AA. Our study sheds light on one of the possible immunological mechanisms of AA.

Melanin plays an important role in the prevention of DNA damage and protection of skin tissues from UV irradiation. However, excessive accumulation of melanin can also cause freckle and melanoma. Thus, we focused on the melanogenesis regulation effect of Tunisian aromatic plants. The melanin synthesis system is known as melanogenesis, and tyrosinase is a key enzyme of melanogenesis (Fang and Setaluri, 1999; Kameyama et al., 1995). Currently, many research efforts have focused on the specific mechanism involved in melanogenesis. It has been reported that ERK1/2 phosphorylation is related to the inhibition of melanogenesis (Kim et al., 2006). Murine melanoma B16 cells were treated with the extract of Tunisian aromatic plants, namely, Capparis spinosa, Erica multiflora and Thymelaea hirsuta, and then cell viability and synthesized melanin content were measured. The C. spinosa and E. multiflora extract increased the synthesized melanin content in B16 cells without cytotoxity. The tyrosinase expression was clearly increased in cells treated with the extracts. The T. hirsuta extract significantly decreased the synthesized melanin content in B16 cells without cytotoxicity. ERK1/2 was immediately phosphorylated in cells just after treatment with the extract. The tyrosinase expression was inhibited in cells treated with the extracts.

Hair growth problems can affect human physical and mental health and are of particular relevance during the aging process. In an effort to resolve such problems, we attempted to find plants having hair growth regulation activity and thus collected plant extracts from Tunisia for bioprospecting purposes. Among them, we investigated the Erica multiflora extract to evaluate the hair growth promotion activity by using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay and cell cycle assay on human dermal papilla cells in vitro and an administration assay on mouse dorsal skin in vivo. The results showed that the Erica multiflora extract promotes dermal papilla cell growth and cell cycle with high activity, and induced hair growth in vivo by induction of anagen phase from telogen phase.

Skin pigmentation is the result of melanogenesis that occurs in melanocytes and/or melanoma cells. Although melanogenesis is necessary for the prevention of DNA damage and cancer caused by UV irradiation, excessive accumulation of melanin can also cause melanoma. Thus, we focused on the antimelanogenesis effect of an extract from Thymelaea hirsuta, a Tunisian herb. Murine melanoma B16 cells were treated with T. hirsuta extract, and then cell viability and synthesized melanin content were measured. We found that the T. hirsuta extract decreased the synthesized melanin content in B16 cells without cytotoxicity. Tyrosinase is a key enzyme of melanogenesis and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation is known to be related to melanogenesis inhibition. To clarify its mechanism, we also determined ERK1/2 phosphorylation and tyrosinase expression level. ERK1/2 was immediately phosphorylated in cells just after treatment with the extract. The tyrosinase expression was inhibited after 24 h of stimulation with the extract. The T. hirsuta extract was fractionated, and we found that one fraction considerably decreased the melanin synthesis in B16 cells and that this fraction contains daphnanes as the main component. This indicates that our findings might be attributable to daphnanes.

© 2014 The Authors. doi: 10.2340/00015555-1807 Journal Compilation © 2014 Acta Dermato-Venereologica. ISSN 0001-5555 Langerhans cell histiocytosis (LCH) is characterised by the clonal proliferation of Langerhans cells (LC) that predominantly occurs in infants and small children, but LCH is rare in adults (1). Generally in children, local therapy would be an appropriate treatment, since patients with localised disease or disease limited to a single organ system have a good prognosis (1). However, the clinical picture of LCH in adults is not fully understood and the optimal treatment for it is still under discussion. In this report, we describe a case of adult onset LCH successfully treated with bi-weekly pegylated interferon (PEG-IFN)α2a and discuss the immunological background focusing on the tumour microenvironment immunologically.

We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice.

Fibroblast growth factor (FGF) 5 inhibits hair growth and induces catagen in mouse hair follicles, in vivo. Given that FGF-5 receptor (FGFR1) is expressed in dermal papilla cells (DPCs), which are known to stimulate outer root sheath cell (ORSC) proliferation, we hypothesized that FGF-5 attenuates DPC-mediated ORSC proliferation. In the present study, DPCs and ORSCs were isolated from rat vibrissae, after which the effects of FGF-5 on proliferation of ORSCs cultured in DPC-conditioned medium were assessed. We first confirmed that FGFR1 was expressed in cultured DPCs and detected FGFR2-4 as well. ORSC proliferation was increased approximately twofold when the cells were cultured in DPC-conditioned medium, and the effect was unaltered by FGF-5. In addition, FGF-5 did not directly inhibit ORSC proliferation; indeed, it actually promoted proliferation of both DPCs and ORSCs. When DPCs were first activated by exposure to FGF-1 and FGF-2, which are expressed in hair follicles during anagen, ORSC proliferation observed in the resultant conditioned medium was substantially greater than in medium conditioned by unstimulated DPCs. The FGF-1-induced enhancement was reversed by FGF-5, diminishing ORSC proliferation to control levels. By contrast, the enhancement of DPC-mediated ORSC proliferation by FGF-2 was not suppressed by FGF-5. Proliferation of ORSCs did not depend on DPC proliferation, nor did FGF-1 directly promote ORSC proliferation. Dermal papillae thus appear to require activation before they will efficiently stimulate hair growth, and FGF-5 appears to inhibit hair growth and induce catagen by blocking that activation.

Introduction The activating receptor, NKG2D, plays crucial role in natural killer (NK) cell-mediated effector function. NKG2D is involved not only in host defense against tumor and viral infection, but also in autoimmune diseases by recognizing stress-induced self-ligands (NKG2DLs). However, the negative feedback regulation of NKG2D has not been fully understood. It has been reported that NK cells undergo rapid apoptosis upon interaction with NK-sensitive tumor cells, suggesting that activation-induced NK cell death can be triggered in certain situations. In this study, we aimed to elucidate underlying mechanism of NK cell death, especially focused on NKG2D-NKG2DLs interaction. Methods NK cells were purified from splenocytes of C57BL/6, perforin-/-, and DAP10-/-/12-/- mice, and cultured with rhIL-2 (1000 U/ml) for 5 days. We used these IL-2-activated NK cells as effector cells and three target cell lines: mouse T cell lymphoma RMA cells (RMA), RMA lacking MHCI expression (RMA-S), and RMA stably expressing an NKG2DL, Rae-1δ (RMA/Rae-1δ). CFSE-labeled NK cells were co-cultured with target cells for 2 hours, and stained with anti-NK1.1 mAb propidium iodide (PI). The percentage of PI-positive cells within CFSE+ NK1.1+ population was measured by flowcytometry. The cell surface expression of Rae-1 on NK cells after co-culture with target cells was evaluated by flowcytometry and confocal microscopy. Results NK cells from WT mice rapidly underwent cell death when co-cultured with Rae-1δ, but not with RMA or RMA-S, suggesting that NKG2D-Rae-1 interaction is involved in NK cell death. NK cells from perforin-/-, and DAP10-/-/12-/- mice did not undergo cell death, highlighting the importance of the NKG2D pathway for NK cell death. However, cross-linking of NKG2D receptor failed to induce NK cell death. To understand underlying the mechanism of NK cell death, we evaluated the cell surface expression of NKG2DLs on NK cells after co-culture with tumor cells. We found that cell surface expression of Rae-1 on NK cells was remarkably induced after co-culture with RMA/Rae-1δ, but no with RMA or RMA-S, implying that these Rae-1-positive NK cells may be lysed by NK cells through NKG2D-induced perforin pathway. The cell surface induction of Rae-1 on NK cells was very rapid (within 5min), and it occurred cell-cell contact dependent manner. Interestingly, NK cells from C57/BL6 mice rapidly became BALB/c allele Rae-1γ-positive after co-culture with RMA/Rae-1γ, indicating that NK cells acquire tumor-derived Rae-1. Consistently, acquisition of Rae-1 by NK cells was confirmed by confocal microscopy. Therefore, NK cells rapidly dress tumor-derived Rae-1 after interaction with tumor cells through intercellular membrane transfer, namely trogocytosis. Trogocytosis of Rae-1 was significantly inhibited in NK cells from DAP10-/-/12-/- mice and by chemical inhibitors of PI3K and Syk, indicating that it requires NKG2D-signaling. To confirm whether Rae-1-dressed NK cells can be recognized and lysed by other NK cells, we used sort-purified Rae-1-dressed NK cells as target cells. Rae-1-dressed NK cells were lysed by WT NK cell in an E/T-ratio dependent manner through NKG2D-induced perforin pathway. Furthermore, adoptively transferred Rae-1-dressed NK cells were significantly eliminated in Rag-1-deficient mice, indicating that Rae-1-dressed NK cells are also recognized and lysed in vivo. Conclusion Upon interaction with NKG2DLs-expressing tumor cells NK cells rapidly dress tumor-derived NKG2DLs, and subsequently undergo fratricide. Our results provide novel insights into activation-induced NK cell death via dynamic intercellular communications. Disclosures: No relevant conflicts of interest to declare.

The intercellular transfer of plasma membrane patches, also known as trogocytosis, has a strong impact on the function and fate of immune cells. We have recently shown that natural killer (NK) cells undergo fratricide following the trogocytosis-mediated acquisition of tumor-derived NKG2D ligands. Malignant cells may therefore employ trogocytosis to escape NKG2D-mediated immune responses.