Abstracts for the 31st annual meeting of the Japanese Society for Investigative Dermatology, May 31 - June 2 2006, Kyoto, Japan

Abstracts for the 31st annual meeting of the Japanese Society for Investigative Dermatology, May 31 - June 2 2006, Kyoto, Japan

Journal of Dermatological Science (2006) 42, 133—198 www.intl.elsevierhealth.com/journals/jods ABSTRACTS Abstracts for the 31st annual meeting of t...

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Journal of Dermatological Science (2006) 42, 133—198

www.intl.elsevierhealth.com/journals/jods

ABSTRACTS

Abstracts for the 31st annual meeting of the Japanese Society for Investigative Dermatology, May 31—June 2 2006, Kyoto, Japan

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Keratinocyte specific abrasion of TAK1 induces abnormal differentiation and apoptosis of epidermis

Role of S1P on plasma cell homing from spleen to bone marrow

Koji Sayama1, Yasushi Hanakawa1, Yuji Shirakata1, Satoshi Hirakawa1, Sho Tokumaru1, Mikiko Tohyama1, Hiroshi Nagai1, Xiuju Dai1, Shizuo Akira2, Koji Hashimoto1 1

Department of Dermatology, Ehime University School of Medicine; 2Research Institute for Microbial Disease, Osaka University Transforming growth factor-b-activated kinase 1 (TAK1) is a member of the MAP kinase family and regulates NF-kB signaling. In addition to TGF-b, TAK1 is involved in IL-1 and TNF-a-induced NF-kB activation. However, TAK1 function has not been studied in keratinocytes. We generated keratinocyte specific TAK1-KO mice by breeding TAK1 flox/flox mice with K5-cre mice. KO mice were macroscopically indistinguishable from their littermates until postnatal day 2-3 (P2-P3), when the skin started becoming rough and wrinkled. This phenotype progressed and the transepidermal water loss increased, and KO mice subsequently dead by P8. Histological analysis showed thickening of epidermis with hyperkeratosis and parakeratosis without granular layer. The thickened epidermis contains focus of keratinocyte apoptosis and intra-epidermal abscess. The supra-basal keratinocytes of KO mice expressed K5 and K14, which are normally confined to the basal layer. Expression of K10 and loricin were absent on the viable epidermal keratinocytes of KO mice. On the other hand, the epidermis of KO mice showed the expression of K16. Staining of Ki67 showed increased proliferation of keratinocytes in the epidermis of KO mice. Furthermore, Ki67 positive keratinocytes were present at the supra-basal layer of the epidermis. Expression of p50 and p65 on the basal layer of epidermis in KO mice decreased indicating that NF-kB signaling was impaired. Keratinocytes from TAK1 flox/flox mice were cultured and transfected with adenovirus vector carrying cre-recombinase, which resulted in keratinocyte apoptosis. In conclusion, TAK1 is essential in the regulation of keratinocyte differentiation and apoptosis.

0923-1811/$30.00 doi:10.1016/j.jdermsci.2006.03.002

Kenji Kabashima1, Jason Cyster2, Yoshiki Tokura1 1 The Department of Dermatology, University of Environmental and Occupational Health; 2University of California, San Francisco, Department of Microbiology and Immunology, San Francisco, USA

Autoimmune skin diseases, such as pemphigus vulgaris, are induced by autoreactive antibody produced by plasma cells. Plasma cells are developed from B cells usually in the germinal center of secondary lymphoid organs like spleen and lymph nodes. Most of plasma cells die in the secondary lymphoid organs shortly, but a fraction of plasma cells home into bone marrow through blood vessels to become long-lived plasma cells producing antibody for a long period. Therefore, the control of plasma cell homing might be a good candidate to control autoimmune skin disease. However, the underlying homing mechanism remains to be elucidated. We first examined that the new immunosuppressant, FTY720, completely inhibited plasma cell egress from spleen to blood vessels after immunization with NP-CGG and alum using ELISPOT assay. Since FTY720 is known to inactivate the effect of sphingosine-1-phosphate (S1P), we examined the effect of FTY720 on plasma cell chemotaxis to S1P. Although wild type plasma cells have good chemotactic activity to S1P by transwell assay, this activity was completely inhibited by FTY720 pretreatment. Then we immunized Blimp-1, a marker of plasma cell, GFP knock-in mice and found that there were two plasma cell subsets, Blimp-1GFPhi+ and GFPint+ in the spleen, but only Blimp-1GFPint+ subset in the blood. Moreover, this Blimp1GFPint+ subset showed that the mRNA level of CXCR4, the ligand of which was SDF-1 enriched in the spleen, was low. And this Blimp-1GFPint+ subset has better chemotactic activity to S1P than the other subset. These data suggest that Blimp-1GFPint+ CXCR4lo+ plasma cell subset can egress from secondary lymphoid tissue into blood, and home to bone marrow depending on the balance of responsiveness to chemoattractants made in separate zones.

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were located downstream of Wnt/b-catenin and/or cAMP/PKA pathways. TOPflash assays showed NF1-knockdown increased Wnt/b-catenin signaling in NF1-knockdown melanocytes. PKA inhibitors further augmented TOPflash reporter activity in NF1knockdown cells, suggesting that Wnt/b-catenin signaling is activated by PKA suppression. Furthermore, b-catenin modulated genes including a transcription factor ITF-2 and cyclin D1 were identified as downstream targets of the up-regulated Wnt/bcatenin signaling in melanocytes. These results suggest that a number of signals that regulate cell proliferation and differentiation as well as melanogenesis might be modified through the loss of non-Ras neurofibromin function in human melanocytes.

Implanted hair follicle stem cells form Schwann cells which support repair of severed peripheral nerves and the spinal cord Yasuyuki Amoh1,2,3, Lingna Li1, Katsumasa Kawahara4, Yuko Hamada2, Kensei Katsuoka2, Robert M. Hoffman1,3 AntiCancer Incorporated, San Diego, USA; 2Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Japan; 3Department of Surgery, University of California, San Diego, CA 92103; 4Department of physiology, Kitasato University School of Medicine, Sagamihara, Japan 1

The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, is also expressed in follicle stem cells as well as their immediate differentiated progeny. The fluorescent protein, GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin driven-GFP stem cells from the transgenic mice are positive for CD34 but negative for keratinocyte marker, keratin 15, suggesting their relatively undifferentiated state. The hair follicle stem cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Equivalent hair follicle stem cells derived from bactin-driven-GFP transgenic mouse, implanted into the gap region of a severed sciatic nerve, greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered indicating the transplanted mice recovered the ability to walk normally. The severed spinal cord partly recovered after injection of hair follicle stem cells. The cord rejoined and the mice partially regained the ability to utilize their hind legs enabling them to walk. These results suggest that hair follicle stem cells provide an important accessible, autologous source of adult stem cells for regenerative medicine. 004 Non-RAS function of neurofibromin regulates the cyclic AMP/ beta-catenin signaling pathway in human melanocytes Minao Furumura, Kunie Kohno, Hiroaki Matsuo, Eishin Morita Department of Dermatology, Faculty of Medicine, Shimane University, Izumo, Japan Neurofibromatosis type 1 (NF1) is a dominant genetic disorder characterized by cafe-au-lait macules and neurofibromatosis. Neurofibromin, the gene product of NF1, has the ability to function as a Ras-GAP. NF1 skin lesions are thought to be induced by increased Ras activity. Several recent studies have demonstrated a novel non-Ras function for neurofibromin, one which can potentiate cAMP signaling by regulating adenylyl cyclase activity. In melanocytes, the cAMP pathway functions as for critical signaling to regulate melanogenesis. However, investigation of a nonRas function for neurofibromin in melanocytes has not been reported. In this study, we investigated the modification of cAMP signaling and its downstream events in NF1-knockdown human melanocytes using siRNAs. cAMP levels were significantly reduced in NF1-knockdown cells. We performed oligo microarrays to ascertain differentially expressed genes and to detect modulated signaling pathways in the NF1-knockdown cells. A number of differentially expressed genes of NF1-knockdown melanocytes

005 Alopecia areata model mouse induced by depletion of regulatory T cells Taisuke Ito, Masahiro Takigawa, Naohiro Seo Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan It has been reported that autoimmune diseases are associated with decreasing of cell number, deficiency of regulatory T cells (Tregs) and a genetic mutation in the foxp3, which is Tregmaster regulation gene. In the alopecia areata-model mouse, C3H/HeJ, the disease onset is inhibited by injection of Tregs, and the number of infiltrated Tregs is decreased in/around hair follicles in anti-CD44v10 antibody-induced hair loss. Therefore, Tregs may play an important role in alopecia areata. In the process of generating antibody against human Tregs in female BALB/c mice (6—8 weeks), the mice also produced antibody against murine Tregs, and all mice showed hair loss on the front head. We used human cDNA of CD4+CD25+ Treg to amplify cDNA of CD220 by conventional PCR technique with gene-specific primers. The PCR product was then ligated into pRc/CMV plasmid at the site between the restriction endonucleases NotI and XbaI, following transformed into bacteria DH5a. The plasmid was then injected intradermally in BALB/c mice. After that, the mice produced antibody against human/murine Tregs, and the number of spleen Tregs was decreased. CD4+ and CD8+ cells immediately proliferated after stimulation with CD3 in vitro. The alopecia lesion of CD225 immunized BALB/c mice showed exceeding infiltration of CD4+, CD8+ and CD56+ cells. In addition, IFN-g, TGF-b and IL-4 were strongly expressed in and around hair follicles compared with control mice. In conclusion, one of the pathogenesis for alopecia areata may be the collapse of immune privilege by the decreasing of Tregs and production of cytokines. 006 Targeted overexpression of VEGF-A or -C in the skin promotes lymph node metastasis and lymphangiogenesis Satoshi Hirakawa1,2, Shohta Kodama3, Kari Alitalo4, Koji Hashimoto1, Michael Detmar2,5 1

Department of Dermatology, Ehime University School of Medicine, Toon, Japan; 2Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA; 3Department of Immunobiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA; 4Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; 5Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland

Abstracts The metastatic spread of tumor cells to sentinel lymph nodes (LN) via lymphatic vessels represents the first step of tumor progression in the majority of human cancers. However, the mechanisms that mediate lymphatic metastasis to sentinel lymph nodes and further spread beyond remain unclear. We found that vascular endothelial growth factor (VEGF)-A and -C potently promote proliferation and migration of human dermal lymphatic endothelial cells in vitro. To directly investigate the importance of VEGF-A and -C in mediating metastasis via the lymphatic system, we generated transgenic mice that express either VEGF-A or VEGF-C, as well as green fluorescent protein, under control of the keratin 14 promoter. These mice were subjected to a standard chemically-induced skin carcinogenesis regimen. VEGF-A transgenic mice, but not VEGF-C transgenic mice, showed strongly enhanced tumor angiogenesis and accelerated and increased development of benign papillomas and of squamous cell carcinomas (SCC), as compared with wild-type controls. However, both VEGF-A and VEGF-C transgenic mice showed strikingly enhanced tumor lymphangiogenesis and sentinel LN metastasis. Importantly, the percentage of sentinel LN metastases that further spread to distant LN was significantly higher in VEGF-A and -C transgenic mice than in wild-type mice. VEGF-A or -C-overexpressing tumors continued to induce lymphatic vessel growth within metastatic LN. Surprisingly, VEGF-A or VEGF-C overexpressing primary tumors induced sentinel LN lymphangiogenesis even before metastasizing. This newly identified mechanism of intranodal lymphangiogenesis likely contributes to lymphatic metastasis and represents a new therapeutic target for the prevention or treatment of cancer metastasis.

007 Hair follicle stem cell-targeted gene transfer and reconstitution system Yoriko Sugiyama-Nakagiri1,2, Masashi Akiyama1, Hiroshi Shimizu1 1 Department of Dermatology Hokkaido University Graduate School of Medicine; 2Kao Corporation Biological Science Laboratories

Gene transfer to hair follicle (HF) epithelium is an attractive approach for not only treating skin diseases, but also many systemic disorders. In order to obtain stable and persistent transgene expression and maximize the beneficial transgene therapeutic effects, the stem cell population which locate in the bulge region of HF should preferentially be targeted. In this study, we attempted to develop a gene transfer system for HF epithelial stem cells. At first, we established a HF stem cell-rich culture system using cells from rat vibrissa bulge cells. In vitro, primary cultures of bulge derived cells display self-renewal characteristics of stem cells even if they are removed from their native niche and induced to proliferate in culture. For persistent and stable transgene expression in HF stem cells, we transferred retroviral vectors encoding reporter genes into cultured HF stem cells. Subsequently, we regenerated an entire HF epithelium and interfollicular epidermis engrafting cultured bulge derived cells mixed with cultured dermal papilla cells into a silicone chamber implanted on to the backs of immunodeficient mice. Four weeks after transplantation, the grafts exhibited tufts of hairs as well as complete interfollicular epidermis. The transgene expression was observed in all skin epithelial compartments including the HF epithelium, sebaceous gland and epidermis. In addition, transgene expression was observed for at least 6 months. We concluded that this hair follicle stem cell-targeted gene transfer and reconstitution system may enable the stable expression of transgene in any part of the cutaneous epithelium and provide long-

135 standing therapeutic benefits if used for gene therapy and gene-function analysis. 008 Virus replication begins in Langerhans cells during transmission of HIV-1 from skin emigrants to T cells Tatsuyoshi Kawamura1, Yuumi Nakamura1, Youichi Ogawa1, Yoshio Koyanagi2, Blauvelt Andrew3 1

Department of Dermatology, University of Yamanashi, Yamanashi, Japan; 2Laboratory of Viral Pathogenesis, Research Center for AIDS, Institute for Virus Research, Kyoto University, Kyoto, Japan; 3Department of Dermatology, Oregon Health & Science University, Portland, OR, USA Mucosal Langerhans cells (LC) or sub-mucosal dendritic cells (DC) are suspected to be the initial cells that support HIV replication following sexual exposure to virus. Recently, novel C-type lectins, langerin and DC-SIGN, expressed on LC and DC, respectively, have been shown to bind HIV. Therefore, we studied relevant host molecular targets utilized for acquisition of HIV infection using a skin explant model. HIVBaL was applied to the abraded epidermal surface of skin explants. After 3 days, emigrant cells from skin explants were co-cultured with allogeneic T cells. Interestingly, pre-incubation of skin explants with anti-CD4 mAb or PSC-RANTES completely blocked subsequent HIV transmission from emigrant cells to T cells, whereas anti-langerin mAb, anti-DC-SIGN mAb or mannan did not. Bead depletion studies demonstrated that LC accounted for more than 95% of the HIV-1 dissemination in this system. When infection levels of single LC or DC within emigrant cells were determined by flow cytometry, HIV p24+ cells were detected only in LC, and not in DC. To further test whether HIV could replicate within LC, skin explants were exposed to HIV variants that were engineered to express GFP during productive infection of cells. Strikingly, GFP+ emigrated cells were detected only in LC, when a CCR5-using HIV (NLCSFV3EGFP), but not a CXCR4-using HIV (NL43EGFP), was applied to skin. When the emigrant cells were co-cultured with T cells, EGFP+ T cells were found neighboring to EGFP+ LC, suggesting the infection-dependent HIV transmission from LC to T cells. Thus, CCR5-mediated productive HIV infection of LC, and not C-type lectin-mediated capture of virus by DC, may provide a biologic basis for understanding susceptibility to initial infection by CCR5-using strains of HIV in humans. 009 CCL27 transgenic mice showed enhanced contact hypersensitivity reaction to fluorescein isothiocyanate Shinji Kagami1, Hidehisa Saeki1, Yuichiro Tsunemi1, Koichiro Nakamura2, Takashi Nakayama3, Osamu Yoshie3, Kunihiko Tamaki1 1

Department of Dermatology, University of Tokyo, Tokyo, Japan; Department of Dermatology, Fukushima Medical University School of Medicine, Fukushima, Japan; 3Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Japan 2

Cutaneous T cell-attracting chemokine (CTACK)/CC chemokine ligand 27 (CCL27) is one of the CC chemokines produced by keratinocytes and suggested to be involved in the pathogenesis of inflammatory skin diseases such as atopic dermatitis and drug eruption. To study the effect of CCL27 produced by keratinocytes during inflammation, we created transgenic mice which overexpressed this chemokine in keratinocytes. We assayed its contact hypersensitivity to oxazolone and fluorescein isothiocyanate.

136 Contact hypersensitivity reaction by repeated challenges with fluorescein isothiocyanate was enhanced in the transgenic mice. Under this condition, the numbers of inflammatory cells, mast cells and CCR10-positive cells were increased in the lesional skin of the transgenic mice. Higher levels of serum IgE were also observed in the transgenic mice and IL-4 mRNA expression was higher and that of IFN-g was lower in the lesional skin. CCL27 modified the inflammation caused by sensitizing reagents by attracting CCR10-positive cells such as plasma cells and CLApositive memory T cells into the lesional skin. Atopic dermatitislike conditions of increased mast cells and serum IgE were observed. Thus, CCL27 may participate in the pathogenesis of skin diseases such as atopic dermatitis by regulating allergic inflammation. 010 Anti-human connective tissue growth factor neutralizing antibody ameliorates transforming growth factor-b-induced mouse fibrosis model Yuka Ikawa1, Miki Kondo1, Sonoko Chujo1, Fumiaki Shirasaki1, Kazuhiko Takehara1, Poh-Sing Ng2 1

Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Nosan Corporation Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteinerich peptide that comprises 4 discrete structural modules and induced in fibroblasts after activation with transforming growth factor-b (TGF-b). To better understand the mechanisms of persistent fibrosis seen in SSc, we have established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF-b transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-b caused persistent fibrosis. From this result, we concluded CTGF plays an important role in the development of skin fibrosis. Since systemic inhibition of TGF-b raises critical safety issues because of the pleiotropic physiological effects of this factor, CTGF may be a potential therapeutic target against skin fibrosis. Therefore, we have investigated whether anti-CTGF neutralizing antibody ameliorates skin fibrosis in our animal model. As the first step, we developed an antibody against the native structure of human CTGF using DNA immunization methods, and succeeded in isolating seven monoclonal antibodies recognizing the native conformation of human CTGF. Then, to examine the anti-fibrosing effects of these antibodies, newborn BALB/c mice received subcutaneous injections of TGF-b for 3 days with either anti-CTGF neutralizing antibodies or control purified immunoglobulin. These antibodies significantly reduced skin fibrosis and collagen contents. The result suggests that anti-CTGF neutralizing antibodies are a feasible strategy to treat skin fibrotic diseases for which there is no specific therapy. 011 Spatial and temporal relevance of class I versus class II presentation of skin-derived soluble antigen in draining lymph nodes Nobuo Kanazawa1,2,3, Hiroaki Azukizawa2,4, Michael Sixt5, Ichiro Katayama4, Fukumi Furukawa1, Yoshiki Miyachi3, Manfred Lutz2 1

Department of Dermatology, Wakayama Medical College, Wakayama, Japan; 2Department of Dermatology, University of Erlangen, Erlangen, Germany; 3Department of Dermatology,

Abstracts Graduate School of Medicine, Kyoto University, Kyoto, Japan; Department of Dermatology, Course of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan; 5 Department of Molecular Medicine, Max Planck Institute of Biochemistry, Martinsried, Germany 4

Dendritic cells (DCs) is responsible for transfer of antigenic information from periphery to draining lymph nodes (dLNs) at the beginning of antigen-specific T cell response. In the steady state, immature DCs are resident in the periphery while both resident immature and continuously-migrating semi-mature DCs are present in dLNs. There exist two ways of antigen delivery from periphery to dLNs; rapid direct flow through lymphatics and DC migration-dependent transfer which need time. Immediately after subcutaneous injection of FITC-conjugated 40 kd OVA protein, immature DCs become more strongly positive for FITC than semi-mature DCs in dLNs. Histologically, injected FITC-OVA reveals the presence of reticular meshwork as a conduit within 1 h and is then captured by CD11b+ DCs. Using DQ-OVA, which become fluorescent after digestion, these DCs are proved to process OVA. Newly-migrated FITChigh DCs appear apart from reticular meshwork 20 h after FITC-OVA administration. CD69 expression on adoptively transferred OVA-specific CD4 T cells starts to be upregulated in dLNs within 4 h after subcutaneous OVA injection. However, by mixed leukocyte reaction using OVA-specific T cell line cells, class IIrestricted OVA presentation by purified CD11c+ DCs in dLNs is not apparent within 4 h compared with that after 20 h (equivalent to 1nM peptide presentation). Interestingly, class Irestricted OVA presentation reaches maximum 1.5 h after OVA injection (equivalent to 0.1 nM peptide presentation) and is downregulated with time. These results suggest that class I presentation is temporally performed by dLN-resident DCs immediately after antigen capture but not by newlymigrated DCs, and that class II presentation needs time either by resident or newly-migrated DCs. 012 Role of Stat3 activation in cutaneous squamous cell carcinoma cell lines Naoko Sumita1, Toshinori Bito1, Koichi Nakajima2, Chikako Nishigori1 1

Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan; 2 Department of Immunology, Osaka City University Graduate School of Medicine, Osaka, Japan Signal transducers and activators of transcription 3 (Stat3) is constitutively activated in various cancers and considered to play important roles in cell growth, survival, differentiation, and transformation. We found that Stat3 is constitutively activated in cutaneous squamous cell carcinoma (SCC) cell lines. To investigate the role of Stat3 in SCC cell lines (HSC-1, 3, and 4), we developed small interfering RNAs (siRNAs) targeting Stat3. siRNAs can be used to silence gene expression in a sequence specific manner without activating nonspecific effects. In this study, a double-stranded siRNA for transient transfection and a vector-based siRNA for stable transfection were used to inhibit Stat3 activation. Transient knockdown of Stat3 did not induce either apoptosis, or reduction of its target genes protein expression (Bcl-2, Bcl-xL and cyclinD1). Transient inhibition of Stat3 did not appear to affect the SCC cells either in proliferating ability or morphologically, whereas stable knockdown of Stat3 induced reduction of its target genes protein expression, the cell growth inhibition and morphology changes. In addition, G0-G1 arrest in cell cycle was observed by flow cytometry. Immuno-

Abstracts cytochemical analysis revealed that the morphological changes were due to the cell differentiation, not apoptosis. Stat3 siRNA transfected SCC cells were not tumorigenic to nude mice. These findings demonstrated that Stat3 plays a crucial role for cell proliferation and tumorigenesis, but not for cell viability in SCC cells. 013 Skin-derived interleukin-7 contributes to the proliferation of lymphocytes in cutaneous T-cell lymphoma Keiichi Yamanaka1,2, Rachael Clark2, Thomas Kupper2, Kenichi Isoda1, Ichirou Kurokawa1, Hitoshi Mizutani1 1

Department of Dermatology, Mie University, Graduate School of Medicine; 2Department of Dermatology, Brigham & Women’s Hospital, Boston, USA Cutaneous T-cell lymphomas (CTCLs) are malignancies of T cells that have a special affinity for the skin. We have previously reported that much of the T cell receptor repertoire is altered in CTCL, and both malignant and non-malignant clones are numerically expanded, presumably in response to T cell trophic cytokines. We therefore examined levels of the T cell trophic cytokines IL-2, IL-4, IL-7, IL-12, IL-13, and IL-15 in plasma in 93 CTCL patients and normal controls. Only IL-7 levels were elevated in CTCL. We next looked at lesional skin from patients with CTCL, and found elevated levels of IL-7 mRNA. Explant cultures of normal and lesional CTCL skin biopsies revealed significantly more IL-7 protein production in CTCL skin. Additionally, cultures of CTCL skin released greater numbers of T cells than normal skin; this was blocked by the addition of an IL-7 neutralizing antibody. Finally, these cultures induced proliferation of normal peripheral skin homing T cells that were added to the cultures. These observations lead us to postulate that IL-7 produced by skin cells contributes to the survival and proliferation of T cells within skin lesions, and is likely the source of elevated circulating IL-7 in CTCL. 014 Engagement of CD47 inhibits the contact hypersensitivity response via the suppression of motility and B7 expression of Langerhans cells Xijun Yu1, Atsushi Fukunaga1, Hiroshi Nagai1, Shuntaro Oniki1, Masamitsu Ichihashi2, Chikako Nishigori1, Tatsuya Horikawa1 1

Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan; 2 Sun Care Institute, Osaka, Japan In the sensitization phase of contact hypersensitivity (CHS), application of haptens on skin stimulates Langerhans cells (LCs) to migrate from the epidermis into the draining lymphoid tissues where they initiate naive T cells by means of MHCantigen complexes. During this migration, LCs undergo maturational changes that involve increases in the expression of cell surface molecules including CD80 and CD86. CD47 is a membrane-associated glycoprotein that suppresses the function of immune cells and that is ubiquitously expressed in many cell types. In this study, we aimed at clarifying the role of CD47 in CHS. We showed that LCs also express CD47, and that ligation of CD47 with its ligand, SHPS-1-Fc fusion protein, in vivo diminishes the development of the CHS response. We further demonstrate that CD47 engagement affects immune functions of LCs. CD47 engagement in vivo significantly inhibits the emigration of LCs from the epidermis into draining lymph nodes following treatment with haptens or TNF-alpha. The emigration of LCs from

137 skin explants into the medium, and the chemotaxis of murine XS52 dendritic cells by MIP-3beta were significantly reduced by treatment with SHPS-1-Fc or an anti-CD47 mAb. Under explant culture system, SHPS-1-Fc treatment suppressed the expression of CD80 and CD86 of LCs. These effects on LCs and CHS response of CD47 ligation were reversed by treatment with a Gi inhibitor, pertussis toxin. These results suggest that the ligation of CD47 inhibits the migration of LC and the expression of B7 costimulatory molecules, which results in inhibition of the contact hypersensitivity response. Drs. Nakayuki Honma (KIRIN Brewery Co., Ltd.) and Takashi Matozaki (Gunma University) contributed to this work. 015 New therapeutic approach targeting T-bet for alopecia areata model C3H/HEJ mice Motonobu Nakamura1, Jun-ichiro Jo2, Yasuhiko Tabata2, Osamu Ishikawa1 1

Department of Dermatology, Gunma University, Maebashi, Japan; 2Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan Alopecia areata is considered to be triggered by a collapse of immune privilege in hair follicles. We previously reported that infiltrating lymphocytes around the hair follicles of alopecia areata were mostly CD4, CCR5 positive with few CD4, CCR4 positive cells. The findings indicate that Th1 cells are dominant in the alopecic lesion. Moreover, local injection of IL-4 was effective for alopecia in C3H/HeJ mice, a mouse model for alopecia areata. Semiquantitative RT-PCR assay demonstrated that there was significantly higher expression of IFN-g in an alopecic skin than in a non-affected skin. Intralesional injection of IL-4 suppressed the enhanced IFN-g expression in the alopecic skin. To elucidate whether or not IFN-g plays an indispensable role in the pathogenesis of alopecia areata, we injected neutralizing antibodies against IFN-g into C3H/HeJ mice. The neutralizing antibodies were significantly more effective for alopecia than control rat IgG. T-bet, a transcription factor expressed in Th1 cells, is important for IFN-g transcription and Th1 differentiation. Antisense T-bet oligonucleotide injections restored the hair elongation in alopecia of C3H/HeJ mice more effectively than a control nonsense oligonucleotide. Furthermore, T-bet siRNA applications conjugated with gelatin showed effects on alopecia in C3H/HeJ mice. Western blotting revealed the diminished T-bet expression after the T-bet siRNA injections. Taken together, new drug delivery system for siRNA conjugated with gelatin can be a novel candidate for promising therapeutic approaches to various human skin diseases and cancers. 016 Kallikrein 8 is involved in skin desquamation through cleaving desmoglein 1 and corneodesmosin Mari Kishibe1, Hidetoshi Takahashi1, Akemi Ishida-Yamamoto1, Shigetaka Yoshida2, Hajime Iizuka1 1 Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan; 2Department of Anatomy I, Asahikawa Medical College, Asahikawa, Japan

The outermost corneocytes are shed from the epidermis surface as a result of proteolytic degradation of corneodesmosomes by epidermal proteases during the desquamation process. It is important to maintain balance between proliferation of

138 epidermal cells and shedding outermost layers. Some proteases of kallikrein family are thought to play important roles for the desquamation. Kallikrein 8 (KLK8/neuropsin), kallikrein type serine protease, has been implicated in the proliferation and the differentiation of epidermal keratinocyte and in the pathogenesis of psoriasis. However, its underlying mechanism remains largely unknown. Here we show that KLK8 is also involved in desquamation. We investigated wild type mice (WT) and KLK gene-disrupted (KLK8/) mice skin before and after TPA application, which induced keratinocyte proliferation to know the function of KLK8. KLK8 mRNA was upregulated along with KLK6 and KLK7 mRNA after TPA application in WT mice. KLK8/mice showed minimum increase of KLK6 and KLK7 transcripts, the proteins and enzymatic activities. These results indicate that KLK8 mRNA is a necessary factor to induce KLK6 and KLK7 mRNA. In addition, KLK8 may be involved in upper stream of activation cascade and regulate other KLK activities. The KLK8/ skin showed more increased number of cell layers of stratum corneum than the WT epidermis. This hyperkeratosis may be based on delay of corneocyte shedding because desmoglein 1 and corneodesmosin were cleaved much less than in WT. We propose that KLK8 induces KLK6 and KLK7, and is involved in desquamation through cleaving desmoglein 1 and corneodesmosin.

017 Focused proteomics of murine epidermis based on quantitative glycome analysis Rie Uematsu1,2, Jun-ichi Furukawa1, Hiroaki Nakagawa1, Yasuro Shinohara1, Kisaburo Deguchi 1, Kenji Monde1, Shin-Ichiro Nishimura1 1

Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan; 2Basic Research Laboratory, Kanebo Cosmetics Inc., Odawara, Japan Analysis of protein glycosylations presents formidable challenges but their determination would generate indispensable insight into biological function. Our interests are to elucidate the quantitative glycomic profiles of murine epidermis and dermis and to unveil their biological significances. A novel glycomic approach for focused proteomics was proposed and evaluated for this purpose. Gross N-glycan profiling of murine epidermis and dermis were elucidated both qualitatively and quantitatively by novel stable isotope coded glycan-selective derivatization reagents (aoWR (H) and aoWR (D)). Glycoproteins carrying unique oligosaccharides were then selectively analyzed following direct tryptic digestion of protein mixtures, affinity enrichment and off-line LC-MALDI-TOF/TOF. The novel derivatization reagents allowed a rapid and sensitive N-glycan profiling on MALDI-TOF in a quantitative manner both intraand inter-samples. The glycomic analysis revealed distinct features of N-glycosylation profile of epidermis and dermis. A feature worthy of mention is that the murine epidermis glycoproteins are characteristic in their high abundance of high-mannose type oligosaccharides. Based on this observation, we performed focused proteomics that carry high-mannose type glycans. We identified 15 glycoproteins with 19 Nglycosylation sites by off-line LC-MALDI-TOF/TOF mass spectrometry. Moreover, the relative quantitation of microheterogeneity of different glycoforms present at each N-glycan binding site was determined. Glycoproteins identified were mainly content of lysosomes, lamellar granules and desmosomes, thus indicating N-glycosylation of tissue-specific glycoproteins may contribute to increase the relative proportion of high-mannose glycans.

Abstracts 018 Elimination of epiplakin by gene targeting results in acceleration of keratinocyte migration in mice Mizuki Goto1, Hideaki Sumiyoshi2, Takao Sakai3, Reinhard Fa ¨ssler3, Shihoka Ohashi4, Eijiro Adacih4, Hidekatsu Yoshioka2, Sakuhei Fujiwara1 1

The Department of Anatomy, Biology and Medicine (Dermatology), Faculty of Medicine, Oita University, Yufu, Japan; 2The Department of Anatomy, Biology and Medicine (Biochemistry), Faculty of Medicine, Oita University, Yufu, Japan; 3The Department of Molecular Medicine, Max-Planck Institute for Biochemistry, Martinsried, Germany; 4The Department of Molecular Morphology, Kitasato University Graduate School of Medicine, Sagamihara, Japan Epiplakin (EPPK) was originally identified as a 552-kDa human epidermal autoantigen that is expressed in various organs including the epidermis, esophagus, hair follicles, and salivary glands. Epiplakin belongs to the plakin family but it has the following unusual features. Epiplakin has repeated domains that are homologous to the B domain, which is one of the plakin repeat domains (PRDs). Epiplakin lacks the coiled-coil rod domain and the aminoterminal domain that are found in all other known members of the plakin family. It seems likely that the carboxy-terminal regions of plakin family members, including PRDs and linker regions, bind to intermediate filaments. EPPK has only one kind of PRD, a B domain, and connecting linker domains, therefore they seem to bundle the intermediate filaments. To identify the function of epiplakin, we generated epiplakin knock-out mice. These mice developed normally, with apparently normal epidermis and hair. Even electron microscopy revealed no differences between wild and EPPK/ mice. Wounds on backs of EPPK/ mice closed more rapidly than those of wild-type mice. At wound edges keratin 6, 16, 17 are expressed in spinous layer of epithelium although keratin 1, 10 are expressed in normal epithelium. In EPPK/ mice, keratinocytes in wounds weakly expressed keratin 6. Outgrowth of keratinocytes from skin explants from knockout mice was enhanced as compared to outgrowth from explants from wild-type mice even in the presence of mitomycin C, suggesting that the difference in keratinocyte outgrowth might be due to a difference in the speed of migration of keratinocytes. These data indicated that epiplakin might regulate the expression and the function of keratin 6 in the wound epidermis and might control the migration of keratinocytes. 019 Effects of calcium concentration on development of tight junction in normal human keratinocytes Takuo Yuki1, Akinori Haratake1, Hisa Koishikawa1, Kazumasa Morita2, Yoshiki Miyachi2, Sintaro Inoue1 1 Basic Research Laboratory, Kanebo Cosmetics Inc., Odawara-shi, Kanagawa, Japan; 2Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan

The tight junction (TJ) is a well-developed intercellular adhesion apparatus found in simple epithelial cells and endothelial cells that is crucial for the barrier function of mammalian skin [1]. In this study, we examined the effects of extracellular calcium (Ca2+) level on the in vitro development of TJ in normal human keratinocytes (NHK). Confluent NHK were transferred to a medium containing either low (0.1 mM) or high (1.8 mM) external Ca2+ concentrations. The permeability barrier function assessed by transepithelial electric resistance and paracellular tracer flux was developed and reached a maximum at 96 h after transferring

Abstracts to the high Ca2+ medium, but did not develop in the low Ca2+. In fact, under the high Ca2+ conditions, typical TJ structures interspersed between desmosomes were confirmed by an electron microscopy. However, occludin and claudin-1 proteins increased time-dependently in a Ca2+-independent manner. Further, immunofluorescence analysis revealed that the NHK in high Ca2+ medium had fully matured intercellular junctions, in which occludin and claudin-1 were aligned in a linear fashion. In contrast, junction maturation scarcely occurred in low Ca2+ medium, with occludin and claudin-1 detected in cell-cell contact sites as distinct rows of dots. We deduced that dysfunction of the permeability barrier in the low Ca2+ medium is due to the lack of maturation of TJ components. Our results indicate that NHK have the ability to produce TJs and establish a permeability barrier. Furthermore, assembly of TJ proteins into functional units is required for effective permeability barrier development.

Reference [1] Furuse M. J Cell Biol 2002;156:1099—111. 020 Role of LIM kinases in normal and psoriatic human epidermis Masaru Honma1,2, S.A. Benitah2, Hajime Iizuka1, F.M. Watt2 1

Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan; 2Keratinocyte Laboratory, Cancer Research UK, London, UK We show that LIM kinases control cell adhesion, compaction and proliferation in human epidermis. LIMK2 is expressed in the epidermal basal layer and signals downstream of the GTPase Rac1 to promote extracellular matrix adhesion and inhibit terminal differentiation. Conversely, LIMK1 is expressed in the upper granular layers and induces cell compaction by phosphorylating and inhibiting cofilin. Expression of LIMK1 is lost in psoriatic lesions and other skin disorders characterized by lack of cell compaction in the differentiating cell layers. In psoriatic lesions downregulation of LIMK1 correlates with upregulation of Myc. Expression of constitutively active cofilin or Myc in reconstituted human epidermis blocks cell compaction. Overexpression of LIMK1 leads to downregulation of Myc, whereas inhibition of Rho kinase, an upstream activator of LIMK1, stimulates Myc expression. Inhibition of Myc by LIMK1 is via inhibition of Stat3 phosphorylation, since constitutively active cofilin or inhibition of Rho kinase results in Stat3 phosphorylation and increased Myc levels, whereas dominant negative Stat3 abolishes the effect. In conclusion, we have uncovered a novel antagonistic relationship between the LIMK1/phosphocofilin and Myc/Stat3 pathways in the differentiating layers of human epidermis and provided evidence that downregulation of LIMK1 contributes to one of the hallmarks of psoriatic epidermal lesions. 021 Nanostructure of stratum corneum intercellular lipid layers in living transglutaminase 1 knock out mice Tatsuya Tsuda1, Noboru Ohta2, Naomi Kunizawa3, Tetsuji Hirao3, Masato Matsuki1, Hitoshi Mizutani4,5, Kiyofumi Yamanishi1,5, Naoto Yagi2, Ichiro Hatta6 1 Department of Dermatology, Hyogo College of Medicine, Nishinomiya, Japan; 2Japan Synchrotron Radiation Research Institute, Kouto, Japan; 3Shiseido Life Science Center, Japan; 4Mie Uni-

139 versity Graduate School of Medicine, Tsu, Japan; 5CREST, JST, Japan; 6Fukui University of Technology, Fukui, Japan Stratum corneum lipids composed of ceramides, fatty acids, and cholesterols are important for maintenance of skin barrier. Those lipids have been imagined to conform regularly arranged stable lamellae in the intercellular spaces of corneocytes according to their polarities, but the model is based on X-ray diffractions of isolated stratum corneum or of reconstituted membrane models. Therefore, the crystal structure of the stratum corneum intercellular lipids in living skin has not been elucidated. In the present study, we first analyzed the structure of the intercellular lipids in dorsal skins of hairless mice in vivo using 5 mm microbeam X-rays, the most powerful synchrotron radiation produced by SPring-8. As the results, X-ray diffraction patterns corresponding to the 13 nm thick layered structures were observed. In contrast, the characteristic X-ray diffraction patterns were hardly detected in transglutaminase 1 knockout mice (TGM1 KO) with severe skin barrier impairment. These results suggest that the stratum corneum intercellular lipids of normal mice are organized as the 13 nm thick layered structures in vivo and those are immature or their ordered arrangement is almost disrupted in TGM1 KO. This advanced technology enables nano-scale analysis of living skin leading a new paradigm, ‘‘nano-dermatology’’. 022 Analysis of ceramide knockout mice III: Causative relationship between disrupted epidermal barrier and inflammation in the dermis Shigetoshi Sano1, Satoshi Itami1, Fumiko Sakamoto2, Masaaki Ito2, Junji Takeda3, Ichiro Katayama1 1 Department of Dermatology, Course of Molecular Medicine, Graduate School of Medicine, Osaka University, Suita, Japan; 2 Department of Dermatology, Course for Molecular and Cellular Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; 3Department of Social and Environmental Medicine, Graduate School of Medicine, Suita, Japan

We have previously reported the generation of keratinocytespecific ceramide knockout mice (referred to as K5. lcb2/), whose epidermis was devoid of serine palmitoyl-CoA transferase (SPT), the key enzyme for the de novo synthesis of ceramides. These mice exhibited a reduction in water retention of the stratum corneum (SC), and ultrastructural study revealed an aberrant alignment of lamella structure in lamellar bodies (LBs). Although the epidermal permeability barrier was normal at birth, recovery from the tape stripping-induced barrier disruption was significantly delayed in K5. lcb2/ mice compared with controls. Delay in repair of their barrier corresponded to retarded secretion of LBs to intercellular space in SC. From two weeks of age, K5. lcb2/ mice exhibited dysfunction of permeability barrier, which became worse as age with a decrease in number of LBs. At the same time, they developed histological abnormalities including hyperkeratosis, acanthosis, follicular keratosis, follicular cysts, and finally alopecia. Simultaneously, K5. lcb2/ mice developed inflammatory reaction in the dermis, including cell infiltrates and vascular proliferation. Taken together, the biosynthesis of ceramides in epidermal keratinocytes is essential for structural integrity of LBs to maintain and repair of permeability barrier upon acute disruption. Furthermore, the primary defect in barrier function of SC resulted in dermatitis, suggesting the causative relationship in inflammatory dermatoses such as atopic dermatitis between disrupted skin barrier and inflammation.

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Abstracts

023

nocytes, BMP-2 suppressed cell density-dependent Wnt-4 induction. The transcriptional activity of T-cell factor/lymphoid enhancing factor (TCF/LEF), which is a target of the canonical Wnt pathway, was upregulated by BMP-2 in both time- and dosedependent manners. However, BMP-2-dependent differentiation of keratinocytes suppressed TCF/LEF transcriptional activity. Conclusion: These results suggest that BMP-2 modulates the expression of molecules involved in Wnt signaling, and activates the canonical Wnt pathway in normal human keratinocytes. Moreover, Wnt signaling may be influenced by the fate of keratinocytes, such as proliferation, migration, and differentiation.

The roles of G2A in human keratinocytes as a receptor for oxidized free fatty acids Tomoyasu Hattori1,2, Hideru Obinata2, Ai Ogawa1,2, Kazuaki Tatei2, Osamu Ishikawa1, Takashi Izumi2 1

The Department of Dermatology, Gunma University Graduate School of Medicine, Gunma, Japan; 2The Department of Molecular Biochemistry, Gunma University Graduate School of Medicine, Gunma, Japan G2A is a G protein-coupled receptor induced by diverse stimuli that cause DNA damage and cellular stress, and oxidized free fatty acids can be produced by many kinds of oxidative stresses. Recently, we reported that G2A is a receptor for oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). As skin is continuously exposed to oxidative stresses such as ultraviolet (UV) irradiation and microorganism infections, G2A is supposed to play some roles in mediating signals of oxidized free fatty acids in skin. We examined the effects of 9-HODE on cultured human keratinocytes and the possible involvement of G2A. G2A mRNA was expressed in normal human epidermal keratinocytes (NHEK) and an immortalized human keratinocyte cell line (HaCaT), and upregulated by UVB irradiation. 9-HODE evoked intracellular calcium mobilization and increased secretion of cytokines such as interleukin (IL)-6, IL-8, and GM-CSF in NHEK cells. These responses were enhanced by overexpression of G2A in HaCaT cells, indicating the involvement of G2A in 9-HODEevoked signalings. Furthermore, 9-HODE induced some morphological changes and inhibited the proliferation of NHEK cells. This growth suppression was mediated by suppression of DNA synthesis and cell cycle arrest in the G0/1-phase, but not by apoptosis. Immunohistochemical analyses demonstrated that G2A was preferentially expressed in the suprabasal layers of epidermis in normal human skin. These results suggest that G2A has biological roles as a receptor for oxidized free fatty acids in skin exposed to oxidative stresses.

025 Effect of adenosine for human hair epithelial cells Yumiko Fujii1, Masato Iino2, Tokuro Iwabuchi2, Ritsuko Ehama2, Yosuke Nakazawa2, Masahiro Tajima2, Hajimu Oura1, Seiji Arase1 1

The University of Tokushima, Department of Dermatology, School of Medicine, Tokushima, Japan; 2Shiseido Research Center, Yokohama, Japan

Lujun Yang, Kenshi Yamasaki, Yuji Shirakata, Xiuju Dai, Sho Tokumaru, Mikiko Tohyama, Yasushi Hanakawa, Koji Sayama, Koji Hashimoto

Purpose: We have already reported that adenosine induced hair growth is partly due to up-regulation of FGF-7 and/or VRGF expression in dermal papilla cells. Because adenosine receptors were also expressed in hair follicle epithelial cells, we studied the effect of adenosine on outer root sheath cells. Methods: Immortalized human outer root sheath cells (ORSC) were incubated with adenosine for 2 h. The mRNA was extracted, and gene expression was analyzed by RT-PCR. Intracellular Ca2+ concentration was assessed by fluorescence spectromicroscope, using the Fura-2/acetoxymethyl ester. Results: RT-PCR analysis showed that VEGF was constitutively expressed in cultured ORSC. Adenosine stimulated VEGF expression in a dose dependent manner. Adenosine is known to have four receptors (A1, A2a, A2b, A3), and VEGF up-regulation was inhibited only by A1 receptor specific antagonist. Adenosine increased the level of intracellular Ca2+ concentration in ORSC biphasically. Calcium ionophore also stimulated VEGF expression. Conclusion: Adenosine was supposed to up-regulate VEGF expression in ORSC via A1 receptor. We speculate that up-regulated VEGF stimulates the proliferation of vascular endothelial cells, which contributes to form vascular network surrounding hair follicles. Thus, adenosine might activate VEGF production not only in dermal papilla cells but ORSC, by which hair growth is activated and maintained.

Department of Dermatology, Ehime University School of Medicine

026

Background: Bone morphogenetic protein-2 (BMP-2) and Wnt are involved in the normal development and tumorigenesis of several organs, and in the development of skin and skin appendages as a morphogen. However, the crosstalk between BMP-2 and the Wnt/ b-catenin signaling pathway is not clear. Objective: We examined BMP-2-dependent expression of Wnt and its receptor frizzled in interfollicular normal human keratinocytes. Methods: The mRNA expression of the Wnt and frizzled families was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or ribonuclease protection assay. b-Catenin expression was measured using RT-PCR and Western blotting. TCF/LEF activity was analyzed using the luciferase reporter assay. Results: We detected the expression of Wnt-2b/13, -4, -5a, 5b, -7a, -7b, and -10a, frizzled-1, -4, -5, -6, -8, -9, and -10, MFRP, and SFRP-1/SARP-2 in keratinocytes. BMP-2 increased Wnt-2b/ 13, -5b, and -7b, and frizzled-6, -8, and -10. Conversely, BMP-2 suppressed Wnt-10a and SFRP-1/SARP-2. Although Wnt-4 expression was not affected by BMP-2 in confluent conditioned kerati-

Hair inducing cells express prominin-1/CD133

024 Expression of Wnt and frizzled in interfollicular keratinocytes, and its regulation by BMP-2

Yuriko Ito1, Tatsuo S. Hamazaki1, Kunihiko Tamaki2, Makoto Asashima3, Hitoshi Okochi1 1

Department of Tissue Regeneration, Research Institute, International Medical Center of Japan; 2Department of Dermatology, Faculty of Medicine, The University of Tokyo; 3Department of Life Science (Biology), Graduate School of Arts and Science, The University of Tokyo Dermal papilla (DP) cells play a crucial role in hair follicle morphogenesis and cycling by contributing to epithelialmesenchymal interaction. It is thought that DP cells manipulate the biological clock. However, it has been difficult to identify a specific surface marker for DP cells. Therefore, to identify such a marker, we focused on prominin-1/CD133 (CD133), a pentaspan transmembrane glycoprotein, expressed on several tissue stem/ progenitor cells. Histological analysis revealed that DP cells express CD133 in the early anagen (active growth phase) stage

Abstracts not only during hair development but also during hair growth after birth periodically. The phenotype of CD133 positive (+) cells in embryonic skin suggests that the cells are derived from the neural crest. The CD133(+) cells were positive for CD44, CD56/NCAM, p75NTR, nestin, and CD90/Thy-1, and negative for Sca-1, CD117/ c-kit, CD45, and CD202/Tie-2. We could exclude the possibility that the CD133(+) cells may be derived from hematopoietic/ endothelial and melanocytic lineages. The CD133(+) cells exerted their multipotency, differentiated into neuronal and mesodermal lineages after 14-days cultivation. Moreover, CD133(+) cells of both embryonic and adult skin could induce the growth of new hairs, in vivo. In summary, DP expresses CD133 in a hair-cycle dependent manner; DP CD133(+) cells are derived from the neural crest; and CD133(+) cells can induce new hairs in vivo. We propose that CD133 is an excellent marker for analyzing DP cells with hairinducing ability. 027 NKG2D ligation without T cell receptor engagement triggers both cytotoxicity and cytokine production in dendritic epidermal T cells Ayano Nitahara, Hideki Shimura, Akiko Ito, Katsuhiro Tomiyama, Masaaki Ito, Kazuhiro Kawai

141 skin-homing ESL++CLA+T cells. To this end, naive CD4+ T cells were activated in the presence of anti-CD3 mAb and IL-12 for 5 days and transferred to the resting culture under either IL-12- or IL-4-rich conditions. Both enzymes were detected intracellularly at similar frequencies on day 5 of activation culture. Nevertheless, these T cells only expressed FucT-IV-dependent epitope (ESL+CLA). When transferred to resting culture with IL-12, FucT-IV expression was dramatically down-regulated, but Fuc-TVII expression remained unchanged. The transition from the ESL+CLA to the ESL++CLA+ phenotype progressively occurred in parallel with down-regulation of FucT-IV expression. In contrast, FucT-VII expression was down-regulated as the T cells were transferred to IL-4-rich resting culture, where FucT-IV expression remained unchanged and the ESL++CLA+ phenotype was not detected. FucTVII expression at early stages of differentiation is unlikely sufficient to fully induce the FucT-VII-dependent epitope, which could be prevented by concomitant expression of FucT-IV. Thus, whether FucT-VII could generate its own ESL epitope on the cell surface of the T cells is largely dependent on both their state of cell activation and their levels of FucT-IV expression, but not a cytokine milieu Down-regulation of FucT-IV needed for further differentiation would be only induced when competition between both enzyme for availability of the same substrate occurs. 029

Division of Dermatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan NKG2D is an activating receptor that recognizes self ligands induced on stressed, infected, or transformed cells. In mice, two NKG2D isoforms (NKG2D-S and NKG2D-L) that associate differentially with DAP10 and DAP12 adaptor proteins exist. Differential expression of these isoforms and adaptor proteins depending on the activating state and cell types determines distinct functional outcomes of NKG2D ligation: direct activation of cytotoxicity in NK cells and cytokine production in activated NK cells, but only costimulation in activated CD8+ T cells. Intraepithelial g d Tcells of the mouse skin, termed dendritic epidermal Tcells (DETCs), were also shown to express NKG2D, but the NKG2D isoform(s) expressed in DETCs have not been determined. Furthermore, functional outcomes of NKG2D ligation in DETCs are largely unknown, although costimulation of DETC-mediated cytotoxicity by NKG2D was demonstrated. Here we show that DETCs constitutively express NKG2D-S, NKG2D-L, DAP10, and DAP12 transcripts as well as cell surface NKG2D protein. Blocking of NKG2D inhibited DETC-mediated cytotoxicity against target cells that do not express T cell receptor ligands. Cross-linking of NKG2D on DETCs induced IFN-g production. These findings demonstrate that DETCs constitutively express NKG2D that acts as a primary activating receptor, and indicate its important role in cutaneous immune surveillance. 028 Dynamic balance between Fuc T-IV and Fuc T-VII is a major check point for the regulation of skin-homing CD4+ T cell differentiation Ryo Takahashi1, Yoshiko Mizukawa2, Tetsuo Shiohara1,2 1

Division of FCM, Kyorin University School of Medicine, Tokyo, Japan; 2Department of Dermatology, Kyorin University School of Medicine, Tokyo, Japan We previously demonstrated that E-selectin ligands expressed on differentiated CLA+ T cells (ESL++) can be generated by a1,3fucosyltransferase (FucT)-VII while that (ESL+) on the early activated CLA T cells can be constructed by FucT-IV. We therefore analyzed expression of both enzymes during differentiation into

Beta 7 integrin is essential for the induction of oral tolerance Hanako Ohmatsu, Takafumi Kadono, Hidehisa Saeki, Kunihiko Tamaki Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan The establishment of antigen-specific immune tolerance leads to the new therapy for various diseases, such as atopic dermatitis, contact dermatitis, autoimmune disease. Oral tolerance is one type of immune tolerance, in which oral administration of antigens prior to immunization reduces the subsequent immune responses. Peyer’s patches and mesenteric lymph nodes are thought to be important for the induction of oral tolerance. The lymphocyte migration to Peyer’s patches is mostly mediated by beta 7 integrin, whereas either L-selectin or beta 7 integrin is required for the migration to mesenteric lymph nodes. In this study, we investigated the mechanisms of oral tolerance using L-selectin knockout (LKO) mice, beta 7 integrin knockout (beta7KO) mice and L-selectin/beta 7 integrin double knockout (L/beta7KO) mice. Mice were fed either with or without ovalbumin (OVA), and were subsequently immunized i.p. with OVA. In wild type mice and LKO mice, OVA-specific antibody titer and the number of antibody forming cells were significantly reduced in OVA-fed mice compared with those in non-fed mice, which suggested that tolerance was successfully induced in both wild type and LKO mice. However, in beta7KO mice and L/beta7KO mice, those numbers were not different between OVA-fed and non-fed mice, indicating that the induction of oral tolerance was equally perturbed in beta7KO and L/beta7KO mice. Cell proliferation assay and the measurement of cytokine production revealed similar results. Thus, beta 7 integrin is essential for the induction of oral tolerance and L-selectin does not have additional roles. These results suggest that the lymphocyte migration to Peyer’s patches rather than mesenteric lymph nodes is important for the induction of oral tolerance. 030 A new strategy for antigen-specific tolerance by induction of tolerogenic dendritic cells Iwao Isomura, Yoichi Shintani, Akimichi Morita

142 Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences The induction of antigen-specific immune tolerance has long been targeted to control allergic or autoimmune diseases without immunosuppressive reagents. Increasing number of reports demonstrated that peripheral tolerance had been induced by modulating dendritic cells (DC). Tolerogenic DC, which can induce peripheral tolerance, are characterized by inhibitory co-stimulatory molecules and IL-10 production. Recently, nuclear factor kappa B (NF-kB) decoy oligodeoxynucleotides (ODN) has been used for heart graft transplantation by inhibiting NF-kB-related gene expression. NF-kB plays significant roles in upregulation of co-stimulatory molecules and immunostimulatory cytokines. We previously have demonstrated that NF-kB decoy ODN could induce antigen-specific peripheral tolerance in DTH through the induction of regulatory T cells (Treg). In this study, we investigated the underlying mechanisms of peripheral tolerance in DC. By topical NF-kB decoy ODN, more LC were observed in the epidermis by 38% with morphologically active characteristics and high expression of MHC class II until 2 days after OVA sensitization. In addition, migration of DC into draining lymph nodes was decreased by 40%. Even after migration of DC, the expression of B7-1, B7-2 were slightly downregulated, and PD-L1 and PD-L2 were significantly upregulated, suggesting NF-kB decoy ODN treatment induces tolerogenic DC. The antigen-specific tolerance could be transferable by isolating CD11c+ DC from the tolerant mice. These findings indicate that the induced tolerance could be mediated by the delay of DC migration and modulation of the surface molecules on DC. Topical application of NF-kB decoy ODN might be a new strategy for inducing tolerogenic DC that contribute to the induction of peripheral tolerance. 031 PGD2 exerts an essential role in chronic allergic inflammation of the skin via CRTH2 receptor Rie Moroi1, Takahiro Satoh1, Yasumasa Kanai1, Hiroo Yokozeki1, Hiroyuki Hirai2, Kinya Nagata2, Kiyoshi Nishioka1, Masataka Nakamura3 1

Department of Dermatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; 2BML, Inc., R and D Center, Tokyo, Japan; 3Human Gene Science Center, Tokyo Medical and Dental University, Tokyo, Japan Biological activities of prostaglandin D2 (PGD2) are thought to be mediated by the classical DP receptor and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells). Despite increasing knowledge about functions of DP, the roles that CRTH2 plays in allergic inflammation in vivo are not fully understood. In the present study, to examine the role of PGD2CRTH2 interaction in development of allergic inflammation, we generated mice that contain a targeted disruption of the CRTH2 gene. We used these mice to characterize the IgE-mediated verylate-phase response (vLPR), a model of chronic allergic inflammation of the skin. The present findings indicate that IgEmediated vLPRs are dependent on PGD2. Cells expressing hematopoietic PGD synthase were found in the dermis of the lesional skin and PGD2 levels were increased at vLPR. Ear swelling responses were suppressed by administration of the PGD synthase inhibitor HQL-79 or the CRTH2 antagonist ramatroban. CRTH2deficient mice failed to develop IgE-mediated vLPRs, which are histologically characterized by a decrease in infiltrative lymphocytes and eosinophils, associated with inhibited production of eotaxin, MDC and RANTES. In addition, the infiltration of basophils that are known to play a critical role in the development of vLPRs was also reduced in the mutant mice. Local production of

Abstracts IL-4 and IFN-g was not affected by lack of CRTH2. These findings indicate that PGD2 signaling via the CRTH2 receptor plays important roles in IgE-mediated cutaneous responses. CRTH2 may represent a novel therapeutic target for the treatment of chronic allergic skin inflammation, such as atopic dermatitis. 032 Disruption of the ICOS-ICOS ligand costimulatory pathway delays cutaneous wound healing Minoru Hasegawa1, Manabu Fujimoto1, Kazuhiko Takahara1, Shinichi Sato2 1 Department of Dermatology, Kanazawa University, Kanazawa, Japan; 2Nagasaki University, Nagasaki, Japan

The skin wound-healing process requires leukocyte recruitment, angiogenesis, and collagen accumulation. Although much is known about the function of inflammatory cells within wounds, the roles of lymphocytes remain unclear. The activation and immune function of lymphocytes are regulated by co-stimulatory molecules. The CD28 homolog inducible costimulator (ICOS) is not expressed by naive T cells but is induced after T cell activation. ICOS is required for proper T cell activation, differentiation, and effector cytokine expression. Evidence suggests that ICOS is essential for Th2 cell function. An only ligand for ICOS, ICOS ligand (ICOSL), is expressed on B cells and in nonlymphoid tissues. We examined the biological roles of ICOSICOSL interactions in skin wound repair using mutant mice. The loss of ICOS or ICOSL significantly delayed wound healing compared with wild type mice. These delayed wound healing was associated with reduced collagen synthesis estimated by hydroxyproline content. Slight decrease in the number of infiltrated neutrophils, macrophages, and Tcells was observed in the wound from ICOS- or ICOSL-deficient mice. The loss of ICOS or ICOSL significantly decreased mRNA expression of Th2 or fibrogenic cytokines such as interleukin (IL)-6, IL-10, and transforming growth factor-b in the skin. Th1 cytokines such as interferong and tumor necrosis factor-a were not influenced by the deficiency of ICOS or ICOSL. IL-4, IL-13, and IL-17 were not detected in any mice. These results demonstrate a distinct role of ICOSICOSL interaction in wound healing and that the delayed wound healing in the absence of these molecules is likely due to decreased production of fibrogenic cytokines from accumulated leukocytes in the wound site. 033 Induction of b-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2 Yasuyuki Sumikawa1, Hideo Asada2, Katsuaki Hoshino3, Hiroaki Azukizawa1, Ichiro Katayama1, Shizuo Akira4, Satoshi Itami1 1

Department of Dermatology, Course of Molecular Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan; 2Department of Dermatology, Nara Medical University, 840 Shijocho, Kashihara, Nara, 6348522, Japan; 3Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehirocho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; 4Department of Host Defense, Research Institute for Microbial Disease, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan The epidermis, which covers the surface of all mammals, serves as a front line of defense against the invasion of pathogenic microbes and acts as a crucial site for innate immune responses. Various antimicrobial molecules are expressed not only on the

Abstracts surfaces of monocytes but also on epithelial cells. b-Defensins (BD), a family of antimicrobial peptides, are produced by several types of epithelial cells including keratinocytes (KC). However, the induction pathways for BD in KC are not fully understood. We hypothesized that bacterial components would trigger the expression of BD in KC through a Toll-like receptor (TLR)MyD88 signaling pathway that plays important roles in innate immunity. Production of TNF-a and IL-1 a following stimulation with LPS or bacterial lipopeptides (BPL) was completely abolished in TLR2&TLR4-doubly KO KC and in MyD88KO KC. Expression of murine BD was up-regulated by BPL in WT KC, while it was attenuated in TLR2KO KC. To evaluate the in vivo role of TLRs in KC, we inoculated S. aureus into the tail skin from TLR2KO mice that had been grafted on the dorsal skin of syngeneic mice. The grafted skin from TLR2KO mice resulted in erosion. We quantitated the amount of S. aureus on the grafted skins by colonization assays. The amount of S. aureus on the TLR2 KO mice skin specimens are about three times more than that on the WT mice skin specimens. Furthermore, we inoculated S. aureus on the grafted skin from MyD88 KO mice and this resulted in severe ulceration of the grafted skin. Although mBD3 was expressed in epidermal KC in upper layer of WTskin, it was not expressed in the epidermis from MyD88 KO skin. These results suggest that mBD3 production via TLR2-MyD88 activation may in part contribute to host defense against S. aureus in vivo.

034 RAB7 interacts with the melanosomal matrix protein GP100/ PMEL17/SILV and regulates its maturation in MMAC human melanoma cells Akinori Kawakami1, Fumio Sakane2, Shinichi Imai2, Hideo Kanoh2, Masae Okura1, Kuninori Hirosaki1, Toshiharu Yamashita1, Kowichi Jimbow1 1 Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan; 2Department of Biochemistry (Secrion II), Sapporo Medical University School of Medicine, Sapporo, Japan

Rab7 is a small GTPase that plays a crucial role in lysosomal/ endosomal traffic in mammalian cells. We have previously identified Rab7 as a melanosome-associated protein involved in the intracellular transport of the melanocyte-specific protein, tyrosinase-related protein 1, from the trans-Golgi network to melanosomes. In order to further identify a potential partner for Rab7-binding, we performed a yeast two-hybrid screening using a cDNA library of the human melanoma cell line MMAc. We found that wild-type Rab7 and its dominant-active mutant (Rab7-Q67L), but not its dominant-negative mutants (Rab7T22N and Rab7-N125I), bound to the carboxyl-terminal onethird of melanosomal matrix protein, gp100/Pmel17/Silv (amino acid 422-661). Although Rab9 also weakly interacted with gp100-422-661 in the two-hybrid assay, other small GTPases examined (Rab2, Rab4, Rab8, Rab22 and Rac1) failed to show the interaction. Rab7-Q67L and wild-type Rab7 were co-localized in the perinuclear region with granules containing stage I-melanosomes where the full-length gp100 was present. On the other hand, Rab7-T22N was diffusely distributed in the cytoplasm, showing no significant co-localization with melanosomes. Next, using Western blot analysis, we quantified proteolytically processed, mature gp100, which appeared in stage II- and later stage-melanosomes, in MMAc cells transfected with Rab7 mutants for 48 h. Interestingly, we found that Rab7-Q67L and, to a lesser extent, wild-type Rab7 increased the amount of the mature gp100. However, Rab7-T22N did not show such effect. These results collectively suggest that Rab7 spe-

143 cifically interacts with gp100, playing an important role in melanogenesis through the regulation of gp100 maturation in melanoma cells. 035 Mutation analysis of Hermansky-Pudlak syndrome TYPE1 among Japanese patients with albinism Tamio Suzuki1, Shiro Ito1, Katsuhiko Inagaki1, Noriyuki Suzuki1, Richard Spritz2, Yasushi Tomita1 1 Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Human Medical Genetics Program, University of Colorado Health Sciences Center, Denver, CO

HPS1 is an autosomal recessive disorder characterized by oculocutaneous albinism (OCA), bleeding tendency, and lysosomal accumulation of ceroid-like material. Seven genetically distinct subtypes of HPS are known in humans; most are rare outside of Puerto Rico. Here, we report the analysis of the HPS1 gene in 24 Japanese OCA patients who lacked mutations in the four genes known to cause non-syndromic OCA (TYR/OCA1, P/OCA2, TYRP1/ OCA3, and MATP/OCA4) and the identification of eight different HPS1 mutations in 10 of these patients, four of which were novel (p.W583X, p.L668P, c.532insC, c.1691delA). An IVS5 + 5G>A splice consensus mutation was particularly frequent, the result of a founder effect for this allele in Japanese patients. And, functional analysis of the p.L668P variant showed that this missense substitution is pathologic. We transfected cells with cDNA expression plasmids encoding either wild-type or p.L668P mutant human HPS1 protein, and analyzed HPS1 and HPS4 protein expression by Western blot assay. Over-expression of normal human HPS1 protein in transfected melan-ep cells fully restored stability of endogenous HPS4 protein. However, over-expression of p.L668P-mutant HPS1 protein failed to restore stability of endogenous HPS4 protein. This result indicates that p.L668P-mutant HPS1 protein is functionally incapable of assembling with HPS4 protein in the BLOC-3 (biogenesis of lysosome-related organelles complex-3). These findings indicate that HPS1 is one of the most common types of OCA in Japanese patients, accounting for approximately 11% of patients studied. 036 BMP-4 signaling regulates kit expression in mouse neural crest cells just before they enter the kit-dependent cycle of melanogenesis Tamihiro Kawakami1, Satoko Kimura1, Yoko Kawa1, Masako Mizoguchi1, Yoshinao Soma1, Masashi Kato2 1 Department of Dermatology, St. Marianna University School of Medicine, Kawasaki, Japan; 2Department of Environmental and Preventive Medicine, Research Institute of Life and Health Science, Chubu University, Aichi, Japan

Genes encoding Kit and Kit ligand (Kitl) play essential roles in the differentiating melanoblasts. We previously established three immortal but distinct cell populations of mouse neural crest (NC) cells. NCCmelb4M5 cells do not express Kit and grow independently of Kitl; they have the potential to differentiate into NCCmelb4 cells, which are Kit-positive melanocyte precursors. NCCmelan5 cells show the characteristics of differentiated melanocytes. We investigated the effects of BMP-4 on these immature melanocyte precursors. Three cell lines showed BMPreceptor expression. BMP-4 up-regulated Kit protein and mRNA expression in most immature NCCmelb4M5 cells. Noggin, a BMP-4 antagonist, dramatically decreased the Kit expression induced by BMP-4. Western blot analysis revealed that extrinsic BMP-4 leads

144 to the phosphorylation of Smads and activates signaling in NCCmelb4M5 cells. We conclude that BMP-4 pathway is activated and is involved in the regulation of Kit expression on most immature melanocyte precursor. We further investigated the influence of BMP-4 in vitro using primary NC cells cultured from wild-type mice. Addition of BMP-4 to the medium increased the number of Kit-positive cells compared to untreated controls. Thus, we have identified BMP-4 as an important factor for prepubertal Kit-negative melanoblasts just before they enter the Kitdependent cycle of melanogenesis. 037 P38 activation is involved via MITF phosphorylation in increased expression of c-KIT in UVB-exposed human melanocyte Yuki Mizutani1, Nobukazu Hayashi1, Makoto Kawashima1, Genji Imokawa1,2 1 Department of Dermatology, Tokyo Women’s Medical University, Tokyo, Japan; 2Skin Science Research Institute, Tokyo, Japan

c-KIT, stem cell factor receptor plays a pivotal role in several pigmentary disorders including UVB melanosis. However, signaling mechanisms of c-KIT expression in melanocytes have not been clarified. To address this question, we determined signaling linkages responsible for increased c-KIT expression in UVBexposed human melanocytes in vitro. Exposure of UVB (40— 80 mJ/cm2) to cultured human melanocytes significantly increased the expression of c-KIT at the gene and protein levels. The UVB-increased expression of c-KIT protein was completely abolished by treatment with inhibitor of p38 (SB 203580), but not affected by inhibitors of MEK (PD98059), JNK (SP600125), Akt (Akt inhibitor III), PKC (Calphostine C) and PKA (H-89). Consistently, UVB exposure markedly stimulated prolonged phosphorylation of p38 with a plateau at 240 min post-irradiation, which was accompanied by a sustained phosphorylation of MITF during an initial comparable timing. The increased MITF phosphorylation was completely abolished by the inhibitor of p38, suggesting one of target molecules by activated p38 to be MITF. In contrast, the inhibitor of MEK rather stimulated MITF phosphorylation in nonUVB exposed human melanocytes, which occurred in concert with the increased expression of c-KIT protein. These findings suggest that p38 dependent-mechanism leading to c-KIT expression in UVB-exposed human melanocytes is mediated via MITF phosphorylation and is modulated by a possible feedback mechanism using MAPK pathway. 038 The presence of soluble form of c-kit and its function in melanocyte

Abstracts brane. It has been observed that stimulants such as phorbol 12myristate-13-acetete (PMA) cleaved the extra cellular domains of c-kit in mast cells and released them into the culture supernatant. However, in melanocytes, released soluble c-kit has not been observed yet. This study detected the soluble form of c-kit in the culture supernatant of melanocyte by Western blot analysis using antibody N-terminal reactive. The culture supernatant was concentrated and stimulated by addition of PMA. The soluble forms of c-kit were detectable after an hour. In addition, the binding assay of SCF/c-kit showed that soluble forms of c-Kit inhibit SCF/c-kit binding in a concentration-dependent manner. These results raise the possibility that the soluble form of c-Kit released into melanocyte might regulate the signaling of SCF/ckit. 039 Skin responses to UV radiation: Pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis Yuji Yamaguchi1,2, Kaoruko Takahashi2, Taketsugu Tadokoro2, Barbara Z Zmudzka3, Janusz Z. Beer3, Satoshi Itami1, Ichiro Katayama1, Vincent J. Hearing2 1 Department of Dermatology, Osaka University Graduate School of Medicine, Osaka, Japan; 2Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; 3Center for Devices and Radiological Health, Food and Drug Administration, Rockville, MD, USA

Melanin plays an important role in protecting the skin against ultraviolet (UV) radiation and melanomas and basal/squamous cell carcinomas occur more frequently in individuals with fair/ light skin. We previously reported that levels of melanin correlated inversely with amounts of DNA damage induced by UV in normal human skin of different racial/ethnic groups. We have now further investigated DNA damage in the upper and lower epidermal layers in various types of skin before and after exposure to UV, and have measured subsequent apoptosis and phosphorylation of p53. The results show that two major mechanisms underlie the increased photocarcinogenesis in fair/light skin. First, UV-induced DNA damage in the lower epidermis (including keratinocyte stem cells and melanocytes) is more effectively prevented in darker skin, suggesting that pigmented epidermis is an efficient UV filter. Second, UV-induced apoptosis is significantly greater in darker skin, which suggests that cells with UV damage may be removed more efficiently in pigmented epidermis. The combination of decreased DNA damage and more efficient removal of UV-damaged cells may play a critical role in the decreased photocarcinogenesis seen in individuals with darker skin.

Shinya Kasamatsu, Kazuhiko Higuchi, Atsushi Ouchi, Takashi Kitahara

040

Biological Science Laboratories, Kao Corporation, Tochigi, Japan

Signaling mechanisims of histon deacetylase inhibitor (HDACi) depsipeptide (FK228) induced apoptosis in acral melanoma cell line

It has been previously reported that c-kit receptor is a receptor tyrosine kinase that binds to its ligand stem cell factor (SCF), autophosphorylates itself, and thereby results in activation of downstream signaling cascades. This signaling serves for various cell proliferations and differentiations, such as melanogenesis, melanocyte proliferation and differentiation. In human skins, UVB-irradiation enhances gene expression of SCF in keratinocyte and activates neighboring melanocyte via c-kit receptors, thereby inducing melanogenesis. It has been suggested that this signaling might play an important role in the development of chronic pigmentation. C-kit is normally found in the cell mem-

Ken Futaki1, Yusuke Furukawa2, Mamitaro Ohtsuki3, Hidemi Nakagawa1, Hiroshi Murata4, Minoru Takata4, Toshiaki Saida4, Genji Imokawa1,5 1 Department of Dermatology, The Jikei University School of Medicine, Tokyo, Japan; 2Division of Stem Cell Regulation, Jichi Medical School, Tochigi, Japan; 3Department of Dermatology, Jichi Medical School, Tochigi, Japan; 4Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan; 5 Skin Science Research Institute, Tokyo, Japan

Abstracts Acral melanoma (ALM) is UV-independent malignant tumor which occurs frequently in Asian people. Here we used an ALM cell and FK228 as HDACi to determine its signalling mechanism. In two non-ALM cell lines, FK228 completely abolished protein expression of MITF at 12 h which was followed by a marked downregulation of CDK2 protein. The marked suppression of MITF expression was accompanied by new expression of apoptotic protein p21 (not expressing in non-treatment) at 12—24 h which suggested that apoptosis occurs due to increased expression of p21 via MITF down-regulation. In contrast, in ALM cells, FK288 elicited a delayed suppression of MITF protein at 48 h. CDK2 protein expression remained unchanged until at least 24 h while the expression of p21 protein had a delayed dynamics with its onset at 48 h. Despite the above expression dynamics of MITF/cell cycle regulatory proteins, their inhibitory effect of FK228 on DNA synthesis (measured by BrdU uptake) was the most marked among three melanoma cell lines tested. The above findings suggest that there is a novel signaling mechanism which is not associated with down-regulation of MITF/p21 lineage in FK288-induced apoptosis of acral melanoma cells. 041 Dynamics of mitf and proliferative signaling molecules during endothelin-1-stimulated intracellular signalings Kayo Sato1, Eijiro Akasaka1, Hajime Nakano1, Katsumi Hanada1, Genji Imokawa1,2

145 1

Department of Dermatology, Hirosaki University School of Medecine, Hirosaki, Japan; 2Skin Science Research Institute, Tokyo, Japan Raf-1 kinase inhibitory protein (RKIP) plays a pivotal role in regulating MAPK cascade by down-regulating MAPKKK activity of activated Raf-1, attributable to growth modulation in variety of cells. In malignant melanoma cells, there is a reciprocal relationship between growth rate and RKIP expression. In non-melanocytic cells, the stimulation by TPA triggers RKIP phosphorylation to elicit the activation of MAPK due to dissociation between RKIP and Raf-1. However, little is still known about intrinsic role of RKIP in modulating cell growth signallings in NHM. In this study, we used ET-1 stimulated MAPK cascade to examine roles of RKIP in regulating NHM proliferation. Western blotting revealed that while RKIP protein expression occurred in non-stimulated NHM, ET-1 (10 nM) did not elicit any significant changes in RKIP protein levels. Although ET-1 stimulation induced a rapid phosphorylation of MEK and subsequently ERK1/2, analysis using immunoprecipitation/Western blotting demonstrated that while in non-stimulated NHM, there was not a detectable level of protein complex consisting of RKIP and Raf-1, the stimulation with ET1 accompanying MAPK activation did not generate the protein complex. These findings suggest that the inhibitory interaction between RKIP and Raf-1 is not attributable to signalling mechanisms underlying the activation of MAPK cascade by ET-1 stimulation in NHM.

1

Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan; 2Skin Science Research Institute, Tokyo, Japan Recently new roles of MITF have been proposed to serve as regulatory factors for Bcl-2 and CDK2 or its inhibitor protein p21. Since these proposals are mainly based upon analysis using mouse melanocytes or melanoma cells, little is still known about proliferative roles of MITF-M in normal human melanocytes. Here we used ET-1 stimulated intracellular signallings to examine proliferative roles of MITF-M in normal human melanocytes. An addition of ET-1 (10 nM) to cultured human melanocytes elicited a marked phosphorylation of MITF with a peak at 5— 20 min post-incubation which was accompanied by preceding phosphorylation of ERK1/2. On the other hand, the expression of MITF-M mRNA transcripts was markedly augmented at 40— 120 min post-incubation which was followed by an increased expression of MITF protein with a peak at 2—3 h post-incubation. The protein expression of CDK2 was augmented at 8—12 h posttreatment with ET-1 which was accompanied by an increase in pRb protein, indicative for CDK2 activity, supporting a stimulation of DNA synthesis revealed by increased incorporation of BrdU. In contrast, the expression of CDK2 inhibitory protein p21 occurred at undetectable levels before and after ET addition. These findings suggest that MITF serves as a regulatory factor for transcription of CDK2 expression rather than for anti-proliferative property dominated by p21 expression in normal human melanocytes. 042 RKIP may not be associated with endothelin-induced activation of MAPK cascade in normal human melanocytes (NHM) Eijiro Akasaka1, Kayo Sato1, Hajime Nakano1, Katsumi Hanada1, Genji Imokawa1,2

043 COL7A1-engineered fibroblasts provide more type VII collagen to BMZ than COL7A1-engineered keratinocytes Maki Goto, Daisuke Sawamura, Kei Ito, Masataka Abe, Wataru Nishie, Kaori Sakai, Akihiko Shibaki, Masashi Akiyama, Hiroshi Shimizu Department of Dermatology, Hokkaido University Graduate School of Medecine, Sapporo, Japan Mutations in the type VII collagen gene (COL7A1) encoding type VII collagen are caused by dystrophic epidermolysis bullosa (DEB), which is an inherited blistering skin disorder. Therapeutic introduction of COL7A1 into skin cells holds significant promise for the treatment of DEB. The purpose of this study was to establish an efficient retroviral transfer method for COL7A1 into DEB epidermal keratinocytes and dermal fibroblasts, and to determine which gene-engineered cells can most efficiently express collagen VII in the skin. Gene transfer using a combination of VSV-G pseudotyped retroviral vector and retronectin could effectively introduce COL7A1 into keratinocytes and fibroblasts from a DEB patient with lack of COL7A1 expression. RT-PCR analysis of the normal human skin showed that quantity of COL7A1 expression in the epidermis was significantly higher than that in the dermis. Subsequently, we have produced skin grafts with the gene-transferred or untreated DEB keratinocytes and fibroblasts, and have transplanted them into nude rats. Interestingly, the series of skin graft experiments showed that the gene-engineered fibroblasts supplied significantly higher amount of collagen VII to the new dermal-epidermal junction than the gene-engineered keratinocytes. These results suggest that fibroblasts may be a better gene therapy target of DEB treatment than keratinocytes.

146

Abstracts

045

3, 10, 17, the mice were treated with 1  104, 105 or 106 tumorlysate-pulsed DCs that were activated with SeV. The antitumor effect was apparently dose-dependent. To assess the optimized route for DC administration, the tumor-burden mice were treated with SeV/DCs by intratumoral, contra-lateral subcutaneous, or intravenous administration. The significant antitumor effect was only recognized in the group of intratumoral administration, and in this case, tumor lysate was not important. In addition, cotreatment of DC therapy and recombinant murine IFN-b protein significantly enhanced the anti-tumor effect for tumor-burden mice. Discussion and conclusion: The intratumoral administration of DCs could induce efficient and optimized antitumor immunity irrespective of pulsing tumor antigens ex vivo, and it is suggested that at least more than 106 DCs in mice are required. Furthermore, the combination therapy, SeV/DCs and IFN-b, could dramatically enhance the anti-tumor effect of DC immunotherapy.

Oncolytic viral therapy for malignant melanoma with herpes simplex virus type 1 mutant HF10 Daisuke Watanabe1,2, Fumi Goshima2, Isamu Mori2,3, Yasuhiko Tamada1, Yoshinari Matsumoto1, Yukihiro Nishiyama2 Department of Dermatology, Aichi Medical University; 2Department of Virology, Graduate School of Medicine, Nagoya University; 3Department of Microbiology and Immunology, Aichi Medical University 1

Replication-competent herpes simplex viruses (HSVs) have shown promise as anti-tumor agent for cancer therapy. We reported that a replication-competent, spontaneous HSV-1 variant, HF10 was effective in treating disseminated peritoneal colon carcinoma and breast cancer in an immunocompetent mouse model. We also reported that intratumoral injection of HF10 in patients with recurrent breast cancer showed tumor cell death with no toxicity in a preliminary study. In this study, we investigated the ability of HF10 to infect and lyse human and murine malignant melanoma cells in vitro and tested its efficacy in immunocompetent animal models of s malignant melanoma. In vitro viral cytotoxity assays and replication assays were performed both in human (G361) and murine (clone M3) cell lines. HF10 was effective in producing cytolytic effects in vitro at various multiplicities of infection (MOI) in all cell lines tested. To assess the therapeutic efficacy of HF10 against subcutaneous or intraperitoneal tumors in vivo, clone M3 were injected into the backs or peritoneal of mice, which were then treated HF10. Tumor volume and survival rate were used as measures of the antitumor effect in the in vivo experiments. In the in vivo study, tumor growth was suppressed and long-term survival rates were observed in HF10-inoculated group. In histological and immunihistochemical analysis, tumor lysis and infiltration of inflammatory cells were observed after HF10 infection and HSV-1 antigen retained until 7 days after infection. These data indicate that HF10 has a remarkable anti-tumor efficacy in malignant melanoma models in mice. 046 Optimization of DC-based cancer immunotherapy using activated SeV/DC for murine B16F10 model of malignant melanoma Satoko Shibata1, Yoshikazu Yonemitsu2, Shinji Okano2, Makoto Inoue3, Mamoru Hasegawa3, Yoichi Moroi1, Masutaka Furue1, Katsuo Sueishi2 1

Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Division of Pathophysiological and Experimental Pathology, Department of Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 3DNAVEC Corporation, Ibaraki, Tsukuba, Japan Background and aim: Dendritic cells (DCs) could be a promising tool for cancer immunotherapy. The early clinical trials showed the therapeutic potential of DC therapy, however, its clinical response is limited. The current issues of DC therapy include that less information for the optimal route for administration and the optimal dose and frequency of DC therapy. In the previous congress, we reported that activated DCs could be generated by transfection of recombinant SeV without other stimulus and induced efficient antitumor immunity against murine melanoma. The aim of the current study is to assess the aforementioned issues using SeV/DC for tumor-burden mice. Methods and results: Female C57BL/6 mice were subcutaneously inoculated with B16F10 melanoma cells on day 0. On day

047 Long-term survival of harlequin ichthyosis patient with a complete lack of ABCA12 function Masashi Akiyama1, Kaori Sakai1, Gerhard Wolff2, Ingrid Hausser3, James R. McMillan4, Daisuke Sawamura1, Hiroshi Shimizu1 1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2Institut fur Humangenetik und Anthropologie, Albert-Ludwigs-Universitat Freiburg, Germany; 3Department of Dermatology, University Clinic Heidelberg, Germany; 4Creative Research Institute Sousei, Hokkaido University, Sapporo, Japan

We recently clarified that serious loss of function of the keratinocyte lipid transporter ABCA12 leads to defective lamellar granule lipid transport in keratinocytes resulting in harlequin ichthyosis (HI: OMIM #242500), one of the most severe and devastating genodermatoses [1]. In this study, we confirmed a novel homozygous deletion mutation, 3270delT in ABCA12, in a female patient with HI. The prognosis of HI was generally thought to be poor with the majority of cases being fatal in the perinatal period. However, the number of HI newborns with a better prognosis increased since administration of oral retinoid, although it has not yet been elucidated whether better prognosis is due to systemic retinoid treatment or due to the nature of respective ABCA12 mutations. The homozygous deletion mutation we identified, leads to the truncation at codon 1090, within the first transmembrane domain complex, losing both ATP-binding cassettes (active sites of the ABCA12 lipid transporter) and is thought to result in a complete loss of protein function. The present patient showed typical clinical features of HI at birth. However, the patient survived beyond the perinatal period with oral retinoid treatment. At age of 8 years, her general condition is good even without oral retinoid treatment although she still shows the clinical features of non-bullous congenital ichthyosiform erythroderma on her entire body surface. These results suggest that even a complete lack of ABCA12 function can lead to HI with an improved prognosis. Reference [1] Akiyama, et al. J Clin Invest 2005.

048 An important role of lymphatic vessels in the control of UVBinduced edema formation and inflammation

Abstracts

147

Kentaro Kajiya1,2,3, Michael Detmar2,3 1

2

Shiseido Life Science Research Center, Yokohama, Japan; Institute of Pharmaceutical Sciences, Swiss Federal Institute of Zurich, Switzerland; 3Department of Dermatology, Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA Exposure of human skin to UVB-irradiation results in epidermal hyperplasia, erythema, vascular hyper permeability, and edema formation. Previous studies have revealed pronounced angiogenesis induced by UVB irradiation and upregulation of angiogenesis factors like vascular endothelial growth factor (VEGF)-A, suggesting that the cutaneous blood vasculature plays a critical role in the mediation of photodamage. In contrast, the role of the lymphatic vasculature in the response to UVB-irradiation has remained unknown. Previously established K14/VEGF-A transgenic mice were treated intraperitoneally with a blocking antibody against the lymphatic specific VEGFR-3 or with control IgG every other day. One day after the first antibody injection, mice were exposed to UVB-irradiation. No difference was observed during the first 4 days. Hematoxylin-eosin stains at day 9 after the UVB irradiation revealed that the ear skin of anti-VEGFR-3 treated mice still displayed the characteristic features of acute photodamage, including epidermal hyperplasia, marked dermal edema, vascular dilation, and inflammatory cell infiltration in the dermis. In contrast, the ear skin of control IgG treated mice already resembled histologically the non-UVB irradiated skin. Immunohistochemistry for CD11b revealed increased macrophage infiltration in the dermis of anti-VFGFR-3 treated mice, as compared with mice treated with control-IgG. Moreover, we found that LYVE-1 positive lymphatic vessels were greatly enlarged in the ear skin of anti-VEGFR-3 treated mice, whereas lymphatic vessels in mice treated with control IgG were collapsed. Our results provide the first evidence that the lymphatic vascular system is functionally involved in the cutaneous response to UVB damage. 049 Keratinocyte growth factor suppresses UVB-induced apoptosis in epidermal keratinocytes

ATM, phospho-p53 and proapoptotic molecule Bax. In conclusion, these findings suggest the existence of a photo protective effect of KGF and create a possible additional therapeutic role of KGF in the prevention of UVB-induced skin carcinogenesis. 050 Cyclophosphamide enhances TNF-a-induced apoptotic cell death in murine vascular endothelial cells Toshio Ohtani1, Tomoyuki Nakamura2, Ken-ichi Toda3, Fukumi Furukawa2 1

Department of Dermatology, Kurasiki Central Hospital, Kurasiki, Japan; 2Department of Dermatology, Wakayama Medical University, Wakayama, Japan; 3Department of Dermatology, Kitano Hospital, Ohsaka, Japan Cyclophosphamide (CPA) is one of the therapeutic agents for systemic inflammatory disorders. Treatment of murine dermal endothelial cells (F-2) with 4-hydroxycyclophosphamide (4-HC), which is active metabolite of CPA, at concentration as low as 10 mM significantly inhibited the cell viability. Keratinocyte (PAM212) and fibroblast (NIH-3T3) were not affected at the same concentration. Characteristic DNA-ladder formation in F-2 treated with 4-HC was demonstrated by agarose gel electrophoresis. By the cell death detection ELISA, 4-HC enhanced TNF-a-induced DNA fragmentation. In addition, 4-HC was shown to elevate TNFa-induced caspase-3 activation using the colorimetric substrates, and Western blots indicated that 4-HC up-regulated the expression of cleaved poly [ADP-ribose] polymerase (PARP). Furthermore, an inhibitor of the caspase-3 blocked the TNF-a and/or 4HC-induced DNA fragmentation. The activation of caspase-9 and the increase in the intracellular expression of pro-apoptotic protein Bax was identified by the treatment of 4-HC, whereas TNF-a was no effect. In contrast, only when treated with TNF-a, the activation of caspase-8 was detected. These results suggest that cyclophosphamide may sensitize endothelial cells to TNF-ainduced apoptosis through a mitochondria-dependent pathway and clinically may contribute to the limitation of inflammatory process.

Xiaoling Wang, Yuji Shirakata, Lujun Yang, Xiuju Dai, Hiroshi Nagai, Mikiko Tohyama, Yasushi Hanakawa, Sho Tokumaru, Koji Sayama, Koji Hashimoto

051

Department of Dermatology, Ehime University School of Medicine

Tetsuya Higuchi1, Hiroo Yokozeki1, Stephan Grabbe2

UV is considered as the most prevalent environmental carcinogen responsible for skin cancer. Effective protective agents become indispensable in the prevention of skin cancer. Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells. KGF promotes epidermal keratinocyte growth. Recently it has been reported KGF has an anti-apoptotic effects. Due to these properties, in this study, we investigated whether KGF could inhibit apoptosis of keratinocytes and protect DNA damage induced by ultraviolet B (UVB) irradiation. Keratinocytes were cultured under serum free condition. KGF was added to the medium and 24 h later UVB (60 mJ/cm2) was irradiated. Cell lysates were harvested at different time points for Western blot. Pre-treatment of KGF suppressed cleaved-PARP, cleaved-caspase-3 and cleaved-caspase-9. Next we investigated annexin V expression in keratinocytes by FACS analysis. Six hours after UVB irradiation, annexin V positive cells were 25% in nontreated cells, whereas they were 15% in KGF-pretreated cells. These results indicate that KGF has an anti-apoptotic effect on UVB-irradiated keratinocytes. We further investigated the modulation of upstream molecule involved in UVB induced DNA damage. KGF down regulated the expression of phospho-ATR/

Establishment of antigen entrapping particle by dextran crystallization

1

Department of Dermatology, Tokyo Medical and Dental University, Tokyo, Japan; 2Department of Dermatology, University of Essen, Essen, Germany Dendritic cells (DCs) are very potent to internalize antigen (Ag) or external materials by endocytosis, macropinocytosis and phagocytosis DC process internalized Ag and present it at the cell surface in association with MHC molecules. DC stimulants such as TNFa or ligands of the family of toll-like receptors, such as lipopolysaccharide (LPS), are well known to strongly activate DCs. In order to induce potent immunity in vivo, DCs that present the Ag to T cells also need to be activated to assume an adequate functional state. By preparing biodegradable particles that conjugate antigen and an appropriate DC activator, it is suggested that the conjugated Ag will be processed by DCs which at the same time will be get activated. Therefore we established biodegradable particles in which both, a model antigen (ovalbumin (OVA) peptide) and a DC activator (LPS), are conjugated to dextrn microspheres. By dextran crystallization, we could obtain stable crystallized dextran spheres (CDS) that entrapped both OVA peptide and LPS

148 We confirmed that CDS were efficiently internalized by bone marrow-derived DCs and that these DCs were activated by the entrapped LPS. By labeling CDS with FITC, we also visually observed the internalization of CDS by cultured DCs. By using OVA-specific T cell receptor transgenic mice, we found CDS were equally potent in stimulating OVA-specific T cell responses in vitro, and induced enhanced and prolonged Ag-specific immune responses in vivo, compared to injection of soluble Ag plus adjuvant. These results suggest the possibility to apply CDS for clinical use for vaccination, e.g. by preparing CDS entrapping tumor antigen. 052 The role of platelets in leukocyte recruitment in chronic contact hypersensitivity induced by repeated application of antigen Risa Tamagawa-Mineoka1, Norito Katoh1, Eiichiro Ueda1, Hideya Takenaka1, Masakazu Kita2, Saburo Kishimoto1 1

Department of Dermatology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan; 2 Department of Microbiology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan Platelets contain many bioactive substances, and are essential for hemostasis. They have also been shown to be important in other processes, including inflammation, but their role in chronic allergic inflammation of skin, such as that occurring in atopic dermatitis (AD), remains unclear. The aim of this study was to investigate the role of platelets in a mouse model of chronic contact hypersensitivity induced by repeated hapten application; the model has characteristics similar to those of AD. Mice were depleted of platelets to a level of about 1000/ml by administration of anti-platelet antibody or busulfan, but levels of circulating leukocytes were unchanged. Ear thickness and swelling, leukocyte infiltration into skin, serum IgE, and scratching behavior significantly decreased in platelet-depleted mice. Gene array analysis of ear skin showed reduced expression of genes associated with Th2-type lymphocytes in platelet-depleted mice. Flow cytometry showed increased expression of P-selectin on platelets and more platelet-leukocyte aggregates in the blood of mice repeatedly challenged with allergen, compared with nonsensitized mice. In platelet-depleted mice, inflammation was restored by infusion of platelets from sensitized mice. Injection of activated platelet supernatant into the ear led to increased leukocyte infiltration. This infiltration was blocked by pretreatment of platelets with PPARg agonists or addition of neutralizing antibodies against MIP-1a, RANTES or TARC to the supernatant. These results suggest that platelets induce recruitment of leukocytes into skin by forming platelet-leukocyte aggregates and secreting chemokines. Therefore, control of platelet activity may be useful for treatment of chronic allergic skin inflammation, such as in AD. 053 IgE-eliciting activity of the recombinant mite allergen DERP 1 is dependent on its proteolytic activity Yuko Kikuchi1,2, Takai Toshiro2, Takeshi Kato2, Takatoshi Kuhara2, Mikiko Ota2, Tomoko Tokura2, Kouichi Mistuishi1, Ko Okumura3, Hideoki Ogawa1, Shigaku Ikeda1 1

Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 2 Atopy Research Center, Juntendo University School of Medicine, Tokyo, Japan; 3Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

Abstracts Background: The majority of atopic dermatitis patients show specific IgE hypersensitivity to house dust mite allergens. Der p 1 is considered to be the most immunodominant house dust mite allergen. We have produced recombinant Der p1 with properties identical to natural Der p 1. Methods: We compared the IgE-eliciting activity and immune response in mice immunized with either (1) recombinant Der p 1 (rDp1) with proteolytic activity (2) inhibitor-treated rDp1, which is enzymatically inactivated with an irreversible cysteine protease-specific inhibitor, E-64 and (3) heat-denatured rDp1, which is structurally denatured and lacking enzymatic activity. In this presentation, rDp1 specific IgE was re-measured in optimized conditions and inhibition tests were performed. Results: Mice immunized with active rDp1 elicited high levels of serum IgE and rDp1-specific IgE and IgG1, whilst mice immunized with E64-treated rDp1 and heat-denatured rDp1 elicited almost no IgE production. Proliferative response of spleen cells stimulated with rDp1 and production of IL-5 by these cells were observed in mice immunized with active rDp1, but not when immunized with E64-treated rDp1, and the highest levels were observed when immunized with heat-denatured rDp1. At low concentrations, the inhibition efficiency of E64-treated Dp1 was lower than Dp1, but at high concentrations, the efficiency was similar. Conclusion: The results indicated that the affinity of rDp1 could be lowered by treatment with E-64 without affecting its structure. In addition, the proteolytic activity of rDp1 is the exclusive factor for induction of potent IgE/Th2 response in mice immunized with rDp1. Moreover, the three types of rDp1, which are different in biochemical function and/or structure, elicit different immune responses. 054 Osteopontin contributes TH2 immune responses: Spontaneous development of atopic dermatitis like lesion and enhanced asthma like response Kazumoto Katagiri, Shoko Arakawa Department of Anatomy, Biology and Medicine (Dermatology), Faculty of Medicine, Oita University, Oita, Japan Osteopontin (OPN) is a multifunctional cytokine that regulates not only metabolism of extracellular matrix including bone formation but also immunity. Analysis of OPN-deficient mice indicates that OPN is essential for host defense to some pathogens, and for development of experimental autoimmune diseases and contact hypersensitivity, all of which are mediated by Th1 response. The reinforcing role of OPN in Th1 immunity is also supported by in vitro study. On the other hand, there are some conflicting results that OPN deficient mice shows no evidence of effects on models of multiple sclerosis and arthritis, and the enhancement of Th1 immunity observed in vitro is caused by contaminated LPS but not OPN itself, which lead the role of OPN in immunity to obscurity. This study was designated to determine the effect of OPN on Th2 immunity in vivo. We found spontaneous development of dermatitis with eosinophil infiltration in transgenic mice expressing OPN mainly in liver under the control of a1-antitrypsin promoter that have high levels of soluble OPN in serum. The dermatitis developed in 60% of mice at 5 month of age. The levels of serum IgE had gradually increased until 10 month of age. Lymph node cells produced large amount of IL-4, IL-5, IL-13 and IFN-g by stimulation with anti-CD3 antibodies. Late onset of the dermatitis suggests that some other factor cooperates with OPN in developing the dermatitis. Furthermore, we found enhanced eosinophil infiltration in the lung in a model of asthma using the OPN-

Abstracts transgenic mice. These results indicate that OPN play an important role for induction of Th2 pathology in vivo. The role of OPN should be reconsidered in the development of immune responses including allergic diseases. 055 Non-pruritic mediators turn into ITCH inducers due to neuronal sensitization in atopic dermatitis

149 real-time PCR. Treatment of T cells with N-acetyl cystein, which partially recovered the IFN-g mRNA and protein production, did not restore T-bet or Txk mRNA but suppressed the augmentation of Gadd45a mRNA. These data suggest that both DEP and FA modulate mRNA expression of several transcription factors that play a role in T cell stimulation or Th1/Th2 deviation. Among them, Gadd45a and/or GILZ genes in DEP- or FA-treated T cells may link the stress response with the diminished IFN-g and IL-10 production.

Miwa Hosogi, Yoshiki Miyachi, Akihiko Ikoma 057 Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan Histamine, substance P, serotonin and bradykinin were applied by iontophoresis to eczematous and non-eczematous skin of 14 patients with atopic dermatitis, and normal skin of 15 healthy volunteers. Application of each substance was done in the same skin area before and 3 h after administration of placebo or antishitamine (olopatadine hydrochloride: h1-blocker). Intensities of itch and pain sensation and areas of flare and weal were measured. Histamine-induced flare was larger in healthy volunteers than in eczema and non-eczema of patients and substance P-induced flare was smaller in non-eczema of patients than in eczema and healthy volunteers. There was otherwise no significant difference in pain, flare and weal. On the other hand, all substances induced significantly more intense itch in eczema than in non-eczema of patients. Even bradykinin induced itch in eczema, though only pain otherwise. Although histamine- and substance P-induced reactions were suppressed almost completely by antihistamines, bradykinin- and serotonin-induced reactions were not, suggesting that bradykinin and serotonin induce itch in eczema independently of histamine. These results implicate that eczematous skin of patients is sensitized for itch and that inflammatory mediators including non-pruritic ones such as bradykinin can selectively intensify their potency due to neuronal sensitization and turn into itch inducers in atopic dermatitis. 056 Molecular events in human T cells treated with diesel exhaust particles or formaldehyde leading to diminished IFN-g and IL-10 production

Akt activation generates dermal hemangioma and skin hypertrophy Masahito Tarutani1, Kazushige Murayama2, Tohru Kimura2, Maya Tomooka3, Megumi Ikeuti2, Satoshi Itami1, Toru Nakano2,3, Ichiro Katayama1 1

Department of Dermatology Course of Molecular Medicine Graduate School of Medicine Osaka University, Osaka, Japan; 2Department of Pathology Graduate School of medicine Osaka University, Osaka, Japan; 3Graduate School of Frontier Biosciences Osaka University, Osaka, Japan PI3 kinase (PI3K)/Akt signal is activated by various growth factors and controls a cell growth and death. In addition, Akt signal activation due to the luck of PTEN participates in the onset of human cancer including skin cancer. However, little is known whether the increase of PI3K/Akt signal is necessary for maintenance of cancer. Therefore we made the transgenic (TG) mice which can control Akt activity from head to foot. This TG mice can control enzyme activity of Akt for 4-hydroxytamoxifen (4OHT) dependence. With this mouse, we examined the role of PI3K/Akt signal in dermis and epidermis. Those mice revealed skin hyperkeratosis and developed subcotaneous hemangioma when activated Akt by applying 4-OHT to mouse skin. After stopping the 4-OHT application, the hyperkeratosis and hemangioma disappeared immediately. The activation of Akt may be necessary for maintenance as well as the onset of hemangioma in skin. 058 Melanoma growth suppression by PLZF is mediated via PBX1

Yoshinori Sasaki, Tomoyuki Ohtani, Yumiko Ito, Masato Mizuashi, Satoshi Nakagawa, Setsuya Aiba Department of Dermatology, Tohoku University, Graduate School of Medicine, Sendai, Japan A series of epidemiological studies and animal and human models have suggested that both diesel exhaust particles (DEP) and formaldehyde (FA) have a role in triggering Th2mediated allergic responses. At first, we demonstrated that FA selectively suppressed IFN-g- and IL-10 mRNA expression and protein production, but not those of IL-4 or IL-5, in human T cells stimulated with anti-CD3/anti-CD28 mAb, which was identical to our previous observations concerning the responses induced by DEP. Therefore, to explore the molecular events underlying the diminished IFN-g and IL-10 production by DEP- or FA-treated T cells, we examined their effects on mRNA expression by T cells stimulated with anti-CD3/anti-CD28 mAb using microarrays and real-time PCR. By real-time PCR, we found that both DEP and FA significantly suppressed mRNA expression of T-bet, Txk and c-Maf but not that of GATA-3, SOCS3, SOCS5, or Gadd45b. The microarrays revealed significant augmentations of two FoxO3a-dependent genes, GILZ and Gadd45a, in addition to several other oxidative stress genes, which was confirmed by

Ken Shiraishi1,2, Kenshi Yamasaki1, Yasushi Hanakawa1, Yuji Shirakata1, Koji Sayama1, Shigeki Higashiyama2, Koji Hashimoto1 1

Department of Dermatology, Ehime University School of Medicine; 2Division of Biochemistry and Molecular Genetics, Ehime University School of Medicine Promyelocytic leukemia zinc finger (PLZF) is a transcriptional repressor and tumor suppressor. We investigated PLZF expression in melanocytes and melanoma cell line. PLZF was expressed in melanocytes but not all of 12 melanoma cell lines. The recovery of PLZF expression using lipofection remarkably suppresses melanoma cell growth. Several target genes regulated by PLZF have been reported, but the precise function of PLZF remains unclear. In the current studies, we searched for candidate target genes of PLZF. We constructed adenovirus vector carrying PLZF, introduced PLZF into melanoma cells and DNA microarray analysis was performed. Pre-B cell leukemia transcription factor 1 (Pbx1), a transcriptional regulator, was one of the prominently suppressed genes. Pbx1 was highly expressed in melanoma cells, and its expression was reduced by transduction with the PLZF gene. Moreover, the growth suppression mediated by PLZF was reversed by enforced expression of Pbx1. Knockdown of Pbx1 by specific

150 small interfering RNAs suppressed melanoma cell growth. We also found that Pbx1 binds HoxB4 and HoxB7. Reverse transcriptionpolymerase chain reaction analysis demonstrated that repression of Pbx1 by PLZF reduces the expression of HoxB4 and HoxB7 and their target genes, including tumor-associated neoangiogenesis factors such as basic fibroblast growth factor, angiopoietin-2, and matrix metalloprotease 9. These findings suggest that deregulation of the expression of Pbx1 and its binding partner Hox due to loss of PLZF expression contribute to the progression or pathogenesis of melanoma. 059 Diacylglycerol kinase a suppresses TNF-a-induced apoptosis of human melanoma cells through activation of NF-kB Kenji Yanagisawa1, Kowichi Jimbow1, Hideo Kanoh2, Fumio Sakane2 1

Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan; 2Second Department of Biochemistry, Sapporo Medical University School of Medicine, Sapporo, Japan Diacylglycerol kinase (DGK) is thought to regulate many cellular processes through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Mammalian DGK exists as a large protein family consisting of 10 isozymes. However, the physiological functions of DGK isozymes in melanoma cells remain unclear. We found that DGKa (type I isozyme) was expressed in several human melanoma cell lines but not in noncancerous melanocytes. To study melanoma-specific functions of DGKa, this isozyme was down-regulated or, conversely, overexpressed in AKI human melanoma cells. The overexpression of wild-type (WT) DGKa considerably suppressed tumor necrosis factor (TNF)-a-induced apoptosis of AKI cells (TUNEL-positive cell: 6.7%), compared with the vector control (20.3%). However, a kinase-dead (KD) mutant failed to affect the extent of the apoptosis (18.2%). Moreover, siRNA-mediated knockdown of DGKa markedly enhanced the apoptosis (10.2%) compared with the control RNA (4.0%). In contrast, overexpression and knockdown of other type I isozymes (DGKs b and g) had no detectable effects on the apoptosis. These results indicate that DGKa is uniquely involved in the suppression of AKI cell apoptosis induced by TNFa. The overexpression of DGKa-WT further enhanced the TNF-astimulated transcription activity of an anti-apoptotic factor, NFkB (2-fold increase), whereas DGKa-KD failed to affect the activity. Conversely, DGKa-knockdown considerably inhibited the TNF-a-dependent NF-kB activity (30% inhibition). Moreover, an NF-kB inhibitor, MG-132, blunted the anti-apoptotic effect of DGKa overexpression. These results collectively indicate that DGKa negatively regulates TNF-a-induced apoptosis through NF-kB activation. 060 Human hair follicle bulge is the stem cell niche: Bioinformatics approach using gene ontology to characterize molecular signature Manabu Ohyama1, Jonathan C. Vogel2, Masayuki Amagai1 1 Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; 2Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Analyses of genome-scale datasets derived from high-throughput technologies such as microarray platform require integrated bioinformatics tools to uncover the biological significance concealed in the data. To demonstrate the advantage of the Gene

Abstracts Ontology based bioinformatics approach for the analysis of microarray datasets, we tried to characterize the gene expression profile of human anagen hair follicle bulge cells using Web-based software. We generated Affymetrix Genechip microarrays from hair follicle bulge and non-bulge control cell populations accurately collected by laser capture microdissection. By comparing the Genechips obtained from individual populations, we identified the list of genes differentially expressed in bulge cells. To convert the divergent expression pattern of each individual gene into integrated biological interpretations, we tried to annotate and analyze those genes by DAVID (Database for Annotation, Visualization and Integrated Discovery) provided by National Institute of Allergy and Infectious Disease, NIH and NetAffyx Gene Ontology Mining Tool supplied by Affymetrix. When Gene Ontology was introduced, those programs clearly demonstrated that biological processes, such as the negative regulation of WNT or BMP signaling pathways involved in hair follicle morphogenesis, were overrepresented in bulge cells, while processes concerning cell proliferation and cell cycle were underrepresented. Thus, the molecular signature of bulge cells reflected the characteristics of the stem cell niche containing keratinocyte stem cells that are maintained in a quiescent state in anagen hair follicles. These findings demonstrate the importance of bioinformatics tools for the biological interpretation of large datasets obtained by highthroughput analyses. 061 Prolonged MHC classII expression and CIITA transcription in human keratinocytes Atsushi Takagi1, Chiharu Nishiyama2, Yusuke Niwa1, Sigaku Ieda1, Hideoki Ogawa1 1 Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 2Atopy Research Center, Juntendo University School of Medicine, Tokyo, Japan

Purpose: The MHC classII transactivator (CIITA) is a transcriptional co-activator that involves in MHC classII gene expression as a master regulator. Human CIITA gene expression is regulated by four promoters (pI, pII, pIII, and pIV) in a cell type specific manner. On the other hand, there is no report analyzing the usage of each CIITA promoter in primary human keratinocytes. Therefore, we monitored the expression level of each type of CIITA mRNA in stimulated human keratinocytes in the present study. Method: Surface expression of MHC classII on cultured normal human keratinocytes (NHK) purchased from KURABO was analyzed by FACS. CIITA mRNA expression level in NHK was analyzed by Real time PCR. Result: FACS analysis showed that IFN-g-stimulation induced HLA-DR expression on NHK surface and the expression level of HLA-DR on NHK was same as those of immuno-related professional APS including B cell line (Raji) and IFN-g-stimulated monocyte cell line (THP-1). The comparable amount of typeIV CIITA mRNA was detected in IFN-g-stimulated NHK and THP-1, and apparent expression of typeIII mRNA was observed in NHK. IFN-g induced HLA-DR expression level on keratinocytes was increased over 72 h, while that of THP-1 reached maximum at 24 h after stimulation. When the time-course of CIITA transcription level was analyzed, the amount of CIITA mRNA in keratinocytes at 24 h after stimulation was significant higher than that of THP-1. Conclusion: The present study demonstrates that CIITA is mainly transcribed from pIV and partly from pIII in primary NHK. In addition, above mentioned results suggest a possibility that long-life of typeIV CIITA mRNA or continuous transactivation of pIV may cause the prolonged expression of MHC classII on NHK as compared with other cell types.

Abstracts 062 Analysis of vaccination therapy with polyarginine-containing protein antigen Hiroshi Mitsui, Takashi Inozume, Naotaka Shibagaki, Shinji Shimada Department of Dermatology, University of Yamanashi, School of Medicine, Yamanashi, Japan We have previously shown that polyarginine (R9)-protein transduction domain (PTD), the most efficacious known PTD, induced more efficient protein transduction to dendritic cells (DC) in vitro than other PTDs studied including TAT-PTD. Immunization with R9-containing antigen (Ag)-treated DC resulted in efficient induction of Ag-specific immune responses mediated by CD8+ and CD4+ T cells, and in a superior antitumor effects. Our recent data demonstrated that R9-PTD-treated DC did not alter their phonotype, and that R9-PTD-protein was transduced by the endocytosis in the early phase. All these results indicated that R9-PTDmediated Ag delivery might facilitate efficient Ag cross-presentation. The purpose of this study was to analyze the immune responses by directly immunization with R9-PTD-containing protein Ag in vivo. For this purpose we generated a fusion proteins comprised of the OVA protein with (rR9-OVA)/without (rOVA) R9PTD. Multiple immunization with rOVA (100 mg) to naive C57BL/6 mice induced only IgG1-dominant anti-OVA Ab production (Th2). In contrast, immunization with rR9-OVA induced higher OVAspecific IgG2 Ab production and SIINFEKL-specific CTL. With this setting, when (OVA-expressing) EG.7-bearing mice were vaccinated with rOVA or rR9-OVA in vivo, tumor growth was temporally suppressed in both groups. However, co-administration with Th1inducible adjuvant (OK432) at peri-tumor site preferentially enhanced antitumor effects only in rR9-OVA-treated group. Our results indicated that efficacious vaccination with R9-PTD-containing Ag might preferentially elicits Tc1 cell activation. Thus, this simple vaccination approach without DC could offers theoretical and practical advantages to those that are in current use. 063 Interleukin-23 and interleukin-27 exert quite different antitumor and vaccine effects on poorly immunogenic mouse melanoma Shuntaro Oniki1, Hiroshi Nagai1, Takayuki Yoshimoto2, Tatuya Horikawa1, Chikako Nishigori1 1

Department of Dermatology, Kobe University Graduate School of Medicine, Hyogo, Japan; 2Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan Recent studies revealed that two novel IL-12-related cytokines, IL-23 and IL-27 have antitumor effects in animal models. However, the effects were mainly evaluated in highly immunogenic tumors, and have not been fully evaluated against non- or poorly immunogenic tumors. In this study, we investigated the antitumor efficacies of IL-23 and IL-27 on poorly immunogenic B16F10 melanoma. To enable of the two cytokines to be directly compared, B16F10 cells were transfected with mouse single chain IL23 or IL-27 cDNA which were cloned into the same expression vector. High level secreting clones of these two cytokines were selected by ELISA or biological assay of CD4+ cells with their culture supernatants (B16/IL-23 and B16/IL-27). In syngeneic mice, B16/IL-23 revealed antitumor effect only after about 20 days of tumor injection into the subcutaneous, and then showed growth inhibition or even regression. In contrast, B16/IL-27 tumors exhibited retardation of tumor growth from the early stage. In vivo depletion assay revealed that the antitumor effect

151 of B16/IL-23 was mainly mediated with CD8+T cells and IFN-g whereas that of B16/IL-27 was mainly with NK cells. Also the antitumor effects of B16/IL-23 and B16/IL-27 were synergistically enhanced by treatment with IL-18 and IL-12, respectively. Furthermore, only B16/IL-23-vaccinated mice developed protective immunity against parental B16F10 tumors. And, when combined with depletion of CD25+ regulatory T cells, most of the parental tumors were rejected. In conclusion, IL-23 and IL-27 exert antitumor effect and vaccine effects on poorly immunogenic melanoma in quite different manners. Our data indicate that IL-23 and IL-27 might play an important role in future cytokine-based approaches for the treatment of poorly immunogenic tumors. 064 Isotype-specific engagement of stimulatory and inhibitory FcgRs regulatesB lymphocyte depletion during CD20 antibody immunotherapy in mice Yasuhito Hamaguchi1, Kazuhiko Takehara1, Thomas Tedder2 1

Department of Dermatology, Kanazawa University School of Medical Science; 2Department of Immunology, Duke University Medical Center, Durham, USA CD20 mAb immunotherapy is effective for lymphoma and autoimmune disease, such as CD20+ cutaneous B cell lymphoma and pemhigus vulgaris. In a mouse model of CD20 immunotherapy, the innate monocyte network depletes B cells through IgG Fc receptor (FcgR)-dependent pathways. Moreover, 12 different mouse antimouse CD20 mAbs exhibit subclass-specific variability in B cell depletion in vivo, with a hierarchy of IgG2a/c > IgG1/ IgG2b > IgG3. To understand the molecular basis for these CD20 Ab subclass differences, bone marrow, blood, spleen and lymph node B cell depletion was assessed in mice deficient or blocked for stimulatory FcgRI, FcgRIII, FcgRIV, or FcR common g chain, or inhibitory FcgRIIB. IgG1 CD20 mAbs induced B cell depletion through preferential, if not exclusive interactions with low-affinity FcgRIII. IgG2b CD20 antibodies interacted preferentially with intermediate-affinity FcgRIV. The potency of IgG2a/c CD20 mAbs may have resulted from FcgRIV interactions, with contributions from high-affinity FcgRI. Regardless, FcgRIV could mediate IgG2a/b/c CD20 mAb-induced depletion in the absence of FcgRI and FcgRIII. By contrast, CD20 mAb-induced B cell depletion by was significantly enhanced by inhibitory FcgRIIBdeficiency. In contrast to other tissues, both FcgR-dependent and -independent pathways contributed to mature bone marrow and circulating B cell depletion by CD20 mAbs. Thus, isotype-specific mAb interactions with distinct FcgRs contributes significantly to the effectiveness of CD20 mAbs in vivo, which may have important clinical implications for CD20 and other mAb-based therapies. 065 ¨gren’s syndrome Identification of specific autoantigens in Sjo by SEREX Yoichi Akita1, Ken-ichi Kozaki2, Kazuo Uchida3, Yoshiaki Kazaoka4, Shigeyoshi Fujiwara5, Shiro Yamada4, Kazuo Shimozato3, Daisuke Watanabe1, Yasuhiko Tamada1, Yoshinari Matsumoto1 1 Department of Dermatology, Aichi Medical University School of Medicine, Aichi, Japan; 2Department of Genome Medicine, Hard Tissue Genome Research Center, Tokyo Medical and Dental University, Tokyo, Japan; 3Second Department of Oral and Maxillofacial Surgery, Aichi Gakuin University School of Dentistry, Aichi, Japan; 4Department of Oral and Maxillofacial Surgery, Aichi

152 Medical University School of Medicine, Aichi, Japan; 5Department of Oral and Maxillofacial Surgery, Japanese Red Cross Nagoya First Hospital, Aichi, Japan We carried out SEREX (serological analysis of antigens by recombinant cDNA expression cloning) using sera from patients with Sjo ¨gren’s syndrome (SjS) and investigated the frequencies of autoantibodies against autoantigens identified by SEREX in sera of healthy individuals (HI) and patients with SjS, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). IFI16 and two kelch-like proteins, KLHL12 and KLHL7, were found to be novel autoantigens in SjS by SEREX. A marked high frequency of anti-IFI16 autoantibodies was observed in the sera of SjS (SjS, 70%; RA, 13%; SLE, 33%; HI, 0%). Interestingly, all serum samples from SjS demonstrated immunoreactivity against one or both of IFI16 and SS-B/La. The presence of autoantibodies against KLHL12 and KLHL7 in sera was significantly specific to SjS (23%, 17%, respectively), not detected in RA, SLE and HI. Furthermore, we confirmed that transcripts of these autoantigens were expressed preferentially in salivary glands and immunoprivileged testes. Our results suggest these autoantigens may be useful as serological markers for the clinical diagnosis of SjS and may play a crucial role as organ-specific autoantigens in the etiopathogenesis of SjS. This study warranted clinical evaluations of autoantibodies against IFI16, KLHL12 and KLHL7 in combination with antiSS-B/La autoantibodies. 066 CINCA syndrome-related mutation of CIAS1 induces cathepsin B-dependent rapid necrosis-like cell death to THP-1 human monocytes Akihiro Fujisawa, Naotomo Kambe, Nobuo Kanazawa, Yoshiki Miyachi Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan Background: CINCA (chronic infantile neurologic, cutaneous, articular syndrome) is known as a severe autoinflammatory disease characterized by neonatal-onset urticaria-like rash, CNS dysfunctions and arthritis. The responsible gene is CIAS1, which has also been associated with 2 less severe but phenotypically similar familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS). Therefore, these 3 disorders are believed to form a same spectrum of the autoinflammatory disease. The product of CIAS1, cryopyrin, mainly localizes in the cytosol of monocytes. Cryopyrin is estimated to recognize specific motif of microorganism with its leucine-rich repeats, but the ligand has still not been identified. However, each diseaserelated variants of cryopyrin is thought to mimic active conformation change induced by a microbial ligand, resulting in the precipitation of NF-kB regulation and induction of IL-1b maturation. Results: In this study, we showed that each disease-related variants of cryopyrin provoked rapid cell death into THP-1 cells. Interestingly, the intensities of cell death induced by CIAS1 variants reflected the disease activity of FCAS, MWS, and CINCA, as well as their abilities of NF-kB regulation. In addition, the characteristic features of cell death were more likely to necrosis, and they were effectively blocked with the specific inhibitor against lysosomal enzyme cathepsin B, CA-074-Me. Conclusion: These results indicates that cryopyrin, when activated, participates in the rapid, cathepsin B-dependent necrosis-like cell death, which can evoke the inflammation to alert to the invasion of microorganism. This is very interesting and reasonable when we think the function of cryopyrin as the intracellular pattern recognition receptor.

Abstracts 067 Production of unique biologically active interleukin-18 species by human mast cell chymase Youichi Omoto1, Kazuya Tokime1, Keiichi Yamanaka1, Tatsuhiko Morioka1, Ichiro Kurokawa1, Hiroko Tsutsui2, Kiyohumi Yamanishi3, Kenji Nakanishi2, Hitoshi Mizutani1 1 Department of Dermatology, Mie University, Graduate School of Medicine, Mie, Japan; 2Department of Immunology & Medical Zoology, Hyogo College of Medicine, Hyogo, Japan; 3Department of Dermatology, Hyogo College of Medicine, Hyogo, Japan

Cutaneous overproduction of interleukin-18 (IL-18) induces atopic dermatitis (AD)-like skin lesions, and IL-18 has been involved in the pathogenesis of AD. IL-18 produced as an inactive pro-form needs processing by specific enzyme for activation. Caspase-1 is a known specific activator of IL-18. However, caspase-1 knockout mice still exhibit biologically active IL-18 species and express AD like features. Additionally, the normal keratinocytes produces proIL-18 but not process or releases functional IL-18 species. Therefore, presence of an alternative IL-18 activation pathway has been suggested. Chymase is a specific enzyme in connective tissueassociated mast cell that accumulates in the skin lesions of AD patients and experimental mouse models of AD. Here, we evaluated the function of human mast cell chymase in IL-18 activation. Recombinant human mast cell chymase rapidly cleaved recombinant proIL-18 at 56-phenylalanine and produced a novel stable IL18 specie with smaller MW (16 kD) than reported IL-18. This p16 showed biological activity, which was abrogated by chymase inhibitors. Mast cell chymase forms an IL-18 paracrine network in skin. The human mast cell chymase and the unique IL-18 peptide will be novel therapeutic targets for AD and IL-18-mediated disorders. 068 Functional analyses of effects of osteopontin on cutaneous mast cells Akiko Nagasaka1, Hiroyuki Matsue1, Rui Aoki1, Susan R Rittling2, Shigeyuki Kon3, Toshimitsu Uede3, Shinnji Shimada1 1 Department of Dermatology, University of Yamanashi, Yamanashi, Japan; 2Department of Genetics, Rutgers University, Piscataway, NJ, USA; 3Institute for Genetic Medicine, Hokkaido University, Hokkaido, Japan

Osteopontin (OPN) is a glycosylated protein found in the extracellular matrix of bone, and is now known to have multiple biological functions. It serves not only as a pleiotropic cytokine for Th1 immunity, but also as a chemokine that attracts many immune cells. Thus, OPN is believed to be involved in many aspects of pathogenesis of immune and inflammatory diseases, including allergic contact dermatitis. On the other hand, mast cells are also involved in many pathological aspects of those diseases by secreting multiple factors. However, it remains unknown whether mast cells produce OPN. We hypothesized that mast cells may secrete OPN and this multifunctional protein may affect mast cell functions. To test this, we used murine fetal skinderived culture mast cells (FSMC) and bone-marrow-derived mast cells (BMMC). Using RT-PCR and ELISA, OPN was detected only in FSMC but not in BMMC at mRNA and protein levels. In the presence of mast cell growth factors, FSMC have been similarly generated from both OPN-deficient (/) and -sufficient (+/+) mice without significant differences of yields, purities, granularities, and viabilities. However, without the growth factors, FSMC derived from / mice showed significant apoptosis, compared to those from +/+ mice. In addition, we found that OPN attracted FSMC in a CD44-dependent fashion and it augmented IgE-mediated degra-

Abstracts nulation by FSMC. These results indicated that cutaneous mast cells secrete OPN, which affects many aspects of mast cell functions including cell survival, chemotaxis, and degranulation. Our study suggests that OPN becomes a new member of multiple mediators secreted by mast cells, and it may exert multiple functions to immune cells including mast cells in many physiological and pathological settings. 069 Human umbilical cord epithelial cells express epidermal keratins and cornified envelope proteins, and show epidermal-like differentiation William Ng1,2, Masayuki Mizoguchi1, Chiharu Nishikawa2, Yasushi Suga1, Hideoki Ogawa2, Shigaku Ikeda1 1 Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 2Atopy Research Centre, Juntendo University School of Medicine, Tokyo, Japan

Human umbilical cord epithelia and the human epidermis are anatomically and developmentally related epithelial tissues. Human umbilical cord epithelia display areas of stratification and has been previously shown to express epidermal related keratins. To further elucidate the biology of human umbilical cord epithelial cells (HUCEC) in relation to human epidermal keratinocytes (HEK), we isolated and expanded HUCEC in culture. In monolayer culture, HUCEC displayed similar morphological characteristics to HEK, and expressed epidermal keratins 5, 14 and 10, with weak or no expression of suprabasal keratins 1 and 2e at the mRNA level. Immunofluorescence of monolayer HUCEC confirmed strong expression of keratins 5 and 14, with occasional positivity for keratin 10 but negative expression for keratins 1 and 2e. In order to study the differentiation program of HUCEC, HUCEC were placed in organotypic cultures consisting of deepidermalized, acellular human dermis with an intact basement membrane that had been placed on a collagen matrix containing early passage human dermal fibroblasts. After 10 days of culture in the presence of an elevated calcium concentration with a period of exposure to air, HUCEC formed a well-differentiated stratified squamous epithelium with a distinct cornified layer, reminiscent of the human epidermis. This was accompanied by the mRNA expression of cornified envelope related proteins filaggrin, involucrin, loricrin and transglutaminase, and their corresponding protein expression was detected in the upper stratified cell layers by immunofluorescence. In order to investigate their differentiation program in vivo, HUCEC in submerged organotypic culture on acellular human dermis were transplanted onto full thickness wounds on athymic mice. 070 Mesenchymal stem cells differentiate into multiple skin cell types and contribute to wound repair

153 isolated from mouse bone marrow and cultured in various conditioned media containing human epidermal growth factor and stained with a keratinocyte marker (keratin 14). We found K14(+) cells (0.4%), namely keratinocytes that transdifferentiated from MSCs. Secondary, we assessed whether MSCs can differentiate into keratinocytes and/or other skin cells in vivo. GFP transgenic mouse-derived MSCs were intravenously injected, then skin wound sites were biopsied and stained with cell specific markers. We detected GFP+ and the cell specific markers for macrophages (CD11b+), myelogenic cells (CD45+), endothelial cells (CD31+) or keratinocytes (keratin+), suggesting that MSCs can differentiate into multiple skin cell types in vivo. Finally, we investigated the contribution of MSCs to wound repair. MSCs injected-mice repaired wound more rapidly than control mice (P < 0.05). Taken together, we have demonstrated that MSCs can differentiate into keratinocytes, and contribute to wound repair via MSCs differentiation into various skin cell types. 071 Characterization of type XII collagen as an adhesive ligand for keratinocytes Hiromi Sengoku, Hironobu Tsugeno, Katsushi Owaribe, Yoshiaki Hirako Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Hemidesmosomes (HDs) are cell-substrate adhesion structures found in stratified and complex epithelia. Integrin alpha6beta4 and type XVII collagen are major transmembrane adhesion receptors of HDs. The extracellular ligand for the integrin is laminin-5, which interacts with type VII collagen thereby connecting HDs to collagen fibrils. We have been characterized HD components using HD-rich fraction isolated from bovine corneas. A group of monoclonal antibodies raised against the HD fraction stain the basement membrane and underlying extracellular matrix in a pattern different from laminin-5 and type VII collagen. These antibodies recognized 1,000- and 350-kDa polypeptides of the HD fraction. To identify the antigen, we purified the polypeptides from corneal stroma. N-terminal sequencing of the purified protein revealed it as type XII collagen. Cell adhesion assay showed that type XII collagen promoted adhesion of a cultured human squamous carcinoma cells (DJM-1). Moreover, when corneal stratified epithelia were dissociated from stroma by EDTA treatment, subpopulation of the type XII collagen molecules remained at the basal side of the epithelia while laminin-5 and type VII collagen were only found at stroma. In contrast, when the epithelia were separated from stroma by collagenase or dispase treatment, type XII collagen was lost from epithelia while laminin-5 and type VII collagen were found to associate to the basal side of the separated epithelial sheets. These results suggest that type XII collagen interacts with HDs in a different way from laminin-5 and type VII collagen.

Mikako Sasaki, Riichiro Abe, Daisuke Inokuma, Satomi Ando, Hiroshi Shimizu

072

Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Ectopic DSG1 compensates for the loss of DSG3-mediated adhesion in DSG3 deficient mice

Mesenchymal stem cells (MSCs) differentiate not only into mesenchymal lineage cells such as adipocytes, osteocytes, chondrocytes but also into various cell lineage including hepatocytes and neural cells. As MSCs can easily be established from bone marrow, MSCs are expected to be applied to various tissue engineering strategies. However, it is currently not known if MSCs can differentiate into keratinocytes. In this study, we assessed whether MSCs can differentiate into keratinocytes. MSCs were

Tsuyoshi Hata1,4, Koji Nishifuji1, Kouji Shimoda2, Taketo Yamada3, Takeji Nishikawa1, Masayuki Amagai1 1 Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; 2Laboratory Animal Center, Keio University School of Medicine, Tokyo, Japan; 3Pathology, Keio University School of Medicine, Tokyo, Japan; 4Fundamental Research Center, KOSE Corporation, Tokyo, Japan

154 We have recently developed an active disease mouse model for pemphigus vulgaris (PV) by adoptive transfer of splenocytes from Dsg3/ mice, which have mix genetic background of C57BL/6J (B6) and 129/Sv, into B6-Rag2/ recipient mice that express Dsg3. However, when we backcrossed the Dsg3/ mice with B6 mice, B6-Dsg3/ mice died out by 4 w/o probably due to severe oral erosions. In this study, to rescue the B6-Dsg3/ mice we generated transgenic mice (K5Dsg1Tg+) expressing ectopic Dsg1a in the lower epithelia under the control of keratin 5 promoter. K5Dsg1Tg+ mice showed positive staining of Dsg1a on the cell surface of keratinocytes in the lower epidermis, oral epithelia and in telogen hair follicles. To evaluate the compensatory effect of ectopic Dsg1a on the loss of adhesive function of Dsg3, K5Dsg1Tg+Dsg3+/+ mice were injected with AK23 anti-Dsg3 IgG mAb that is capable of inducing blisters. While AK23 mAb induced severe weight loss and telogen hair loss in wild type mice, these changes were significantly suppressed in K5Dsg1Tg+ mice. The degree of the suppression was correlated with the expression level of ectopic Dsg1a Furthermore, when K5Dsg1Tg+ mice were bred to B6-Dsg3/ mice, the survival rates of K5Dsg1Tg+Dsg3/ mice improved to 50% (3/6) at 6 w/o while those of B6-Dsg3/ mice were 0% (0/56). K5Dsg1Tg+Dsg3/ mice didn’t show apparent oral suprabasilar acantholysis nor telogen hair loss. These findings indicated that ectopic Dsg1a in the lower epithelia could compensate in vivo for the impaired Dsg3-mediated adhesion induced both by pathogenic antibody and genetic ablation. K5Dsg1Tg+Dsg3/ mice will enable us to obtain PV model mice with B6 genetic background for further defined immunological analysis. 073 Alternation of p120ctn phosphorylation and of p120ctn binding to Desmoglein 3 (Dsg3) induced by anti-Dsg3 monoclonal antibodies Yuki Kawasaki1, Yumi Aoyama1, Kazuyuki Tsunoda2, Masayuki Amagai2, Yasuo Kitajima1 1

Department of Dermatology, Gifu University School of Medicine, Gifu, Japan; 2Department of Dermatology, Keio University School of Medicine, Tokyo, Japan We have previously reported that pemphigus vulgaris (PV)-IgG induced phosphorylation of Dsg3, decreased Dsg3 on cell surface, and formed Dsg3-depleted desmosomes in cultured keratinocytes. Furthermore the cell treatment with the potent pathogenic monoclonal antibody against Dsg3 also decreased the amount of Dsg3 in cultured keratinocytes. Four monoclonal antibodies against Dsg3 (AK mAbs) which have different epitopes and activities to disrupt of intracellular adhesion in keratinocytes, induced decrease of Dsg3 in different levels. Although its precise mechanisms have been unclear, we have proposed the involvement of any intracellular signal transductions by binding of autoantibodies to Dsg3. Here we examined whether each AK mAbs alters phosphorylation and association of Dsg3 and p120catenin (p120), which we recently found to binds cytoplasmic region of Dsg3. Cell lysates of AK mAbs-treated human squamous cell line, were immunopricipitated with PV-IgG and then subjected to Western blotting. AK mAbs did not induce tyrosine phosphorylation of Dsg3, but other tyrosine-phosphorylated proteins were detected. Those phosphorylated proteins were detected with anti-p120 antibody and anti-phospho p120 antibody indicating phosphorylation of Dsg3-associated p120. Phosphorylated p120 revealed to vary in size according to the different AK mAbs treatments, indicating binding of AK mAbs to different epitopes induced phosphorylation at different sites of p120 and/or of different p120 isoforms. These results suggest that

Abstracts each AK mAb activates different signaling pathways by binding to different epitopes, which might be a clue to reveal regulation of cell-cell adhesion in keratinocytes. 074 Control mechanism in keratinocyte differentiation via P51 Eisaku Ogawa, Ryuhei Okuyama, Yoshiyuki Kusakari, Teie Egawa, Akira Hashimoto, Hiroshi Watanabe, Hachiro Tagami, Setsuya Aiba Departmet of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan p51, a homolog of the tumor suppressor p53, is chiefly expressed in epithelial tissue, including epidermis. Similar to p53, p51 affects cell death whereas it plays important roles in development of epithelial tissues and maintenance of immature epithelial cells involving stem cells. However, it remains to be elucidated how p51 controls epithelial cells. In order to clarify p51 functions, we have focused on effects of p51 on keratinocyte differentiation. We examined differentiated cell characters when p51 was overexpressed in primary mouse keratinocyte. p51 suppressed loricrin and filaggrin expressed in upper spinous and granular layers. On the other hand, against expectation p51 increased the amount of keratin 1 which start to be expressed from suprabasal layer. Although basal keratinocytes have high amount of p51, they do not start keratin 1 induction. This discrepancy suggests existence of suppressive factor(s) against keratain 1 induction specifically in basal keratinocytes, not suprabasal layer. Here we present our analysis about control of keratinocyte differentiation though p51 and the suppressive factor(s). 075 Control mechanism in keratinocyte commitment via notch and P51 Ryuhei Okuyama, Eisaku Ogawa, Yoshiyuki Kusakari, Teie Egawa, Akira Hashimoto, Hiroshi Watanabe, Hachiro Tagami, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan Notch family receptors play crucial roles in cell fate determination in various cell systems. Mammalian Notch, in particular, was shown to play multiple roles in maintaining cells as undifferentiated progenitors in some tissues and promoting differentiation in others. In epidermis, Notch1 is highly expressed in basal and spinous layers, and its functional importance in these cells is supported by ample lines of evidence. However, its observed high expression in the basal cells characterized by active proliferation and high integrins expression was inconsistent with the evidence that the activation of Notch1 causes growth arrest and downregulation of integrins expression in keratinocytes, which suggests existence of suppressive factor(s) against Notch function specifically in basal keratinocytes. p51, p53 homolog, is expressed chiefly in basal keratinocytes, and is suggested as not only a marker for keratinocyte stem cells but also a prerequisite for the epidermal development. We demonstrate that p51 maintains undifferentiated cell characters through suppression of Notch activity. Notch activity was decreased by p51 overexpression, whereas its activity was increased together with p51 down-regulation by specific siRNA. Moreover, co-expression of p51 with Notch1 into keratinocytes showed that p51 restored undifferentiated cell characters against Notch1 expression. Our data demonstrate that p51 keeps keratinocytes in undifferentiated state through the inhibition of Notch signal, and the

Abstracts balance between these two molecules tightly connects with determining keratinocyte cell commitment. 076 SCCA1 plays critical roles in UV stress responses and regulates keratinocyte death and proliferation Chika Katagiri1, Jotaro Nakanishi1, Eiji Yoshinaga2, Seiichi Izaki2, Toshihiko Hibino1 Shiseido Life Science Research Center; 2Department of Dermatology, Saitama Medical Center, Saitama Medical University 1

Our previous study revealed that squamous cell carcinoma antigen 1 (SCCA1) plays a critical role in UV-induced apoptosis in human keratinocytes via the suppression of c-Jun N-terminal kinase (JNK). In the present study, we further investigate the relationship between SCCA1 and JNK isoforms or other mitogen activated protein kinases (MAPK). In addition, we report the roles of SCCA1 in cell death and proliferation. First we examined effects of SCCA1 on the MAPK family members, including JNK13, ERK1/2, and p38a kinases. In the presence of SCCA1, JNK1 activity on c-Jun phosphorylation was markedly reduced. In contrast, kinase activities of JNK2, JNK3, ERK1/2 or p38a were not affected by SCCA1. SCCA1 was detected in the keratinocyte extract precipitated with anti-JNK1 antibody after UV irradiation, indicating the binding of SCCA1 with JNK1. In SCCA knockdown HaCaT cells established with siRNA, UV-induced apoptosis strongly increased. In this condition, c-Jun phosphorylation was significantly decreased. Furthermore, we established 20 individual clones that stably express SCCA1 using 3T3/J2 cells (1—2800fold expression). SCCA1-expressing clones showed a strong correlation between the expression levels and the suppression of UVinduced apoptosis. Interstingly, they also showed a strong correlation between the expression levels and the proliferation. In psoriasis vulgaris, SCCA1 and JNK1 dramatically increased in the upper layers of the lesional epidermis, whereas JNK2 or JNK3 showed little changes. Our data suggest that the up-regulation of SCCA1 in the psoriatic epidermis suppresses death-inducing JNK1 activity and also contributes to the keratinocyte proliferation. SCCA1 may play important roles in the pathogenesis of psoriasis. 077 CCL27/CTACK production induced by TNF-alpha is suppressed by IL-17 via inhibition of IRF1 transcriptional activity Mikiko Tohyama, Yasushi Hanakawa, Eriko Tan, Teruko Tsuda, Sho Tokumaru, Yuji Shirakata, Koji Sayama, Koji Hashimoto The Department of Dermatology, Ehime University School of Medicine, Ehime, Japan CCL27/CTACK is produced specifically by epidermal keratinocytes, and the production is enhanced by TNF-a stimulation. Interestingly, the CCL27 mRNA expression is not augmented in the epidermis of psoriasis, although it is well known that TNF-a plays an important role in psoriasis pathologically. Since IL-17 mRNA is detected in the epidermis of psoriasis skin but not normal skin, we hypothesized that IL-17 suppress CCL27 production. At first, we studied the mechanism of CCL27 production induced by TNF-a A transfection of mutant IkBa completely suppressed CCL27 production caused by TNF-a, indicating that the activation of NF-kB is essential for CCL27 mRNA expression. However, CCL27 mRNA started to increase 4 h after stimulation with TNF-a, and pretreatment of cultured keratinocytes with cyclohexamide completely suppressed the CCL27 mRNA expression. These findings suggest that another transcription factor induced by the activa-

155 tion of NF-kB is involved in the expression of CCL27 mRNA. An addition of IL-17 strongly suppressed CCL27 production in response to TNF-a. Previously, a significant suppression of RANTES production was reported under the same condition, and it has been suggested that transcriptional activity of IRF1 was involved. The present study showed that NF-kB mediates IRF1 production and translocation to the nucleus, and IL-17 suppressed IRF1 translocation to nucleus. To test the involvement of IRF1 in CCL27 production, we examined the effect of IRF1 siRNA on CCL27 production after stimulation with TNF-a Addition of IRF1 siRNA significantly suppressed CCL27 production. Taken together, NF-kB/IRF1 signaling pathway is involved in the production of CCL27 induced by TNF-a, and IL-17 suppresses CCL27 production via inhibition of IRF1 transcriptional activity. 078 Pivotal role of PPARg/C/EBPa signaling pathway in 1, 25-dihydroxyvitamin D3-induced keratinocyte differentiation Xiuju Dai, Yuji Shirakata, Sho Tokumaru, Yasushi Hanakawa, Mikiko Tohyama, Lujun Yang, Koji Sayama, Koji Hashimoto Department of Dermatology, Ehime University School of Medicine The active Vitamin D3 regulates cell proliferation and differentiation. In epidermal keratinocytes, 1, 25-dihydroxyvitamin D3 (1, 25(OH)2D3) suppresses cell proliferation and induces keratinocyte differentiation upon binding to its receptor. Peroxisome proliferation-activated receptors (PPARs) are ligand-activated transcription factors that regulate the expression of target genes involved in many cellular functions including cell proliferation, differentiation and immune/inflammation response. Among the members of PPARs, PPARg activation has been demonstrated to stimulate keratinocytes differentiation and induce involucrin gene. The purpose of this study is to investigate whether PPARg/C/EBPa signaling pathway is involved in keratinocyte differentiation induced by 1, 25(OH)2D3. Keratinocytes were cultured under serum free condition. mRNA expression of involucrin and loricrin was analyzed by RT-PCR. To suppress PPARg activity, we construct adenovirus carrying the dominant negative form of PPARg (Ax-dn-PPARg), which blocks the binding of ligand to PPARg and inhibits its transcription activation.1, 25(OH)2D3 induced rapid expression of PPARg, not other members of PPAR family, as well as C/EBPa, the downstream molecule of PPARg. The induction of PPARg and C/EBPa preceded the expression of differentiation markers, involucrin and loricrin in 1, 25(OH)2D3treated cultured normal human keratinocytes. Transfection of Ax-dn-PPARg suppressed the induction of involucrin and loricrin by 1, 25(OH)2D3. Furthermore, treatment with HX-531, the inhibitor of PPARg/RXR, also inhibited 1, 25(OH)2D3-induced involucrin expression. Taken together, for the first time, our study demonstrates that PPARg/C/EBPa signaling pathway plays a pivotal role in VD3-induced kratinocyte differentiation. 079 A clue for Epstein-Barr Virus (EBV) reactivation in cutaneous lesions determined by a novel, non-invasive technique for latent EBV infection Takenobu Yamamoto, Kazuhide Tsuji, Daisuke Suzuki, Keiji Iwatsuki Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Background: Because EBV-associated cutaneous disorders usually occur in childhood, it is not easy to obtain a skin biopsy.

156 Purpose: To establish a non-invasive, diagnostic method for latent EBV infection, and investigate EBV reactivation in the skin lesions. Materials and methods: Seven crust samples with EBV-associated cutaneous lesions and 23 crust samples of control diseases were used. The presence of latent EBV infection was diagnosed by routine in situ hybridization for EBV-encoded small RNA (EBER). Biopsy specimens from patients with or without latent EBV infection were also used. Total RNA was extracted from the crusts and control materials, and converted to cDNA. In order to detect the latency-associated EBV gene transcripts, the cDNA samples were amplified by PCR using specific primer sets for EBER1 and BARTs, respectively. In order to detect EBV reactivation, BZLF1 mRNA was also examined by PCR. Results: Intact RNAs were successfully extracted from the crusts as well as control materials. EBER1 and BARTs mRNAs were detected in the EBV-associated cutaneous diseases. The sensitivity and specificity of our assay for latent EBV infection was 100% and 95.8% for EBER1 mRNA, and 85.7% and 100% for BARTs mRNA, respectively. Although no BZLF1 mRNA was found in the skin lesions from patients with hydroa vacciniforme, we could detect it in the skin lesions of a high risk group, including hypersensitivity to mosquito bites, severe HV and NK/T cell lymphoma. Conclusions: We detected latency-associated transcripts of EBV in the crusts of EBV-associated cutaneous diseases with a high specificity and sensitivity. Using this non-invasive method, we obtained a clue for the presence of EBV reactivation (BZLF1 mRNA) in the skin lesions of patients with a high malignant potential. 080 Invasion activity of a Mycobacterium leprae peptide presented by the Escherichia coli AIDA autotransporter Maho Kanoh, Takao Fujimura, Naoya Sato, Mikio Masuzawa, Kensei Katsuoka Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan In leprosy, percutaneous thick contact and respiratory route are thought to be the principal route of infection with M. leprae. However, the details still remains unclear. Mycobacterium cell entry protein (MceP) is a protein encoded by mce1 region of the M. tuberculosis complex and is associated with epithelial cell entry. We found the region in M. leprae that have highly homologous with the mce1 of M. tuberculosis and recombinantly expressed it in E. coli. The recombinant Mce1A protein of M. leprae (r-lep-Mce1A) had an activity of entry into epithelial cells and suggested to be an important virulence factor associated with epithelial cell entry. Our previous study using r-lep-Mce1A which were deleted at N-terminus and/or C-terminus amino acid residues of full length 47kDa Mce1A indicated the active sequence associated with epithelial cell entry was located 316—921 bp of mce1A region. In our new study, we expressed the active sequence in E. coli on their surface as a fusion to the Adhesin Involved in Diffuse Adherence (AIDA) autotransporter translocator and tested whether they invade epithelial cells. DNA fragments of mce1A region (316—531, 532—753, 754— 921 bp) were cloned into pMK90 vector and expressed in UT4400 E. coli The recombinant E. coli were added to the BEAS-2B cell monolayer compared to UT4400. Following incubation, the cells were washed and the invasion to the cells of the recombinant E. coli was determined by electron microscopy and colony forming units. The recombinant E. coli expressing 316—531 bp on their surface showed their ability to invade BEAS-2B cells whereas UT4400 did not. It suggests that expression of a part of mce1A

Abstracts region (positions 316 to 531) of M. leprae on the surface conferred on a nonpathogenic E. coli an ability to invade epithelial cells. 081 Mechanisms of intraepidermal neurite formation in atopic dermatitis Mitsutoshi Tominaga1, Sumiko Ozawa2, Kenji Takamori1,2 1 Institute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan; 2 Department of Dermatology, Juntendo University Urayasu Hospital, Chiba, Japan

Pruritus is a common symptom in various dermatosis. The pruritus of atopic dermatitis (AD), which is characterized by dry skin, is uncontrolled by the administration of antihistamines. Therefore we investigated the mechanism of pruritus of AD in relation to the nerve fibers in the epidermis. Our previous studies revealed that many nerve fibers existed in epidermis of AD and xerosis, raising the possibility that neuritogenesis in epidermis is related to itching in the skin. However, the precise mechanisms of intraepidermal neurite formation are poorly understood. In this study, we analyzed the mechanisms of neurite formation using NC/Nga mice as a model of AD. The mouse spontaneously manifests ADlike skin lesions with marked elevation in plasma levels of total IgE when they are raised in air-unregulated conventional circumstance. Immunohistochemical studies revealed that many intraepidermal neurites (PGP9.5-positive) were observed in specificpathogen free (SPF)-NC/Nga mice when compared to ICR mice. The density of intraepidermal neurites in conventional (Conv)NC/Nga mice was significantly higher than that of SPF-NC/Nga mice. Expression of NGF and amphiregulin (AR) was increased in epidermis of Conv-NC/Nga mice in comparison with SPF-NC/Nga mice. Gelatinase activity was remarkably increased in epidermis of Conv-NC/Nga mice over ICR and SPF-NC/Nga mice. Furthermore expression of E-cadherin and ZO-1 was decreased in epidermis of Conv-NC/Nga mice than that of ICR and SPF-NC/Nga mice. These results suggest that NGF, AR and gelatinase is related to intraepidermal neurite formation in Conv-NC/Nga mice affected AD, and downregulation of E-cadherin and ZO-1 expressions in epidermis of the mice may give the spaces in among keratinocytes for neurite formation. 082 Somatic mosaicism of CIAS1 recognized in a cinca syndrome patient Naotomo Kambe, Akihiro Fujisawa, Yoshiki Miyachi Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan Objective: Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM #607115) is a severe inflammatory disease with mutation in CIAS1, which has also been associated with 2 dominantly inherited disorders, familial cold autoinflammatory syndrome (MIM #120100) and Muckle-Wells syndrome (MIM #191900). However, CIAS1 mutations have only been detected in half of the patients and it remains unclear which genes are responsible for the remaining patients. A patient: The patient was a 15-year-old boy born by normal vaginal delivery with no consanguinity. He had persistent urticaria-like rash since the neonatal period, chronic arthritis, contraction of the knee joints and growth retardation (height 8.5 SD). He had CNS dysfunctions, such as chronic meningitis,

Abstracts papilledema and sensorineural deafness, but showed normal mental development. Laboratory findings showed persistent elevations of CRP and marked leukocytosis. Results: Our patient has one heterologous SNP 587G>A (S196N) and one heterologous mutation 1709A > G (Y570C) in exon 3 of CIAS1. The latter mutation was found to occur as somatic mosaicism. PBMCs from the patient produced a large amount of IL-1b in the absence of stimulation, unlike those from normal controls or from his mother with S196N polymorphism. In addition, Y570C (with or without S196N) induced ASC-dependent NF-kB activation, unlike WT or S196N alone. Conclusion: From this patient, we have learned that somatic mosaicism of CIAS1 is sufficient to cause the clinical manifestations of CINCA syndrome although the clinical phenotype is less severe. This case also reveals that somatic mosaicism is one reason why CIAS1 mutations have not been detected in some CINCA syndrome cases. 083 Complete loss of Langerhans cells in the lesional skin in acrodermatitis enteropathica with novel SLC39A4 mutation Yuumi Nakamura1, Tatsuyoshi Kawamura1, Youichi Ogawa1, Masao Furuhashi1, Hajime Nakano2, Katsumi Hanada2, Shinji Shimada1 1 Department of Dermatology, University of Yamanashi, Yamanashi, Japan; 2Department of Dermatology, Hirosaki University School of Medicine, Aomori, Japan

Acrodermatitis enteropathica (AE) is a rare autosomal recessive disease that manifests with low serum zinc levels and acral dermatitis. Recently, SLC39A4 gene has been revealed to encode a ZIP4 protein (zinc transporter) and several SLC39A4 mutations have been demonstrated in AE patients. In this study, we identified a novel premature termination codon mutation in SLC39A4 gene in a case of AE. Sequencing of PCR products spanning exon 9 revealed a homozygous 1438 G to T. To date, the pathogenesis of immune dysfunction in AE remains largely unknown. Interestingly, histological examinations in specimens of AE lesions demonstrated individual necrosis of dendritic cells in the epidermis. We hypothesized that Langerhans cells (LC) undergo extensive apoptosis in AE lesions. To justify this hypothesis, we examined various immunohistochemistry with specimens of AE lesions. Control skin specimens were taken from the patients with atopic dermatitis and psoriasis. We found that: (1) Langerin antigens on LC were completely absent in the epidermis of AE lesion, but not in controls. (2) HLA-DR antigens on LC were also absent in AE specimens, but were present on the surface of dermal mononuclear cells. (3) cleaved caspase-3 positive dendritic cells were detected in the epidermis of AE lesion, but not in controls. (4) TGFb1 expression levels were severely decreased in the epidermis of AE lesion, when compared to controls. Taken together, these results indicate that LC destruction in AE occurs as a result of apoptosis presumably due to the absence of TGFb1 which is a critical endogenous growth factor of LC survival. We have noted the absence of LC in lesional skin in AE, thus providing evidence that this defect may be an important mechanism involved in AErelated immune dysfunction. 084 NY-ESO-1 protein-based immunotherapy for malignant melanoma: Tumor antigen-specific CTL induction and suppressive phenomenon Kazuhide Tsuji1, Toshihisa Hamada1, Yayoi Tokuyama1, Osamu Yamasaki1, Eiichi Sato2, Akiko Uenaka3, Eiichi Nakayama3, Keiji Iwatsuki1

157 1

Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Pathology, Tokyo Medical University, Tokyo, Japan; 3Immunology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

Hydrophobized polysaccharide, pullulan (CHP) is a novel, simple protein delivery system, which is designed to form complexes with soluble protein molecules. The protein formulated in CHP enter dendritic cells (DCs) by phagocytosis, and then the truncated protein is processed through a pathway similar to that for endogenous proteins. DCs sensitized by these complexes can prime and boost both antigen-specific CD4+T cells and CD8+T cells. The NY-ESO-1 human tumor antigen is a cancer-testis antigen expressed by various neoplasms, including melanoma. We report a case of malignant melanoma treated with the NYESO-1 protein formulated in CHP (CHP/ESO). A 50-year-old man with stage IV (T4N3M1c) malignant melanoma was challenged by s.c. injections of CHP/ESO vaccine every 2 weeks, according to Code of Ethics. A month after treatment, bullous formation was observed on the metastatic lesions. Histological examination confirmed the necrotic change of melanoma cells. TUNEL assay also confirmed the apoptotic death of tumor cells. CHP/ESO vaccine induced a gradual increase of NY-ESO-1-specific IgG1 and high levels of serum IFN-g. NY-ESO-1-specific CD8+T cells were demonstrated 29 days after immunization by IFN-g secretion assay. In this case, there was a significant increase of IL-4 and IL-5 in the blister fluid, compared to the serum and NY-ESO-1 expression was diminished in the remnant tumor. Despite having received a cancer vaccine, tumors continued to grow and the patient died of pulmonary failure due to multiple metastasis. Our observations indicate the occurrence of NY-ESO-1-specific immune response and immune suppression simultaneously, which may result in tumor progression and immune escape phenomenon. 085 Serum levels of 8-isoprostane, a biomarker of oxidative stress, are evaluated in patients with systemic sclerosis Fumihide Ogawa1, Toshihide Hara1, Eiji Muroi1, Kazuhiro Shimizu1, Minoru Hasegawa2, Kazuhiko Takehara2, Shinichi Sato1 1

Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; 2Department of Dermatology, Kanazawa University Graduate School of Medical Science Purpose: Oxidative stress is one of the important factors that contribute to tissue damage in systemic sclerosis (SSc). Since 8-isoprostane is produced in vivo by direct free radical peroxidation of arachidonic acid, it has been considered as an ideal, reliable biomarker of oxidative injury. To determine the clinical significance of 8-isoprostane in SSc, we investigated the prevalence and clinical correlation of serum 8-isoprostane levels in SSc patients. Methods: Serum 8-isoprostane levels were examined in 67 patients with SSc by ELISA. Renal vascular damage was determined as a pulsatility index (PI) in the renal interlobar arteries by color-flow Doppler ultrasonography. Results: Serum 8-isoprostane levels were significantly elevated by 75-fold in SSc patients compared to normal controls ( p < 0.0001). Furthermore, 8-isoprostane levels correlated negatively with pulmonary function, including %VC (r = 0.42, p < 0.005) and %DLco (r = 0.45, p < 0.005), and correlated positively with renal vascular resistance, which was determined as the PI value. Serum 8-isoprostane levels also correlated positively with serum IgG levels (r = 0.43, p < 0.001) and levels of

158 anti-agalactosyl IgG autoantibody (r = 0.6, p < 0.0005). Finally, serum 8-isoprostane levels in dead SSc patients were significantly 3.2-fold higher than in alive SSc patients ( p < 0.01). Conclusion: These results suggest that 8-isoprostane is a useful serological marker for evaluating oxidative injury and the disease severity in SSc. 086 The effect of non-steroidal anti-inflammatory drugs on absorption of wheat allergen Hiroaki Matsuo1, Sakae Honda1, Sakae Kaneko1, Yoshio Tsujino1, Minao Furumura1, Kunie Kohno1, Mikiko Kawaguchi2, Tsutomu Honjoh3, Kenichi Aburatani3, Eishin Morita1

Abstracts ICAM-1 expression. Although the skin from TSK/+ mice showed upregulated expression of fibrogenic Th2 cytokines, including interleukin (IL)-4 and IL-6, compared to wild type mice, that were significantly suppressed by loss of ICAM-1 expression. Proalpha2 (I) collagen and TGF-b mRNA expression was remarkably augmented on TSK/+ fibroblasts stimulated with IL-4 and ICAM-1 deficiency attenuated this expression. Interestingly, in the presence of IL-4, surface ICAM-1 expression was increased 2-fold in cultured skin fibroblasts from TSK/+ mice compared to wild type fibroblasts. Therefore, ICAM-1 may play critical roles in the production of fibrogenic cytokines and collagens in the skin from TSK/+ mice, suggesting that ICAM-1 is a potential therapeutic target for human systemic sclerosis. 088

1

Department of Dermatology, Shimane University School of Medicine, Izumo, Japan; 2Department of Nutrition Management, Shimane University Hospital, Izumo, Japan; 3Morinaga Institute of Biological Science, Inc., Yokohama, Japan

The involvement of reactive oxygen species in tissue injury of immune complex disease

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is a distinct form of a common wheat allergy induced by the combination of wheat ingestion and physical exercise or dosage of aspirin. Recently we demonstrated that levels of serum gliadin, causative allergen in WDEIA, increase in well accordance with the allergic symptoms during the provocation test. We also showed that aspirin-induced increases of the serum gliadin level occur not only in patients with WDEIA but also in healthy subjects without symptom. The aim of this study was to investigate the effect of NSAIDs on serum levels of wheat gliadin in healthy subjects. Aspirin (100, 200, 500, or 1000 mg) was administered to subject 30 min before ingestion of wheat product (udon 120 g). Venous blood was taken 0, 15, 30, 60, 120, and 180 after ingestion of wheat. The serum concentration of gliadin was measured using a sandwich ELISA (Morinaga Institute of Biological Science Inc.). Although there is a marked individual difference in the degree of effect, aspirin dose-dependently increased the concentration of serum gliadin. The effects of Loxonin (120 mg), Voltaren (50 mg), and Mobic (15 mg) were also examined using the same method as for aspirin. Loxionin and Voltaren increased the levels of serum gliadin as well as aspirin, however Mobic which is cyclooxygenase2 selective inhibitor had little effect on the serum gliadin level. Our results suggested that NSAIDs-induced facilitation of allergen absorption may elicit symptoms of food allergy in some cases.

Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

087 Intercellular adhesion molecule-1 deficiency attenuates the development of skin fibrosis in the tight-skin mouse Yukiyo Matsushita1, Minoru Hasegawa1, Manabu Fujimoto1, Kazuhiko Takehara1, Shinichi Sato2 1 Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Department of Dermatology, Nagasaki University Graduate School of Biomedical Science, Nagasaki, Japan

Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive extracellular matrix (ECM) deposition in the skin and other visceral organs, although the molecular basis for SSc is unknown. To directly assess roles of adhesion molecules in skin fibrosis, tight-skin (TSK/+) mice lacking L-selectin and/or intercellular adhesion molecule (ICAM)-1 were generated. Deficiency of ICAM-1 but not L-selectin significantly suppressed (48%) the development of skin sclerosis in TSK/+ mice. Similarly, the development of skin fibrosis in TSK/+ mice was also prevented by treatment with antisense oligonucleotides that selectively inhibit

Kazuhiro Shimizu, Fumihide Ogawa, Shinichi Sato, SangJae Bae

The formation and local deposition of immune complexes are thought to be important inflammatory mediators which can cause acute tissue injuries. The diseases caused by immune complexes are recognized as immune complex diseases. Although the clarification of molecular mechanisms has not established completely, reactive oxygen species are assumed as executing factors in the tissue injuries of immune complex diseases. Among reactive oxygen species, superoxide and nitric oxide are emitted in such inflammatory sites and these two molecules can immediately produce peroxynitrite which is thought to be very destructive. In other way, superoxide produce hydroxy radical in the metabolic process. Above-mentioned molecules are thought to be involved in the tissue injuries of immune complex disease. In order to clarify the involvement of these molecules in tissue injury of immune complex disease, we chose cutaneous reverse passive Arthus reaction in mice as an experimental model, which was produced by the intradermal injection of antibody followed immediately by intravenous infection of antigen. Rabbit immunoglobulin G (IgG) antichicken egg albumin antibodies was injected intradermally as an antibody, and chicken egg albumin was given intravenously as an antigen. The intradermal injection of purified polyclonal rabbit IgG followed by intravenous installation of chicken egg albumin served as a negative control. Mice used for experiments were C57BL/6 female mice and 12—16 weeks old. Using this model, we recognized the involvement of these molecules in the inflammatory development. Furthermore, their exact contributions to tissue injury are now being analyzed using nitric oxide synthase inhibitor, superoxide dismutase and scavenger of hydroxy radical. 089 Differential expression and function of Scavenger receptors by mouse skin- and bone marrow-derived mast cells Masaaki Matsumoto, Kana Wada, Mitsunori Ikeda, Hajime Kodama Department of Dermatology, Kochi Medical School, Kochi, Japan Not only oxidized low density lipoprotein (ox-LDL) but also mast cells play a significant role in the pathogenesis of xanthelasma and tuberous xanthoma. Human mast cells are divided into subclasses based on their neutral protease composition: mast

Abstracts cells containing both tryptase and chymase (MCTC) and mast cells with only tryptase (MCT). We previously reported that MCTC predominated in xanthelasma lesions and MCT predominated in tuberous xanthoma lesions. However, the each functional role of MCTC and MCT in the xanthoma lesions is unknown. Mouse skinderived mast cells (SMC) and mouse bone marrow-derived mast cells (BMMC) were used as a model of MCTC and MCT, respectively. Special attention was paid to scavenger receptor expressions, degranulation and cytokine production in mast cells by ox-LDL stimulations. SMC highly expressed scavenge receptor class B (CD36), class E (LOX-1) and class F (SREC) as compared with BMMC. Upon ox-LDL stimulations, SMC released more b-hexosaminidase than BMMC, whereas BMMC produced more IL-6 than SMC. Since activated mast cells enhance LDL uptake by macrophages and IL-6 activates macrophages to produce MCP-1, differential scavenger receptor expression and different degree of degranulation and cytokine production in MCTC and MCT reflect a clinical phenotype of xanthoma lesions. 090 Involvement of cutaneous mast cells in herpes simplex virus infection Rui Aoki1, Hiroyuki Matsue1, Akiko Nagasaka1, Fumi Goshima2, Yukihiro Nishiyama2, Shinji Shimada1 1

Department of Dermatology, University of Yamanashi, Yamanashi, Japan; 2Laboratory of Virology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan Mast cells have long been considered as major effector cells in type I-allergic responses. Recent studies, however, have revealed the additional importance of mast cells in host defense against pathogens, especially bacteria. Although we have recently reported that cutaneous mast cells express functional Toll-like receptors (TLRs) that recognize viral components, it remains largely unknown whether mast cells are involved in host defense against viruses. We hypothesized that degranulation by mast cells may be induced upon herpes simplex virus (HSV) infection, thus being involved in the inflammatory and immune responses against the virus. To test this, we used murine fetal skin-derived cultured mast cells (FSMC). Rapid (within 10 min) degranulation by FSMC was induced in a virus number-dependent fashion without affecting cell viability, when HSV1 (KH7 strain) or HSV2 (186 strain) was added to FSMC cultures (b-hexosaminidase assay). The viral components were detected within 3 h after the infection (immunohistochemistry). Upon inoculation of 186 strain (5  106 PFU) intradermally into B6 mice, the dermal infiltrates and significant increase in the number of degranulated mast cells were observed (toluidine blue staining). These results indicate that cutaneous mast cells degranulate upon HSV infection, thus participating in the induction of inflammatory responses and the subsequent acquired immune responses against the virus. 091 Mast cells contribute to bacterial clearance by producing IL-12related molecules during polymicrobial infections Nobuhiro Nakano, Chiharu Nishiyama, Ko Okumura, Hideoki Ogawa Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan Interleukin (IL)-12 and IL-23 are heterodimeric cytokines that mutually share identical p40 subunit. IL-12/23, which are mainly produced by dendritic cells and macrophages, are important

159 regulators of Th1 responses and critically required for host defense against bacterial infections. In this study, we demonstrated that mast cells, which are known to be a source of Th2 type cytokines, also produce IL-12/23. The transcription of IL-12-related genes, p40, p35, and p19, in mouse bone marrow-derived mast cells (BMMCs) were induced by stimulation with interferon (IFN)-g and lipopolysaccharide (LPS). The amount of p40 and IL-12p70 (p35/p40) protein secreted from BMMCs by LPS stimulation was similar as that of peritoneal macrophages, and was augmented by preincubation with IFN-g. In contrast, cross-linking of FceRI did not induce IL-12-related molecules production in mast cells. Furthermore, in the cecal ligation and puncture-induced acute septic peritonitis model, the p40 level in the peritoneal cavity of genetically mast cell-deficient W/Wv mice following the surgery was significantly lower than that of the control WBB6F1 +/+ mice. The reconstitution of W/Wv mice by wild-type (WT)-BMMC rescued p40 production, while the reconstitution by p40/-BMMC had no effect on reduced p40 level in peritoneal cavity of W/Wv mice. The survival rate of W/Wv mice that were reconstituted with p40/  -BMMC following the surgery was significantly decrease compared with that of the control mice and WT-BMMC reconstituted W/Wv mice. These results suggested that mast cells contribute to the bacterial clearance by producing IL-12/23p40 during polymicrobial infections as well as dendritic cells and macrophages. 092 Serotonin activates human monocytes and prolongs their lifespan via the 5-HT1 receptor Fujiko Soga, Norito Katoh, Tomoko Inoue, Saburo Kishimoto Department of Dermatology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kyoto, Japan It has been known that activated platelets release variety of mediators, which contribute to allergic inflammation. However, the role of platelets in allergic inflammation of the skin remains unclear. Serotonin (5-hydroxytryptamine, 5-HT) is one of the prototypic mediators derived from activated platelets. Since activation of platelets should often take place in the lesions of atopic dermatitis (AD) because of excoriation due to scratching and the engagement of FceRI on their surface with antigens, we examined the effect of serotonin on function and lifespan of human monocytes which play a critical role on the chronicity of atopic dermatitis. Treatment of monocytes with 10 mM of 5-HTupregulated the expression of co-stimulatory molecule such as CD80 and CD86 and enhanced the capacity to stimulate allogeneic CD4+ T cells. Furthermore 5-HT induced production of IL-6 and TNF-a in LPS-stimulated monocytes. These finding suggest that 5-HT activates monocytes. In addition, 5-HT reduced apoptosis induced by serum deprivation or CD95/Fas ligation in human monocytes in a dose dependent manner. The inhibitory effects of 5-HT on monocyte apoptosis were antagonized by 5-HT1R antagonist, but not by 5-HT2R and 5-HT3R antagonists, and were mimicked by an 5-HT1R agonist. 5-HT also up-regulated Bcl-2 and Mcl-1 expression, and inhibited caspase-3 activation. 5-HT and 5-HT1R agonist, but not 5-HT2R and 5-HT3R agonists, induced ERK1/2 phosphorylation. Taken together, these findings support 5-HT activates monocytes via the 5-HT1 receptor and inhibits their apoptosis, thereby contributing to chronic inflammation. 093 Low responsiveness of murine contact hypersensitivity through grafted skin is mediated by regulatory T cells Ryutaro Yoshiki, Kazunari Sugita, Kenji Atarashi, Takatoshi Shimauchi, Kenji Kabashima, Yoshiki Tokura

160

Abstracts Health,

Department of Dermatology, Kochi Medical School, Nankoku, Japan

Background: It is empirically known that contact dermatitis can not develop through full-thickness skin grafted areas. This phenomenon was confirmed in our murine model, which demonstrated that the suppression of sensitization phase occurs via only grafted local areas. However, the immunological mechanisms underlying this unresponsiveness remain to be clarified. To address the mechanisms, we focused on CD4+CD25+ regulatory T cells that may play a role in the sensitization phase. Method: Full-thickness skin graft was performed in BALB/c mice, and a hapten was applied onto the grafted area for sensitization. Five days after sensitization, lymph nodes and spleens were taken from the mice. Then, CD4+CD25+ and CD4+CD25 subsets were purified by MACS. Each subset was transferred into syngeneic naı¨ve mice, which were sensitized with the same hapten and challenged with the hapten onto the earlobes. Result: In comparison with control mice, the CD4+CD25+ T cells exhibited a less ear swelling response. This suggests that CD4+CD25+ regulatory T cells play a role in the suppression via grafted skin.

Scavenger receptors (SRs) were originally found to play a role in the uptake of modified lipoproteins by macrophages. Recently the functions of SRs in innate immunity have been revealed. However, it is unknown whether SRs are expressed in epidermal keratinocytes. We examined the expression and the function of scavenger receptor expressed by endothelial cells (SREC)-I, a type F SR, in cultured normal human epidermal keratinocytes (NHEKs). NHEKs were shown to express SREC-I by Western blot and immunofluorescence staining. The expression was decreased in the differentiated NHEKs induced by high calcium concentration. NHEKs were not transformed to foam cells by the incubation with oxidized low density lipoprotein, a natural ligand for SREC-I. Furthermore, NHEKs secreted interleukin-8 by the stimulation of oxidized low density lipoprotein. Lipopolysaccharides of bacterial origin stimulated interleukin-8 secretion from NHEKs through SREC-I. These results suggested that NHEKs detected the bacterial invasion though SREC-I and that NHEKs functioned as front line cells of the innate immunity against bacterial infections by promoting the infiltration of neutrophils to the epidermis through interleukin-8 secretion.

University of Occupational and Environmental Department of Dermatology, Kitakyushu, Japan

094 Cytokine profile and redox status of lymph node cells in contact dermatitis induced by low concentration of DNFB 1

1

1

Kaori Takada , Sachio Kanamori , Katsumi Kaneko , Seiji Kawana1, Kazumi Iida2 1

The Department of Dermatology, Nippon Medical school, Tokyo, Japan; 2The Research Institute of Vaccine Therapy for Tumors and Infectious Diseases, Nippon Medical School, Tokyo, Japan

096 Staphylococcal enterotoxin B enhances a flare-up reaction of murine contact hypersensitivity through an increase of interferon-g production Akiko Miyata, Masaru Natsuaki, Kiyofumi Yamanishi Department of Dermatology, Hyogo College of Medicine, Nishinomiya, Japan

Conventionally in experiment of allergic contact dermatitis, it was used that the concentration of hapten is higher than threshold level, which induce contact dermatitis by a single painting. In this experiment, we examined whether dermatitis is inducible by repeated application of hapten, which concentration is lower than threshold level, and the changes in cytokines profiles and redox status before and after dermatitis is induced. Methods: C57BL/6 mice were shaved on their back, and painted with 25 ml of 0.1% DNFB in acetone:olive oil (at a ratio of 4:1) twice a week for 1—3 weeks. Lymph node cells (LNC) were obtained from regional lymph nodes on 4 days after the last paint. Normal B6 spleen cells were used as antigen-presenting cells (APC) after irradiation at 30 Gy and pulsed with haptens. The hapten-pulsed APC were cultured with LNC for 3 days. Then the cytokines (IL-4, 5, 10 and IFN-g) in that cultured supernatant were assayed. The redox status of LNC was examined by the staining of monochlorobimane. Results: Contact dermatitis was induced by 3 weeks application of low concentration of hapten. Hapten specific cells were appeared in regional lymph nodes on the 1st week without dermatitis. Only IFN-g was produced by hapten specifically, but not IL-4, 5 or 10. IFN-g production was increased until 2nd week, and decreased thereafter on 3rd week even the dermatitis was induced. It was also observed that the ratio of reductive cells in regional lymph nodes was highest on the 2nd week, just before the dermatitis was induced.

We have established a model of chronic contact dermatitis, in which BALB/c mice sensitized and repeatedly challenged with a hapten, dinitrofluorobenzene (DNFB), on each ear develop twophased ear swelling responses, immediate and delayed-type reactions. In this model, we found that an intravenous injection of a specific hapten causes a delayed-type flare-up reaction in the ear and an injection with staphylococcal enterotoxin B (SEB), a potent superantigenic bacterial exotoxin, further enhanced the flare-up reaction. In the present study, we examined the role of cytokines in the SEB-enhanced flare-up reaction. BALB/c mice were sensitized on the dorsal skin, then challenged 4 times on each left ear with DNFB, and injected with SEB. Cervical lymph node cells of those mice were cultured with a specific hapten in vitro and their proliferative responses were assayed using 3Hthymidine incorporation. The production of cytokines IFN-g, IL-4, IL-5 and IL-13 from those cells were also assessed. As the results, SEB injection markedly enhanced 3H-thymidine incorporation and production of IFN-g in those cells. In contrast, the production of IL-4, IL-5 or IL-13 was decreased or unchanged. These results suggest that down-regulation of Th2 and up-regulation of Th1 in the cytokine profile are involved in the SEB-enhanced flare-up reaction of contact hypersensitivity. Thus, the mechanisms might be involved in exacerbation of eczema in association with staphylococcal infection.

095

Increased production and secretion of glial cell line-derived neurotrophic factor from atopic dermatitis skin lesions

097

Expression and function of Scavenger receptor expressed by endothelial cells (SREC)-I in normal human epidermal keratinocytes

Kazuya Tokime1, Ritsuko Katoh-Semba2, Keiichi Yamanaka1, Akira Mizoguchi3, Hitoshi Mizutani1

Mitsunori Ikeda, Tomoya Takata, Masaaki Matsumoto, Masahiro Seike, Masato Iwagaki, Hajime Kodama

1 Department of Dermatology, Mie University, Graduate School of Medicine, Tsu, Japan; 2Institute for Developmental Research,

Abstracts Aichi Human Service Center, Kasugai, Japan; 3Department of Anatomy, Mie University, Graduate School of Medicine, Tsu, Japan Glial Cell Line-Derived Neurotrophic Factor (GDNF) is a factor to promote survival and morphological differentiation of embryonic midbrain dopaminergic neurons. GDNF supports nigral dopaminergic neurons. Recently, GDNF was revealed being a neurotrophic factor for a variety of neurons. The patients with chronic inflammatory skin disease especially atopic dermatitis (AD) complain severe itching and express severe damage of skin due to intensive excoriation. The overgrowth and hypersensitivity of sensory nerves has been observed in AD skin lesions, and importance of neurotrophins has been proposed. Different from in central nervous system, the functions of GDNF in itching dermatitis is unclear. In this report, we analyzed mRNA expression and protein production for GDNF in inflamed skin from AD model mice. GDNF was produced and secreted from in the epidermis, and mainly localized in the basal layer of the epidermis. Furthermore, we have detected thick cutaneous innervation of nerve fibers in AD mice compared to normal controls. Taken together, GDNF produced and released from the inflammatory skin lesions has important roles in forming AD skin lesions. 098 Suppressive effects of steroid and Vitamin D derivatives used for psoriasis therapy on superantigen (SPEA)-induced lymphocyte-blastogenesis Kae Arai1, Yukari Ohkubo1, Toshihiko Hirano2, Ryoji Tsuboi1 1 Department of Dermatology, Tokyo Medical University; 2Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Science

Glucocorticoids (GCs) and Vitamin D3 derivatives (VD3s) are used topically for psoriasis treatment. However, large individual differences are observed in clinical efficacies of GC and VD3. It has been suggested that Hemolytic streptococci infection is implicated in the pathogenesis of psoriasis. Moreover, H. streptococciderived superantigens such as SPEA may modify clinical GC efficacies. We examined the effects of betamethasone butyrate propionate (BBP) and VD3s against concanavalin A (conA)- or SPEA-induced blastogenesis of peripheral-blood mononuclear cells (PBMCs) obtained from 17 healthy subjects. PBMCs were suspended in RPIM1640 medium, and then incubated for 96h in the presence of con A or SPEA and therapeutic drug. The drug concentrations giving 50% inhibition of the blastogenesis (IC50s) were estimated. The median (range) of the IC50 values for maxacarcitol (MC), tacalcitol (Tac), and BBP against con A-induced PBMC blastogenesis were 17 (0.007—5000), 1514 (2—3997), and 0.07 (0.001— 223) ng/ml, respectively. In contrast, the median (range) of the IC50 values for MC, Tac, and BBP against SPEA-induced PBMC blastogenesis were 2 (0.05—3349), 90 (1—322), and 292 (0.001— 1172) ng/ml, respectively. The IC50 values for BBP evaluated in SPEA-stimulated PBMCs were significantly higher than those evaluated in con A-stimulated PBMCs ( p = 0.0245). Whereas, the IC50 values for MC and Tac were not significantly different between con A stimulated- and SPEA stimulated-PBMCs The attenuated efficacy of BBP by the SPEA stimulation was recovered by use of MC in addition to BBP. These observations raised the possibility that SPEA induced by H. streptococci infection attenuates clinical BBP efficacy, whereas VD3s are effective in such cases in psoriasis.

161 099 CD4+CD25+Foxp3+ regulatory T cells induced by plasmacytoid DC produce high amount of IFN-G and IL-5 in atopic dermatitis patients Hideo Hashizume, Takahiro Horibe, Yasushi Yoshinari, Natsuho Ito, Taisuke Ito, Masahiro Takigawa Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan In humans, at least two dendritic cell (DC) subsets are identified based on their origin, phenotype and function: CD11c+ CD123 myeloid DC (mDC) and CD11c CD123+ CD62L+ plasmacytoid DC (pDC). Recent accumulating findings suggest that both DC subsets also have tolerogenic potential to dampen immune responses by promotion of T cells with regulatory activity (Treg) as well as potential of Th1- and Th2-cell differentiation. However, DC induced Treg population has never been investigated under disease conditions in terms of the pathogenic role. The present study compared the phenotypic characteristics and suppressive function of CD4+CD25highFoxp3+ Treg generated by pDC between atopic dermatitis patients and normal individuals. While Treg possessed comparable potential to inhibit proliferation of lymphocytes between the two groups, we found that Treg in AD had much higher potential to produce IFN-g and IL-5 than those in normal subjects. Foxp3+IL-5+ cells in close associations with pDC accumulated in skin lesions in AD, but not in normal skin. Our observations suggest that atopic pDC generate an aberrant type of CD4+CD25high Treg to release IFN-g and IL-5, which might modulate atopic inflammation with tissue eosinophilia. 100 Vasoactive intestinal peptide and inflamatory cytokines enhance vascular endothelial growth factor production from epidermal keratinocytes Maki Kakurai1, Naoka Umemoto1, Yukiko Kobayashi2,3, Tae Inoue4, Mamitaro Ohtsuki2, Yusuke Furukawa3, Toshio Demitsu1 1

Department of Dermatology, Jichi Medical University, Omiya Medical Center, Saitama, Japan; 2Department of Dermatology, Jichi Medical University, School of Medicine, Tochigi, Japan; 3 Division of Molecular Hematopoiesis, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan; 4Akita University, School of Medicine, Akita, Japan Vascular Endothelial Growth Factor (VEGF) can induce angiogenesis. VEGF has been implicated in the pathologic angiogenesis observed in psoriatic lesions. Recent evidences suggest an active role of vasoactive intestinal peptide (VIP) in the pathogenesis of inflammatory skin disease such as psoriasis. We already presented VIP and cytokines (IFN-g/TNF-a) induced the release of VEGF on kerationcytes. The aim of this study is to confirm whether VIP induces the production of VEGF in kerationcytes. VEGF transcript was detected in normal human epidermal keratinocytes (NHEK), and a human epidermal keratinocyte cell line DJM-1, using reverse transcription-polymerase chain reaction. Then we investigated the effects of VIP on VEGF production by kerationcytes. DJM-1 cells were incubated for 24 h in the presence of VIP (1 mM). Western blot analysis revealed that intracellular VEGF level was increased by VIP at 3 h and 12 h. These results suggest that several inflammatory cytokines and VIP from mast cells and nerve ending are capable of inducing VEGF production from epidermal keratiocytes, which might be involved in the pathogenesis of inflammatory dermatoses such as psoriasis.

162

Abstracts

101

significantly different from that of wild-type. DETC in b7 integrin deficient mice did not express aE integrin on their surfaces, indicating they were retained in the epidermis using adhesion molecules other than aE/b7 integrin. As the number of DETC was maintained in b7 integrin deficient mice, their capability of repairing wounds was not perturbed. These data suggest that aE/b7 integrin is dispensable for the maintenance of DETC.

Epidermal regulation of AQP3 expression and HA synthesis (1): The reciprocal regulation by keratinocytes in spongiotic dermatitis Tomoyuki Ohtani1, Ryoko Saito1, Tetsuya Sayo2, Yoko Endo2, Yoshinori Sugiyama2, Shintaro Inoue2, Setsuya Aiba1 1 Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan; 2Basic Research Laboratory, Kanebo Cosmetics Inc., Odawara, Japan

T-cell-mediated keratinocyte apoptosis with E-cadherin cleavage plays a key pathogenic role in eczematous dermatitis. However, it remains unanswered how spongiosis retains water in the intercellular space of epidermis. Recently, we have reported that IFNgamma-treated human keratinocytes significantly augmented the production of hyaluronic acid (HA), which has profound effects on the distribution and movement of water, and that keratinocytes express aquaporin 3 (AQP3) which belongs to a family of water channels and is up-regulated by osmotic stress. In this study, to clarify the pathomechanism of spongiosis, we examined the expression of HA and AQP3 in 20 cases of spongiotic dermatitis and 10 of normal skin by immunohistochemical staining with hyaluronic acid binding protein (HABP) and anti-AQP3 antibody. The lesion of spongiotic dermatitis significantly augmented the immunoreactivity with HABP, while AQP3 expression was down-regulated around spongiotic vesicles. The reciprocal regulation of HA production and AP3 expression was confirmed by Texas Red-labeled HABP and FITC-labeled anti-AQP3 Ab. Since IL1beta, TNFalpha, and IFNgamma play crucial roles in the efferent phase of contact dermatitis, we examined the effects of these cytokines on HA production and AQP3 expression by keratinocytes in vitro. IL-1beta and IFNgamma significantly augmented HA production by keratinocytes, while TNFalpha down-regulated it. In contrast, the immunoblotting demonstrated that both IL1beta and TNFalpha suppressed AQP3 expression, while IFNgamma significantly augmented it. These studies suggest that the proinflammatory cytokines produced by infiltrating lymphocytes in contact dermatitis regulate HA production and AQP3 expression, which may lead to the intercellular edema in spongiosis. 102 aE/b7 integrin is dispensable for the maintenance of dendritic epidermal T cells (DETC) Takafumi Kadono, Hanako Ohmatsu, Yuri Masui, Kunihiko Tamaki

103 Influence of an endocrine disrupture, tributyltin (TBT) on the mucosal immune system Takahisa Uchida1, Masatosi Nakazawa2, Mutsuhiko Minami2, Kazuo Takahasi1, Zenro Ikezawa1 1

Department Environmental Immuno-Dermatology, Yokohama City University, Yokohama, Japan; 2Department of Immunology, Yokohama City University, Yokohama, Japan Purpose: TBT has been well known as an endocrine disrupture, and used for antifoulants on ships. TBT is absorped into human body by eating fish and shellfishes. We investigated whether TBT effects on cells of Peyer’s patch (PP) or mesenteric lymph nodes (MLN), and oral tolerance. Material and method: After C57BL/6 mice were fed with or without TBT diet for two weeks, we isolated the cells from the PP and MLN, and investigated a number and percentage of T/B, NK, NKTcell, and dendric cell (DC). Cytokine (IFN-g, IL-4, IL-10, TGFb) production from PP or MLN cells stimulated with ConA or LPS was assessed by ELISA. Next, mice were orally administered 5mg of OVA for induction of oral tolerance after 2 weeks of feeding with TBT. A week later, the mice were immunized with OVA precipitated in Freund’s complete adjuvant (FCA) on foot pad. Two week later, the mice were bled for analysis of amounts of OVA specific IgG1 and IgG2a. The cells from inguinal lymph nodes were isolated and cultured with OVA for 48 h. Culture supernatants were tested for IFN-g, IL-4, IL-10 or TGF-b production. Lymphocyte proliferation was assessed by incorporation of 3H-TdR. Result: The number of T cells (CD4, CD8, and CD4CD25), B cells, NK/NKT cells, and DC was significantly decreased in mice treated with TBT, while the percentage of them was unchanged. IFN-g and IL-10 production in the supernatants from PP or MLN cells were decreased in TBT fed mice. In the immunized mice, the amount of OVA specific IgG1 was increased in TBT treated mice, but the mount of OVA specific IgG2a was comparable with that of untreated mice. Conclusion: These results suggest that TBT affected on PP and MLN to reduce mucosal immune response and increase systemic Th2 response.

Department of Dermatology, University of Tokyo, Tokyo, Japan Dendritic epidermal T cells (DETC) are unique mouse intraepithelial lymphocytes that exhibit distinct gd TCR. Studies using mice deficient in gd T cells have revealed critical roles of DETC in skin inflammation, wound healing, and tumor surveillance. In addition to their invariant g3 TCR, the development of DETC requires IL-15 signaling, but how these DETC migrate to skin and sustain themselves is not well known. One remarkable feature of DETC is the expression of aE/b7 integrin on their surfaces. aE/b7 integrin is an adhesion molecule and is supposed to mediate the binding between DETC and epithelial cells through the interaction with E-cadherin. When the number of DETC was examined in aE integrin deficient mice, it was reported that the number was reduced to less than 1/3 compared to that of wild-type. In this study, we examined the number of DETC in mice deficient in b7 integrin, which forms heterodimer with aE integrin. Unexpectedly, the number of DETC in b7 integrin deficient mice was not

104 Evaluation of changes of cell surface thiols as a new biomarker for in vitro sensitization assay: The comparison between cell lines Morihiko Hirota1, Mie Suzuki1, Saori Kagatani2, Yosinori Sasaki2, Masato Mizuashi2, Shigenobu Hagino1, Setsuya Aiba2, Hiroshi Itagaki1 1

Shiseido Safety and Analytical Research Center, Yokohama, Japan; 2Department Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan Objective: In the process of allergic contact dermatitis, dendritic cells (DCs) including Langerhans cells, which have potent antigen-presenting function, play crucial rules in the induction phase of skin sensitization. For predicting the sensitization

Abstracts potential of chemical in vitro, it is one of useful steps to detect the activation of antigen-presenting cells including DCs by haptens. Now, we take interest in the changes of cell surface thiols by haptens as one of triggers of the activation of DCs and its possibility as a biomarker for in vitro sensitization test. In this study, we evaluated 2 cell lines for selecting of indicator cell as replacement of DCs that is difficult to prepare for routine use. Materials and methods: Human monocytic cell lines, THP-1 and U-937 were treated by chemicals for 2 h. Cells were washed and incubated with nonpermerable thiol reactive compound Alexa flour C5 maleimide (AFM) for 30 min. After having been washed again, cells were analyzed by flow cytometry. Results and discussion: In most cases, the cell surface thiols on THP-1 and U-937 decreased after treatment with the allergens. But some sensitizers did not change the cell surface thiols or increase them. And by the treatment of DPCP, the cell surface thiols on THP-1 and U-937 increased significantly. When the significant decrease of cell surface thiols were applied as tentative criterion, the accuracy of in vitro assay using THP-1 versus in vivo assay was a little better than that of in vitro assay using U937. These results suggest THP-1 cells are good indicator cell lines for in vitro sensitization assay using the changes of cell surface thiols as a biomarker. References [1] Aiba, et al. J Invest Dermatol 2003;120:390—9. [2] Mizuashi, et al., J Invest Dermatol 2005;124:579—86.

105 Effect of cyclophosphamide on the tolerance induced by DNFB oral administration Satoko Tatewaki1, Masatoshi Nakazawa2, Naomi Hongo2, Chie Hotta2, Kazuo Takahashi1, Mutsuhiko Minami2, Zenro Ikezawa1 1 Enviromental Immuno-Dermatology, Yokohama City University, Yokohama, Japan; 2Deptartment of Immunology, Graduate School of Medicine, Yokohama

Objective: It is well known that hapten specific oral tolerance is induced by oral administration of hapten. In addition, it is known that CD4+CD25+ regulatory T cells (Treg) participate momentously in induction of hapten specific oral tolerance. We reported that enhancement of the CH reactions by Cyclophosphamide (Cy) treatment is attributed to the decrease in the number, and the function of T reg. We examined whether and how Cy affected on the induction of oral tolerance and induced oral tolerance against hapten. Method: C57BL/6 mice (6 to 8 weeks of age, female) were administrated 300 mL of 0.1% 2, 4-dinitrofluorobenzene (DNFB) by orally. Ten days after, mice were sensitized epicutaneously by application of 50 mL of 0.5% DNFB onto shaved abdominal skin. Seven days after the sensitization, mice were challenged with 10 mL of a nonirritant concentration of 0.25% DNFB onto the right ear. Two days before oral administration of DNFB or 2 days before sensitization, Cy was injected ip. into mice at the dose of 200 mg/ kg. Ear swelling was measured at 24 h or 48 h after challenge. We measured proliferation of hapten specific T lymphocytes and cytokine production at 7 days after challenge. Result: CH reactions in both Cy-treated groups were significantly greater than in oral tolerance group induced by DNFB administration without Cy treatment. DNFB specific T lymphocytes proliferation was partially recovered in Cy-treated group at two days before oral administration. In Cy treated group at 2 days

163 before sensitization, DNFB specific T lymphocytes proliferation was recovered to the level of that of positive control group. Cy treatment may affect on both CD4+CD25+ Treg and DNFB specific regulatory T cells induced by DNFB administration to failure the oral tolerance. 106 Hapten specific tolerance was sufficiently induced by nasal administration of dinitrobenzen sulfonate Kazuko Nakamura1, Masatoshi Nakazawa2, Kazuo Takahashi1, Michiko Aihara1, Mutsuhiko Minami2, Zenro Ikezawa1 1

Department of Environmental Immuno-Dermatology, Yokohama City University Graduate School of Medicine, Yokohama, Japan; 2 Department of Immunology, Yokohama City University of Medicine Introduction: Understanding the mechanisms of mucosal tolerance might lead to control allergic diseases. We investigated the mechanism of the intranasal tolerance to hapten dinitrobenzen sulfonate (DNBS). Materials and methods: BALB/c mice received 0.075% DNBS intranasally 3 times continuously from day 0 to day 2. Mice were sensitized with 0.5% Dinitrofluorobenzene (DNFB) on day 9 and challenged with 0.25% DNFB on day 16. Ear thickness was measured and hapten specific T lymphocyte proliferation and cytokine production were assessed after the challenge. We performed transfer experiments and investigated inhibitory effect of T lymphocytes from mice intranarsally administered with DNBS on ear swelling in vivo. Next, we investigated whether T lymphocytes from mice intranasally treated with DNBS can inhibit the T cell proliferation from DNFB sensitized mice in vitro. To investigate the role of IL-10 and TGF-beta, we added anti-IL-10 and anti-TGF-beta mAbs to the assay plates. Results and discussion: Intranasal administration of DNBS induced inhibition of CHS response to DNFB and T cell proliferation response to DNBS. Decreased amount of IFN-g and increased amount of IL-10 was produced by T cells from mice intranasally treated with DNBS. The mice transferred with CD4+ T lymphocytes isolated from the mice intranasally administered with DNBS were reduced CHS response to DNFB. These CD4+ T lymphocytes also inhibited DNBS specific T lymphocyte proliferation in vitro. Adding neutralizing anti-IL-10 and/or anti-TGF-beta mAbs partially recovered the T cell proliferation against DNBS. These data indicated that intranasal tolerance against hapten was mediated by CD4+ T lymphocytes partially with secretion of the immunosuppressiove cytokines, such as IL-10 and TGF-beta. 107 The cross-link between the reduced cell surface thiols and the oxidized GSH/GSSG ratio in human dendritic cells treated with haptens Saori Kagatani1, Yoshinori Sasaki1, Masato Mizuashi1, Morihiko Hirota2, Mie Suzuki2, Hiroshi Itagaki2, Setsuya Aiba1 1 Department of Dermatology, Graduate School of Tohoku University, Sendai, Japan; 2Shiseido Safety and Analytical Center, Yokohama, Japan

Although we and other groups demonstrated that p38 mitogenactivated protein kinases (MAPK) plays a crucial role in the activation of monocyte-derived dendritic cells (MoDC) by contact sensitizers such as NiCl2 and 2, 4-dinitrochlorobenzene (DNCB), the upstream signals of p38 MAPK remain undetermined. Recently, we have demonstrated that the imbalanced ratio of the oxidized (GSSG) versus reduced (GSH) form of cellular

164 glutathione plays a crucial role in triggering DC maturation by sensitizers. This observation raises the next question of how the imbalance of the intracellular GSH/GSSG ratio is induced by sensitizers. Since it has been reported that treatment with exogenous nonpermeable GSSG, which results in a significant decrease of exofacial cell membrane thiol groups and the intracellular GSH content, phosphorylates p38 MAPK, we examined the effects of several sensitizers and irritants on the exofacial cell membrane thiol groups of MoDC. In this study, MoDC were treated with DNCB, NiCl2, formaldehyde, and SLS for 2 h, incubated with a nonpermeable thiol-reactive compound, Alexa fluor 488 C5 maleimide (AFM), or anti-phospho-p38 MAPK after fixation and permeabilization. The cells were washed and analyzed by flow cytometry. DNCB, NiCl2, and formaldehyde significantly reduced cell-surface thiols at non-toxic concentrations while SLS did not. In parallel with the decreased cell-surface thiols, these three sensitizers phosphorylated p38 MAPK in MoDC. These data suggest cross-links between the intracellular redox balance and the cellsurface thiol groups, and between the activation of p38 MAPK and the intracellular or cell surface redox imbalance. 108 Relationship between human genetic polymorphisms and drug sensitivity in psoriasis vulgaris Michiya Yamaguchi, Yumiko Takahata, Hirotaka Deguchi, Makoto Ichimiya, Masahiko Muto Department of Dermatology and Biomolecular Recognition, Yamaguchi University School of Medicine, Ube, Japan It is considered that psoriasis vulgaris (PV) is an immunological skin disorder caused by the concerted action of multiple genetic and environmental factors. However, the pathogenesis of PV has not been cleared. It has been reported that various genes locating near HLA-C is analyzed especially because strong association between PV and HLA-Cw6 has been well known. Furthermore, it is suggested that Tcell which has natural killer receptor (NKR) is necessary for the pathogenesis in PV. It was also revealed that NK-T cell is recognized in plaque lesion of psoriasis immunohistochemically (Nickoloff et al., 1999). We analyzed relationship between KIR (killer cell immunoglobulin-like receptor) polymorphisms and drug sensitivity by using representative drugs (cyclosporine and etretinate) for PV. 109 CD4+CD25+ adult T-cell leukemia/lymphoma cells express CTLA-4, but have no function of regulatory T cells Takatoshi Shimauchi, Kenji Kabashima, Yoshiki Tokura Department of Dermatology, University of Occupational and Environmental Health, Japan Adult T cell leukemia/lymphoma (ATL) cells share the CD4+CD25+ surface expression molecule phenotype with regulatory T cells (Treg). However, it is still controversial whether ATL cells belong to Treg. Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is one of the specific markers for Treg. In this study, we examined the 17 ATL patients with skin involvement. Flow cytometric analysis of the expression of CTLA-4 on CD4+CD25+ cells or Foxp3 on CD4+ cells in peripheral blood mononuclear cells (PBMC) demonstrated that circulating tumor cells from two patients with the leukemic change highly expressed CTLA-4 as compared to those from the normal healthy donors. ATL cells isolated from skin tumors of other three cases also bore high CTLA-4 expression. Moreover, an immunohistochemical study also demonstrated that, in five out of the nine examined patients, skin-infiltrating

Abstracts tumor cells were positive for CTLA-4. Furthermore, after 12 h activation of crude PBMC with anti-CD3/CD28 antibodies, an expression of CTLA-4 on the CD4+CD25+ cells was significantly higher than that from normal healthy donors. Although, there was no difference of IL-10 concentration of those supernatants between the patients and normal controls by ELISA assay. Finally, to evaluate the function of Treg, we prepared the skin-infiltrating tumor cells or CD4+CD25+ T cells derived from the patient with high CTLA-4 expression by immunomagnetic beads. These tumor cells, however, did not suppress allogenic mixed lymphocytes reaction, suggesting that those tumor cells have no Treg function. Therefore, our study demonstrated that ATL tumor cells express CTLA-4, especially upon activation, skin infiltration or leukemic change, but have no function of Treg. 110 Analysis of the effects of prion protein on TNF-a production by human monocyte derived dendritic cells Shinichi Hashimoto1, Koichiro Nakamura1, Fumio Kaneko1, Hideki Fujita2, Makoto Sugaya2, Kunihiko Tamaki2 1

Department of Dermatology, Fukushima Medical University, Fukushima, Japan; 2Department of Dermatology, Tokyo University, Tokyo, Japan Prion disease is a transmissible spongiform encephalopathies. Although prion protein (PrPc) is dominantly expressed on brain, prion is also expressed in peripheral tissues. We have recently published PrPc expression on epidermal Langerhans cells (LC). We examined the effects of PrPc (106-126) on TNF-a production by monocyte derived denritic cells (MoDCs). MoDCs were generated under the stimulation of IL-4 and GM-CSF. MoDCs spontaneously produced TNF-a, and PrPc significantly enhanced this TNF-a production by MoDCs (from 148.7 pg/ml to 299.9 pg/ml). PrPcinduced TNF-a production by MoDCs was partially inhibited by genistein, curcumin, PD98059 and wortmannin (inhibition rate: 43.8%, 83.7%, 80.3%, 88.3%). Immunoblot analysis confirmed the results. These results indicate that NFkB and PI3kinase pathways are involved in PrPc-induced TNF-a production by human MoDCs. 111 Dexamethasone negatively regulates Syk in mast cells by up-regulating src-like adaptor protein (SLAP) Takaaki Hiragun1,2, Ze Peng2, Michael A. Beaven2, Michihiro Hide1 1

Department of Dermatology, Programs for Biomedical Research, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 2 Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, USA The glucocorticoid, dexamethasone, suppresses antigen-induced degranulation, cytokine production, and intermediate signaling events in RBL-2H3 mast cells, although the exact mechanisms are uncertain. By microarray analysis, we discovered that the expression of an inhibitory adaptor protein, src-like adaptor protein (SLAP), was up-regulated elevenfold in dexamethasone-treated RBL-2H3 cells. SLAP is a known inhibitor of T-cell signaling and interacts with the tyrosine kinase, ZAP70. Exposure of RBL-2H3 mast cells to dexamethasone markedly increased expression of SLAP. Cells so exposed or made to over-express SLAP exhibited reduced antigen-stimulated phosphorylation of Syk (a cognate of ZAP70), phospholipase Cg, and extracellular regulated protein kinase (ERK). Calcium mobilization, calcium-dependent degranulation and ERK-dependent release of arachidonic acid were suppressed as well. Small interfering RNA directed against SLAP

Abstracts blocked the induction of SLAP and reversed the inhibitory effects of dexamethasone on phosphorylation of Syk and phospholipase Cg but not downstream events which are likely suppressed by upregulation of Dok-1 and MKP-1. The induction of these inhibitory regulators suggests that such induction is an important component of the immunosuppressive activity of dexamethasone in mast cells. 112 Phenotypic and numerical examination on the dendritic cells in dermatopathic lymphadenitis associated with cutaneous T cell lymphoma Kenji Asagoe, Masashi Komatsubara, Daisuke Suzuki, Kazuhide Tsuji, Keiji Iwatsuki Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Background: It has been disclosed that enlarged paracortex areas of dermatopathic lymphadenitis are composed of interdigitating dendritic cells (DCs), although their phenotypes and maturation stages have not been fully understood. Furthermore, little is known about the subset of DCs in dermatopathic lymphadenitis associated with different underlying diseases. Objective: To reveal the subset and phenotype of DCs in dermatopathic lymphadenitis associated with cutaneous T cell lymphoma (CTCL). Materials and methods: Paraffin-embedded and frozen sections of reactive swollen lymph nodes (LNs) were obtained from 6 patients with CTCLs, and analyzed immunohistochemically to observe the distribution of DCs with various phenotypes. S100+ or factor XIII+ DCs were co-stained with anti-Fascin, CD1a, CD83, DC-LAMP, CD123, CD68, or CD14 antibodies (Abs) for the detailed characterization. Anti-Langerin Ab was used for the further phenotypic analysis of CD1a+ cells. Results: S-100+ DCs increased in number in LNs depending on the severity of CTCL. Most S-100+ DCs co-expressed Fascin, a third co-expressed CD1a, and a variable number of S-100+ DCs coexpressed DC-LAMP depending on the patients. However, S-100+ DCs rarely co-expressed CD83 or histiocytic markers, and showed mostly different distribution from the cells with these two markers. On the other hand, CD123+ plasmacytoid DCs (pDCs) gathered around the high endothelial venules. Furthermore, some of the factor XIII-positive cells co-expressed CD1a and some others co-expressed CD68. Conclusions: LN DCs in dermatopathic lymphadenitis of CTCLs increased and were composed at least 3 types of DCs, including a major epidermis-derived DCs with various maturation stages, and a minor dermal-derived DCs and blood-derived pDCs. 113 Phenotypes of dendritic cells distributed in the surrounding areas of psoriatic plaques Mayumi Komine, Tomonori Takekoshi, Naoki Sakurai, Yosaku Minatani, Hidehisa Saeki, Akihiko Asahina, Kunihiko Tamaki Department of Dermatology, University of Tokyo, Tokyo, Japan Psoriatic plaques usually have more active phenotype in the expanding borders of the plaques, which suggests that early events occur in the border lesion or the surrounding perilesional areas of the plaques. In order to investigate the very early events happening in the plaque forming steps, we studied the immuno-

165 logical phenotypes of the infiltrating cells in the border and the surrounding perilesional areas of the psoriatic plaques. Biopsies including lesional and perilesional skin were taken from seven psoriatic patients, and immunohistochemical studies were performed in order to characterize the infiltrating dendritic cells using antibodies against CD1a, CD11c, CD11b, CD80, CD86, HLADR, langerin and CD83. CD83 positive cells were infiltrating in the epidermal dermal junction, which were CD11c, HLADR positive, but CD1a or langerin negative. These cells are considered dermal dendritic cells. Only few BDCA2 positive plasmacytoid DCs were seen in the papillary dermis of the lesion and the perivascular infiltrates in the dermis. CD3 positive T lymphocytes were densely seen in the lesion, but sparse in the perilesional skin. These results suggest that the CD83 positive dermal dendritic cells in the perilesional skin may play some part in the formation of psoriatic plaques. 114 A newly identified function of herbal medicines in dendritic cells by screening analysis-the application of amomum seed to tumor immunity Hajime Fukui1,2, Seika Mitsui1,2, Mitsuhiko Nose3, Hajime Mizukami1, Akimichi Morita2 1 Department of Phamacognosy, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya, Japan; 2Department of Dermatology, Graduate School of Medicine, Nagoya City University, Nagoya, Japan; 3Department of Pharmacognosy, Pharmaceutical Science, Meijo University, Nagoya, Japan

Dendritic cells (DCs) play major roles in the regulation of immune responses including tumor immunity and peripheral tolerance. In the present study, we have identified a novel function of herbal medicines in DCs by a screening analysis. We have selected 100 herbal medicines which are mainly contained in 210 Chinese medicine and approved by Ministry of Health, Labor and Welfare. The ethanol extracts were prepared from 100 herbal medicines. The murine epidermal-derived Langerhans cell line, XS106 was employed to screen 100 extracts by analyzing MHC class II expression. Syukusya (amomum seed), Chorei (polyporus sclerotium), and Syazenshi (plantago seed) highly activated XS106 and were selected for the further analysis. In the next step, fresh-isolated bone marrow derived DCs (BM-DC) were examined for the surface phenotypes, i.e., MHC class II, CD 80, and CD86 and IL-12 p70 production. BM-DC treated with Syukusya exhibited activated phenotypes and secreted IL-12p70 significantly. The activation level was almost similar to that of LPS (100 ng/mL). Finally, a tumor model with EG7 (EL4-OVA transfectant) was employed to examine antitumor effects with Syukusya. Pretreatment with subcutanous injection of BM-DC treated with Syukusya and OVA peptide significantly inhibited the growth of tumor cells and prolonged the survival time as compared with the controls. The inhibition rate was comparable to that of LPS. These results indicate that Syukusya has a capacity to highly activate DCs and to be feasible for DC vaccination. 115 Expression of the co-stimulator ICOS and ICOS ligand in patients with psoriasis vulgaris and pustulosis palmaris et plantaris Satomi Kobayashi1, Hiroko Ogiuchi2, Makoto Kawashima1, Takehiko Uchiyama3, Junji Yagi3 1

Department of Dermatology, Tokyo Women’s Medical University, Tokyo, Japan; 2Department of Oral and Maxillofacial Surgery,

166 Tokyo Women’s Medical University, Tokyo, Japan; 3Department of Microbiology and Immunology, Tokyo Women’s Medical University, Tokyo, Japan Inducible co-stimulator (ICOS) is one of CD28 family members that binds to its ligand, B7h, on antigen presenting cells and provides co-stimulatory signals to augment T cell proliferation and various cytokine productions. We investigated ICOS expression on peripheral T cell subsets from patients with psoriasis vulgaris, pustulosis palmaris et plantaris, and healthy individuals by flow cytometry. Additionally, distribution of ICOS+ T cells in psoriatic lesional skin was assessed immunohistochemically. ICOS expression on peripheral T cells was increased in patients with psoriasis vulgaris compared to healthy individuals and pustulosis palmaris et plantaris. Higher percentages of ICOS+ T cells on CD4+ were observed in the patients with higher scores of PASI, and ICOS expressions both on CD4+ and CD8+ T cells were effectively suppressed in the patients under oral cyclosporin treatment. Immunohistochemical study revealed that ICOS+ T cells were infiltrated in dermal papilla in psoriatic lesional skin. Some of the infiltrated T cells in psoriatic lesional skin express CD25, but not CD69 or CD40L. We also established human ICOS mouse IgG Fc fusion protein, and B7h expression was assessed in lesional skin from the patients with psoriasis vulgaris and pustulosis palmaris et plantaris. B7h was expressed on keratinocytes in basal layer partially, and also on some infiltrated cells adhered to basal membrane zone in psoriatic lesional skin, but not in normal skin. These findings suggested that ICOS would be one of the activation markers of Tcells in relatively early stage, and activated Tcells at skin lesions could recruit into the peripheral blood in patients with psoriasis. Furthermore, B7h would be expressed also on keratinocytes under the condition of inflammation. 116 Exogenous cholesterol overload dysregulates Fc epsilon receptor 1 mediated signaling in human monocyte derived dendritic cells Hiroyuki Murota, Shun Kitaba, Mayuko Izumi, Mika Terao, Ichirou Katayama Department of Dermatology, Course of Molecular Medicine Graduate School of Medicine, Osaka University, Japan Altered epidermal barrier function might result in induction of a various kind of dermatoses, including atopic dermatitis (AD). Recent studies showed that reduced amount of total ceramides are responsible for functional abnormalities of the skin of AD. On the one hand, amount of cholesterol composition is controversial among individual studies. Moreover, it has been reported that utilization of physiological lipid mixture improves barrier repair effectively. These results indicated that cholesterol is also required for barrier recovery. To examine the effect of exogenous application of cholesterol, AD patients were treated with 10% (w/ w) cholesterol ointment. Unexpectedly, cholesterol treatment is efficacious to eliminate of clinical manifestations of AD. This phenomenon made us to hypothesize that cholesterol might possesse anti-inflammatory effect. Dendritic cells are known to contribute to maintain the late phase inflammatory reaction in AD by the interaction of allergen-specific IgE and its high-affinity receptor, Fc epsilon receptor 1 (FceR1). FceR1 utilizes lipid raft to initiate the downstream signal transduction, and exogenous cholesterol administration leads to instability of lipid raft. Taken together, there is possibility that external application of cholesterol might induce dysfunction of the FceR1-mediated signaling in dendritic cells. To verify above hypothesis, monocyte derived dendritic cells (MoDC) from healthy volunteers and patients with AD were treated with cholesterol in vitro. As expected, choles-

Abstracts terol loaded MoDC has demonstrated abnormal localization of FceR1alpha, and lost the induction of TARC following mite antigen and IgE stimulation. These results indicated that cholesterol might have potential to be a novel therapeutic intervention for AD. 117 Skin application of ketoprofen induces immunosuppression via CD25+CD4+ regulatory T cells Kenji Atarashi1, Kenji Kabashima1, Satoshi Imai2, Katsuhiko Akiyama2, Yoshiki Tokura1 1 Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan; 2Dermal Physiology and Toxicology Team, Fundamental Research Laboratories, Hisamitsu Pharmaceutical Co., Inc. Tsukuba, Japan

Ketoprofen (KP) is a non-steroidal anti-inflammatory drug that inhibits biosynthesis of prostaglandin. We investigated the effect of topical skin application of KP on the development of murine contact hypersensitivity (CHS), focusing on the role of CD25+CD4+ regulatory T cell (Tr) as a suppressor. Mice were sensitized with picryl chloride (PCl) on the shaved abdomen and challenged with PCl on the earlobes. In this system, the sensitizing site (abdomen) or the back was simultaneously painted with KP to examine the effect of topical KP. Both KP application procedures suppressed the CHS responses, suggesting that the depressed response occurs as a result of systemic immunosuppression. Lymph node cells (LNC) obtained from mice treated with PCl or oxazolone in conjunction with KP were transferred into naive mice, which were sensitized and challenged with PCl. Mice transferred with LNC from PCl + KP-treated mice exhibited a lower CHS response than the non-transferred mice, whereas the response of mice transferred with LNC from oxazolone + KP-treated mice was not inhibited. LNC from mice treated with PCl+KP were separated to CD25+CD4+ cells and CD25-depleted cells by MACS. Naive mice that were transferred CD25+CD4+ cells and sensitized/challenged with PCl showed low responsiveness, whereas transference of CD25-depleted cells was not suppressive. These results suggest that the CHS inhibition induced by topical skin application of KP is systemic and haptein-specific immunosuppression that is mediated by Tr. 118 Counterreguration between TOII-like IgE-mediated responses in mast cell

receptor

2

and

Yoshiko Mizukawa, Tetsuo Shiohara Department of Dermatology, Kyorin University School of Medicine, Tokyo, Japan IgE-mediated mast cell activation has been regarded as a major mechanism responsible for immediate-type hypersensitivity (ITH). Since the discovery of TLR 2 and 4 expression by mast cells, it is becoming evident that contribution of mast cells to ITH extends far beyond their accepted IgE-dependent mechanisms. We have previously demonstrated that the TLR2-dependent rapid footpad swelling corresponding to ITH response is elicited after hapten exposure to the footpad; this ITH response in the footpad increased dramatically at the time when serum IgE did not increase, and after chronic exposure there was a decrease in ITH response despite an increase in serum IgE. These phenomenon could be reproduced in the reconstituted model; reconstitution of the footpad with TLR2/ bone marrow-derived mast cells (BMMC) failed to mount early footpad swelling compared with those with wild-type BMMC. Pretreatment of BMMC with hapten-

Abstracts specific IgE could inhibit the early footpad swelling, while no significant inhibition was detected using not specific for the hapten. To better understand the interactions between TLR2 and IgE/Fc e RI pathway, TLR2/ mice were sensitized on the footpad and repeatedly elicited in the same way with same hapten. A rapid footpad swelling became detectable even in TLR2/ mice. Because local injection of IgE into the footpad of adult mice usually induces the enhancement of early swelling reactions, we next performed the local injection of IgE in mastcell-knock-in mice. When TNP-specific IgE was injected one day before hapten exposure, IgE did not enhance the hapten-induced ITH reactions in mast-cell-knock-in mice. These results indicate that reciprocal crosstalk between TLR2 and IgE/Fc e RI pathway can regulate mast cell activation.

119 Antimicrobial peptides human beta-defensin-3 and -4 activate mast cells through a G protein-coupled phospholipase C pathway Xuejun Chen1,2,3, Francois Niyonsaba2, Hiroko Ushio2, Ko Okumura4, Yasushi Suga1, Shigaku Ikeda1, Hideoki Ogawa2 1 Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 2Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan; 3Department of Dermatology, Sichuan Provincial People’s Hospital, Chengdu, Sichuan, China; 4Department of Immunolology, Juntendo University School of Medicine, Tokyo, Japan

Antimicrobial peptides human beta-defensins (hBDs) are mainly produced by epithelium of several organs including skin and lungs, and play a crucial role in innate immunity by killing invading microorganisms. Besides their microbicidal activities, hBDs activate several inflammatory and immune cells. Since hBDs are generated by tissues where mast cells are abundantly present, we hypothesized that these peptides may activate mast cells. Thus, in the present study, we evaluated the activities of hBD-3 and hBD-4 on mast cells by using rat peritoneal mast cells, a representative of connective tissue mast cell type. Mast cell degranulation and prostaglandin D2 production were investigated through beta-hexosaminidase release assay and enzyme immunoassay, respectively. Intracellular Ca2+ mobilization was studied using a spectrofluorometer. Mast cell migration was performed using tissue culture inserts, where as the induction of skin inflammation was determined by extravasation of Evans blue dye. The results showed that both hBD3 and hBD-4 induced mast cell degranulation, prostaglandin D2 production and intracellular Ca2+ mobilization in a dose-dependent fashion. Furthermore, the abilities of hBD-3 and hBD-4 to elicit mast cell degranulation and prostaglandin D2 generation were inhibited by pertussis toxin and U-73122, inhibitors of G protein and phospholipase C, respectively. We further revealed that both hBD-3 and hBD-4 elicited mast cell chemotaxis rather than chemokinesis. Moreover, these peptides increased vascular permeability in vivo. These findings imply that, besides their antimicrobial activities, hBDs may participate in allergic and/or inflammatory reactions by recruiting and activating mast cells, and increasing vascular permeability. 120 IL-17 suppresses TNF-a-induced CCL27 production through induction of cyclooxygenase-2 in human keratinocytes Naoko Kanda, Shinichi Watanabe Department of Dermatology, Teikyo University School of Medicine, Tokyo, Japan

167 Background: A chemokine CCL27 attracts skin-homing T cells. CCL27 production by keratinocytes is dependent on NF-kB activity and enhanced in lesions with atopic dermatitis, psoriasis vulgaris or allergic contact dermatitis. IL-17 is released from activated memory T cells and modulates skin inflammation. We recently found that a chemical mediator prostaglandin E2 suppressed CCL27 production in keratinocytes. Objective: We examined in vitro effects of IL-17 on TNF-ainduced CCL27 production in human keratinocytes. Methods: Keratinocytes were incubated with TNF-a and/or IL-17. CCL27 production was analyzed by ELISA and RT-PCR. Cyclooxygenase-2 (COX-2) promoter and NF-kB activities were analyzed by luciferase assays. Results: IL-17 suppressed TNF-a-induced CCL27 secretion and mRNA expression and NF-kB activity in keratinocytes. COX-2 inhibitor NS398 counteracted the effects of IL-17, and COX-2 product, prostaglandin E2 prevented the counteraction by NS398. IL-17 alone or synergistically with TNF-a, increased prostaglandin E2 release from keratinocytes and the increase was suppressed by NS398. IL-17 alone or synergistically with TNF-a, increased COX-2 mRNA and protein levels, promoter activity and mRNA stability. Stimulatory effects of IL-17 on COX-2 expression were suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase. IL-17 alone or synergistically with TNF-a, induced dual phosphorylation of p38 MAPK and ERK. Conclusion: IL-17 may suppress TNF-a-induced CCL27 production by inhibiting NF-kB via induction of COX-2. The induction of COX-2 may be mediated by activation of p38 MAPK and ERK. Tcellderived IL-17 may alleviate T cell skin infiltration via inhibition of CCL27 production. 121 Elevated serum BAFF levels in patients with systemic sclerosis (SSc): Enhanced BAFF signaling in SSc B lymphocytes Takashi Matsushita1, Minoru Hasegawa1, Manabu Fujimoto1, Shinichi Sato2, Kazuhiko Takehara1 1

Department of Dermatology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan Objective: To determine serum levels of B cell activating factor belonging to the tumor necrosis factor family (BAFF), a potent B cell survival factor, and clinical association in patients with systemic sclerosis (SSc). Methods: Serum BAFF levels from 83 SSc patients were examined by ELISA. For a longitudinal study, 131 sera from 21 SSc patients were analyzed. BAFF mRNA expression in the skin was quantified by real-time RT-PCR. BAFF receptor (BAFF-R) expression on CD19+ B cells was assessed by flow cytometry. IgG and IL-6 production by isolated B cells was examined by ELISA. Results: Serum BAFF levels were elevated in SSc patients compared to healthy controls and correlated positively with the extent of skin fibrosis. In a longitudinal study, 21 SSc patients were classified as follows: 7 patients with decreasing BAFF levels; 11 with unchanged levels; and 3 with increasing levels. Decreasing BAFF levels were accompanied by regression of skin sclerosis, whereas increasing levels were associated with new onset or worsening of organ involvement. BAFF mRNA expression was up-regulated in the affected skin from early patients with diffuse cutaneous SSc. BAFF-R expression on B cells was increased in SSc patients relative to healthy controls. Furthermore, SSc B cells exhibited an enhanced ability to produce IgG and IL-6 by BAFF stimulation.

168 Conclusion: These results suggest that BAFF and its signaling in B cells contribute to B cell abnormalities and disease development in SSc. 122 Changes in substance P and neutral endopeptidase induced by skin scratching Junichi Yamaoka, Seiji Kawana 1

Department of Dermatology, Nippon Medical School, Tokyo, Japan Background: Skin scratching is one of the biggest aggravating factors of pruritic skin diseases such as atopic dermatitis or prurigo. To investigate effects of skin scratching on the molecular and cellular basis, we established mice model for skin scratching and got several new findings about elongation of nerves and change in nerve growth factor (NGF) after skin scratching. Neuropeptides, bradykinin and/or cytokines secreted after skin scratching may possibly play important roles in the consequent inflammation and the acquisition of hypersensitivity. However, it’s not clear when, where and how are these molecules secreted and degradated. Therefore, in this study, we investigated changes in substance P (SP) and its degradating enzyme: neutral endopeptidase (NEP). Methods: Constant scratching stimulations were given on the back skin of anesthetized mice. Then, skin tissues were taken from mice and were examined by immunohistochemistry, ELISA and real time PCR. Changes in enzymatic activity of NEP in skin tissue homogenates were also measured. Results and discussions: SP-positive nerve fibers decreased rapidly soon after skin scratching. This may reflect the secretion of SP from nerves. This response lasted for few days and disappeared later. ELISA revealed that the level of SP protein in skin homogenates decreased transiently after skin scratching. Like this, the enzymatic activity of NEP also decreased transiently and then returned to the basal level. After several days, expression of NEP mRNA started to rise. These findings suggest that secreted SP may be degradated rapidly with NEP. In addition, expressions of NEP and SP receptor NK-1R had almost identical localizations. Thus, NK-1R and NEP may be controlled within a delicate balance. 123 Protection by UV endonuclease against photoaging through the suppression of inflammatory mediator production Yasunobu Ochiai1, Kei Obayashi1, Yuri Okano1, Daniel B. Yarosh2, Hitoshi Masaki1,3 Cosmos Technical Center Co., Ltd. Tokyo, Japan; 2AGI Dermatics. NY, The United States of America; 3Nikko Chemicals Co., Ltd. 1

Background: Ultraviolet (UV) irradiation to the skin causes inflammation and functional alterations, and finally leads to photoaging. UVB in particular easily damages DNA, yielding cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts through photo-chemical reactions. DNA damage is an important initiator of UV-induced inflammation because UVB irradiation of xeroderma pigmentosum group A gene-deficient mice, which are deficient in repair of DNA damage, significantly increases prostaglandin E2 (PGE2) production compared to the wild-type mice [1]. Therefore, we investigated the effects of UV endonuclease, an enzyme that specifically recognizes and initiates repair of CPD, on cultured skin cells to clarify the relationship among DNA damage, inflammation, and subsequent change of the cutaneous function.

Abstracts Method: The UV endonuclease was encapsulated in liposomes for delivery to the cells. Normal human keratinocytes pretreated with liposomal UV endonuclease were irradiated with UVB and PGE2 content in the cultured medium was determined by ELISA. In addition, the effect of PGE2 on the expression of extracellular matrix genes in normal human fibroblasts was examined by RTPCR, because this is one of the indicators of skin response to wounds. Results and discussion: UV induced the secretion of PGE2 in keratinocytes. Treatment of UVB-irradiated keratinocytes with UV endonuclease decreased PGE2 content significantly in the culture medium. Furthermore, fibroblasts treated with PGE2 decreased the expression of collagen genes. These results indicate that UV endonuclease protects against photoaging through the suppression of at least one inflammatory mediator by enhancing DNA repair. Reference [1] Kuwamoto K, et al. J Invest Dermatol 2000.

124 Analysis of the function of lipopolysaccharide on chemokine production by HaCaT cells Koichiro Nakamura, Noritaka Oyama, Fumio Kaneko Department of Dermatology, Fukushima Medical University, Fukushima, Japan TARC/CCL17, CTACK/CCL27, IP-10/CXCL10 are chemokines that differentially attract and activates different leukocytes. We have previously shown that HaCaTcells, keratinocytes cell lines, produce chemokines under the stimulation of IFN-g and TNF-a. In atopic dermatitis and psoriasis, different chemokines play a role in the pathogensis. However, the role of innate immunity via toll like receptors in chemokines production has not been fully understood. We investigated the effects of lipopolysaccharide (LPS) on chemokine production by HaCaT cells. HaCaT cells spontaneously produced various chemokines (CCL17, CCL27, CXCL10), and this was differentially upregulated by different doses of LPS. The maximal effects of LPS on CCL17, CCL27 and CXCL10 production was observed at 0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml, respectively (CCL17: 1255.8 pg/ml, CCL27: 1306.2pg/ml, CXCL10: 2405.5 pg/ml) at 24 h culture. RT-PCR analysis also confirmed this. These results indicate that innate immune function can regulate chemokine production by HaCaT cells, and may play a role in the modulation of cutaneous inflammation. 125 Induction of keratinocyte Th2 chemokine production, dermal eosinophil infiltration, and late phase reaction in barrierdisrupted skin Ayako Uyemura, Kenji Kabashima, Miwa Kobayashi, Tomoko Mori, Kenji Atarashi, Yoshiki Tokura Department of Dermatology, University of Occupational and Environmental Health, Japan Skin barrier disruption is known to stimulate keratinocytes to produce cytokines, such as IL-1a and TNF-a However, the physiological significance of barrier disruption in skin immune milieu remains to be clarified. We investigated the effects of barrier disruption on Th1/Th2 balance and skin inflammation in earlobes of BALB/c and C57BL/6J (B6) mice. Twenty-four hours

Abstracts after tape stripping or acetone rubbing, epidermal cell suspensions were prepared and cultured for 48 h. The concentration of IP-10, TARC, MDC and RANTES in the culture supernatants was measured by ELISA. Epidermal cells from BALB/c mice treated with tape stripping and acetone rubbing produced higher levels of TARC and MDC (Th2 chemokines) than those of untreated mice, whereas IP-10 (Th1 chemokine) production was not affected by barrier disruption. Next, we examined the histology of barrier disrupted skin. Earlobes treated with tape stripping showed infiltration of eosinophils in the dermis. Eosinophils appeared in the dermis 6 h after treatment, and reached their peak 24 h later. BALB/c mice, a Th2-polarized strain, had higher number of eosinophils than B6 mice. Finally, we applied the FITC hapten-induced dermatitis model to address the physiological importance of barrier disruption. BALB/c mice were sensitized with 1% FITC, and earlobes were provoked by painting of 0.5% FITC after tape stripping. The earlobes of barrier disrupted mice swollen more severely than those of untreated mice at 4—8 h after challenge, which corresponds to the late phase reaction. These findings suggest that barrier disruption induces Th2 chemokine production by keratinocytes, infiltration of eosinophils, and enhancement of late phase reaction, implying that dry skin or scratching exacerbates atopic dermatitis. 126 Gene expression and localization of TNF-a converting enzyme during wound healing in rat skin Masakazu Kawaguchi, Yoshihiko Mitsuhashi, Shigeo Kondo Department of Dermatology, Yamagata University School of Medicine The TNF-a converting enzyme (TACE) is a metalloprotease-disintegrin that releases soluble TNF-a from cells by cleaving within the extracellular domain of membrane-bound pro-TNF-a. Furthermore, TACE is reported to cleave several other substrates including TGF-a, HB-EGF, L-selectin, TNF receptors, Notch, fractalkine. In this study, we examined the expression of TACE mRNA during wound healing by in situ hybridization analysis. The expression of TACE was seen in hair follicles. Expression level of TACE mRNA was increased anagen phase compared with telogen phase. In cutaneous wound healing model, expression level of TACE mRNA was increased in epidermis and dermis compared with normal skin. In addition, in vitro and organ culture model, TACE/ MMP inhibitor and specific TACE inhibitor caused suppression of keratinocyte migration. To study the regulation of TACE, we examined the expression of TACE mRNA in cultured keratinocyte treated with TPA, EGF, TGF-a, HGF, TGF-b, TNF-a, IFN-g using real-time PCR.

127 Fibroblast growth factor-6 effect on the integrin-associated protein expression in melanoma cells Maki Kawashima, Takenori Takahashi, Yasuhiro Kawachi, Fujio Otsuka Department of Dermatology, University of Tsukuba, Ibaraki, Japan Fibroblast growth factor (FGF) family plays many important roles in cellular developmental events including the proliferation and the migration. Whereas FGF-2 has been shown to serve as the most potent effector, the relevance of other FGF members has still been unclear. FGF-6, a protooncogene product, is

169 expressed in both epithelial and mesenchymal derivatives and has autocrine and/or paracrine roles. We previously evaluated FGF-6 function on melanoma cell proliferation and apoptosis. The present investigation showed that FGF-6 elicited the integrin-associated protein (IAP, CD47) expression on human melanoma cell surface. IAP is a member of the immunoglobulin superfamily and binds to its receptor, SHPS-1, which is widely expressed and is particularly abundant in neural and myeloid tissues. Recent studies suggest that the IAP-SHPS-1 interaction leads to the dephosphorylation of SHPS-1 and dissociation of SHP-2 from intracellular SHPS-1 region, resulting in the inhibition of cell migration. Thus, IAP and SHPS-1 may be important molecules for the malignant behavior of certain cell types. Although the regulation of SHPS-1 expression has been shown in several tissues and cells including breast cancer, the regulation of IAP expression has hardly been reported. Our results might imply the participation of FGF-6 in the invasive action of melanoma cells. 128 Histamine receptor (HR1, HR2, HR3) profiles in skin lesions of MRL/lpr mice Takashi Yoshimasu, Takeshi Nishide, Fukumi Furukawa Department of Dermatology, Wakayama Medical University, Wakayama, Japan It is supposed that the development of skin lesions of LE is associated with histamine and histamine receptors (HR). Th1 cells express HR1 and induce IFN-g Th2 cells are associated with HR2 and they induce IL-10. Purpose: We determined cytokine profiles in skin lesions of MRL/lpr (l) mice by RT-PCR method. Furthermore, we assessed the relationship between the skin lesions and histamine receptors by using immunohistochemical analysis and RT-PCR method. Methods: We sacrificed the MRL/l mice (1 to 5-months) and MRL/n mice (5-month-old). Skin samples were obtained from the upper backs of all examined mice, and non-lesional skins of MRL/l mice (5-month-old) were also obtained. Results: Skin lesions were clinically and histologically seen in MRL/l mice of 5-month-old. The skin lesions of MRL/l mice showed TNF-a and IL-10 but not IFN-g by RT-RCR method. The expressions of HR1, HR2 and HR3 were equally seen in hair follicles and around sebaceous glands of all MRL/l mice (1 to 5-months) and MRL/n mice by immunohistochemical staining. The expressions of HR1 and HR3 were strongly seen in the skin of 1-month-old MRL/l mice by RT-PCR method, however the expressions were decreased in aged mice of MRL/l, irrespective of presence or absence of skin lesions. The expression of HR2 was increased in the skin of MRL/l mice at 2-months compared with those at 1-month. The expression of HR2 was gradually increased in skin lesions of MRL/l mouse with age but the expression was decreased in non-lesional skin of MRL/l mouse (5-months). All expressions of HR1, HR2 and HR3 were decreased in MRL/n mice compared with those of MRL/l mice. Conclusion: It is suggested that the skin lesions of MRL/l mice are associated with Th2 type immune response which include HR2 expression, not HR1 or HR3 expression. 129 Anti-P53 antibody is a novel autoantibody related to limited cutaneous systemic sclerosis Toshihide Hara, Fumihide Ogawa, Shinichi Sato Department of Dermatology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

170 The tumor suppressor protein p53 plays an important role in the control of cell proliferation, DNA repair, angiogenesis, and apoptosis. Recently, the presence of autoantibody to the p53 nuclear protein (anti-p53 antibody) has been reported in various autoimmune diseases. In this study, we assessed the prevalence and clinical significance of anti-p53 antibody in patients with systemic sclerosis (SSc). Enzyme-linked immunosorbent assay revealed that serum anti-p53 antibody levels were significantly higher in SSc patients (n = 70) than healthy controls (n = 21; p < 0.01)). Remarkably, anti-p53 antibody levels were significantly higher in patients with limited cutaneous systemic sclerosis than those with diffuse cutaneous systemic sclerosis ( p < 0.05). Regarding its clinical correlation, the frequency of anti-p53 antibody positivity correlated positively with percentage vital capacity ( p < 0.05). The presence of anti-p53 antibody was confirmed in sera from SSc patients by Western blotting. These results suggest that anti-p53 antibody is a novel autoantibody associated with the milder form of SSc. 130 CTGF maintains TGF-b-induced skin fibrosis by stimulating alpha2(1) collagen (COL1A2) expression Miki Kondo, Fumiaki Shirasaki, Kazuhiko Takehara Department of Dermatology, Kanazawa University Graduate School of Medical Science, Ishikawa, Japan Systemic sclerosis (SSc) is characterized by an excessive production of extracellular matrix and understood to develop by the excessive action of some growth factors. To better understand the role of these cytokines in the process of SSc in vivo, we previously established an animal model of skin fibrosis induced by exogenous injection of transforming growth factor-b and connective tissue growth factor (CTGF) into newborn mice. In this model, TGF-b transiently induced subcutaneous fibrosis in the first 3 days and serial 4 days injection of CTGF caused persistent fibrosis. Proto-oncogene product c-Myc plays important roles in cell growth, death and differentiation. c-Myc was reported to suppress alpha2(1) collagen (COL1A2) transcription and to be regulated by TGF-b. Therefore, c-Myc may contribute to skin fibrosis induced by TGF-b and CTGF. In this study, we investigated the action of TGF-b and CTGF on cultured fibroblasts to define the mechanism of the regulation of COL1A2 expression. TGF-b increased COL1A2 mRNA expression by 50%, although CTGF alone did not affect COL1A2 expression. When cultured fibroblasts were treated with both TGF-b and CTGF, 2fold increase of COL1A2 mRNA expression was observed. The additional effect of CTGF on the TGF-b-induced COL1A2 promoter activation was also observed. c-Myc suppressed the TGF-binduced COL1A2 promoter activation. And c-Myc was continuously reduced by the stimulation with TGF-b and CTGF. These results suggested that c-Myc, which is regulated both TGF-b and CTGF, suppresses the TGF-b-induced COL1A2 promoter activation, indicating that c-Myc regulates the basal transcriptional activity of COL1A2 promoter. Furthermore, we are investigating the expression of c-Myc in fibroblasts derived from SSc. 131 Microarray analysis of PU.1-mediated mast cell- and monocytespecific gene expression Yusuke Niwa1, Chiharu Nishiyama2, Nobuhiro Nakano2, Asuka Kamei3, Hisanori Kato3, Shigaku Ikeda1, Hideoki Ogawa2 1 The Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 2Atopy Research Center, Juntendo Uni-

Abstracts versity School of Medicine, Tokyo, Japan; 3Graduate School of Agricultural and Life Science, The University of Tokyo, Tokyo, Japan Purpose: We have previously reported that overproduction of PU.1 in bone marrow-derived mast cells (BMMC) causes functional and morphological changes toward monocytes. In brief, cell surface expression of c-kit was reduced, expression of CD11b, CD11c, and MHC classII was induced, IgE-mediated degranulation reaction was down-regulated, and response to lipopolysaccharide via TLR-4 was up-regulated. In this study, we analyzed gene expression of BMMC overproducing PU.1 by using microarray to further elucidate the mechanism of PU.1-mediated gene regulation. Methods: Total RNAs were extracted from following three samples; PU.1-overproducing BMMC that was prepared by using retrovirus expression system, control BMMC, and bone marrowderived dendritic cell (BMDC), and were reverse transcribed to cDNA. We analyzed gene expression of each cDNA by using microarray (Affymetrix). Results: In BMDC, the transcriptional level of IRF-4 and IRF-8 which form hetero-dimeric complexes with PU.1 were markedly higher, and the transcriptional level of GATA-1and -2, which involve in mast cell-specific gene expression, were significantly lower as compared with those of BMMC. Overproduction of PU.1 did not cause apparent effects on expression level of these transcription factors. Now, we are continuously referring to microarray data to identify the genes under the control of PU.1, and quantitatively analyzing the expression level of each molecule. 132 EM703, the new derivative of erythromycin, inhibits transcription of type I collagen through CCAAT box in normal and scleroderma fibroblasts Hideyuki Ikeda1, Toshiaki Sunazuka2, Satoshi Omura2, Hiromi Suzuki1, Soji Yamazaki1, Atsushi Hatamochi1 1

Department of Dermatology, Dokkyo University School of Medicine, Tokyo, Japan; 2Kitazato Institute, Kitazato University, Tokyo, Japan Systemic sclerosis (SSc) is a fibrotic disorder characterized by excessive accumulation of collagen in the skin and a variety of internal organs. The accumulation of collagen is thought to result from enhanced transcription of collagen in fibroblasts. None of treatment modalities for patients with SSc has brought satisfactory improvement. We previously presented transcriptional inhibition of type I collagen gene expression in normal and scleroderma fibroblasts by EM703. In this time we investigated the mechanism for the inhibition of transcription of type I collagen. Methods: Three normal and SSc fibroblasts were cultured with routine methods. When the cell layers had become confluent, the cells were incubated for 48 h at 37 8C in DMEM containing 1% FBS, supplemented with 107 M to 105 M of EM703. Amounts of collagen were measured using ELISA for type I collagen. Northern blots analysis, luciferase assay, electrophoretic gel mobility shift assay were also performed. Results: EM703 reduced collagen production (to 40% in maximum) and mRNA levels of a1(I) collagen (to 30% in maximum) in a dose dependent manner in normal and SSc fibroblasts. COL1A1 transcription was down-regulated by addition of EM703 (105 M) to 30% by the Luciferase assay using various length of type I collagen promoter even deleted to only 133 bp. Interestingly EM703 do not inhibit COL1A1 transcription by the Luciferase assay using 332 bp promoter with point mutation of the CCAAT box. Nuclear extracts from fibroblasts treated with EM703 reduced the

Abstracts CCAAT binding factor (CBF) binding activity. These results suggest that EM703 inhibits transcription of type I collagen through the CCAAT box accompanying reduced the CBF binding activity in normal and scleroderma fibroblasts. 133 Expression of p63 in sweat gland neoplasms Seiji Maeshima1,2 1

The Second Department of Pathology, Osaka Medical College, Osaka, Japan; 2Maeshima Clinic Purpose: The purpose of this study was to investigate the pattern of p63 immunoreactivity in the sweat gland neoplasms. The transcription factor p63 is a recently identified homologue of the tumor suppressor p53. Unlike p53, which is dispensable for normal development, p63 is essential in the development of epithelia and is mainly expressed by basal and myoepithelial cells. Materials and methods: Immunohistochemical analysis for p63 was performed on formalin-fixed, paraffin-embedded archival tissue from 21 benign sweat gland tumors (eccrine poroma: 10, clear cell hidradenoma: 4, eccrine cystadenoma: 2, apocrine cystadenoma: 1, apocrine tublar adenoma: 2, hidradenoma papilliferum: 1, parauretheal cyst: 1. Results: In normal skin, p63 was expressed in the basal and suprabasal cells of epidermis and cutaneous appendages, including the basal and myoepithelial cells of sweat glands. All ecrine poromas showed strong p63 expression without the luminal cells. In clear cell hidradenomas, dark spindle cells showed strong p63 expression but clear cells didn’t. In contrast, the myoepithelial cells and the outer layer of cuboidal epithelium of the ductal structures of the eccrine cystadenoma, apocrine cystadenoma, apocrine tublar adenoma, hidradenoma papilliferum and parauretheal cyst expressed p63, but the inner layer of cylindrical epithelium didn’t. These facts are effective to know the direction where the neoplasm cell differentiates. 134 Serine protease-like HTRA1 is specifically upregulated in keloid legions Naomi Matoh 1, Motoko Naitoh2,3, Mika Ikeda2, Tomio Tsukie1, Hirofumi Shirane4, Toshinori Tanaka4, Hiroshi Kubota3, Kazuhiro Nagata3 1 Department of Plastic and Reconstructive Surgery, Kobe General Hospital, Kobe, Japan; 2Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Department of Molecular and Cellular Biology, Institute for Frontier Medical Science, Kyoto University, Kyoto; 4 Department of Clinical Pathology, Kobe General Hospital, Kobe, Japan

Background: Keloids are a dermal fibrotic disease whose etiology remains unknown and for which there is no successful treatment. We employed cDNA microarray analysis to examine gene expression in keloid lesions and control skin. We found that 32 genes among the 9, 182 tested were strongly up-regulated in keloid lesions, of which HtrA1 was focused on this report. Roles of HtrA1 in modulating the pathology of various diseases including osteoarthritis, ovarian cancer and malignant melanoma have been suggested. Aim: To clarify the role of HtrA1 in keloid pathogenesis, the expression pattern of HtrA1 in keloid lesions was analyzed.

171 Methods: Total RNA was isolated from keloid legions of six patients and two normal control skin samples by single-step method. The expression levels of HtrA1 were examined by using Northern blot analysis. Keloid, hypertrophic scar and normal skin tissue samples were fixed with paraformaldehyde, and paraffin sections were obtained. Immunohistochemical analysis was performed with anti HtrA1 polyclonal antibody. Results and discussion: As determined by Northern blotting, the mRNA level of HtrA1 was markedly elevated in all keloid legions, compared to normal skin. Immunohistochemical analysis revealed that the fibroblast-like cells abundant in the margin of keloid lesions were strongly stained with anti HtrA1 antibody. In contrast, no HtrA1 staining was observed in the fibroblast of normal skin. Interestingly, no significant staining was detected in hypertrophyic scar lesions, despite the clinical aspects similar to keloids. HtrA1 may contribute to the development of keloid lesions by remodeling keloid-specific components of extracellular matrix or cell surface molecule.

135 Genome-wide association study of psoriasis using polymorphic microsatellite markers Tomotaka Mabuchi1, Akira Oka2, Yoshinori Umezawa1, Takashi Matsuyama1, Yumiko Kubota3, Juichiro Nakayama3, Tadashi Terui4, Maki Ozawa5, Shinichiro Yasumoto6, Takashi Hashimoto6, Shigaku Ikeda7, Yoshinari Matsumoto8, Hirohiko Sueki9, Masafumi Iijima9, Hidetoshi Inoko2, Akira Ozawa1 1 Department of Dermatology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan; 2Department of Molecular Life Science, Tokai University School of Medicine, Kanagawa, Japan; 3Department of Dermatology, Fukuoka University School of Medicine, Fukuoka, Japan; 4Department of Dermatology, Nihon University School of Medicine, Tokyo, Japan; 5 Department of Dermatology, Tohoku University Graduate School of Medicine, Miyagi, Japan; 6Department of Dermatology, Kurume University School of Medicine, Fukuoka, Japan; 7Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan; 8Department of Dermatology, Aichi Medical University School of Medicine, Aichi, Japan; 9Department of Dermatology, Showa University School of Medicine, Tokyo, Japan

Genome-wide studies of psoriasis has made remarkable progress recently. These studies have identified many psoriasis susceptibility loci on several chromosomes. However, psoriasis susceptibility genes have not identified. In this study, we have systematically performed association analysis of psoriasis to define all psoriasis susceptibility loci. The subjects investigated in this study were unrelated Japanese patients with psoriasis vulgaris. Independent 125 samples were pooled for the 1st, 2nd and 3rd sets Similarly, independent 125 unrelated healthy individuals as controls were pooled for the 1st, 2nd and 3rd sets. We used the 1st set pooled DNA for the 1st screening. After that, 2nd and 3rd sets were used for the 2nd and 3rd screening to avoid false positive associations. As the result, putative psoriasis candidate loci were narrowed down to 16 regions. Summary of the results will be reported in this meeting. 136 Effect of light-emitting diode irradiation on skin Satoshi Morita, Miho Narita, Noritake Kitatani, Tomohiro Terauchi Naris Cosmetics Co., Ltd., OSAKA, Japan

172 Light-emitting diode (LED) is a photomodulator that emits monochromatic light stably. Effects of LED treatments are wavelengthdependant, and several types of treatments are reported in the field of cosmetic dermatology. For example, blue LED is employed in treatment for acne, and red LED accelerates wound healing and has anti-irritant and anti-inflammatory effects. Red LED is also known to be effective in treatment for wrinkles and skin pigmentations. Moreover, in vitro studies show that visible LED enhances proliferation of cells, synthesis of collagen, and production of ATP. As for red, blue, and green LEDs, the mechanisms of such effects have been shown at the molecular level. Mechanism of near-infrared LED treatment, on the other hand, has not been well studied. The purpose of our study was to evaluate the effects of near-infrared LED on skin. We investigated the changes in gene expression after the near-infrared LED irradiations. A reconstructed skin model was irradiated with near-infrared LED at 905nm, and its gene expression was compared with that of sham-irradiated skin model using DNA microarray. The result showed that LED irradiation up-regulated HIST genes and the genes related to cell proliferation, such as TUGCP3, KIF11, TTK, and KPNA5. In contrast, MMP10 and genes related to apoptosis, such as CASP2 and TRPM2 were down-regulated. Furthermore, our experiment with cultured keratinocytes and fibroblasts showed that LED irradiation stimulated cell proliferation, collagenogenesis, and cell migration. These results suggest that near-infrared LED irradiation is effective for treatment of aging skin. 137 TGF-b/SMAD signaling is functional in human eosinophils Mirei Kanzaki1,2, Naotaka Shibagaki1, Hiroshi Mitsui1, Takashi Inozume1, Atsushi Okamoto2, Yoh Dobashi3, Hideoki Ogawa4, Shinji Shimada1, Atsuhito Nakao2,4 1 Department of Dermatology, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan; 2Department of Immunology, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan; 3 The First Department of Pathology, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan; 4Atopy Research Center, Juntendo University School of Medicine, Tokyo, Japan

There is a paradoxical finding that eosinophils are frequently accumulated at the sites of allergic inflammation and tissue fibrosis where TGF-b, a negative regulator of eosinophil survival, is upregulated, but yet eosinophil accumulation proceeds unchecked. These observations have prompted us to hypothesize that TGF-b signaling in eosinophils may be impaired. We thus sought to examine the expression and function of Smad proteins in eosinophils. Eosinophils were isolated from peripheral blood of normal donors and expression and activation of endogenous Smad proteins in eosinophils were examined by RT-PCR and Western blot. Smad function in transcription of TGF-b target genes was investigated using a dominant negative form of Smad2/3. Effects of TGF-b on eosinophil survival were evaluated by cell viability assay. Human eosinophils expressed mRNAs and proteins of TGF-b type I and type II receptors, Smad2, Smad3, and Smad4. TGF-b induced phosphorylation of Smad2 in eosinophils, which was blocked by SB431542, a selective inhibitor of TGF-b type I receptor kinase activity. A cell membrane-permeable form of mutant Smad3 protein, that has a dominant negative effect on Smad2/3, suppressed TGF-b-induced Smad7 and cjun mRNA expression in eosinophils. Finally, we detected expression of TGF-b mRNA in eosinophils, which was enhanced upon stimulation with TGF-b. However, blockade of endogenous TGFb signaling by SB431542 did not affect GM-CSF-mediated prevention of eosinophil apoptosis whereas exogenously added TGF-

Abstracts b did inhibit it. Human eosinophils have intact Smad signaling pathway leading to major TGF-b target gene expression and can respond to exogenous, but not endogenous, TGF-b in vitro Thus, if any, eosinophils might become resistant to TGF-b in in vivo circumstances. 138 Involvement of FGF family members and their receptors in fullthickness skin wound healing: A study on the possible role of FGF23 Toru Imamura Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST) The highly ordered process of wound healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide growth factors. To gain additional insight into these issues, we quantified the absolute copy numbers of mRNAs encoding all the fibroblast growth factors (FGFs), their receptors (FGFRs) and two other growth factors in the dorsal skin of young and aged mice during the healing of full-thickness skin excisional wounds. In young adult mice (8 weeks old), FGF7, 10 and 22 mRNAs were all strongly expressed in healthy skin, and levels of FGF7 and 10 but not 22 increased 2- to 3.5-fold over differing time courses after wounding. The levels of FGF9, 16, 18 and especially 23 mRNAs were moderate or low in healthy skin but increased 2- to 33-fold after wounding. Among the four FGFRs, expression of only FGFR1 mRNA was augmented during wound healing. Notably, in aged mice (35 weeks old), where healing proceeded more slowly than in the young, both the basal and wound-induced mRNA expression of most of these genes was reduced. While these results confirm the established notion that keratinocytespecific, FGFR2IIIB ligands (FGF7, 10 and 22) are important for wound healing, they also indicate the potential importance of further study of the involvement of FGF9, 16, 18 and 23 in the wound healing process. Although FGF23 has been shown to play important pathophysiological roles in hypophosphatemic diseases, its involvement in wound healing has not been studied. Our results of its activity on epidermal and dermal cells will be discussed. 139 A novel effect of diosgenin on skin aging Yayoi Tada1, Naoko Kanda1, Akinori Haratake2, Megumi Tobiishi2, Hideyo Uchiwa2, Shinichi Watanabe2 1 Department of Dermatology, Teikyo University School of Medicine; 2Basic Research Laboratory, Kanebo Cosmetics Inc.

Background: Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health food for climacteric in USA. The extracts contain large amounts of diosgenin. We recently found that diosgenin enhanced DNA synthesis of TESTSKIN. Objective: In this study, we examined in vivo effects of oral diosgenin on climacteric-mimetic skin and investigated the mechanisms for the enhancement of DNA synthesis by diosgenin in keratinocytes in vitro. Methods: Ovariectomized mice were orally given 0.01, 0.02, or 0.04% of diosgenin in AIN-93G five times per week for 20 weeks. Epidermal thickness and weight of subcutaneous or internal fat tissues and uteri were measured before and after the treatment. Adult human keratinocytes were preincubated with inhibitors of signaling pathways or antisense oligonucleotides against estrogen

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has a deleterious effect on blood vessels, which is markedly associated with endothelial apoptosis. We therefore explored whether or not S1P modulates H2O2-induced apoptotic responses in cultured bovine aortic endothelial cells (BAEC). Western blot analyses using a subtype-specific antibody for the cleaved form of caspase-3 revealed that treatment with H2O2 (750 mM, for 6 h) induces cleavage of caspase-3 protein (2.9 folds of cleaved caspase-3 fragment formation), indicating that these cells undergo apoptotic processes under the H2O2 treatment. When BAEC had been pre-treated with S1P (1 mM for 30 min) prior to H2O2 treatment, the degree of cleaved caspase-3 fragment formation was attenuated (77% versus H2O2 alone). Conversely, pretreatment with S1P significantly decreased the degrees of H2O2-induced DNA fragment formation by 22% ( p < 0.01). Collectively, these data suggest that the platelet-derived lipid growth factor S1P may protect vascular endothelial cells from oxidative stress-induced apoptotic cell death, possibly representing novel actions of this bioactive lipid on vasculature during wound healing processes.

Analyses of signal transduction in human keratinocyte stimulated by basic fibroblast growth factor

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Yoko Sogabe, Masatoshi Abe, Osamu Ishikawa

Fibroblast growth factor-2 elicits epithelioid sarcoma cells invasion into extracellular matrix

receptor b (ERb) or GPR30, then treated with diosgenin for 20 h, then pulsed with BrdU for 4 h. BrdU uptake was examined by ELISA. Results: In ovariectomized mice, epidermal thickness is reduced and subcutaneous or internal fat tissues were more accumulated compared to those in sham-operated mice. Diosgenin intake improved the reduced skin thickness in the ovariectomized mice without altering accumulation of fat tissues. Diosgenin did not bind nuclear ER. Diosgenin did not increase uterine weight in mice. Diosgenin (109 to 108 M) increased BrdU uptake and intracellular cAMP level in adult human keratiocytes in vitro. These effects of diosgenin were blocked by adenylate cyclase inhibitor but not by antisense oligonucleotides against ERb or GPR30. Conclusion: Diosgenin may enhance DNA synthesis and improve reduced proliferation of keratinocytes via inducing cAMP signal without involvement of ERb or GPR30.

The Department of Dermatology, Gunma University Graduate School of Medicine, Masbashi, Japan Topical application of human recombinant basic fibroblast growth factor (bFGF) promotes wound healing. bFGF, however, has been reported to have little in vitro effects on keratinocyte compared to other cell types such as endothelial cells or fibroblasts. Normal human keratinocytes formed lamellipodia only when they were stimulated with bFGF on the collagen-coated coverslips. Under these conditions, vinculin was expressed and GTP-loaded Rac (an activated form) was participated in that phenomenon. These results strongly suggest that integrina2b1 plays a crucial role in this morphological change. We are examining the expressions of integrina2b1and FAK activation in bFGF-stimulated keratinocytes on collagen-coated coverslips by immunofluorescence microscopy, and the expression of integrina2b1, vinculin, FAK activation and induction of actin stress fiber in the same way after treatment of the neutralizing antibody for integrina2b1 or Rac1 inhibitor, pulldown assay to detect GTP-loaded Rac and cdc42 (an activated form), and the effects of bFGF on keratinocyte migration. These in vitro studies may clarify a part of intracellular signal transduction that bFGF exerts stimulatory effect on keratinocyte migration under the presence of type I collagen as a scaffold.

Takenori Takahashi, Hiroko Sato, Yasuhiro Fujisawa, Fujio Otsuka Department of Dermatology, University of Tsukuba, Ibaraki, Japan

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in bovine artery vascular endothelial cells

Epithelioid sarcoma (ES), a rare malignant neoplasm of uncertain origin, shows a high metastasis rate (45%). This aggressive soft tissue tumor expresses an epithelial membrane antigen and possesses the epithelial cell characteristics. It has been shown that the mitogenic activity of ES cells is subject to the modulation of many cytokines such as epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)-1 and FGF-2. Little is however known so far about the factor affecting a motogenic activity of ES cells. The present study explored the effect of diverse growth factors on the invasion potential of human ES cells into extracellular matrix. Modified Boyden chamber elucidated the strongest effect of FGF-2 and the slight effect of hepatocyte growth factor/scatter factor (HGF/SF) on the invasion of ES cells into Matrigel while EGF, TGF-alpha, PDGFA, FGF-1, FGF-5, FGF-6, FGF-7 failed to augment the ES cells invasion activity. FGF-2 enhanced the expression level of integrin alpha3, the adhesion to type 1 collagen and fibronectin of ES cells, and also activated their focal adhesion kinase signaling. ES cells used expressed FGF receptor (FGFR)-1 IIIb and FGFR-2 IIIb. The FGFR neutralizing monoclonal antibody inhibited the FGF-2 effects on ES cells.

Tetsuya Moriue1, Kozo Yoneda1, Hiroaki Kosaka2, Yasuo Kubota1, Junsuke Igarashi2

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Department of Dermatology, Kagawa University, Takamatsu, Japan; 2Department of Cardiovascular Physiology, Kagawa University, Takamatsu, Japan

Sphingosine 1-phosphate (S1P) is a platelet-derived biologically active lipid mediator that modulates a wide array of essential vascular functions, including regulation of blood pressure, cellular proliferation and angiogenic responses to ultimately promote wound healing processes of injured skin tissue. S1P binds to S1P1 receptors and subsequently activates several key protein kinase pathways within vascular endothelial cells, including MAP kinase as well as PI3-K/Akt/eNOS pathways. In various pathophysiological conditions, including wound healing, oxidative stress

Increased apotosis and reduced proliferation in human melanoma cells treated with g-tocopherol Shizuko Kobayashi, Tatsuya Watanabe, Aiko Hashimoto, Yuriko Moriyama Molecular Physiology, Kyoritsu University of Pharmacy, Tokyo, Japan Background: A role for g-tocopherol (g-Toc) has not been fully explored, because g-Toc is no more effective than a-Toc as an antioxidant and is more readily metabolized than a-Toc. Recent studies have shown that g-Toc inhibits prostate cancer cell proliferation, suggesting the potent antitumor activity of g-Toc. In

174 this study, we examined the effects of g-Toc on proliferation and apoptosis in human melanoma cells by in vitro and ex vivo experiments. Methods: Human melanoma cell lines (HMV-I) were incubated with a-Toc or g-Toc (50—200 mM) for 24 and 48 h and cell numbers were counted. The induction of apoptosis was detected by methods of FACS and DNA ladder, and measurements of caspase-3 and -9 activities. For ex vivo experiment, HMV-1 cells (1  106) were injected to nude mice (BALB/c nu/nu) and test compounds were topically applied on these mouse skin 3 times/ week. After 2 weeks, tumors in the mouse skin were measured the weight and size and their tissues were stained with HE or TUNEL. Results and discussion: g-Toc, but not a-Toc, significantly inhibited cell proliferation in human melanoma cells. Moreover, g-Toc induced apoptosis in the cells by the activation of caspase-3 and -9, and involvement of caspase-dependent pathways. The results of this ex vivo experiment showed that g-Toc topical application tended to decrease the size/weight of tumors and increase the TUNEL positive cells in the melanoma-transplanted skin. Thus, these results indicate that g-Toc can up-regulate the induction of apoptosis. Our results suggest that topical application of g-Toc may be efficacious in preventing or reducing human melanoma.

144 Expression of senescence-related genes in skin tumors Yasutoshi Hida, Yoshiaki Kubo, Kazutoshi Murao, Seiji Arase Department of Dermatology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Premature senescence induced by stress signals, especially oncogenic stress, is supposed to be one of important safeguard programs against tumor formations in normal cells. Tumors would arise if some genetic or epigenetic events could overcome senescent programs in the cells. Unlike experiments in vitro, skin tumors in vivo have not been well studied so far. In this study, we have investigated expression of senescence-related genes of p14, p16, p21, p27 and SIRT1 in skin tumors, including squamous cell carcinoma (SCC), basal cell carcinoma (BCC), Bowen’s disease (BD), and seborrheic keratosis (SK). Protein products of p14, p16, p21 and p27 regulate cell cycle negatively in response to stress stimuli in vitro, and are associated with senescence. SIRT1, a mammalian homolog of yeast Sir2 that increases longevity, is a deacetylase associated with cell cycle. We extracted total RNA from each 6 cases of SCC, BCC, BD, SK, and normal epidermis in total 30 cases, and examined expression of senescence-related genes in them by semi-quantitative RT-PCR analysis. No obvious common features of gene expression could be found in SCC, BCC, and BD. However, all of SK showed strong expression of SIRT1 compared with others. Similar patterns were observed on expression of p27 that could be induced by SIRT1. Next, we examined protein expression of SIRT1 and p27 in each 20 cases of SK and normal skin by immunohistochemical analysis. More increased numbers of positive cells of SIRT1 and p27 were found in SK than normal skin. In addition, cases of SK whose epidermal portions are thinner showed stronger expression of SIRT1 and p27 by both procedures. Thus, we suggest that overexpression of SIRT1 and p27 might have some relevance to the tumorigenesis in the early stage of SK. 145 Expression of connexin 26 by human malignant melanoma cells and endothelial cells around melanoma cell nests

Abstracts Hideo Asada1, Mamiko Katsuragi1, Hironori Niizeki1, Mikio Masuzawa2, Masahiro Tsutsumi3, Hiroshi Nojima4, Sachiko Miyagawa1 1

Department of Dermatology, Nara Medical University, Kashihara, Japan; 2Department of Dermatology, Kitasato University School of Medicine, Sagamihara, Japan; 3Department of Oncological Pathology, Nara Medical University, Kashihara, Japan; 4 Department of Molecular Genetics, Institute for Microbial Diseases, Osaka University, Suita, Japan Connexins (Cx) form the intercellular channels of the gap junction and play an integral part in controlling the growth of various tumor cells as well as that of normal cells. Previously, we demonstrated that Cx26 is upregulated in BL6 cells (a highly metastatic subline of B16 mouse melanoma cells) compared to F10 (a weakly metastatic subline), and that ectopic over-expression of the Cx26 gene in F10 cells caused them to spontaneously metastasize to similar levels as observed with BL6 cells. Here, to investigate the role Cx26 plays in melanomatous metastasis in the patients, we used immunostaining to examine Cx26 expression in primary and metastatic melanoma lesions and in the endothelial cells around the melanoma cell nests. We studied 33 tissue samples of primary and metastatic melanomas obtained from 16 patients (male, 10; female, 6; age, 24—78), as well as 3 squamous cell carcinoma (SCC), 5 basal cell carcinoma (BCC), 5 nevocellular nevi (NCN) and 5 normal skin samples. We also examined Cx26 mRNA and protein expression in the cultured normal endothelial cells and hemangiosarcoma cell line. Immunohistochemistry demonstrated that Cx26 was clearly expressed by the endothelial cells of the small vessels surrounding the melanoma cell nests as well as the melanoma cells. However, SCC, BCC and NCN tumor cells did not express Cx26, while Cx26 was found to be expressed in the endothelial cells around SCC, but not in those around BCC or NCN. Cx26 mRNA and protein expression was detected in cultured endothelial cells and hemangiosarcoma cell line. Cx26 may contribute to the metastasis of melanoma by facilitating communication between melanoma cells and their surrounding endothelial cells. 146 Signal transduction in a novel acral melanoma cell line, SMYMPRGP, showing cyclin D1 amplification Hiroshi Murata, Minoru Takata, Atsuko Ashida, Yuko Takagi, Maki Yamaura, Toshiaki Saida Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan We recently established a new melanoma cell-line, SMYM-PRGP, from the macular part of a primary melanoma on the sole. The cells, which show spindle or multipolar morphology, are now maintained with MCDB 153 containing 2% FBS with the supplementation of bFGF, SCF, ET1, GRO-alpha, TNF-alpha, EGF, transferring, insulin, selenium and CaCl2 at 37 8C under 5% O2/5% CO2 atmosphere. Genomic analyses showed that this cell line had wild-type BRAF, copy-number loss of CDKN2A/CDKN2B and the amplification of cyclin D1. RT-PCR analyses revealed the mRNA expression of ETRA, ETRB, c-Kit, and EGF receptors as well as SCF, TGF-alpha and FGF2. Among the growth factors supplemented in the maintenance medium, ET1 plus CaCl2 showed the strongest effect on cell proliferation. This growth stimulation could be blocked with the selective ETRB antagonist BQ788, but not with ETRA antagonist BQ123. Western blot analysis showed weak phosphorylation of serine 473 of the Akt protein, whereas phosphorylated-ERK1/2 proteins were not detected. Phosphorylation of JNK and p38MAPK proteins was also very weak. Interestingly,

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Genomic altertions in primary cutaneous melanomas detected by comparative genomic hybridization: 6P gains may predict poor outcome

A2058 and HMV-I cells subcutaneously and treated with either LAQ824, or CRA, or combination in vivo. Combination treatment showed 61% and 82% tumor growth inhibition in A2058 and HMV-I as compared to control. Expression of RAR-beta2 in two human melanoma cell lines was silenced by histone acetylation and methylation and that the RAR-beta2 expression is repressed by epigenetic mechanism, and the HDAC inhibitor LAQ824 may restore retinoid sensitivity by reverting its epigenetic silencing. Difference of antitumor effects between two human melanoma cell lines might be associated with the induction of SM22 by the HDAC inhibitor LAQ824. In conclusion, the HDAC inhibitor LAQ824 has a synergistic antitumor activity in combination with retinoids on melanoma cells in vitro and in vivo. Combination of HDAC inhibitors and retinoids represents a novel therapeutic approach in patients with malignant melanoma.

Takeshi Namiki1,2, Toshiyuki Izumo2, Masashi Ishikawa2, Takahiro Satoh1, Hiroo Yokozeki1, Yasuhiko Kaneko2

149

however, marked phosphorylation of ERK1/2 proteins was observed when cells were cultured without growth factor supplementation. Addition of ET1 and CaCl2, while inducing marked cell proliferation, rapidly decreased ERK1/2 phosphorylation within 10 min. Although a PI3K inhibitor LY294002 inhibited the cell proliferation, an AKT blocker SH-6 showed no effect. These results suggest the complex growth signaling pathways in this particular acral melanoma cell line with cyclin D1 amplification, and warrant further investigation. 147

Department of Dermatology, Tokyo Medical and Dental University, Tokyo, Japan; 2Saitama Cancer Center, Saitama, Japan

Ultraviolet light is not required for the acquisition of the BRAFV600E mutation in melanocytic nevi

Genomic alterations of cutaneous melanoma have been examined by various methods including cytogenetics and comparative genomic hybridization (CGH), but prognostic significance of these alterations largely remains to be elucidated. To clarify the possible correlations of genomic alterations with clinical and histological features, we performed CGH analyses on 20 primary cutaneous melanomas which were classified into four groups: T1 (<1.0 mm in thickness), T2 (1.01—2.0 mm), T3 (2.01— 4.0 mm) and T4 (>4.0 mm) according to the melanoma staging system of the AJCC. Three of our 20 melanomas were classified as T1, six as T2, two as T3 and nine as T4. We used laser capture microdissection for 11 tumors and manual microdissection for 9 tumors. Eight out of 11 laser capture microdissected tumors were classified as T1 or T2. There was no difference in average number of chromosomal aberrations among the four groups. While other aberrations were equally distributed among the four groups, 6p gains were found only in T4 (P = 0.015). The mean age of patients with 6p gains was 79.3 years, 19.3 years older than that of patients without 6p gains (P < 0.01). Patients with 6p gains had a lower overall survival rate than those without them (P = 0.0002). These results suggested that chromosomal aberrations had already occurred even in thin tumors, and that 6p gains may have a prognostic value.

Nami Nakato1, Minoru Takata1, Hiroshi Murata1, Akihide Fujimoto2, Naohito Hatta3, Toshiaki Saida1

1

148 Different antitumor effects of combination of retinoic acid with the histone deacetylase inhibitor LAQ824 in malignant melanoma models

1 Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan; 2Department of Dermatology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 3Dermatology Division, Toyama Prefectural Central hospital, Toyama, Japan

To investigate whether the frequency of the BRAFV600E in melanocytic nevi is associated with sun exposure patterns, we examined the mutation in 120 acquired melanocytic nevi excised from various anatomic sites including glabrous skin, as well as 62 congenital nevi. We used a new mutation detection kit based on the shifted termination assay, called Mutector, which was able to detect only 5% of heterozygous mutant cells within the samples. We detected the mutation in 105/120 (87.5%) acquired nevi and 43/62 (69.4%) congenital nevi. Notably, we found the mutation in 35/43 (81.4%) acquired nevi excised from glabrous skin and genitalia. Interestingly, in congenital nevi, the BRAFV600E mutation was much less frequent in medium-sized (1.5 cm) than in small (<1.5 cm) nevi (6/20, 30.0% versus 37/42, 88.1%; p < 0.0001 by the chi-square test). We found NRAS mutations at codon 61 in 9 of 14 medium-sized congenital nevi, which possessed wild type BRAF, suggesting a different histogenesis between small and medium-sized congenital nevi. These results clearly show that UV-light is not necessarily required for the acquisition of the BRAFV600E mutation, and suggest that nonmutagenic effects of UV-light to melanocytes may be more important in the development of melanocytic nevi.

Yukihiko Kato1, Roberto Pili2, Ryoji Tsuboi1 1

Tokyo Medical University, Department of dermatology, Tokyo, Japan; 2Johns-Hopkins University Chromatin remodeling agents such as the histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in different tumor types and to reverse epigenetic repression of certain. In the current study we tested that antitumor effect of the HDAC inhibitor LAQ824 on human melanoma cell lines in vitro and in vivo. Treatment of LAQ824 showed a dose-dependent inhibitory effect on human melanoma A2058 and HMV-I in a clonogenic assay. These cell lines were relatively resistance to treatment with either 13 cis-retinoic acid (CRA). Upon treatment with combination of LAQ824 and CRA, however, a synergistic inhibitory effect was achieved. The antitumor effects for A2058 and HMV-I cell lines were the induction of cell cycle arrest and apoptosis, respectively. Thus, nude mice were injected with

150 Disruption of ID2 reveals major difference in chemical carcinogenesis Atsushi Tokuriki1, Masanobu Kumakiri1, Yoshihumi Yokota2 1

Department of Dermatology, School of Medicine, University of Fukui, Fukui, Japan; 2Department of Molecular Genetics, School of Medicine, University of Fukui, Fukui, Japan Id proteins function as negative regulators of basic helix-loophelix (bHLH) transcriptional factors that specifically regulate gene expression during cell fate determination. To date, four individual Id genes, Id1-Id4, have been identified in mammals. From the ability of Id proteins to inhibit differentiation of certain lineages and their potential to stimulate proliferation, these proteins could have key roles in the cancer process. Bolstered

176 by overexpression data in a range of human cancers, investigators have suggested that id2 proteins are likely to have pivotal roles in the initiation and progression of cancers. Here we evaluate the role of Id2 in a clinically relevant tumor model system using a twostep chemical carcinogenesis protocol. Remarkably, we find that Id2 null mice are more susceptible to skin tumorigenesis compared to their wild-type counterparts in spite of overexpression of Id2 mRNA in tumors occurring on wild-type mice. Cutaneous neoplasms in Id2 null mice show increased proliferation. We also identify that Id2 null mice possess fewer Langerhans cells and skin-specific gd T lymphocytes. We conclude that the lack of these cells in charge of tumor immunity result in tumor susceptibleness in Id2 null mice. 151 Establishment of stable and inducible in vitro cellular model for loricrin keratoderma Kozo Yoneda1, Toshio Demitsu2, Motomu Manabe3, Kozo Nakai1, Tetsuya Moriue1, Yasuo Kubota1 1

The Department of Dermatology, Kagawa University, Kagawa, Japan; 2Department of Dermatology, Jichi Medical School; 3 Department of Dermatology, Akita University School of Medicine Loricrin is one of the major constituents of the epidermal cornified cell envelope. Recently, heterozygous loricrin gene mutations have been identified in two dominantly inherited skin diseases, the ichthyotic variant of Vohwinkel syndrome and progressive symmetric erythrokeratoderma, collectively termed loricrin keratoderma. Although the gene defects underlying loricrin keratoderma have been identified, the mechanisms that contribute to the keratoderma of the mutant loricrin have not been fully delineated. We have previously shown that wild loricrin constructs, when transiently transfected into keratinocytes, lead to features of apoptosis. As the apparent transfection rate was so low with this system, further kinds of studies were hindered. To bypass this problem, we generated stable keratinocyte cell lines that express wild or mutant loricrin using an ecdysone-inducible system. HaCaT cells were first stably transfected with pVgRXR and cells in which protein expression was well regulated by muristerone A (one type of ecdysone) were selected. The selected cells were then transfected with pINDwild loricrin and pIND-mutant loricrin. Zeocin and G418 resistant clones were then screened for transgene expression induced with muristerone A. The amount of wild loricrin and mutant loricrin was dependent on the dose of muristerone A. In conclusion, we created a stable and inducible cellular model of loricrin keratoderma. Using this model, we intend to explore the molecular mechanisms that contribute to abnormal keratinization observed in loricrin keratoderma. 152 Functional characterization of class II PI3-kinase in epidermal differentiation via targeted gene disruption and RNA interference Kazutoshi Harada1,2, Amy Truong2, Ti Cai2, Shinji Shimada1, Paul Khavari1

Abstracts has been suggested by PI3K pharmacologic blockade, which abolishes differentiation, and by C2beta overexpression in vitro, which induces keratinocyte differentiation. To define the role of PI3K C2beta in epidermal differentiation, we performed two independent approaches to genetic loss-of-function, RNA interference and generation of knockout mice. In vitro, RNAimediated C2beta knockdown failed to exert major effects on calcium-induced differentiation or cell proliferation. Mice generated from embryonic stem cells in which the C2beta gene had been deleted by homologous recombination also failed to display abnormal differentiation. C2beta-null murine keratinocytes express filaggrin, a marker of keratinocyte differentiation with addition of calcium in vitro. These mice exhibited normal wound healing and normal skin barrier function by TEWL assay. Consistent with this, multiple transgenic mice lines expressing epidermis-targeted C2beta also failed to display any disruptions in epidermal homeostasis in vivo, suggesting that prior in vitro overexpression studies were not in the range of physiologic relevance. These data indicate that C2beta is dispensable for normal epidermal homeostasis and differentiation; however, potential redundancy with the other class II PI3K expressed in epidermis, C2alpha, cannot be excluded and current efforts are directed at achieving simultaneous loss of function of both isoforms. 153 Cytokine gene expressions in chronic UV-exposed skin Daiki Murase, Kazuhiko Higuchi, Atsushi Ohuchi, Takashi Kitahara Biological Science Laboratories, Kao Corporation, Tochigi, Japan Background: Aging as well as chronic sun exposure are major causes of changes in skin conditions. It has been reported that there is an increase in gene expression of melanogenesis-related cytokines in chronic pigmentation, such as endothelin and SCF, both of which are derived from keratinocyte. In this study, human epidermal tissues with and without chronic UV exposure were compared by using gene expression analysis to investigate the effect of chronic UV exposure on the changes in gene expression. In addition, as a marker of chronic UV damage in the skin, mtDNA deletion was analyzed to clarify the relationship between the chronic UV damage and the changes in gene expression. Method: Epidermal tissues were sampled by a suction blister technique from the lateral side (UV-exposed site) and the medial side (less UV-exposed site) of forearms of healthy male volunteers. Total RNA and DNA were extracted from these samples, and gene expressions and mtDNA deletion were analyzed using realtime PCR. Result/discussion: Through gene expression analysis, a significant increase in the quantities of SCF and IL-1a mRNA was determined in the tissues from the UV-exposed site as compared to the tissues from the less UV-exposed site. In addition, the analysis of mtDNA deletion revealed a significant induction in the UV-exposed site. These results indicate the correlation of these cytokine expressions with chronic UV damage, raising the possibility that continuous increases of these cytokine expressions might be involved in chronic UV-induced changes of skin conditions such as pigmentation.

1

Department of Dermatology, University of Yamanashi, Yamanashi, Japan; 2Program of Epithelial Biology, Stanford University, Stanford, USA Phosphoinositide 3-kinase (PI3K) gene family members affect proliferation, apoptosis and differentiation in a variety of tissues. A role for the class II PI3K C2beta in epidermal homeostasis

154 DFS70/LEDGF, a major autoantigen of atopic dermatitis, locates in the keratohyalin granules Kazumitsu Sugiura1, Yoshinao Muro 1, Yuji Nishizawa2, Miyako Okamoto1, Toshimichi Shinohara3, Jiro Usukura2, Yasushi Tomita1

Abstracts 1

Department of Dermatology, Nagoya University, Graduate School of Medicine, Nagoya, Japan; 2Department of Anatomy, Nagoya University, Graduate School of Medicine, Nagoya, Japan; 3 Department of Ophthalmorgy, University of Nebraska Medical Center, Omaha, USA Background: We identified DFS70 for the first time by immunoscreenig with the serum from an interstitial cystitis and reported that this protein was a major autoantigen found in about 30% of atopic dermatitis (AD) patients. This protein also was identified and named independently as LEDGF by Shinohara et al. DFS70/ LEDGF localized exclusively in nucleus in various cultured cells and was involved in various cellular processes including DNAbinding transcriptional co-activator. How these autoanitibodies or the autoantigens are involved in the pathogenesis of AD has not been studied. Aim and methods: To elucidate the physiological and pathological role of DFS70/LEDGF in skin, we for the first time tried to determine the precise localization of DFS70/LEDGF in the epidermis. We performed immunocytocheimistry on normal human skin and electron microscopic immunocytochemistry on it. Results: We found that DFS70/LEDGF localized not only in the nucleus but also in the cytoplasm, that its nuclear localization was more dominant in the basal layer than in the granular layer, vice versa was its cytoplasmic distribution in the both layers. And we found electron microscopically that the molecule accumulated in keratohyalin granules (KG) in the granular layer as well as the stratum corneum. Discussion: It is intriguing that localization of DFS70/LEDGF in vivo epidermis is remarkably different from in vitro cultured cells. DFS70/LEDGF locates in specific organelle of epidermis, KG. The finding that the unique destination of DFS70/LEDGF from nucleus to KG coincident with terminal differentiation of keratinocytes, suggests a possible important function of DFS70/LEDGF in KG, and presents novel implication of a nuclear autoantigen of DFS70/ LEDGF in AD. 155 The effect of trichloroacetic acid and glycoric acid on PDGF and VEGF expression in the skin Nozomi Yonei1, Toshio Ohtani2, Yuki Yamamoto1, Fukumi Furukawa1 1 Department of Dermatology, School of Medicine, Wakayama Medical University, Wakayama, Japan; 2Department of Dermatology, Kurashiki Central Hospital, Kurashiki, Japan

Trichloroacetic acid (TCA) and glycoric acid (GA) are widely used as agents for chemical peeling. In this study, we assessed PDGF and VEGF mRNA expression in TCA, or GA-treated keratinocytes and performed ELISA using cell culture supernatants for better understanding of the wound healing mechanisms. Keratinocytes (Pam 212) were grown using serum-free DMEM for 12 h, and treated by TCA (at concentration of 0%, 0.005%, 0.05%), or GA (at 0 mM, 1 mM, 10 mM) for 3 h. In TCA-treated keratinocytes, PDGF mRNA level tends to increase at higher concentration, but shows no significant differences. In GA-treated keratinocytes, PDGF mRNA level was decreased in a dose-dependent manner. No change in VEGF mRNA expression was observed. To determine the amounts of PDGF and VEGF secreted from cultured keratinocytes, we carried out ELISA. Murine keratinocytes were cultured in DMEM containing same as the above-mentioned concentration of TCA or GA and 2% fetal calf serum for 24 h, and the cell culture supernatants were used for ELISA. In TCA-treated keratinocytes, PDGF increase in the medium was not confirmed, although PDGF expression was increased at mRNA level. There was no difference in VEGF production as in mRNA expression. In GA-treated keratinocytes, PDGF production

177 was significantly decreased as in mRNA expression. There was no significant difference in VEGF production. We also examined the expression of these growth factors in the skin obtained from a healthy volunteer at 2 days after treatment of 40% or 60%TCA. We found that PDGF mRNA, but not VEGF mRNA, increased after treatment of 60%TCA in comparison with 40%TCA.PDGF derived from keratinocytes might be an important component during wound healing in TCA-treated skin. 156 Identification and functional analysis of a mouse homologue of SASPase Takeshi Matsui1, Yoko Ida1, Fumie Kisumi1, Masaki Hata1, Sayaka Katahira1, Kazumasa Morita2, Yoshiki Miyachi2, Shoichiro Tsukita3 1

KAN Research Institute; 2Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto, Japan Proteases play an important role in epidermal differentiation. During high throughput in situ hybridization screening of mouse epidermis, we identified a mouse homologue of human Skin ASpartic Protease (SASPase), a recently identified epidermisspecific retroviral-like aspartic protease as a clone exclusively expressed in the granular layer of the epidermis [1]. Antibody against mouse SASPase (mSASP) detected 32- and 15-kDa bands in stratum corneum extract (SCE) of mouse epidermis. We purified endogenous 32-kDa (mSASP32) and 15-kDa (mSASP15) forms of mSASP from mouse SCE and determined their amino acid sequences. To confirm that mSASP15 is an enzymatically active form, we bacterially produced mSASP15 via the autoprocessing activity of GST-mSASP32 and purified to near homogeneity. Purified recombinant mSASP15 (rmSASP15) cleaved the quenched fluorogenic peptide substrate, the sequence of which is derived from a natural processing site for mSASP32. The cleavage of the peptide substrate by rmSASP15 was maximal at pH5.77, which is close to the pH at the epidermal surface. We also present data of analyses of mSASP-deficient mice that could help in understanding the role of proteases in epidermal differentiation.

Reference [1] Bernard, et al. J Invest Dermatol 2005;125:278—87.

157 A unique monoclonal antibody 29a stains the cytoplasm of amniotic epithelia and cutaneous basement membrane Mitsuhito Ota1, Daikuke Sawamura1, Koichi Yokota1, Masamichi Ueda2, Yuji Horiguchi3, Kazuo Kodama1, Maki Goto1, Hiroshi Shimizu1 1

Department of Dermatology, Hokkaido University Graduate School Medicine, Sapporo, Japan; 2Institute for Virus Research, Kyoto University, Kyoto, Japan; 3Department of Dermatology, Osaka Red Cross Hospital, Osaka, Japan The basic function of epithelia is to provide a boundary between tissue and its external environment. The basement membrane (BM) of many epithelia is a specialized extracellular matrix that serves many functions, of which the most obvious is the attachment of the epithelia to underlying mesenchyme. Novel molecules which play a crucial role in normal and abnormal conditions are though to be expressed in the epithelial, and identification of such molecules may lead to significant progress in epithelial

178

Abstracts

biology and furthermore in dermatological research. In this study, we raised a monoclonal antibody to detect a novel epithelial molecular component. We have produced a mouse monoclonal antibody using normal human amniotic tissue as an immunogen. The monoclonal antibody was immunohistochemically screened, and the target antigen was cloned using an immunoscreening method. In the course of the screening, we identified unique antibody staining patterns within the cytoplasm of a subset of amniotic cells at intervals within the normal placental epithelia. By immunoscreening, we identified this candidate gene as laminin receptor (LR). By dot—blot analysis, this antibody reacted with recombinant LR. The same localization of the antigen and LR was proved by a double staining immunofluorescence test in the placenta. This monoclonal antibody unexpectedly demonstrated linear staining within the dermal-epidermal junction of normal human skin but failed to react within the keratinocyte cytoplasm. In conclusion, we have produced a novel monoclonal antibody that recognizes an LR-related molecule, which demonstrated a unique staining pattern. This monoclonal antibody might be a useful tool for further investigations into the epithelial tissues and the cutaneous BM. 158 TRPV1 activation is involved in the acceleration of epidermal turnover after chemical peeling with glycolic acid 1

2

2

Sumiko Denda , Kei Negishi , Nobuharu Kushikata , Shingo Wakamatsu2, Mitsuhiro Denda1, Toshihiko Hibino1 1

T-cadherin enhances cell-ECM adhesion by increasing surface b1 integrin expression. In this study, we aimed to further clarify the roles of T-cadherin in the regulation of surface b1 integrin expression. In cultured cutaneous squamous carcinoma cells, internalization of b1 integrin was induced by serum starvation, and overexpression of T-cadherin inhibited the internalization. The internalization of b1 integrin is likely mediated by caveolae because of its sensitivity to cholesterol depletion or tyrosine kinases inhibition. Actually, overexpression of T-cadherin reduced endocytosis of cholera toxin, which is a ligand for ganglioside GM1 and is known to be a maker of caveolae-dependent endocytosis. Moreover, genistein, a tyrosine kinase inhibitor, suppressed both internalization of b1 integrin and endocytosis of cholera toxin, and simultaneously suppressed tyrosine-phosphorylation of EGF receptor. Similarly, T-cadherin overexpressing cells exhibited reduced phospho-tyrosine content of EGF receptor. Furthermore, the internalization of b1 integrin was also suppressed by PD168393 or AG1478, both of which are specific inhibitors of EGF receptor. Finally, the accumulation of surface b1 integrin on T-cadherin-overexpressing cells was abolished thorough tyrosinephosphorylation of EGF receptor by EGF treatment. These results suggest that surface b1 integrin expression is mediated by Tcadherin through caveolae-dependent endocytosis, which is likely dependent on tyrosine-phosphorylation of EGF receptor. 160 hBD-1 promotes keratinocyte migration through HB-EGF/EGFR transactivation mechanism

Life Science Research Center, Shiseido Co., Ltd., Yokohama, Japan; 2Institute of Aoyama Women’s Medicine, Tokyo Women’s Medical University, Tokyo, Japan

Sho Tokumaru, Koji Sayama, Yasushi Hanakawa, Yuji Shirakata, Xiuju Dai, Lujun Yang, Mikiko Tohyama, Satoshi Hirakawa, Koji Hashimoto

Superficial chemical peeling has become a popular method for producing facial rejuvenation. However, there are few studies reporting the molecular mechanism of this procedure. We investigated the effects of topical application of glycolic acid (GA) on a skin equivalent model. Within 10 min after GA application, ATP was released into the culture medium. Then after 24 h BrdUincorporation into keratinocytes and expression of differentiation marker proteins were increased. These effects were dependent on the pH of GA, and were inhibited by antagonists for TRPV1, an acid-sensitive ion channel. Inhibitors for ATP receptors (P2X and P2Y) reduced the epidermal proliferation. In human skin, histological studies revealed that GA application stimulated epidermal proliferation; ATP release was detected using the organ culture of human skin. These data suggests that one of the mechanisms of GA peeling is a growth response of the epidermis to noxious stimuli. This response may be mediated by TRPV1 activation and ATP release. Activation of P2 receptors by the released ATP may also be involved in this mechanism.

Department of Dermatology, Ehime University School of Medicine

159 T-cadherin enhances cell-surface b1 integrin expression through suppression of tyrosine-phosphorylation of EGF receptor Yohei Mukoyama1,2, Atsushi Utani2, Seiya Matsui2, Yoshiki Miyachi2, Norihisa Matsuyoshi2

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. It has been reported that antimicrovial agents, human beta defensins (hBD) are chemotactic for dendritic cells, T-lymphocyte and mast cells. However the effect of hBDs on keratinocyte migration are not clearly identified. Several groups reported that ligands for G-protein-coupled receptor (GPCR) induced EGF receptor (EGFR) transactivation by HB-EGF shedding (the proteolytic conversion of membrane-anchored HB-EGF to the soluble one, which binds to EGFR and results in EGFR activation). Last year we reported that antimicrovial agents, LL-37-induced keratinocyte migration occurs via EGFR transactivation. We hypothesized that hBD1-induced keratinocyte migration is mediated by EGFR transactivation. We examined the effect of hBD1 on keratinocyte migration by Boyden chamber assay. hBD1 induced keratinocyte migration 1.3-fold at 1microgram/ml optimally. Addition of hBD1 (1 mg/ml) induced the phosphorylation of EGFR with a maximum at 30 min. This EGFR phosphorylation is inhibited almost completely by OSU-8, an HB-EGF converting proteinase inhibitor, CRM197, an inhibitor for soluble HB-EGF, anti-EGFR antibody and AG1478, EGFR kinase-specific inhibitor. Furthermore, these inhibitors also suppressed hBD1-induced keratinocyte migration. In conclusion, hBD1-induced keratinocyte migration requires HB-EGF/EGFR transactivation mechanism.

Drug Discovery Research, Research Laboratories, Kyoto R&D Center, Maruho Co., Ltd., Kyoto, Japan; 2Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

161

T-cadherin is specifically localized at the basal cells, but its functions are not fully understood. We previously reported that

The Department of Dermatology, Okayama University Medical School, Okayama, Japan

1

WNT expression in normal skin and psoriasis Gen Nakanishi, Song-Nan Lin, Keiji Iwatsuki

Abstracts Human Wnt genes encode 19 family members of secretory glycoproteins which regulate distinct developmental processes. During embryonic development, the expression of Wnt proteins is important in patterning through control of cell proliferation and determination of stem cell fate. Wnt proteins are involved in the maintenance of many normal tissue structures as well as tumor formation. There are some reports about Wnt gene expression and its function in murine hair follicle development and metastasis of malignant melanoma. Wnt gene expression, however, has not been examined in inflammatory skin diseases. In this study, we characterized a Wnt gene expression profile in psoriasis. RT-PCR analysis using degenerate Wnt primers detected Wnt3 mRNA and Wnt4 mRNA in psoriatic skin. Immunohistochemical analysis demonstrated that Wnt3 was expressed in normal human epidermis, but not in psoriatic skin by goat polyclonal anti-Wnt3 antibody (N-15) (Santa Cruz Biotechnology). In addition, immunohistochemical analysis showed specific signals in adult mouse epidermis, but not in TPA treated mouse skin. Wnt3 was also detected in newborn mouse interfollicular epidermis, whereas, no Wnt4 was detected by goat polyclonal anti-Wnt4 antibody. Wnt3 over-expressed in CHO cells was recognized by Western blot analysis using anti-Wnt3 antibody (N-15). In conclusion, we confirmed at least one anti-Wnt3 antibody that can be used in immunohisotchemical analysis and in Western blot analysis and we found that the expression of Wnt3 protein was decreased in psoriatic epidermis compared with human normal epidermis. 162 Inhibition of inducible nitric oxide synthase transcription by prolonged exposure to high glucose in the human keratinocyte cell line HaCaT Kozo Nakai1, Tetsuya Moriue1, Kozo Yoneda1, Yasuo Kubota1, Hiroaki Kosaka2, Masahide Urushihara3 1 Department of Dermatology, Faculty of Medicine, Kagawa University, Kagawa, Japan; 2Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, Kagawa, Japan; 3 Department of Research Equipment, Faculty of Medicine, Kagawa University, Kagawa, Japan

Background: In human skin, inducible nitric oxide synthase (iNOS) appears to be a key enzyme during wound healing and has roles in protection from infection. We speculated that diabetic skin complications such as delayed wound healing and skin infection were due to iNOS activity altered by high glucose in keratinocytes. Objectives: The purpose of this study was to see how high levels of glucose affect iNOS activity in the human keratinocyte cell line (HaCaT). Methods: HaCaT cells were exposed to high glucose for 1 day or 10 days. We measured nitric oxide (NO) end product nitrite in the culture medium using the Griess reagent, and intracellular tetrahydrobiopterin (BH4, a cofactor of NOS) content by using HPLC, analysed the expression level of iNOS mRNA by the RT-PCR method and evaluated the DNA binding activity of nuclear factor kappa B (NF-kB) by EIA. Results: Stimulation with IFN-g, TNF-a and LPS induced iNOS mRNA and increased NO production in HaCaT cells. Short-term exposure (1 day) to a high level of glucose increased BH4 and iNOS activity at the post-translational level. However, long-term exposure (10 days) to high glucose reduced NF-kB binding activity and inhibits iNOS transcription and its activity. Conclusions: Long-term treatment with high glucose reduced NF-kB activity and inhibited iNOS transcription and NO production, implying the involvement of a deficiency in NO synthesis in both skin infection and impaired wound healing in diabetic patients.

179 163 Interaction of BPAG1e with intermediate filaments Tadashi Karashima, Takashi Hashimoto Department of Dermatology, Kurume University, Fukuoka, Japan Bullous pemphigod antigen 1e (BPAG1e) is a component of hemidesmosomes and the target of antisera with the autoimmune blistering disease, bullous pemphigoid. BPAG1e belongs to the plakin family of cytolinker proteins, which also includes desmoplakin, plectin, envoplakin, periplakin and epiplakin. Most plakins that share certain functional domains such as the actin-binding domain, plakin domain, coiled-coil rod domain and C terminus plakin repeat domain (PRD). These proteins contribute to the maintenance of tissue integrity by interacting with cytoskeletal filaments and anchoring them to each other and to membrane junction complexes. Its N terminal domain interacts with the plasma membrane and its C terminus interacts with intermediate filaments (IFs). It has been shown that the PRD of plectin and desmoplakin plays critical role in the direct binding of IFs. Attempts to identify the binding sites for IFs on BPAG1e have not fully developed so far. The aim of our experiments was to identify the binding sites for IFs on BPAG1e, and to carry out a more detailed study of the interaction of BPAG1e with IFs. By transient transfection of COS7 cells and primary human epidermal keratinocytes with deletion mutants of BPAG1e, we mapped sequences required for keratin5/keratin14 (K5/K14) IFs interaction to the region of the linker motif of BPAG1e C terminus. Co-transfection assay of constructs containing the C terminus of plectin and BPAG1e suggested that the plectin C-terminus may stabilize the interaction of the BPAG1e C terminus with IFs. We conclude that the plectin C terminus domain plays an important role in linking BPAG1e to keratin 5/14 IF networks. 164 Pathogenesis of ACNE through TLR expressing in keratinocytes Miki Nakanishi, Noriyasu Imai, Hiroko Kimura, Takuji Masunaga Fundamental Research Center, KOSE Corporation, Tokyo, Japan Acne vulgaris, a common skin disease, is a chronic inflammatory disorder which most people experience at adolescence. It is generally known that Propionibacterium acnes, a Gram-positive bacterium, located in sebum or at infundibulum, is related to the manifestation of the disease. However, its etiology is very complicated and multifactorial. Since Toll-like receptors (TLRs) were identified to be important molecules in innate immune system, it has been reported that P. acnes induces the secretion of inflammatory cytokines from TLR2-expressing monocytes at the acne lesion as a new development pathway of acne. Our purpose of the present study is to clarify the response of keratinocytes against P. acnes to verify the hypothesis that the initial step of the acne pathogenesis is the secretion of inflammatory cytokines by keratinocytes via TLR2 since (1) P. acnes contacts to keratinocytes first and (2) keratinocytes also express TLR2. First, TLR2 expression was confirmed in keratinocytes at both gene and protein levels. Then, the increased secretion of inflammatory cytokines (IL-8, IL-1a) by the stimulation with P. acnes was demonstrated. Blocking experiment with anti-TLR2 antibody indicated its mechanism is TLR2 dependent. Thus, it is proposed that keratinocytes produces inflammatory cytokines via TLR2 and those lead to the infiltration of inflammatory cells. Furthermore, these cytokines are suggested to be related to the follicular keratosis at the acne lesion, which is an important event in the acne

180 pathophysiology, as they affect the proliferation and differentiation of keratinocytes. The investigation regarding the thickening of epidermis using reconstructed skin equivalent is in progress. 165 Expression pattern of DSG1 in stratum corneum and its involvement in desquamation Yoshikazu Naoe, Tsuyoshi Hata, Hiroko Kimura, Takuji Masunaga Fundamental Research Center, KOSE Corporation, Tokyo, Japan Stratum corneum (SC), maintained by adhesive and desquamative mechanisms via desmosome (DS), plays important roles for skin moisturization and barrier function. The degradation of DS leads to the loss of adhesion in SC, resulted in shedding corneocytes from the skin surface. Desmoglein 1 (Dsg1), a desmosomal protein, is expressed in the suprabasal layer of the epidermis. However, its expression and function in SC have remained unclear. In this study, we investigated the expression pattern of Dsg1 in SC to disclose the relationship between the degradation of DS and the roles of SC. To evaluate the expression pattern of Dsg1 in SC, the corneocytes obtained by tape stripping were subjected to immunofluorescence and immunoelectron microscopy with anti-Dsg1 antibody. As a result, the cells from lower layer of the upper arm showed dot-like staining pattern throughout the surface of the cells, whereas the positive staining was observed only near the edge of cells from the upper layer of the upper arm. On the other hand, the staining pattern in cells from uppermost layer of the face showed the similar pattern to that of lower layer of the upper arm. These results suggest that Dsg1 is degraded to attenuate the adhesion between the lower and upper cells in the process of desquamation, and that the regulation of Dsg1 degradation is diverse in different regions of body. Present immunocytochemical analysis of the Dsg1 expression demonstrated one part of the desquamation process of SC. Our findings give a clue to clarify the alteration, function and roles of DS in SC. 166 Epidermal regulation of AQP3 expression and hyaluronan synthesis (2): Retinoic acid up-regulates AQP3 expression in cultured keratinocytes Yoshinori Sugiyama1, Kohei Yamazaki1, Ayumi Kusaka1, Setsuya Aiba2, Shintaro Inoue1 1 Basic Research Laboratory, Kanebo Cosmeteics Inc.; 2Department of Dermatology, Tohoku University Graduate School of Medicine

The aquaporins are a family of water channel proteins with at least 13 known members in human tissues. Aquaporin-3 (AQP3), highly expressed in the epidermis, permeates small solutes such as glycerol and urea as well as water. Impairments in the hydration, elasticity, barrier recovery, and wound healing of skin in AQP3 null mice indicate that AQP3 plays important roles in skin physiology. Many features of AQP3 remain unclear, however, including the factors which regulate its expression in the epidermis. In the present study, we treated a monolayer of normal human keratinocytes and skin equivalent cultures with retinoic acid (RA) and then analyzed AQP3 expression by an RNase protection assay and immunoblotting. The stimulation of growing keratinocytes cultured in low-calcium medium with RA (0.03—1 mM) for 24 h induced distinct and dose-dependent increases in AQP3 expression at both mRNA and protein levels. The induction of the expression was detected by as early as 2 h

Abstracts after the addition of RA. Furthermore, the up-regulation of AQP3 expression by RA was also detected in differentiated cells cultured in high-calcium medium and in the epidermal cells of skinequivalent cultures at both the mRNA and protein levels. In separate experiments, mRNA expression of AQP3 and a RAregulated gene, hyaluronan synthase 3 (HAS3), in cultured keratinocytes was analyzed by Real-Time PCR. Both mRNAs were up-regulated by addition of RA (0.03—3 mM) in a similar dosedependent manner, and induced by as early as 2 h after the addition. These results indicate that RA is a regulatory factor for AQP3 expression in cultured human keratinocytes. Transcriptional activation mediated by nuclear RA receptors is thought to be involved in the up-regulation of AQP3 and HAS3 gene expression by RA. 167 Dynamics of matrix adhesion-hemidesmosomes and focal contacts Daisuke Tsuruta1, Toshiyuki Ozawa2, Hiromi Kobayashi 1, Teruichi Harada2, Kazuo Ikeda3, Naoki Oiso4, Akira Kawada4, Hopkinson Susan5, Jones Jonathan5 1

Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan; 2Department of Plastic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan; 3Department of Anatomy, Department of Plastic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan; 4Department of Dermatology, Kinki University Medical School, Osaka, Japan; 5Northwestern University, The Feinberg School of Medicine, Chicago, IL, USA Interaction between cells and the extracellular matrix (ECM) impacts various cellular functions. The focal contacts (FCs) are regions of close interaction between cells and the ECM in mesenchymal cells. Recently, FCs are reported to be highly dynamic structure by us and other groups. Until recently, hemidesmosomes (HDs) are believed to be stable anchoring structure in epithelial cells. However, recently, we showed that HDs are also significantly dynamic. Recently, keratinocyte (KC) also has been reported to have dynamic FCs. Thus, KC has dual adhesion system composed of HDs and FCs. Moreover, we showed that FCs in endothelial cells complex with intermediate filament and adhere laminin, suggesting that KCs also may well have such a dual adhesion system between FCs and HDs. In this presentation, we first show dynamics of HDs in live epithelial cells and that of FCs in endothelial cells. Then, we show the dynamics of FCs probed with GFP-tagged actinin-1 in live KCs (HaCat cells) and in live fibroblasts (FBs, 3T3 cells) and compare dynamics between them. As a result, the speed of FCs in KCs was significantly slower than in FBs (81.72  53.51 nm/min versus 116  85.53 nm/min). The size of FCs in KCs was significantly smaller than in FBs (3.06  1.74 mm2 versus 3.96  2.14 mm2). The direction of the movements is quite different; that of FCs in KCs are relatively regular, but in FBs are rather random. The difference of mobile fraction (91.14  11.61% versus 80.38  21.53%) and that of t1/2 value (73.86  39.22 s versus 99  15.00 s) in the FRAP analyses between KCs and FBs was not significant. The difference of velocity and size of FCs between KCs and FBs may suggest the communication of FCs and HDs in KCs. 168 Autoflouorescence of corneocytes and relationship between beta structure in its proteins Norio Fujiwara1, Naoki Asakawa2, Yoshio Inoue2

Abstracts 1

POLA Chemical Industries, Inc. Yokohama, Japan; 2Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Japan It is known skin emit autofluorescence (AF). There are some reports about source of AF. One refers about collagen, another insists on amino acids in proliferating keratinocytes. But, it is not clear about isolated corneocyte AF. Then, this research aimed at clarifying the property of corneocyte AF. Experiment and result: [1] By fluorescence microscopy (UVA 365 nm) we observed isolated corneocyte of 143 Japanese women aged 20’s to 50’s. And evaluation of each AF strength was performed by brightness image analysis. As a result, all volunteers’ corneocytes had blue-white AF. In addition, we found the significant differences in individual AF. [2] On the hypothesis free amino acids or sebum caused AF, we removed these from corneocytes by distilled water or organic solvents. But, AF scarcely changed. [3] On the hypothesis of protein conformation, SDS, urea, or KOH affected these corneocytes. As a result, these AF remarkably decreased. [4] Fibroin is known having alpha-to-beta transitions. These corneocytes have similarity in AF property. Especially fibroin shows alpha-to-beta transitions in humidity with heat. Thus, we hypothesized beta structure emit AF. As a result, under humidity with heat (37 8C RH100% 72 h or 121 8C 20 min) these corneocytes AF remarkably increased. [5] Congo red stain-polarized microscopy is used for detection of beta structure in tissue. Using this, polarized light probably caused by beta structure was observed in the cross section of corneocytes of strong AF group than weak group. And corneocytes applied humidity with heat showed too. Conclusion: We found some interesting that isolated corneocytes have AF, variation exist in AF strength, and that beta structure may emit AF as amount of beta structure. Now with solid-NMR research is in progress. 169 P120ctn is associated with Desmoglein1 and 3, and localized at desmosomes in cultured human keratinocytes Miho Kanno, Yumi Aoyama, Yukari Yamamoto, Miki Nagai, Yasuo Kitajima Department of Dermatology, Gifu University School of Medicine, Gifu, Japan Back ground: p120ctn is one of catenin family proteins and binds to intracytoplasmic domain of cadherins through its arm repeat domain. Several lines of evidence indicate that p120ctn stabilizes cadherin at the cell surface and regulates the cell adhesion. We showed that p120-ctn was associated with Desmoglein (Dsg) 1 and 3 in cultured keratinocyte, and also localized at desmosome by immuno-electron microscopy. Purpose: The present purpose is to identify the p120-ctn association domain of Dsg3 and to confirm whether p120-ctn co-localized with Dsg3 in keratinocyte. Method: The flag-tagged wild type (wt) and carboxyl-terminal deletion mutant forms of Dsg3 were transiently expressed with HA tagged p120-ctn into HEK 293 cells. Cell lysate was subjected to immunoprecipitation. Western blotting was performed by antiflag antibody to detect the association of these proteins. Transiently expressed wt, mutated Dsg3 and p120-ctn in DJM-1 cell were subjected to immunocytochemistry.

181 Result: p120-ctn was found to bind to the membrane proximal region including IA region (641—714AA) of cytoplasmic Dsg3. An additional deletion in this region prevented the association with p120-ctn and abolished the localization to cell membrane. Dsg3 and p120-ctn recombinant proteins were co-localized in keratinocyte by imunno-florescence. Discussion: These data suggest that p120-ctn associates with intracellular domain of Dsg3 and that p120-ctn is a new member of desmosomal protein, which may implicated in regulation of cell adhesion at desmosome. 170 Cutaneous changes induced by experimental repeated barrier disruption Yukiko Matsunaga1, Haruhi Iwaki 2, Maki Kaneko 2, Makoto Tsunenaga 2, Shinji Inomata2, Motoki Ooguri 1, Naomi Kunizawa1, Toyonobu Yamashita1, Yuji Masuda1, Satoshi Amano 1 1 Shiseido Life Science Research Center; 2Shiseido Cosmetic Ingredient & Medicament Development Center

We recently found that chronic barrier disruption induces wrinkle formation in hairless mice. In this study, we investigated cutaneous changes induced by repeated barrier disruption at human facial skin. Methods: Facial rough skin was induced by excessive washing with soap (5 times per day for 7 days) during October or November in 26 healthy male volunteers (average age: 43 years). Skin condition, including water content in stratum corneum, transepidermal water loss (TEWL), sebum content, elasticity, surface texture, wrinkles and the thickness of epidermis and stratum corneum, was evaluated at 0, 5, 7, 14 and 21 days after the first wash. Results: As compared to the value at day 0, water content in stratum corneum decreased by 40% at day 14. Conversely, TEWL was increased 2.3-fold at day 7, but had reverted to normal by day 14. The sebum content at day 7 was increased 9.6-fold over that at day 0, peaked at 16-fold on day 14 and showed a 4.6-fold increase even at day 21. As for skin elasticity, the Uf value, corresponding to extensibility, was significantly decreased at days 5 and 7. However, the Ur/Uf ratio, corresponding to skin elasticity, did not change significantly throughout the experiment. The area and volume of wrinkles were significantly increased at day 7, being closely correlated with the changes of TEWL. The thickness of epidermis and stratum corneum at day 7 was increased to 1.3 times that at day 0 and had not recovered to normal by day 21. Conclusion: These data suggest that repeated removal of skin surface lipids induces barrier disruption, followed by acanthosis and hyperkeratosis, leading to a reduction of extensibility or softness. These cutaneous changes increase the appearance of wrinkles and worsen the degree of wrinkling. 171 In vivo determination of site and age variations in water concentration depth profiles in the stratum corneum by Raman spectroscopy Mariko Egawa, Tetsuji Hirao, Motoji Takahashi Life Science Research Center, SHISEIDO Co., LTD. Although there were many reports on water content of the stratum corneum (SC), it was difficult to discuss water concentration depth profiles in those reports. In this study, we applied Raman spectroscopy to determine site and age variations in water concentration depth profiles in the SC as well as effect of hydration. Raman spectra were recorded at 2 mm intervals from the

182 skin surface towards deeper using a confocal Raman microspectrometer: Model 3510 (River Diagnostics) with laser power at the skin surface of 17.0—19.0 mW (671 nm) and a measurement time of 1 s per spectrum in the 2600—4000 cm1 region. Healthy volunteers (20’s—70’s) were enrolled to examine site and age variations. Water concentration gradient, from 20 to 30% (water/ wet tissue) at the outermost layer in SC to 60—70% at the deeper layer, was observed, although the profiles were different among the sites. A characteristic depth profile was observed at palm, which has the large part with 30—40% of water content in the middle SC. Site variations in SC apparent thickness calculated from water concentration depth profiles was as follows in the order of thickness: palm, back of the hand, forearm, upperarm, and cheek. At female forearm, there was a positive correlation between age and SC apparent thickness and water content in the middle part of SC was decreased with age. In addition, application of water to the skin resulted in penetration of water throughout SC and its swelling. After stopping hydration, water evaporated gradually from upper part of SC with time. These results suggested that variation in water concentration depth profiles in SC could be determined in vivo by Raman spectroscopy. 172 Significance of barrier repair in non-lesional dry skin of atopic dermatitis: Efficacy of a topically applied ceramide cream Takahiro Matsuki1, Sayuri Sato1, Hayato Matuski2, Kimihiro Kiyokane2, Genji Imokawa3 1 Deparment of Dermatology, Sanno Hospital; 2Department of Dermatology, Osaka Medical University, Osaka, Japan; 3Skin Science Research Institute, Tochigi, Japan

Atopic dermatitis (AD) can be considered a barrier disease. Thus, replenishing the barrier function of the non-lesional skin of AD to healthy control level seems to be a key for preventing the refractory nature of the dermatitis. In this study, we topically applied a synthetic ceramide (CER) containing cream to the nonlesional skin of AD patients for 4 weeks and evaluated its efficacy in comparison with untreated skin. Comparison during the 4 weeks of therapy demonstrated that while the CER cream significantly reduced dry skin properties (accompanied by significant decreases in TEWL and increases in capacitance values) at 3 and 4 weeks compared with pretreatment, non-treated skin also had a significant reduction in dry skin properties (but no significant decrease in TEWL or increase in capacitance values). There were significant differences in dryness and scaling as well as in the TEWL and capacitance values at 3 and 4 weeks between the CER cream-treated and the non-treated skin. Comparison of TEWL and capacitance values revealed that whereas those two parameters of the non-treated skin remained within the levels of the mild to moderate AD skin at 3 and 4 weeks, those of the CER creamtreated skin were distributed into the levels of healthy control skin. Because properties of dry skin, such as dryness and scaling, do not necessarily reflect the intensity of barrier disruption, measurement of the barrier function in concert with clinical observations is required for precise clinical evaluation during skin treatment of AD patients. Our results further demonstrate that the use of skin care products is an essential requirement for repairing the barrier function. 173 A study on the skin concentration after percutaneous administration of glycolic acid Masaaki Hasegawa1, Akiko Miyake2, Naoki Tominaga3, Fumio Matsuno3, Tatsuya Okumura1, Tohru Okamoto2

Abstracts 1

Safety & Analytical Research Center, SHISEIDO CO., LTD., Yokohama, Japan; 2Material Science Research Center, SHISEIDO CO., LTD., Yokohama, Japan; 3Product Development Center, SHISEIDO CO., LTD., Yokohama, Japan Object: Chemical peeling (CP) is one of the esthetic dermatological techniques employed for the treatment of acne or hyperpigmentation. It accelerates the exfoliation and regeneration of the hony layer. However, little has been known on the skin concentations of CP agents after application. Therefore, using 14C-glycolic acid (GA) as a representative of CP agents, we studied the skin concentations after percutaneous administration of various concentrations of GA at different pHs. Method: The study was carried out with 10% to 50% GA formulations at the pH range of 1.3 to 3.8. Each GA formulation was applied to the dosal skin of either hairless mice or guinea pigs for 10 min. After the given time, the radioactivities in the hony layer and epidermis or dermis were measured and converted to GA concentrations by calculation. Result: In the case of 40% or 10% GA formulation, the skin concentations of GA increased sharply at the pHs lower than 2.0. At the same pH, GA penetration was increased in a dose-dependentry. Their were observed in both hairless mouse and guinea pig. There was no particular difference in these animal species. The skin concentations of GA increased immediately after the application but it decreased sharply in an hour. After that it eliminated gradually in 14 days. In the other pHs, the skin concentations of GA decreased gradually immediately after the applications, hardly any difference was observed in any pHs in 7 days after the application. Conclusion: From these, it was concluded that the SC of GA is dependent on the pH rather than on the concentration in the formulation. This study suggests that it is important to choose an appropriate pH for the CP with GA in accordance with the severity of the legions.

174 Differentiation disturbance in keratinocytes lacking the epidermal fatty acid binding protein gene Teie Egawa, Ryuhei Okuyama, Yoshiyuki Kusakari, Eisaku Ogawa, Akira Hashimoto, Hiroshi Watanabe, Hachiro Tagami, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan Fatty acids metabolism is tightly connected with pathomechanism of psoriasis. Psoriatic patients are said to absorb much meat whereas their symptom is sometimes improved by fish-oil intake. Fatty acid binding protein (FABP) is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system within cytoplasm. Among the FABP family, epidermal-type FABP (E-FABP) is solely expressed in keratinocyte. E-FABP is overexpressed in psoriatic epidermis, but its specific role in psoriasis is not yet established. In order to clarify its functions, we compared cell characters of E-FABP null mice with those of wild littermates. The amount of fatty acids was decreased in E-FABP null epidermis and the absorption of fatty acids, especially linoleic acid, was also down-regulated in the null keratinocytes, which demonstrate tight connection of fatty acid metabolism with E-FABP in keratinocytes. Next, we analyzed keratinocyte proliferation and differentiation in E-FABP null mice. In spite of the overexpressed in psoriasis, the E-FABP null keratinocytes showed no difference compared to wild type cells in cell proliferation. On the other hand, the E-FABP null keratinocytes showed decreased induction of keratin 1 and involucrin, which are expressed in spinous layers. Involucrin down-

Abstracts regulation in the E-FABP null keratinocytes reminds us increased expression of involucrin in psoriasis. Furthermore, E-FABP null keratinocytes revealed down-regulation of NF-kB activity that is reported to induce keratin 1 and involucrin. Our analyses suggest that E-FABP controls keratinocyte differentiation via NF-kB and that its overexpression may affect the differentiation in psoriatic keratinocytes. 175 The effects of ISP-I treatment on stratum corneum barrier function Koji Mizukoshi1, Hiroshi Ooshima1, Katsuo Matsumoto1, Ryouji Hirose2, Tetsuro Fujita3 1 POLA Chemical Industries, Inc, Yokohama, Japan; 2Mitsui Sugar Co., Ltd. Development & Research Section, Kobe, Japan; 3 Research Institute for Production Development, Kyoto, Japan

Serine palmitoyl transferase (SPT) is a rate-limiting enzyme for the de novo synthesis of ceramides and its deficiency leads to SC barrier dysfunction with an increased TEWL, and invites a fatal condition for life. On the other hand, the effects of SPT inhibitionin vivo have not been fully studied. In this study, therefore, the hairless mouse dorsal skin was treated with ISP-I, myriocin a specific inhibitor of SPT, and its effects on SC barrier function and the SC properties following various barrier perturbations were examined. Barrier malfunction was predicted when ISP-I was applied to the mouse skin before or after the 2MED UVB irradiation where the scaling and the extent of TEWL increase were observed at 72 h post irradiation. However, it was significantly suppressed, that is, TEWL was kept low by the seven continuous days ISP-I treatment before or after UVB irradiation. In another barrier perturbations such as acetone treatment or tape stripping, ISP-I treatment before the perturbations did not show any effects, but the treatment after perturbations resulted in the delayed barrier recovery. To understand the contradictory result, the mouse skin was simply treated with ISP-I and factors or components which contribute to the properties of SC were analyzed. The result was also contradictory. That is, the lower TEWL which represented the improvement of barrier function was observed even though the amount of ceramides was decreased. The number of SC layers and the dry weight of stratum cornem were increased in the ISP-I treated mice skin. From these results, it was suggested ISP-1 treatment transiently improved the UVB irradiated SC barrier function because of the physically multilayered SC even though the ceramides production was suppressed by SPT inhibition. 176 Induction of hair follicle apoptosis by peroxides

183 and hair loss, we studied their effect on mouse hair follicles and mouse hair cycles. At first, it was observed that linolein hydroperoxides, one of the lipid peroxides, induced apoptosis in hair follicles, and that topical application of linolein hydroperoxides lead to the early onset of the catagen phase. Therefore, it was suggested that the induction of apoptosis by lipid hydroperoxides caused a modulation of the hair cycle. Furthermore, in order to reveal the mechanism of induced apoptosis by linolein hydroperoxides, we analyzed the expression of apoptosis related genes in keratinocyte treated with linolein hydroperoxides using quantitative RT-PCR. In the result, it was observed that the expression of apoptosis related genes, such as p53, Bcl2, Bax, Caspase-8, -9, -10 had changed. Therefore, it was suggested that apoptosis was induced by two signaling cascades, one is via death receptor and the other is via mitochondria. 177 Expression of keratinocyte lipid transporter ABCA12 in developing human epidermis Yasuko Yamanaka1, Masashi Akiyama1, Kaori Sakai1, Yoriko Sugiyama-Nakagiri1,2, McMillan James R.1, Mitsuhito Ota1, Daisuke Sawamura1, Hiroshi Shimizu1 1 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2Kao Corporation Biological Science Laboratories, Tochigi, Japan

We clarified that ABCA12 works as an epidermal keratinocyte lipid transporter, and that a defective ABCA12 results in a loss of the skin lipid barrier, leading to harlequin ichthyosis, one of the most devastating genodermatoses [1]. In the present study, expression of ABCA12 was studied using immunofluorescent staining in human embryonal and fetal skin samples of 49—122 days estimated gestational age (EGA). As control, we also studied expression of transglutaminase1 (TGase1) that is known to catalyze cross-linking of several precursor proteins in the formation of cornified cell envelope at the terminal differentiation of keratinocytes. In the early two-layered epidermis (49 days EGA), ABCA12 and TGase1 immunostainings were both positive in the periderm cells. In the three-layered epidermis (76 days EGA) and in four- or more-layered epidermis (108—122 days EGA), ABCA12 and TGase1 were expressed in the periderm cells. After keratinization occurred (newborn skin), ABCA12 was expressed in the granular and cornified layers, while TGase1 was restricted to upper granular and cornified layers. From the expression of cornified cell envelope-associated proteins, we previously suggested that the process of periderm regression is similar to keratinization [1]. The present findings also support this idea. In addition, ABCA12 expression in the periderm suggested the possibility that ABCA12 works in lipid transport in the periderm.

Atsushi Naito, Yasushi Koike, Teruhiko Yoshino, Motoyasu Ohdera

Reference

Biological Science Research Center, Research&Technology Headquarters, LION Corporation, Kanagawa, Japan

[1] Akiyama, et al. J Invest Dermatol 1999.

It is said that no less than 10 million men are troubled with hair loss or thinning hair. Causes of male pattern baldness are androgen and hereditary predisposition, but it has been suggested that stress, irregular diet and high secretion levels of sebaceous lipid, also influence the progression of baldness. We focused on the lipid peroxides which are conversed from sebum or free fatty acid on the skin of the scalp by external stimulus such as the sun’s violet rays and also by hydrogen peroxide which is an ingredient in recent-prescribed hair-dyes. In order to reveal the relationship between these lipid peroxides

178 Induction of differentiation to melanocytes from stem cells derived from subcutaneous adipose tissue Hirohiko Akamatsu1, Seiji Hasegawa2, Naoki Yamamoto3,4, Satoru Nakata2, Kayoko Matsunaga1 1 Department of Dermatology, Fujita Health University School of Medicine, Toyoake, Japan; 2Research Laboratories, Nippon

184 Menard Cosmetic Co., Ltd., Nagoya, Japan; 3Laboratory of Molecularbiology, Joint Research Laboratory, Fujita Health University School of Medicine, Toyoake, Japan; 4Division of Cell Biology, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan Purpose: We isolated tissue stem cell-candidate cells from subcutaneous adipose tissue of ICR mouse using panning method based on p75NTR, and suggested at the meeting of this association last year a possibility to induce the differentiation of the obtained cells to adipose cells, smooth muscle cells, nerve cells and fibroblasts. This time, we investigated a possibility of inducing the differentiation of tissue stem cell-candidate cells obtained from the subcutaneous adipose tissue of C57BL/6 mouse to differentiate into melanocyte. We also investigated the induction of differentiation into adipose cell, smooth muscle cell, nerve cell and fibroblast. Results & discussion: As a result of immunohistological observation, we found the cell in which p75NTR considered as an epithelial stem cell marker was expressed in the subcutaneous adipose tissue as in the case of ICR mouse. This p75NTR-positive cells were isolated by the cell sorter. After incubation in various differentiation-inducing media, the differentiation into adipose cells, smooth muscle cells and fibroblasts was demonstrated. Furthermore, the differentiation into nerve cells and melanocyte-like cells was observed after addition of SCF, endothelin, etc. to the medium. Conclusion: It was suggested that multi-functional tissue stem cell-candidate cells are present in the subcutaneous adipose of C57BL/6 mouse as in the case of ICR mouse and that this cell could be induced to differentiate into melanocyte-like cell. Detailed cell analysis is going on at present. 179 Long term culture of mouse vibrissal dermal papilla cells and de novo hair follicle induction Aki Osada1, Tokuro Iwabuchi2, Jiro Kishimoto2, Tatsuo S. Hamazaki1, Hitoshi Okochi1 1

Department of Tissue Regeneration, Research Institute, International Medical Center of Japan, Tokyo, Japan; 2Skin Biology Research Labs., Life Science Research Center, SHISEIDO CO., LTD. For de novo hair follicle induction, large numbers of dermal papilla cells are needed for transplantation with epidermal cells. However, dermal papilla cells under normal culture conditions do not proliferate well and lose their hair-inducing capacity after more than 10 passages. Therefore, simpler and more stable culture methods for dermal papilla cells have been long desired. The outgrowth of dermal papilla cells was markedly stimulated when explants of mouse vibrissa dermal papillae were cultured with 10%FBS-DMEM including bFGF. Moreover, this effect was maintained during serial cultivations, and more than 30 passage cultivations were established. To examine their follicle-inducing ability, these established dermal papilla cells were combined with epidermal cells and transplanted subcutaneously into athymic mice. New hair follicles were induced when dissociated dermal papilla cells at earlier passages (under passage 4) were injected with epidermal cells isolated from newborn or embryonic mice skin, but the cells from higher passages could not induce follicles, as previously reported. More recently, we generated spheroids of dermal papilla cells and injected them with epidermal cells. Surprisingly, the spheroids made from the higher passage cells (more than 10 passages) did induce new hair follicles. Moreover, the expression of several genes

Abstracts specific for dermal papillae in vivo was elevated in the spheroids compared with that in adhered dermal papilla cells. These results suggest that bFGF is essential for dermal papilla cell culture and that spheroid formation partially represents the intact dermal papilla, resulting in hair follicle induction even by highly passaged cells. 180 Eyelash growth and structure of eyelid Moe Tsutsumi1, Tsutomu Souma1, Yosuke Nakazawa1, Masahiro Tajima1, Kazumi Tsurukiri2 1 SHISEIDO Research center, Yokohama, Japan; 2Tsurukiri KeiseiHifuka, Tokyo, Japan

Purpose: Mascara has become 300 million yen business because many women are interested in long and thick eyelashes. However, feature of eyelashes remains incompletely understood. In this study, we performed the basic research about the growth of eyelashes and histochemical study of eyelids in human. Methods: 2—2.7 cm regions from medial angle of eye were cut with scissors leaving 2 mm eyelashes. The images of these areas were taken by a digital microscope (VH-6300, Keyence). The length of eyelashes was measured on the images and we calculated the hair-cycle and growth rate. We defined the anagen eyelash as growing more 0.08mm per day and calculated the anagen ratio. Folmalin-fixed, celloidin embedded human tissue was sectioned vertically. Thirty micrometer-thick sections were stained with Azan staining. Results: The hair-cycle of eyelashes was 118  16 days (average  S.D.), anagen was 39  6 days. The growth rate of eyelashes was 0.18  0.018 mm per day and the length of eyelashes significantly correlated with both of the anagen term and the growth rate. The anagen ratio was 15—40%. It has seasonal changes and statistically highest in April. We observed much blue region in eyelash area compared to upper eyelid skin by Azan staining, suggesting that the eyelash area has collagenal connective tissue rich structure. It was also observed the eyelash area was surrounded by some orbicularis oculi muscle. It has been stated that the eyelash area has the similar structure to the upper eyelid skin, but we found out a clear difference between those two areas. Conclusion: These data suggested that eyelashes are strongly holding by the connective tissue. Therefore, eyelash club hair might hardly fall off. 181 Signal sequence trap screening for the patterning molecules in early head ectodermal differentiation Akiko Arakawa1, Yoshiki Sasai2, Yoshiki Miyachi1 1

The Department of Dermatology, University of Kyoto, Kyoto, Japan; 2The Neurogenesis and Organogenesis Group RIKEN Center for Developmental Biology, Kobe, Japan Secreted proteins such as growth factors and extracellular matrix components play critical roles in the formation and differentiation of multicellular organisms. A number of family members of BMPs, FGFs and Wnts transmit differentiation signals from cell to cells. The extracellular matrix and adhesion molecules also coordinate cell state. Furthermore, secreted proteins can be effective therapeutic agents or targets for antagonistic or agonistic therapy. Directing of secreted proteins to the cell membrane is mediated by a short hydrophobic stretch of amino acids, termed the signal sequence. The signal sequence trap (SST) is a

Abstracts screening method to identify cDNAs containing signal sequences that encode secreted proteins. Dr. Kitamura and his colleagues (Tokyo University) recently developed an efficient SST system based on retrovirus-mediated expression screening (SST-REX). To search for regulatory factors acting in early head development, we applied this SST-REX method to the cDNAs derived from chicken embryonic head ectoderm. We screened 1.94X10(6) clones and successfully isolated 114 clones by one-step screening. Thus, SST-REX will be a potential strategy for systemic screening of molecular key player of epidermal differentiation. 182 Functional analysis of pten-deficient mice in melanocytes Tae Inoue1, Takehiko Sasaki3, Hiroyuki Kishimoto2, Masatake Osawa 4, Tetsunobu Kimura5, Satoshi Itami6, Tak Wah Mak8, Toru Nakano7, Motomu Manabe1, Akira Suzuki2 1

Department of Dermatology and Plastic Surgery, Devision of Sensory Organ, Akita University School of Medicine, Japan; 2 Department of Molecular Biology, Akita University School of Medicine, Akita, Japan; 3Department of Microbiology, Akita University School of Medicine, Akita, Japan; 4Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan; 5Sapporo Institute of Dermatopathology, Sapporo, Japan; 6Department of Dermatology, Graduate School of Medicine, Osaka University, Suita, Japan; 7The Campbell Family Institute of Breast Cancer Research, and Departments of Immunology and Medical Biophysics, University of Toronto, Toronto, Ont., Canada; 8Department of Pathology, Medical School and Graduate School of Frontier Biosciences, Osaka University, Suita, Japan PTEN is a tumor suppressor gene that is mutated in many human sporadic cancers and in hereditary tumor susceptibility disorders such as Cowden disease. PTEN is a multi-functional phosphatase whose major substrate is phosphatidylinositol-3,4,5-triphosphate (PIP3), a lipid second messenger molecule. PIP3 activates numerous downstream targets, including the serine-threonine kinase PKB/Akt which is involved in anti-apoptosis, proliferation, and oncogenesis. By using its lipid phosphatase activity to dephosphorylate PIP3, PTEN negatively regulates the PI3K-PKB/Akt pathway and thus exerts tumor suppression. Although PTEN mutation was observed in low frequency in primary melanomas (<10%) and in melanoma cell lines (<30—40%), loss of PTEN protein expression occurs in 8% of nevi and 63% of primary melanomas, indicating that inactivation of PTEN protein may be a feature of progression from malanocytic nevi to primary melanoma. Now, we used the Cre-loxP system to generate a melanocytes-specific mutation of Pten (DctCrePten) in mice to analyze the function of Pten in melanocytes. 183

185 acterize the biological processes involved in the development of melanocytes from follicular stem cells and their differentiation. In this study we further characterized bulge-derived cells by expressing of Wnt signaling pathway, e.g., Wnt3a. The bulge regions were isolated under a dissecting microscope from mouse vibrissal hair follicles of adult Dct-lacZ transgenic mice. In this animal system, lacZ gene is driven by the Dct promoter and the melanocytic stem cells are expected to express b-galactosidase by regulating the transcription of the Dct gene. We used recombinant mouse Wnt3a in order to examine the influence of Wnt3a to b-galactosidase positive cells. Infection of recombinant adenovirus expressing Wnt3a affected the growth of b-galactosidase positive cells, regulating both growth proliferation and inhibition of adult melanocyte stem cells. 184 The expressions of melanosome associated rabs are upregulated in melanocytes but downregulated in nevus cells by MSH Tsuneyoshi Kamo, Yoko Funasaka, Chikako Nishigori Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan Melanosomes are transported from the perinucleus to the peripheral region of melanocytes, eventually being transferred to adjacent keratinocytes. Rab proteins are monomeric GTPases of the Ras superfamily. In the active state, Rabs recruit a diverse group of proteins termed effector proteins to the cytoplasmic leaflet of the membrane, and recruitment of effector proteins might enable Rabs to control the main steps in vesicular transport. We have previously reported that Rab3A and Rab8A are associated with melanosomes in mouse B16 melanoma cells. Recently another secretion related Rab protein, Rab27A has been shown to bind with melanophilin/ Slac-2a which associates with myosin Va. MSH is a well known physiologic stimulator of melanin synthesis and melanosome transport in the actin-rich region of the dendrite extremities. We examined the effect of MSH on Rab expression as well as configuration using cultured normal human melanocytes and nevus cells, the latter are believed to have a tendency to retain melanin. MSH upregulated the expression of Rab3A, Rab8A, and Rab27A in melanocytes with the dendrite elongation and enhanced tyrosinase expression. In contrast to melanocytes, nevus cells are downregulated in their expression of Rabs and tyrosinase. The determination of the melanocortin-1 receptor (MC-1R) sequence showed no difference between these cell types. These results suggest that GTPase molecules involved in melanosome transport are coordinately regulated with melanin synthesis by MSH stimulation, however these are downregulated in nevus cells which are poor in the ability of melanosome transport, and these differences were not due to the variance of MC1-R gene itself, but rather the variance of signal transduction in the downstream of MSH receptor.

The growth and differentiation control of melanocyte stem cells from mouse vibrissal hair bulge by Wnt signaling pathway

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Akihiro Tominaga1, Toshiharu Yamashita1, Ichiro Ono1, Takahiro Kunisada2, Kowichi Jimbow1

Production and examination of transgenic mice that have KITV620A mutation in human piebaldism with progressive depigmentation

1

Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan; 2Structure and Organ Formation Control, Regeneration Medicine and Bioethics, Gifu University, Gifu, Japan The nature and origin of adult melanocyte stem cells are not yet fully understood. We previously reported our success in separating and identifying melanocyte stem cells from the bulge region of mouse vibrissal hair follicles. The goal of our study is to char-

Hiroko Tosaki1, Takahiro Kunisada2, Yasuo Kitajima1 1 Department of Dermatology, Gifu University School of Medicine, Gifu, Japan; 2Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu, Japan

Piebaldism is an autosomal dominant genetic pigmentary disorder, characterized by congenital white hair and patches located

186

Abstracts

on the forehead, anterior trunk, and extremities. Most patients with piebaldism have mutation of the KIT gene encoding a tyrosine kinase receptor that included in the pigment cell development. The white hair and patches of the patients had already been completed when giving birth and have not been expanded usually throughout life. The stability of pigmented spots was applies to the case in KITW or KitlSl mutant mice. However a novel piebaldism case that showed mother and daughter with progressive depigmentation having a novel Val620Ala mutation in the KIT gene, has reported in 2001. Aiming to reproduce the model of this Val620Ala KIT mutation and explore unknown function of KIT signaling to maintain pigmented melanocytes in the skin or more specifically the integrity of post natal skin melanocyte stem cell system, we produced a transgenic mouse expressing Val620Ala KIT. These mice well mimic the white spotting pattern of patients, however, no change of white spot pattern was observed after birth, even after increasing the transgene expression by various means. Here, we elucidated unexpectedly extremely stable maintenance of melanocyte stem cell system under the stringent conditions for KIT signaling.

of matrilysin in human melanomas, in acquired melanocytic nevi, and in Spitz nevi, and analysed the data in connection with the clinicopathological factors. The percentage of melanoma samples positive for matrilysin as detected by immunohistochemistry was 85.7% (30/35). Matrilysin was mainly overexpressed at the invasive front of the tumours and in metastatic lesions. In situ hybridisation demonstrated that melanoma cells almost selectively express matrilysin mRNA. In contrast, matrilysin was not detected in common nevi or in Spitz nevi. Overexpression of matrilysin in primary melanomas was associated with the presence of metastases, tumour thickness, and staging ( p < 0.001, p = 0.023, 0.014, respectively). The 5-year overall survival was 40% for matrilysin-positive cases and 100% for matrilysin-negative cases among primary melanoma specimen. We found matrilysin overexpression in primary and metastatic melanomas. We further demonstrated that matrilysin staining intensity was associated with invasion of primary melanoma lesion and metastases. Our observations indicate that matrilysin may be associated with melanoma progression, and may enhance melanoma tumour cell invasion. Therefore, matrilysin may be potentially valuable as a prognostic indicator to predict the clinical behavior of melanoma.

186 Light and electron microscopic study to a novel asymmetrical ripple-like reticular hypermelanosis 1

2

1

1

Naoki Oiso , Daisuke Tsuruta , Tomoko Oota , Masuki Yoshida , Masamitsu Ishii2, Akira Kawada1 1

Department of Dermatology, Kinki University School of Medicine, Osaka-Sayama, Japan; 2Department of Dermatology, Osaka City University School of Medicine, Osaka, Japan

188 Bidens pilosa suppresses interleukin-1b-induced cyclooxygenase-2 expression through the inhibition of mapk phosphorylation in fibroblasts Nobuyo Yoshida, Kanekura Takuro, Yuko Higashi, Tamotsu Kanzaki Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

The congenital pigmentation abnormalities are derived from migration and distribution of melanocytes, formation and melanization of melanosomes in melanocytes, and transport of melanin from melanocytes to keratinocytes. Variety disorders characterized by a reticulate hypermelanosis have been reported. Here, we report a three-year-old girl with asymptomatic and asymmetrical ripple-like reticular hypermelanosis. Skin biopsies were taken from bilateral upper back: hyperpigmented lesion and normal skin colored lesion. With hematoxylineosin stain, focally aggregated melanocytes were shown on both biopsy specimens. With Fontana-Masson stain, typical two abnormalities were exhibited; one was absence of melanin granules on the hair follicles and another was asymmetrical distribution of melanin granules on the epidermis. With electron microscopy, immature melanosomes were more present than expected. We point out the possibility that the novel pigmentation disorder stem from abnormal melanocyte distribution, immature biogenesis of melanosomes and aberrant transportation of melanin.

Bidens pilosa (BP) Linn. var. radiata is a plant used in traditional folk medicine. It is clinically effective in various diseases; the pathogenesis of most of these involves cyclooxygenase (COX)-2. To investigate the mechanism on which the clinical effectiveness of BP is based, we examined its effects on COX-2 expression and its major product, prostaglandin (PG) E2, under conditions of inflammation. We induced inflammation in normal human dermal fibroblasts with interleukin (IL)-1b and examined the effects of BP on COX-2 expression and PGE2 production using Western blotting and competitive enzyme-immunoassay, respectively. The functional involvements of MAPKs ERK1/2, p38, and JNK in COX-2 expression were also examined by Western blotting. IL-1b-induced COX-2 expression was regulated by MAPK pathways, especially by p38. BP inhibited the phosphorylation of MAPKs, COX-2 expression, and subsequent PGE2 production. The physiological activities and clinical effectiveness of BP observed under diverse conditions may be partly attributable to its ability to inhibit MAPK activity, COX-2 expression, and subsequent PGE2 production.

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189

Matrilysin (matrix metalloproteinase-7) overexpression in human cutaneous melanoma

Quantitative analysis of Malassezia and measurement of specific IgE antibody in antimycotic-treated atopic dermatitis patients

Kanade Kawasaki1, Tamihiro Kawakami1, Hidenori Watabe1, Masako Mizoguchi1, Yoshinao Soma1, Fumio Ito2 1

Department of Dermatology, St. Marianna University School of Medicine, Kawasaki, Japan; 2Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Japan Matrilysin (MMP-7), a member of the matrix metalloproteinases (MMP) family of proteins, is expressed in various types of malignant tumours. There have been no previous studies of the correlation between expression of matrilysin and human cutaneous melanoma. We evaluated the production and tissue localisation

Mami Tajima1, Takashi Sugita2, Tomonobu Ito1, Akemi Nishikawa3, Ryoji Tsuboi1 1 Department of Dermatology, Tokyo Medical University, Tokyo, Japan; 2Departments of Microbiology, Meiji Pharmaceutical University, Tokyo, Japan; 3Departments of Immunobiology, Meiji Pharmaceutical University, Tokyo, Japan

Malassezia species, normally colonizing the skin surface of healthy individuals, have been involved as either the cause or exacerbating factor in a number of skin conditions including

Abstracts atopic dermatitis (AD). We found three novel Malassezia spp. among the nine species detected in human hosts. The present investigation was conducted to measure the change in the numbers of Malassezia organisms and values of Malassezia-specific IgE antibodies before and after the topical treatment of intractable AD patients using ketoconazole 2% cream. Twelve moderate or severe adult AD patients with face and neck lesions were enrolled in this study. Any concurrent treatment for AD involving topical steroids and tacrolimus was continued. Fungal DNA was extracted directly from the samples collected by tape stripping the surface of the lesional skin. We analyzed the quantity of Malassezia spp. by real-time PCR assay (ABI 7500), for which we prepared TaqMan probes designed from Malassezia 26S or IGS rRNA. M. globosa and M. restricta-specific IgE antibodies were originally prepared as an Ala-STAT system. As a result, the quantity of Malassezia spp. in all of the samples decreased after topical application of the antimycotic, and 70% of the AD patients showed clinical improvement in erythema and scaling. The serum levels of total IgE antibodies, M. globosa and M. restricta-specific IgE antibodies, decreased as well. These results indicate that removal of the organisms by antimycotic agent results in an improvement in the clinical manifestations of intractable AD and suggests that Malassezia spp. constitute one of the exacerbating factors of AD.

187 Tokyo, Japan; 4Department of Dermatology, Tokyo Woman’s Medical University Medical Center East, Tokyo, Japan

Shuji Fukagawa, Takeshi Nakahara, Junichi Hachisuka, Yoichi Moroi, Kazunori Urabe, Masutaka Furue

There are some problems in PASI (Psoriasis Area and Severity Index) score. Calculation of PASI is complicated, the scores fluctuate among different evaluators, etc. In order to evaluate severity more objectively, we developed a computer program which automatically calculate the area and redness intensity from photographs. This program extracts the lesion having a certain amount higher ‘a’ value of Lab color index, which shows redness, from the normal skin. The score of severity is defined as a product of the area of involvement and average difference of ‘a’ value between the lesion and the normal skin. We call this score as CASI (Computer-assisted Area and Severity Index) score. Comparison of CASI score with the result of manual extraction by a dermatologist as a standard revealed the sensitivity was 71.3  14.3% and the specificity was 97.9  1.1%. The correlation coefficient of the CASI and the PASI score was 0.92. We assume the improvement in the accuracy of extraction will enable objective evaluation only by photography. Next in order to evaluate the relationship between the severity and QOL, PASI score and Skindex-16 were compared. Skindex-16 is a measurement of impaired QOL for patients with skin disease composed of three scales: Symptoms, Emotions, and Functioning. The higher measurement means the more impaired QOL. Total QOL scores for 16 items tended to be impaired in patients with high PASI score. In the meantime, a correlation between severity and each QOL scale was low. This is because there are some cases with high scores in Emotions and Functioning irrespective of low PASI score. We would like to emphasize the QOL evaluation is also essential as well as the exact and objective evaluation of severity for an assessment of patients with psoriasis.

Department of Dermatology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

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190 Dendritic cell-based immunotherapy for melanoma patients with cyclophosphamide pretreatment

From 2002, twelve patients with stage IV melanoma received the DC vaccine and were assessed for effectiveness and safety in our department. Some patients showed DTH responses, vitiligo, erythema (tumor resection lesion) and temporary tumor shrinkage, but all metastatic lesions progressed. Furthermore, melanoma progressed very rapidly in some patients. It was recently reported that CD4+CD25+ regulatory T cells (T-reg) accumulated in the peripheral blood of patients and mice with various carcinomas and they inhibited anti-tumor immune responses. Therefore, depletion of T-reg can be an attractive strategy for tumorspecific immunotherapy. Low doses of cyclophosphamide (Cy) has been shown to reduce CD4+CD25+ T cell function in mice and in cancer patients, and to restore the efficacy of adoptive immunotherapy in experimental tumors. We administrated Cy on two melanoma patients. Both patients were diagnosed as PD in clinical responses, but CD4+ cells decreased in a dose dependent manner. We also examined the profile of CD4+CD25+ regulatory T cells. 191 Quantitative measurement of severity by computer in patients with psoriasis vulgaris, and consideration of severity and QOL by skindex-16 Ayako Miyake1, Masayuki Kimoto1, Hiroshi Oka2, Hitoshi Iyatomi3, Koichi Ogawa3, Masaru Tanaka4 1

Department of Dermatology, Keio University School of Medicine, Tokyo, Japan; 2Department of Information and Computer Science, Faculty of Science and Technology, Keio University, Yokohama, Japan; 3Department of Electronics Electrical and Computer Engineering, Faculty of Engineering, Hosei University,

Characterization of scalp and hair of the female with each period Yosuke Nakazawa1, Yuko Komori2, Kanako Kio2, Yuki Shimada2, Junko Tajima2, Nao Murayama2, Kazuo Tsuchida2, Masato Iino1, Masahiro Tajima1 1 Shiseido Research Center, Kanagawa, Japan; 2Shiseido Institute of Beauty Sciences, Tokyo, Japan

Purpose: In recent years, the women who have worries of baldness and fallen hair are seen in young generation in addition to middle age. Therefore, we examined the conditions of scalp and hair of the female with each period. Methods: Two hundred and ninety-nine Japanese female teens-sixties were recruited. Their hairs were evaluated hardness, thickness and volume by two beauticians. The scalp sebum was measured by the absorption-transcription method. The sebum was absorbed to the filter paper. The image was taken by the digital microscope and was measured as the ratio of the sebum adsorption area. The hairs in quadrangle of 5 mm  5 mm near the vertex area was cut with scissors. The scalp was taken of an image with the digital microscope. Hair diameter and density were measured on the image. The degree of baldness was evaluated on the photograph. Results: Hardness, thickness and volume of the hair correlated with hair diameter. In twenty-forties, there were much sebum and fifties-sixties were about 60% of twenties The percentage of the thick hair that the diameter not less than 80 mm was reached an average of about 67% at thirties, it declined to 64% at forties, 57% at fifties and 50% at sixties. The hair density was an average of more than about 230/cm2 to thirties, but declined to 223/cm2 at forties, 206/cm2 at fifties and 199/cm2

188 at sixties. The ratio of baldness female was about 16% at thirties, but it increased to 32% at forties, 54% at fifties and 57% at sixties. Conclusion: Sebum, hair diameter and density of the female reached a peak at twenties-thirties and declined from fortiesfifties. These results were similar to research result in the past. But the conditions of scalp and hair of young generation to be more aggravated than before. 193 Effects of macrolides on IL-8 release via activation of proteaseactivated receptor 2 (PAR2) in cultured normal human keratinocytes

Abstracts Methods: We reviewed whether we cannot use cell activator action and oxygen supply acceleration action to tissue to have of PEP in wound healing acceleration. We made cutaneous ulcer in back of the C57BL/Ks-J (db/db) laboratory mouse which was (in vivo) diabetes mellitus model laboratory mouse. We applied 3%, 20% PEP liquid once a day and measured contraction rate of cutaneous ulcer and reviewed re-epithelization rate, a granulation rate, blood vessel hyperplasy rate histologically. Furthermore, we compared the effect with the FGF spray which was Fibrast spray. We used a (in vitro) human skin origin fibroblast immortalization stock (Mori SV) and human skin origin normal fibroblast (TIG109) and reviewed influence of PEP which gave it to fibroblastic breeding.

Chika Ishikawa, Tatsuya Tsuda, Yoshiaki Lin, Kiyofumi Yamanishi

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Department of Dermatology, Hyogo College of Medicine, Nishinomiya, Japan

The effect of raloxifene (selective estrogen receptor modulator) therapy on the skin of Japanese postmenopausal women

Protease-activated receptor 2 (PAR2) is a novel G-protein-coupled receptor with seven transmembrane domains. Serine proteases, such as mast cell tryptase, cleave PAR2 and expose a tethered ligand (SLIGKV), which binds and activates the receptor. PAR2 is expressed in the epidermis and possibly involved in various inflammatory conditions via stimulating IL-8 release from keratinocytes. Besides antibiotic action, macrolides show immunomodulatory activities and are sometimes useful for treatment of panbronchitis and some inflammatory skin diseases. To elucidate the mechanisms of the anti-inflammatory actions of macrolides, we examined the effects of EM and RXM on PAR2-mediated IL-8 release in cultured normal human epidermal keratinocytes (NHEK). NHEK were pre-incubated with 10 mM EM or 10 mM RXM for 24 h, and then treated for 48 h with an agonist peptide of PAR2 (100 mM SLIGKV-NH2), its reverse peptide (100 mM VKGILS-NH2), 100 ng/ml IL-1b or 100 ng/ml IL-18. IL-8 release was induced by SLIGKV-NH2 or IL-1b, but neither by IL-18 nor VKGILS-NH2. In the presence of both IL-1b and SLIGKV-NH2, IL-8 production was synergistically induced. In the presence of EM or RXM, SLIGKV-NH2- and/or IL-1b-induced IL-8 release was decreased in dose-dependent manner. These results suggest that these macrolides modulate skin immune response via PAR2 activation system in keratinocytes.

Tetsuo Sasaki1, Yaku Tanaka 2, Itsuo Gorai2 1

Department of Dermatology, International University of Health and Welfare Atami Hospital, Atami, Japan; 2Department of Obstetrics and Gynecology, International University of Health and Welfare Atami Hospital, Atami, Japan

Research about the effect of phosphoenolpuruvic acid (PEP) for wound healing

The effect of raloxifene (selective estrogen receptor modulator) therapy on the skin has not been described yet. In order to clarify its effects on the skin we now investigated the skin of Japanese postmenopausal women under raloxifene therapy. In comparison those who received treatment with hormone(s) (estrogens conjugated or estradiol with or without medroxyprogesterone acetate) or alfacalcidol only were also included in this study with informed consent. The skin on the cheek, extensor aspect of the forearm and dorsum of the hand was examined by pinching and using the skin elasticity meter (Cutometer) every 4 to 5 weeks. Twelve women who have received one of the three kinds of treatments mentioned above over 6 months and have been examined over four times during the treatment period so far were analyzed. Six women have received treatment with raloxifene, five with the hormone(s) and one with alfacalcidol. They were from 50 to 76 years old. The observation period was from 27 to 39 weeks. The skin examination was carried out from four to nine times during the period. No significant changes have been found in subjective assessment by the patients, skin score by the pinching method and the skin elasticity measurement. Since no negative effects on the skin have been observed yet, further follow up study will be warranted until one year.

Yuji Inoue1, Hironobu Ihn1, Hiroki Kitaoka2, Sakiko Fukuda2, Emi Oomori2, Tetumi Irie2

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1

Department of Dermatology, Kumamoto University Hospital; Department of Clinical Chemistry and Informatics, Graduate School of Pharmaceutical Sciences, Kumamoto, Japan 2

Background: High-energy phosphate bond such as ATP does not transmit with cap formation. However, Hamasaki discovered that phosphoenolpyruvic acid (PEP) which is full of the highest energy phosphorylation slightly transmitted in facilitated transport with cap formation in vivo. PEP added in the cap formation outside is taken in promptly into a cell, and it is converted into ATP and activates a cell. 2-PG is Enolase, and it is metabolized in glycolytic pathway and becomes PEP of high-energy compound and we convert energy of PEP into ADP by pyruvate kinase reaction and produce the ATP which is an energy source of cellular universality. PEP is phosphorylation which is full of the most energy in vivo. PEP is only phosphorylation which can transmit the cap formation. 2.3-DPG which are metabolic product of PEP can promote oxygen supply to tissue by decreasing haemoglobin and affinity of oxygen.

Influence of cyclosporin on production of extracellular matrix proteins and proliferation of human dermal fibroblasts Masatoshi Abe, Yoko Sogabe, Yoko Yokoyama, Tomoko Shyuto, Hirohisa Ishibuchi, Osamu Ishikawa Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan The clinical efficacy of cyclosporin (CyA) in the treatment of sever psoriasis is well established. The fact that CyA monotherapy without any topical treatment can bring the lesional dermal improvement suggests that CyA may be involved in the remodeling of extracellular matrix. The aim of this study is to determine whether CyA may modify the connective tissue metabolism of human fibroblast or not. CyA altered the fibroblast morphology but not tubulin formation on collagen-coated coverslips with serum or IL-6 stimulation. While in the absence of IL-6, the production of type I collagen and TIMP-1 decreased at CyA

Abstracts 1000 ng/ml, in the presence of IL-6, these production decreased at CyA 1000 and 100 ng/ml after 24 and 48 h CyA stimulation. In the absence of IL-6, the production of MMP-1 gradually decreased at CyA dose dependent manner at 24 h. In contrast, the production of MMP-1 decreased at CyA 1000 ng/ml only at 48 h in the absence of IL-6 and at 24, 48 h in the presence of IL-6. Regardless of the presence or absence of IL-6, the production of MMP-2 decreased at CyA 1000 and 100 ng/ml at 24 and 48 h, and the production of MMP-9 was not changed at any concentration at 24 and 48 h. In the absence of IL-6, the fibroblast proliferation was suppressed at CyA 100 and 10 ng/ml at any time, while in the presence of IL-6, the fibroblast proliferation was suppressed at CyA 100 to 1 ng/ml at 24 and 48 h. In conclusion, this preliminary study indicates that CyA may influence the connective tissue metabolism and proliferation of human dermal fibroblast. This preliminary study may suggest the possibility that CyA improves psoriatic skin by regulation of the remodeling of extracellular matrix as well as other inflammatory cells. 197 Knockdown of Skp2 by siRNA inhibits melanoma cell growth in vitro and in vivo Yoshiyuki Katagiri, Yutaka Hozumi, Shigeo Kondo Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan Low levels of p27Kip1 expression are associated with poor prognosis in various malignancies including malignant melanoma. Recently, it has been reported that S phase kinase-interacting protein 2 (Skp2), the specific ubiquitin ligase subunit that targets p27Kip1 for degradation, was overexpressed and was inversely related to p27Kip1 levels in malignant melanoma with poor prognosis. We investigated whether small interfering RNA (siRNA)mediated gene silencing of Skp2 can be employed in order to inhibit p27Kip1 down-regulation and suppress melanoma cell growth as a consequence in vitro and in vivo. We constructed a plasmid vector, which synthesizes siRNAs to determine the effects of decreasing the high constitutive levels of Skp2 protein in melanoma cells. Western blot and real-time RT-PCR were performed to examine the decreases of Skp2 protein and mRNA in vitro. Furthermore, melanoma cells were injected into the back of nude mice subcutaneously to examine the suppression of tumorigenicity targeting Skp2 gene silencing in vivo. Skp2 protein was decreased and the p27Kip1 protein was accumulated in Skp2 siRNA transfected melanoma cells. Skp2 siRNA inhibited the cell growth of melanoma cells in vitro. Moreover, Skp2 siRNA also suppressed tumor proliferation in vivo. Our results suggest that siRNA-mediated gene silencing of Skp2 can be a potent tool of cancer gene therapy for suppression of p27Kip1 degradation in malignant melanoma. 198 Incidence of atopic dermatitis in nursery school children–—A follow-up study, Kyushu University Ishigaki Atopic Dermatitis Study (KIDS) Noriko Fukiwake1, Norihiro Furusyo2,3, Norihiko Kubo2,3, Hiroaki Takeoka2,3, Kazuhiro Toyoda2,3, Keisuke Morita1, Satoko Shibata 1, Takeshi Nakahara1, Makiko Kido1, Sayaka Hayashida1, Yoichi Moroi1, Kazunori Urabe1, Jun Hayashi 2,3, Masutaka Furue1 1

Department of Dermatology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of General Medicine, Kyushu University Hospital, Fukuoka, Japan; 3Department of Environmental Medicine and Infectious Disease, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan

189 Atopic dermatitis (AD) is a multifactorial disease that usually decreases the quality of life of affected patients. Clinically, the majority of patients with infantile and childhood AD is believed to spontaneously regress. However, few studies have actually addressed and confirmed this assumption using population-based cohort study methodology. We monitored the incidence of AD and serum total IgE levels annually among nursery school children in Ishigaki Island, Okinawa, Japan, from 2001 to 2004. A total of 1731 children were enrolled. The prevalence of AD ranged from 3.7 to 11% in each year, with no significant difference between boys and girls. Eight hundred and sixty-nine children were examined at least twice. 71.6% (53/74) of AD patients regressed spontaneously. In contrast, 5.5% (44/795) of non-AD individuals developed AD during the 3-year follow-up, that is, new-onset AD occurred at a rate of 3.67%/person year. We divided all cases into 4 groups, the continuous AD group, the regressed AD group, the newly-developed AD group, and the non-AD group, and compared the alterations of total IgE level. Although total IgE levels increased in all the groups, the increases were greater and more rapid in children with continuous AD than those in the other groups. In conclusion, the spontaneous regression was occurred in the high percentage of AD patients in nursery school children, resulting in the confirmation of our assumption. Considering the spontaneous regression and de novo development, the clinical course of AD is clearly extremely diverse in childhood. 199 Some EBA sera react with distinct epitopes on the central collagenous domains, but not the NC1 and NC2 domains, of type VII collagen Norito Ishii1, Mariko Yoshida1, Toshihiro Tanaka2, Akemi Ishida-Yamamoto3, Takashi Hashimoto1 1 Department of Dermatology, Kurume University School of Medicine, Kurume, Japan; 2Department of Dermatology, Shiga University of Medical Science, Shiga, Japan; 3Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan

The sera of epidermolysis bullosa acquisita (EBA) react with type VII collagen, a major component of anchoring fibrils, in which the major epitopes have been considered to be present in the Nterminal non collagenous (NC)-1 domain. We previously reported that most EBA sera reacted with the NC1 domain, while a few sera reacted with the NC2 domain by immunoblotting using bacterial recombinant proteins of the NC1 and NC2 domains of type VII collagen. The purpose of the present study is to determine the ultrastructural localization of epitopes which were recognized by some selected EBA sera, showing no reaction with either NC1 or NC2 domain. Postembedding immunoelectron microscopy was performed. In normal skin, reaction products for the sera without reaction with either NC1 or NC2 domains were all found in the dermis, 0—360 nm below the lamina densa. These distribution histograms were different from those of the sera that reacted with NC1 and/or NC2 domains. In recessive dystrophic epidermolysis bullosa skin, depositions of IgG of patients’ sera were absent. 200 Plasminogen system mediates the C-terminal cleavage of 120-kDa LAD-1 producing a 97-kDa polypeptide Yoshiaki Hirako1, Detlef Zillikens2, Katsushi Owaribe1 1 Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan; 2Department of Dermatology, University of Luebeck, Luebeck, Germany

190 BP180 (bullous pemphigoid antigen 180)/type XVII collagen is a type II-oriented transmembrane glycoprotein of the hemidesmosome, a cell-matrix adhesion structure. In cultured keratinocytes, the ectodomain of BP180 can be cleaved within the membrane-proximal 16th non-collagenous A (NC16A) domain, producing a 120-kDa soluble molecule (LAD-1). This N-terminal cleavage is mediated by ADAMs (a disintegrin and metalloprotease). On the other hand, a 97-kDa extracellular fragment of BP180 (LABD97) is isolated from human epidermis. The epidermal 97-kDa fragment is produced by a cleavage at the C-terminus of BP180 in addition to the N-terminal cleavage in the NC16A domain. The present study addressed the question which protease(s) cleave(s) at the C-terminus. In a first set of experiments, we observed that the 120-kDa fragment, obtained from cultured keratinocytes may degrade to a 97-kDa polypeptide, which shares biochemical characteristics with epidermal 97-kDa fragment. Interestingly, the C-terminal degradation was only observed when the 120-kDa fragment-rich fraction was prepared using culture medium from keratinocytes that had been plated more than 3 days ago. Moreover, this degradation was abolished by serine protease inhibitors. From the result of an in vitro assay, we found that, at least, two factors are involved in the C-terminal cleavage. Urokinase-type plasminogen activator and plasmin, both of which are serine proteases, could reproduce the enzymatic activities of these factors. Our result suggests that the C-terminal cleavage of the hemidesmosomal adhesion molecule controlled by plasminogen system may be a mechanism facilitating basal keratinocytes motility during wound healing. 201 COL7A1 mutation analysis in 22 Japanese families with dystrophic epidermolysis bullosa Daisuke Sawamura, Maki Goto, Kana Yasukawa, Kazuko Sato-Matsumura, Hideki Nakamura, Kei Ito, Hiroyuki Nakamura, Yuki Tomita, Hiroshi Shimizu Department of Dermatology, Hokkaido University Graduate School of Medecine, Sapporo, Japan Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disorder, which is clinically characterized by mucocutaneous blistering in response to minor trauma, followed by scarring and nail dystrophy caused by mutations in the type VII collagen gene (COL7A1). DEB is inherited in either an autosomal dominant (DDEB) or recessive (RDEB) fashion. DDEB basically results from a glycine substitution mutation within the collagenous domain on one COL7A1 allele, while a combination of mutations such as premature termination codon, missense, and splice-site mutations on both alleles causes RDEB. This study performed mutation analysis in 22 distinct Japanese DEB families (5 DDEB and 17 RDEB). The result demonstrated 33 pathogenic COL7A1 mutations with 18 novel mutations, which included 6 missense, 5 nonsense, 1 deletion, 2 insertion, 1 indel, and 3 splice-site mutations. We confirmed that Japanese COL7A1 mutations were basically family specific, although 3 mutations 5818delC, 6573 + 1G > C, and E2857X were recurrent based on previous reports. Furthermore, Q2827X mutation found in two unrelated families would be regarded as a candidate recurrent Japanese COL7A1 mutation. The study furthers our understanding of both the clinical and allelic heterogeneity displayed in Japanese DEB patients. 202 Kindlin-1 and -2 expression in normal human and Kindler Syndrome patients’ skin

Abstracts McMillan James1,2, Masashi Akiyama2, Kana Yasukawa2, Hideki Nakamura2, Maki Goto2, Daisuke Sawamura2, McGrath John3, Hiroshi Shimizu2 1

Creative Research Institute Sousei, Sapporo, Japan; 2Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan; 3Genetic Skin Disease Group, St John’s Institute of Dermatology, King’s College London, London, UK Kindler syndrome (KS; OMIM173650) is an autosomal recessive skin disorder associated with trauma-induced blisters and increased epithelial malignancy risk. Defects in the protein kindlin-1 encoded by the gene KIND1 (or C20ORF42) underlie this disease. We have undertaken a detailed immunohistochemical and immuno-ultrastructural study in skin biopsies and cultured keratinocytes from control (n = 4) and KS skin (n = 4) to examine the expression of kindlin-1 and kindlin-2 (MIG-2, a related member of the kindlin superfamily). Three novel polyclonal kindlin-1 and one kindlin-2 antisera were assessed. In normal human skin sections, kindlin-1 and -2 both showed diffuse peripheral expression that included dermal fibroblasts and the keratinocyte cytoplasm overlying the dermal-epidermal junction (DEJ). By immunogold electron microscopy, both kindlin-1 and kindlin-2 were diffusely localized to the periphery of the keratinocyte cytoplasm. However, in cultured keratinocytes kindlin-1 localized to distinct focal contacts (FC) whereas kindlin-2 localized to areas of the cell periphery at the cellsubstratum contact. In KS patient skin, there was absent or reduced kindlin-1 staining, which was most marked in the two KS patients that harbored previously identified KIND1 nonsense mutations. However, kindlin-2 expression in KS patients was similar to control skin. Kindlin-1 and -2 staining patterns highlight the distinct functions that these proteins play in cytoskeletal linkage. Kindlin-1 staining in KS patients’ skin suggests either a complex kindlin-1 isoform expression pattern or molecular heterogeneity in the underlying cause of this disease in KS patients. 203 ATX-S10(Na)-PDT shows more potent effect on collagen metabolism of human normal and scleroderma dermal fibroblasts than ALA-PDT Hidetoshi Takahashi1, Susumu Nakajima2, Isao Sakata3, Hajime Iizuka1 1

Department of Dermatology, Asahikawa Medical College, Asahikawa, Japan; 2Mori Memorial Hospital; 3Photochemical Co., Ltd Topical photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) is useful for sclerosis in scleroderma patients. Recent study revealed that PDT with a novel photosensitizer (ATX-S10(Na)), shows more potent effects for various skin diseases than ALA-PDT. The effect of ATX-S10(Na)-PDT on fibroblasts is still unknown. Using dermal fibroblasts derived from normal and scleroderma patients, and mouse skin in vivo, we compared the effects of ATX-S10(Na)-PDT and ALA-PDT on the production of metalloproteinases (MMPs), MMP inhibitors (tissue inhibitors of metalloproteinases: TIPM), and collagen synthesis. Fibroblasts from normal, scleroderma patients or mice skin were treated with ATX-S10(Na)-PDT or ALA-PDT. After the PDT treatments, the expression of MMPs, TIMPs, and collagen was assayed using ELISA and reverse transcription-PCR (RT-PCR). ATX-S10(Na)-PDT more potently increased the expression of MMP-1 and MMP-3 than ALA-PDT in protein and mRNA levels in both normal and scleroderma fibroblasts. Furthermore, similar potent induction of MMP-1 and MMP-3

Abstracts

191

was observed in both normal and scleroderma fibroblasts by PDT. The increased expression of MMP-1, MMP-3, and decreased expression of collagen I was also confirmed by mRNA levels in both normal and scleroderma fibroblasts. In mice skin the effect of PDT for MMPs and collagen 1 was also detected and the effect was more potent in ATX-S10(Na)-PDT. In contrast, MMP-2, TIMP-1, TIMP-2 and collagen 3 expression was not affected by the ATX-S10(Na)-PDT or ALA-PDT treatment. ATXS10(Na)-PDT is more potent modulator for dermal matrix components than ALA-PDT and might be useful for scleroderma patients. 204 UVA1 (340—400 NM) induces apoptosis in malignant B cells and plasma cells Keiko Kobayashi, Yoko Yasuda, Akimichi Morita Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan In Europe, extracorporeal photochemotherapy (ECP) has been used for the treatment of erythrodermic CTCL, etc. However, one concern in ECP is that 8-MOP is returned to the circulation of the patient, which may cause phototoxic effect, liver dysfunction and cataract. UVA1 radiation (340—400 nm) is effective for the treatment of T cell-mediated skin diseases including CTCL. In the previous study, we have shown that the induction of apoptosis in skin-infiltrating T-cells is the major mechanism of action operative in UVA-1 phototherapy. UVA1-ECP would prevent this adverse effect induced by ECP. In order to develop ECP, we investigated thether UVA1 radiation would induce apoptosis in malignant B cells and plasma cells as well as malignant T cells. Cells from different malignant pre B cell lines including BALL1and 697, mature B cell lines including Daudi, Radi, P3HR-1, KIS1, NOl-3, Ly8, SP49, SU-DHL-10, CB##, MEC-2, MO1043, WSU-CLL and SACHI were employed. For UVA-1 irradiation, UVA-1 Sellamed 2000 System Dr. Sellmaier (Sellas, Gevelsberg, Germany) irradiation device was used. Freshly isolated malignant B cells were irradiated in vitro with UVA1. Cells were harvested for 24 h and subjected to FACS analysis by using Annexin V method. Positive apoptosis was shown as 40—60%in pre B cell lines and 10—50% in mature B cell lines, 20—50% in plasma cell lines at the dose of 30 J/cm2 of UVA1. From these observations, UVA1 induces apoptosis in malignant B cells and plasma cells as well as malignant T cells, although the rates of apoptosis were variable each cell line. The further study should be required to identify the underlying mechanisms to determine the susceptibility towards UVA1 radiation. 205 TACE(ADAM17) activator, 4-aminophenylmercuric te(APMA) inhibits photoaging in mouse

aceta-

Mika Terao, Hiroyuki Murota, Ichiro Katayama Department of Dermatology, University of Osaka, Osaka, Japan Background: Photoaging of the skin is characterized by degeneration of dermal extracellular matrix, which commonly occurs to almost every organism. However, little is known about pathogenesis of this UV-induced degenerative change. Tumor necrosis factor (TNF)/TNF receptor (TNFR) system is known to control degradation of extracellular matrix. TNFRp55 signaling is assumed to play an important role in photoaging pathway because of the previous two findings: (1) TNF derived from UV irradiated keratinocyte and fibroblast induced expression of collagenase; (2)

UV activates downstream signaling via TNFR with ligand-independent manner. Objective: In this study, we focused on cell surface TNFRp55 as a target of anti-photoaging. TNF activating converting enzyme (TACE/ADAM17), which is a sheddase of TNFRp55, forms soluble TNFRp55 (sTNFRp55). As sTNFRp55 is known to neutralize the effect of TNF, we investigated whether activation of TACE can be a candidate of anti-photoaging agent. Methods: TACE is known to be activated by 4-aminophenylmercuric acetate (APMA) specifically. To evaluate the antiphotoaging effect of APMA, HaCaT and photoaging model mouse (SKH mouse exposed to UVB at a dose of 180mJ/cm2 every other day for 12 days) were treated with APMA and histopathological and molecular biological analysis were performed. Results: Application of APMA to cultured cell dose dependently increased concentration of sTNFRp55 of cultured medium and also suppressed phosphorylation of JNK pathway in vitro. Degradation of collagen fiber was suppressed in UV-irradiated SKH mouse treated with APMA topically compared with control mouse. Conclusion: Taken together, these results indicated that APMA might be one of a candidate of anti-photoaging agent. 206 Induction of keratinocyte apoptosis by photosensitizing chemicals plus UVA Masako Kurita1, Takatoshi Shimauchi2, Miwa Kobayashi2, Yoshiki Tokura2 1 Occupaional Health Training Center, University of Occupational and Environmental Health, Kitakyusyu, Japan; 2Department of Dermatology, University of Occupational and Environmental Health, Kitakyusyu, Japan

The capacity of photosensitizing chemicals plus ultraviolet A (UVA) to induce apoptosis is a hallmark to evaluate their phototoxic and photoallergic potentials. We examined the ability to induce keratinocyte apoptosis of various chemicals that are known as causative agents of photocontact dermatitis and drug photosensitivity. HaCaT keratinocytes were incubated with tetrachlorosalicylanilied (TCSA), bithionol, diphenhydramine, chlorpromazine, and 6-methylcoumarin at 107 to 104 M and irradiated with UVA at 4 J/cm2 Apoptosis and necrosis were assessed by enumeration of Annexin V+ 7-AAD and Annexin V+7-AAD+ cells, respectively, by flow cytometry. The expression of apoptosis-related molecules, caspase-3 and poly (ADP-ribose) polymerase (PARP), was tested by flow cytometric and Western blotting analyses. When compared to non-irradiated cells, UVA irradiation induced significant apoptosis in TCSA, bithionol, and chlorpromazine at 105 M, while necrosis occurred in these chemicals mostly at 104 M. Neither apoptosis nor necrosis was seen in diphenhydramine or 6-methylcoumarin. Caspase-3 and PARP were activated in HaCaT cells phototreated with TCSA or chalorpromazine. Given that apoptotic cells are easily presented by antigen-presenting cells, the ability of photosensitizers to induce apoptosis may indicate the photoallergic potential of the chemicals. We suggest that our method is useful for screening of not only phototoxicity but also photoallergenicity of chemicals. 207 Induction of eosinophil-infiltrating drug photoallergy in mice Daisuke Nishio, Kenji Kabashima, Yoshiki Tokura The Department of Dermatology, School of Medicine, University of Occupational and Environmental Health, Fukuoka, Japan

192

Abstracts

Drug photoallergy is one of the common adverse effects, which is clinicallyrecognized as photosensitivity dermatitis. We established a murine model of this hypersensitivity by administration of representative photosensitizing drug, afloqualone (AQ), in combination with UVA irradiation. AKR/J mice were sensitized with AQ (2 mg/kg/mouse) and subsequent irradiation of the shaved abdomen with UVA (12 J/cm2). This sensitization procedure was repeated 2, 4, 6, 8, 10 or 12 times (twice a week). Three days after the last immunization, they were challenged by a subcutaneous injection of AQ solution and irradiation of the same site with UVA (12 J/cm2). Mice receiving more than 10 times of sensitization exhibited a massive infiltrate of eosinophils and lymphocytes at the challenged site. Both afloqualone and UVA were required for this reaction, and the eosinophilic infiltrate was specific to AQ. AKR/J mice were high responders of this sensitivity, because BALB/c mice showed no substantial cell infiltrate. Transfer study was performed with immune lymph node cells (LNCs) and spleen cells from AKR/J mice sensitized with afloqualone plus UVA 10 times. The sensitivity was successfully transferred with 5—8  107 cells into naive mice. At least CD4+ T cells were responsible for this sensitivity, since 1  107 CD4+ cells alone induced a high level of sensitivity reaction. Culture supernatants from LNCs of AQ-photosensitized mice contained higher levels of IL-5 and IL-4 than those from naive mice. It is suggested that eosinophilic drug photoallergy is mediated by Th2 cells and this model is useful for exploring the mechanism of eosinophilinfiltrating photosensitivity.

nective tissue alteration. We hypothesized that connective tissue alteration followed by repeated mild inflammation may lead to photoaging, and thus studied the behavior of neutrophils, as inflammatory cells releasing and producing several proteinases, in the process of photoaging. Methods: The dorsal skin of 5-week-old female hairless mice (Hos: HR-1) was irradiated with ultraviolet B 3 times/week for 10 weeks, with the first week under the erythema dose at 50 mJ/ cm2/d, followed by 9 weeks at 100 mJ/cm2/d. Skins were collected at 6, 24 and 48 h after UVB exposure at the end of 5 and 10 weeks. Each skin sample was examined under light microscopy (LM) and electron microscopy (EM). Results and conclusions: Neutrophil infiltration was observed by LM and EM. Neutrophil infiltration in dermis was observed after facilitation of mast cell degranulation. Under EM, most neutrophils migrated around mast cells. This study confirmed neutrophil infiltration in skin repeatedly exposed to UVB. These data suggest that mast cell degranulation is involved in neutrophil migration in a photoaging model. As a whole, these results indicate that neutrophils and mast cells may play a role in cutaneous photoaging.

208

Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Localization of lysozyme in sun-exposed skin Eiji Yoshinaga1, Naoki Oiso1, Shigeru Kawara1, Akira Kawada1, Minoru Akiyama2, Shingo Tajima2 1 Department of Dermatology, Kinki University School of Medicine, Osaka, Japan; 2Department of Dermatology, National Defense Medical College, Saitama, Japan

Actinic keratosis (AK) is a precancerous skin tumor that develops predominantly on elderly sun-exposed skin such as face and dorsum of hand. In the dermis of lesions of AK solar elastosis is usually observed. Lysozyme is one of polysaccharide digestive enzymes which localizes in lysosome and it is confirmed to bind tropoelastin by Mecham et al. In this study we investigated the localization of lysozyme in extra-lesional portion of AK specimen taken from 16 patients aged 7 to 91 years immunohistochemically. Immunohistochemical staining revealed the deposition of lysozyme on the degenerated elastic component and infiltrating cells in the dermis. Furthermore, in some specimens lysozyme deposition was detected in upper dermis even when solar elastosis was not present. It is supposed that lysozyme may be a valuable marker for cutaneous photoaging more sensitive than solar elastosis. 209 Behavior of neutrophils and mast cells in a hairless mouse photoaging model Hirotaka Takeuchi1, Hiroshi Watanabe2 1 POLA Chemical Industries, INC, Yokohama, Japan; 2School of Nursing, Yamagata University Faculty of Medicine, Yamagata, Japan

Aims: Photoaging in UV-exposed skin reportedly results from decreases in skin elasticity and changes to connective tissue components such as collagen and elastic fibers. Elastase, reactive oxygen species and matrix metalloproteinase participate in con-

210 Narrow-band UVB radiation has the suppressive effects on delayed-type hypersensitivity and contact hypersensitivity Yoichi Shintani, Iwao Isomura, Akimichi Morita

Narrow-band UVB therapy is more often used to treat refractory skin diseases, e.g., psoriasis and atopic dermatitis than PUVA therapy. Action mechanisms responsible for the efficacy of narrow-band UVB are mostly thought to induce the apoptosis in pathogenetically relevant cells. However, PUVA therapy or narrow-band UVB therapy generally induces a relatively long remission period likely 4—6 months in patients with psoriasis. This could not be explained simply by the induction of apoptosis. Therefore, besides the induction of apoptosis, a role of regulatory T cells should be considered. Recently, the evidence that UVB radiation induces regulatory Tcells to suppress contact hypersensitivity has been reported. Therefore, we have asked whether narrow-band UVB, would suppress delayed-type hypersensitivity (DTH) and contact hypersensitivity (CHS) by inducing regulatory T cells. The induction of CHS to DNFB was suppressed by narrow-band UVB exposure in a dose-dependent manner in a mouse model. The significant suppression was observed from 1000 mJ/cm2 to 3000 mJ/cm2 at the p level of 0.05. The suppressive effect obtained with 1000 mJ/cm2 of narrowband UVB was almost similar to that obtained with 100 mJ/cm2 of broad-band UVB. The induction of DTH was similarly suppressed by narrow-band UVB (1000 mJ/cm2). The suppressive effects either of CHS or of DHT could be transferable to naı¨ve mice by transferring lymph node cells from the tolerant mice. These data indicate narrow-band UVB induces tolerance to CHS and DTH. For the further study, we are currently investigating the underlying mechanisms of the tolerance induce by narrow-band UVB. 211 Establishment of Rapid detection method of KSHV DNA by loopmediated isothermal amplification (LAMP) method Tomoe Kuhara1,2, Tetsushi Yoshikawa2, Daisuke Watanabe1, Masaru Ihira2, Yasuhiko Tamada1, Yoshizou Asano2, Yoshinari Matsumoto1

Abstracts 1

The Department of Dermatology, Aichi Medical University, Aichi, Japan; 2Department of Pediatrics, Fujita Health University, Aichi, Japan Background: Novel DNA amplification method termed loop mediated isothermal amplification (LAMP) has been recently developed. It has several advantages such as rapidity (complete the method about 30 min) and convenience (thermal cycler is not required) in comparison to conventional PCR method. Aims of this study are to establish a rapid Kaposi’s sarcoma-associated herpesvirus (KSHV) DNA detection system by using LAMP method. Methods: DNA extracted from KSHV infected cells (BCP-1 cells) was used to establish the procedure. Primers were designed to amplify KSHV minor capsid protein gene. In order to confirm the specificity of the method, DNAs of human herpes simplex viruses (HHV-1, 2), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpes virus 6A, 6B (HHV-6A, 6B), human herpes virus 7 (HHV-7) were amplified. PCR product including target sequences was cloned into pGEM-T vector. The plasmid was used to determine the sensitivity of the method. Results: Although KSHV DNA was amplified in the DNA extracted from KSHV infected cells, no amplified product was detected in DNAs extracted from other herpesviruses infected cells. Sensitivity of the method was 100 copies/reaction. Conclusion: LAMP method for detection of KSHV DNA was established.

193 Fumiaki Nakayama, Akiko Kawana, Makoto Akashi The Department of Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba, Japan Heparan sulfate proteoglycan (HSPG) is one of the increased extracellular matrix components in the radiation-induced fibrotic tissues. It plays an important role in the local concentration of several growth factors with heparin-binding properties. However, little is known about how the expression of HSPG is regulated in the impaired tissues. In this study, we succeeded in inducing the increase of HSPG expression in the a-SMA positive myofibroblasts by culturing the in vitro skin model after X-ray irradiation and showed that the anti-oxidative enzymes such as catalase also increased in this skin model. Transfection of the Catalase gene into keratinocyte cell line, HaCaTcells, resulted in the increase of HSPG expression, whereas vector-based small interfering RNAs targeted against Catalase gene downregulated the expression of HSPG. The transcript levels of 11 glycosyltransferases were determined in irradiated skin model and catalase transfectants. The level of catalase expression was most correlated with the amount of EXTL2, enabling to initiate heparan sulfate synthesis. Exogenous catalase induced the expression of HSPG in artificial skin. These findings suggest that catalase may control the level of HSPG expression in radiation-induced skin fibrosis and H2O2 may also be involved in HSPG expression.

212 214 The subsets of cytotoxic T lymphocytes infiltrating in herpetic vesicles: Comparison with superficial bacterial infections Shin Morizane, Daisuke Suzuki, Kazuhide Tsuji, Takashi Oono, Keiji Iwatsuki Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Herpes simplex (HS), herpes zoster (HZ), varicella and hydroa vacciniforme (HV) are characterized by umbilicated vesicle formation. In this study, we investigated immunohistochemically to confirm common immune responses in herpetic vesicles and compare them with other skin disorders. Phenotyping of infiltrating cells was carried out in biopsy specimens from HS, HZ, varicella and HV, in comparison with nonviral contact dermatitis and bacterial folliculitis. Viral antigens and EBV-encoded small nuclear RNA (EBER) were detected by immunostaining and by in situ hybridization, respectively. Infiltrating CTLs expressing granzyme B and granulysin were determined by double immunostaining using a confocal laser scanning microscope. In all herpetic vesicles, the corresponding viral antigens were observed. In all HV lesions studied, EBER+ T cells were detected in 5—10% of the dermal infiltrates composed mostly of T cells without CD56 expression. CTLs expressing granzyme B and granulysin were present in both herpetic and HV lesions, ranging from 10 to 25% of dermal infiltrates, whereas biopsy specimens from nonviral contact dermatitis contained less than 1%. In biopsy specimens from folliculitis, granulysin-producing cells were observed. Furthermore, confocal laser microscopic examination demonstrated that both CD4+ and CD8+ T cells expressed granzyme B and granulysin in herpetic and HV lesion. In biopsy specimens from folliculitis, CD4+ T cells produced granulysin. The subsets of cytotoxic T lymphocytes infiltrating in herpetic vesicles are different from them of superficial bacterial infections. It seems to show the difference of immune response between viral and bacterial infection. 213 The regulation mechanism of heparan sulfate proteoglycans in the radiation-induced cutaneous injuries

Type I and type III collagen gene expression are enhanced in cultured dermal fibroblasts by the new erythromycin analog EM201 Hiromi Suzuki1, Toshiaki Sunazuka2, Satoshi Omura2, Yohei Kitamura1, Takahiro Yoshida1, Hideyuki Ikeda1, Soji Yamazaki1, Atsushi Hatamochi1 1

Department of Dermatology, Dokkyo University School of Medicine, Tochigi, Japan; 2Kitazato Institute, Kitazato University, Tokyo, Japan

Thin of dermis is a principal histological change of skin atrophic disorders or aged skin. It is reported that amount of collagen in the skin is decreased during aging. It is also known that productions of type I and type III collagen are decreased and collagenase expression is increased in in vitro and in vivo aged fibroblasts. Macrolides have been reported to show various pharmacological activities in addition to antimicrobial activity including anti inflammatory activity, inhibition of tumor angiogenesis, inhibition of the expression of vascular endothelial growth factor (VEGF), immuno-modulation of keratinocytes and inhibition of the in vitro proliferation of malignant tumor cells, etc. We would like to present effects of the new erythromycin analog EM201 on collagen I and III production in cultured dermal fibroblasts. Methods: Dermal fibroblasts were cultured with routine methods. When the cell layers had become confluent, the cells were incubated for 48 h at 37 in DMEM containing 0.2 FBS, supplemented with from 109M to 105 M of EM201. Amounts of collagen were measured using ELISA for type I collagen. Total RNA was isolated from cultured fibroblasts by extraction in guanidium isothiocyanate then Northern blots analysis were performed using 32P labeled cDNA of a1(I) collagen, a1(III) collagen and GAPDH. Results: EM201 enhanced collagen production and mRNA levels of a1(I) collagen (to 200% in maximum) and a1(III) collagen (to 170% in maximum) in a dose dependent manner in cultured dermal fibroblasts. These results suggest that EM201 has potential to improve the thin of dermis of skin atrophic disorders or aged skin in future.

194

Abstracts

215

A3G756 peptide for skin wound healing. First, we show that SB203580, the selective inhibitor of p38 mitogen-activated kinase (p38MAPK), inhibited kereatinocyte migration induced by A3G756. Next, inhibitory studies using functionally blocking antibodies against integrin subunits show that incubation with the anti-a5 and -b1 integrin antibodies significantly decreased the A3G756-induced cell migration. Furthermore, microspheres covered with A3G756 bound to syndecan-4, could cluster b1 integrin and vinculin. These results suggested that cell stimulated by A3G756 may migrate on fibronectin produced by keratinocytes. For in vivo study, cutaneous wound was made with mouse. Topical application of A3G756 to cutaneous wound accelerated reepithelialization. The A3G756-induced wound repair was blocked by treatment with SB203580. Our studies indicated that A3G756induced early closure of the cutaneous wound required p38MAPK activation. By those data, it is indicated that the A3G756 peptide is likely a potential therapeutic reagent for chronic cutaneous ulcer.

Puva enhance gene expression of collagenase and inhibits gene expression of type I and type III collagen in fibroblasts in vitro Youhei Kitamura, Hiromi Suzuki, Takahiro Yoshida, Hideyuki Ikeda, Mitio Hashikabe, Youichiro Hamasaki, Soji Yamazaki, Atsushi Hatamochi Department of Dermatology, Dokkyo University School of Medicine, Tochigi, Japan UVA irradiation is known to induces collagenase in human dermal fibroblasts in vitro and in vivo. Recent studies have demonstrated that PUVA therapy is effective to sclerotic skin of scleroderma. We investigated that effects of UVA irradiation on gene expression of collagenase, typeI collagen and type III collagen in cultured normal and scleroderma fibroblasts under the presence of 8-methoxypsoralen. Methods: Dermal fibroblasts were cultured with routine methods. When the cell layers had become confluent, the cells were incubated for 24 h at 37 8C in DMEM containing 10% FBS, supplemented with 4  106 M 8-methoxypsoralen. Subsequently cells were irradiated with from 0.3 to 4.8 J/cm2 of UVA. Amounts of collagen were measured using ELISA for typeI collagen. Total RNA was isolated from cultured fibroblasts by extraction in guanidium isothiocyanate then Northern blots analysis were performed using 32P labeled cDNA of a1(I) collagen, a1(III) collagen and GAPDH. Results: UVA irradiation reduced collagen production and mRNA levels of a1(I) collagen (to 9%in maximum) and a1(III) collagen (to 12%in maximum), and enhanced gene expression of collagenase (to 2840% in maximum) in a dose dependent manner in normal and SSc fibroblasts under the presence of 8methoxypsoralen in vitro. These results suggest that direct effect of UVA irradiation under the presence of 8-methoxypsoralen to fibroblasts of collagen and collagenase expression is one of the mechanism for effectiveness to sclerotic skin of scleroderma by PUVA therapy. 216 Promotion of skin wound healing by laminin-5, the basement membrane protein Yutaka Momota1, Yoshitoshi Kasuya2, Fumiharu Yokoyama3, Eri Araki4, Takeshi Togou5, Mitsuko Furuya6, Hiroshi Shinkai7, Toshiroh Iwasaki8, Atsushi Utani 4 1 Department of Veterinary Internal Medicine, Faculty of Agriculture, Iwate University, Morioka, Japan; 2Department of Biochemistry and Molecular Pharmacology, Graduate School of Medicine, Chiba University, Chiba, Japan; 3School of pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan; 4 Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 5Department of Plastic and Reconstructive Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 6Department of Molecular Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan; 7 Department of Dermatology, Graduate School of Medicine, Chiba University, Chiba, Japan; 8Department of Veterinary Internal Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan

Deposition of precursor laminin-5 over the wound surface is early event necessary for keratinocyte migration in reepithelialization. We reported that the laminin-5 a3 chain C-terminal globular module 4 (a3 LG4) and the synthetic peptide A3G756 (KNSFMALYLSKGRLVFALG) were shown to stimulate keratinocyte migration. In the present study, we demonstrate the usefulness of the

217 Extracellular matrix related genes regulation by phospholipase D signaling in human dermal fibroblasts Masayoshi Yamanaka, Osamu Ishikawa Department of Dermatology, Gunma University Graduate School of Medicine, Gunma, Japan Phospholipase D (PLD) hydrolyses phosphatidylcholine to phosphatidic acid (PA) and choline. PA is the most critical metabolite generated by PLD, and can be converted to diacylglycerol (DAG) or lyso-PA, both of which have a second messenger function that may contribute to the effects of PLD Although several authors report a role of PLD in cell proliferation and cancer, a role for PLD in extracellular matrix (ECM) metabolism has been rarely investigated. Recently, Yamamoto et al. [1] reported that PLD signaling was involved in ventricular fibrosis and N-methylethanolamine (MEA, inhibitor of PLD activation) prevented the fibrosis in a model rat. Our hypothesis is that PLD signaling is also involved in fibrotic skin diseases such as keloid and scleroderma, and MEA may have therapeutic effects on skin fibrosis. To determine how influence MEA on ECM accumulation in human fibroblasts, we investigated the effects of MEA on mRNA levels of MMP-1 and alpha 2 (I) collagen in normal fibroblasts by real time PCR. MEA treatment markedly up-regulated MMP-1 gene expression after 24 h and down-regulated alpha 2 (I) collagen gene expression after 48 h. Together, these observations suggest that MEA inhibits ECM deposition. We compared the mRNA levels of PLD1 and PLD2 among normal, scleroderma and keloid fibroblasts by real time PCR. Unexpectedly, the mRNA levels of PLD1 and PLD2 was decreased in scleroderma and keloid fibroblasts. Reference [1] Yamamoto, et al., European Heart Journal 25, 2004.

218 Tenascin expression in human skin after chemical peeling used trichloroacetic acids and phenol Yuki Yamamoto, Koji Uede, Fukumi Furukawa Department of Dermatology, Wakayama Medical University, Wakayama, Japan Tenascin (TN) is a large extracellular matrix glycoprotein. In normal human skin, TN is present in the papillary dermis and

Abstracts near the basement membranes surrounding blood vessels and epidermal adnexa, and is strongly upregulated in hyperproliferative skin disease such as psoriasis, epidermal tumors, excisional wounds and the ulcers. Many factors have been described to modulate TN expression such as IL-4, TNFa and IFNg. To better understand the wound healing mechanisms of chemical peeling, we investigated the human skin treated with phenol or trichloroacetic acid (TCA). Forty percent, 60% TCA and phenol were applied once on the inside of the upper arm of 3 healthy Japanese adults, and 4 mm biopsy specimens were obtained at 2, 6, and 12 h, and at 1, 2 and 7 days. Monoclonal antitenascin MAB1927 (Chemi-Con, CA, U.S.A.) were used. Expression patterns of TN in the skin on day 1, was strongly upregulated into the reticular dermis in the 60%, 40% TCA and phenol-treated skins. Seven days after peeling, in the 40% TCA-treated skins TN expression is seen into the whole epidermis and the whole dermis. But in the 60% TCA and phenol treated skins it seen only the whole dermis. TN is upregulated in wound heeling process induced by phenol or TCA which is now used for chemical peeling. And we conclude that keratinocyte-derived factors may regulate dermal tenascin expression during wound healing.

195 Department of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan

Deletion analysis of humana 1 (I) collagen gene 50 flanking region for the transcriptional activity using human dermal fibroblasts

Although the pathogenesis of systemic sclerosis is unknown, a fascinating hypothesis called a 2-step process of fibrosis has been proposed. The key cytokines are transforming growth factor-b (TGF-b), which stimulates fibroblasts first, and connective tissue growth factor (CTGF), which acts to maintain tissue fibrosis. Following this hypothesis, a new therapeutic strategy focusing not TGF-b but CTGF may be promising choice for the treatment of systemic sclerosis. In the present study, we tried silencing of CTGF production by human dermal fibroblasts using RNAi method. We designed two different siRNA for CTGF-specific oligonucleotides. We transfected a couple of siRNA with Lipofectamine 2000 (Invitrogen Technologies (Carlsbad, CA)) then cultured for 72 and 144 h. Immunoblotting study confirmed that cells transfected with one siRNA with CTGF-specific oligonucleotides markedly reduced of production of CTGF in human fibroblasts after 144 h. Then, further experiments are being carried to test the effects of CTGF silencing on the connective tissue metabolism of human dermal fibroblasts. We measured the production of type I collagen, matrix metalloproteinase (MMP)-1 and tissue inhibitor of metalloproteinase (TIMP)-1. Although we faces a lot of problem such as the delivery system as well as medical ethics, this preliminary study suggests the possibility that the therapeutic use of RNAi is promising for the treatment of systemic sclerosis in the future.

Takahiro Yoshida, Hideyuki Ikeda, Hiromi Suzuki, Youhei Kitamura, Soji Yamazaki, Atsushi Hatamochi

221

Department of Dermatology, Dokkyo University School of Medicine

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

The regulation of type I collagen gene expression is one of the important role in formation of fibrosis or skin atrophy. However, Deletion analysis of humana1(I) collagen gene (COL1A1) 50 flanking region for the transcription level of humanCOL1A1 is not satisfactory performed. We present deletion analysis of COL1A1 50 flanking region for the transcriptional activity with luciferase assay using chimeric genes with various deletion constructs of the human COL1A1 50 flanking region fused to the luciferase gene. Methods: Human dermal fibroblasts were cultured with routine methods. Cells were transfected employing the FuGene 6 transfection reagent (Roche Applied Science). Fibroblasts were plated and cultured to 50% confluence in 60-mm dishes and then transfected with 1.0 mg of COL1A1 deletion constracts of 2, 300 bp, 804 bp, 610 bp, 332 bp, 165 bp and 133 bp fused to the luciferase gene and with 20 ng of CMV renilla luciferase constract (Promega). After 48 h, cells were harvested and lysates were prepared using passive lysis buffer (Promega). luciferase activity in equal aliquots was determined by Dual Luciferase Reporter Assay System (Promega). Results: Relative luciferase activity were not so much changed when DNA containing 804 bp or 610bp of COL1A1 were transfected, whereas when DNA containing 332 bp of COL1A1 were transfected, relative luciferase activity were remarkably reduced. These results suggest that there are cis-DNA elements which activate transcription of COL1A1 between 310 and 610 of the COL1A1 5, flanking region. Now the work is process of more precise deletion analysis of that region.

Norihoro Fujimoto1, Joseph Terlizzi 2, Raymond Brittingham2, Andrzej Fertala2, Jouni Uitto2

219

1 The Department of Dermatology, National Defense Medical College, Saitama, Japan; 2Thomas Jefferson University, Philadelphia, USA

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellularmatrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin1D, measured by biosensor assay, revealed a Kd of 5.71  108 M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.

220 Attempt to silence of CTGF production by human dermal fibroblasts using rnai method Hirohisa Ishibuchi, Masatoshi Abe, Yoko Sogabe, Yoko Yokoyama, Osamu Ishikawa

222 SLRPs are abundantly expressed by palmoplantar fibroblasts and may contribute to the body site-specific morphogenesis of collagen bundles

196 Masahito Yasuda1, Yoshiki Miyachi2, Kenzo Takahashi2, Osamu Ishikawa1 1

Departments of Dermatology, Gunma University Graduate School of Medicine, Maebashi, Japan; 2Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto, Japan Human skin shows various morphological characteristics and acquires unique physiological properties depending on the body-sites. The distinct skin phenotypes of various body sites have been essentially explained from the variance of epidermal keratinocytes and the presence of skin appendages, however, the contribution of dermal fibroblasts to the skin variance has been poorly understood. We previously demonstrated that trunk fibroblasts expressed fibronectin more dominantly than palmoplantar or oral mucosa fibroblasts. Consequently, we tried to identify clones that are abundantly or specifically expressed by palmoplantar fibroblasts. We identified three cDNA clones encoding decorin, chemokine receptor 11 and tenascin X by a subtraction cDNA screening. Decorin and tenascin X act on the bundling of collagen and elastin fibrils. Electron microscopic examination of dermal collagen bundles of rat skin revealed that the diameter of collagen fibrils (73  14 nm) in paw skin is significantly thinner and more scattering than those in back skin (80  13 nm). Palmoplantar fibroblasts also expressed other SLRPs, such as biglycan and lumican, more abundantly than trunk skin fibroblasts. On the other hand, thrombspondin 2 and dermatopontin were expressed more massively by trunk fibroblasts. Spatial transcriptional regulation of extracellular matrix proteins associated with collagen fibrils should play a key role in the morphogenesis variety of collagen fibrils at various body sites.

223 Automatic three-dimensional analysis of human facial skin in vivo by swept source optical coherence tomography Shingo Sakai1, Yasuaki Hori2, Masayuki Matsumoto3, Tomoko Sugawara1, Yoshiaki Yasuno2, Shuichi Makita2, Masahiro Yamanari2, Violeta Dimitrova Madjarova2, Masahide Itoh2, Toyohiko Yatagai2 1

Basic Research Laboratory, Kanebo Cosmetics Inc.; 2Computational Optics Group, University of Tsukuba; 3Cosmetics Laboratory, Kanebo Cosmetics Inc. Dermatologists have yet to answer many important questions about the relationships between surface morphologies and the internal structures of the skin. Time-domain optical coherence tomography (TD-OCT) has so far been useful for non-invasive histological observations of these relationships. However, the considerable time required to take measurements by threedimensional scanning prevents investigators from feasibly applying TD-OCT for three-dimensional morphological analyses. As a possible solution, several groups have proposed the application of swept-source OCT (SS-OCT), a Fourier domain OCT which can remarkably shorten measurement times by detecting the wavelength resolutions of backscattering light. In this study we threedimensionally segmented and measured the facial skin automatically in vivo. The forehead areas (4 mm  4 mm) of five males were scanned by a 1.3 mm SS-OCT in 2 s. Sequential image fields of 1024 (A-scan)  200 (B-scan)  200(C-scan) pixels were reconstructed three-dimensionally. We applied an algorithm to extract characteristic parameters and morphological structures of infundibula based on volume. Our algorithm provided parameters such as the mean epidermal thickness, infundibulum density, and dermal attenuation coefficient. The values obtained were almost

Abstracts the same as those reported in earlier studies. Better still, the algorithm segmented the epidermal layer and infundibulum of the sebaceous follicle three-dimensionally, thereby allowing us to examine the relationship between the surface morphology and distribution of the infundibulum. SS-OCT will be practically suitable for three-dimensional analyses of the skin. This method will serve important purposes not only in dermatology, but also cosmetic science and surgery. 224 An immunohistochemical study of cytokeratin expression in nevus sebaceous Ichiro Kurokawa1, Keisuke Nishimura1, Arata Hakamada1, Ken-ichi Isoda1, Kei-ichi Yamanaka1, Hitoshi Mizutani1, Airo Tsubura2 1

Department of Dermatology, Mie Graduate University School of Medicine, Tsu, Japan; 2Department of Pathology, Kansai Medical University, Moriguchi, Japan Background: Cytokeratin (CK) expression in nevus sebaceous (NS) remains unclear according to the stage. Objectives: To elucidate CK expression in NS, an immunohistochemical study was performed using 10 anti-keratin antibodies. Materials & methods: Anti-keratin antibodies used were 34betaB4 (CK1), LP5K (CK7), LP3K (CK8), LHP1 (CK10), LL002 (CK14), LHK15 (CK15), LL025 (CK16), E3 (CK17), 5D3 (CK18) and b170 (CK19). Ten cases of NS were studied. The age of the patients ranged 3 to 61 year old. There were five males and five females. There were five cases of secondary tumors (basal cell carcinoman: n = 5, syringocystadenoma papilliferum: n = 1, sebaceous epithelioma: n = 1) Our immunohistochemical study used the labeled streptoavidin-biotin (LSAB) method. Results: In the first stage of NS, CK 14 was expressed not only basal cells but parabasal cells in the epidermis, it was expressed in the whole layers in the infundibulum. In the second stage of NS, raised CK 14 expression was found, and declined CK1 and 10 expressions were found. Primary hair germ structure was positive for CK14. In the third stage of NS, CK 14, 17 and 19 expressions were detected in secondary tumors. No differences in CK expression were observed in eccrine sweat glands. Conclusions: CK 14 expression was increased, and CK 1 and 10 expressions were decreased in NS, and CK 14, 17 and 19 were expressed in secondary tumors. These results suggested that these keratin disorders are related to the histogenesis of NS and secondary tumors. 225 Primary cutaneous large B-cell lymphomas: Clinicopathologic analysis in a large series of patients Kazuo Kodama1, Cesare Massone2, Andreas Chott3, Dieter Metze4, Lorenzo Cerroni2, Helmut Kerl2 1

Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 2Department of Dermatology, Medical University of Graz, Graz, Austria; 3Departments of Dermatology, University of Mu ¨ nster, Germany; 4Department of Pathology, Medical University of Vienna, Austria Subject: In the new WHO/EORTC classification of cutaneous lymphomas, primary cutaneous large B-cell lymphomas (PCLBCLs) are divided into 3 groups: ‘‘leg-type’’; follicle center lymphoma, diffuse type (‘‘FCL type’’); ‘‘other’’. However, at present there are no clinicopathologic data supporting the inclusions of PCLBCLs into the 3 aforementioned groups. In addition, prognostic factors for these patients are largely unknown.

Abstracts Method: We studied a large number of PCLBCLs in order to test the validity of the classification, and to identify prognostic factors for these patients. Ninety-three cases of PCLBCL were analyzed for clinicopathologic features and expression of several markers. Result: Patients were classified into the following categories: ‘‘FCL type’’: 44 cases; ‘‘leg-type’’: 40 cases. ‘‘other’’: 9 cases. Statistical analyses showed that the groups of ‘‘leg-type’’ and ‘‘FCL type’’ were clearly distinct in terms of clinicopathologic features and survival. As compare with ‘‘FCL type’’, the cases of ‘‘leg-type’’ statistically revealed that the age distribution was higher, the location on the leg was predominant, expression of MUM-1 and FOX-P1 were higher, and the prognosis was worse. Six prognostic factors were identified as follows; location on the leg, age > 70, round cell morphology, Bcl-2 expression, MUM-1 expression, and FOX-P1 expression. The group of ‘‘other’’ had features in-between those of ‘‘leg-type’’ and ‘‘FCL type’’. Conclusion: Our study shows that accurate morphologic and phenotypic analyses allow to stratify most patients into the prognostically different categories of ‘‘leg-type’’ and ‘‘FCL type’’. The definition of a third category of ‘‘other’’ requires further studies to clarify whether these cases show indeed distinct clinicopathologic features. 226 Immunohistiochemical study of mid-dermal elastolysis with precedent erythematous changes Takane Suda1, Hiroyuki Hara1, Mari Yoshitake1, Tadashi Terui1, Tetsuya Obayashi2, Tomoyuki Nakamura2 1 Department of Dermatology, Nihon University School of Medicine, Tokyo, Japan; 2Department of Medical Chemistry, Graduate School of Medicine, and Center for Molecular Biology and Genetics, Kyoto University, Kyoto, Japan

Background: Elastic fibers are essential extracellular matrix macromolecules comprising elastin cores and its surrounding fibrillin-rich microfibrils. They endow critical properties of elasticity and resilience in the skin. Fibulin-5, one of microfibrils, has been recently found as one of secreted extracellular matrix proteins that function as a scaffold for newly-synthesized elastic fibers. However, the spacial and temporal localization of fibulin-5 in human skin is not known fully. Objective: We report a 43-year-old Japanese woman presenting with round erythema and subsequent wrinklings that met the clinico-pathological criteria for mid-dermal elastolysis. Methods: The distribution of elastin, CD68, MMP-9 and fibulin5 was investigated immunohistochemically. Skin samples were obtained from both erythematous and wrinkled lesions. Results: Immunohistochemical study revealed numerous CD68+ and MMP-9+ histiocytes and giant cells partly in the erythema. The elastin immunoreactivity disappeared from the mid dermis adjacent to the granulomatous area. Faint fibrillar reactivity of fibulin-5 was found in the deep dermis, colocalized with the elastic fiber components. In the wrinkled skin, there were few CD68+ cells. Elastin immunoreactivity disappeared from the mid dermis. Fibulin-5 colocalized with the elastic fiber components in the deep dermis, which were not fibrillar, but rather short and fragmented. Conclusion: Mid-dermal elastolysis may be initiated by MMP-9 produced by histiocytes and giant cells through its degradation of elastic fibers. In the deep dermis of the wrinkled skin, the fragmented expression of fibulin-5 was associated with the incomplete reproduction of the elastic fibers.

197 227 Attenuated ubiquitination in the molluscum bodies of agminated mollusca contagiosa in a patient with malignant lymphoma Toshio Demitsu 1, Kozo Yoneda2, Naoka Umemoto1, Maki Kakurai1, Junji Nishida3, Chieko Sadahira2, Yasuo Kubota2 1 Department of Dermatology, Jichi Medical School Omiya Medical Center, Saitama, Japan; 2Department of Dermatology, Kagawa University Faculty of Medicine, Kagawa, Japan; 3Department of Hematology, Jichi Medical School Omiya Medical Center, Saitama, Japan

We describe a case of agminated mollusca contagiosa in a patient with underlying malignant lymphoma and show the results of the ubiquitination of molluscum bodies by an immunohistochemical method. A 41-year-old woman visited us with multiple papules on the face and neck. She had also noted remittent fever and cervical node swelling. On physical examination, disseminated, umbilicated papules were found on the face, neck, and trunk. Histology of a papule revealed intracytoplasmic inclusion bodies in the hypertrophic epidermis. Histopathology of her cervical lymph node confirmed the diagnosis of composite lymphoma of Lennert’s lymphoma and Hodgkin’s disease. Recently, we have reported that ubiquitination is involved in the elimination of molluscum bodies. To investigate the ubiquitination of the recalcitrant molluscum contagiosum in the immunocompromised host, we have examined the distribution of ubiquitin in the molluscum bodies in the patient using immunohistochemical stainings. As a result, we found attenuated ubiquitination in molluscum bodies in the present case. In contrast, we observed an increased expression of ubiquitin in molluscum bodies from several healthy infants. The ubiquitinproteasome protein degradation pathway is comprised of ubiquitin, a three-enzyme ubiquitination complex, the intracellular protein ubiquitination targets, and the proteasome that is the organelle of protein degradation. The gene encoding ubiquitin ligase is mutated in some breast and ovarian cancer cell lines. Similarly, attenuated ubiquitination of molluscum bodies might be due to a decreased activity of ubiquitin ligase in our case. Attenuated ubiquitination of molluscum bodies might be involved in the development of persistent, widespread, and recalcitrant mollusca contagiosa.

228 Two cases of drug-induced papuloerythroderma: Confirmation by in vitro T cell responses and mediation by Th2 cells Kazunari Sugita, Daiki Nakashima, Ryutaro Yoshiki, Chizuko Koga, Kenji Kabashima, Yoshiki Tokura Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan The etiology of papuloerythroderma is unknown in the vast majority of patient, although the association with cutaneous lymphoma and internal malignancies have been suggested. We describe the first two cases of drug-induced papuloerythroderma, focusing on the reactive T cells. Case 1: A 70-year-old man had a cerebral infarction and had been treated with aspirin for 10 years. Clinical examination revealed widespread eythroderma with coalescent solid papules. Lymphocyte stimulation test (SI; 4.48 normal, less than 1.8) and an oral challenge test were positive with aspirin. Twenty-four hours after the oral provocation test, the patient had a higher percentage of CCR4+CD4+ Th2 cells than CXCR3+ CD4+ Th1 cells. Aspirin was discontinued and his eruption

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Abstracts

was improved. After clinical improvement, the percentage of Th2 cells was decreased. Case 2: A 79-year-old man had been treated for cardiac dysfunction. Four months after additional administration of furosemide, his eruption was remarkably deteriorated. Clinical examination revealed widespread eythroderma with coalescent solid papules predominantly on the trunk and extremities. An oral challenge test and lymphocyte stimulation test (SI; 9.08 normal, less than 1.8) were positive with furosemide. Forty-eight hours after the oral provocation test, the patient had a higher percentage of CCR4+CD4+ Th2 cells than CXCR3+ CD4+ Th1 cells. Furosemide was discontinued and he eruption was improved. Our cases clearly demonstrated that aspirin or furosemide can cause papuloerythroderma, a usually chronic eruption. It is suggested that papuloerythroderma occurs as a drug eruption and presumably drug-reactive Th2 cells play an essential role in the pathogenesis of this characteristic disorder.

cells and Vb5+, Vb9+, Vb+13, and Vb17+CD8+T cell in the peripheral blood, which indicated SEB-mediated T cell activation. Stimulation of PBMC in the recovery phase with SEB and lipopolysaccharide (LPS) induced a synergy of TNFa, IFNg and IL-6 productions, comparing to stimulation with SEB alone or LPS alone. Toxic shock syndrome is a multisystem disorder caused by superantigens such as toxic shock syndrome toxin-1 and enterotoxins, secreted from Staphylococcus aureus. Although the present case did not fulfill the clinical criteria for toxic shock syndrome, a synergistic action of SEB and LPS might cause the severe septic shock.

229

Yusuke Asano, Yoko Kano, Tetsuo Shiohara

A synergistic action of enterotoxin B and lipopolysaccharide in a case of toxic shock syndrome

The Department of Dermatology, Kyorin university, Tokyo, Japan

Osamu Yamasaki1, Kazuhide Tsuji1, Norihiro Suzuki1, Daisuke Suzuki1, Keiji Iwatsuki1, Narushi Sugiyama2, Wataru Danjou3 1

Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Department of Plastic and Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 3Department of Emergency Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayana, Japan An 80-year-old woman was admitted to the high care unit because of a mixed-depth, flame burn of 20% body surface areas. Despite twice excisions and grafting, necrotic ulcers with methicillinresistant Staphylococcus aureus (MRSA) colonization remained. On the 7th day after operation, she developed a shock with high fever and oliguria. A few days later, an erythematous rash was evident in the trunk and legs. Blood examination revealed evidence of disseminated intravascular coagulation and liver and renal dysfunctions. A blood culture revealed MRSA and Klebsiella pneumoniae. A serum endotoxin level was slightly increased. The septic symptoms were delayed, but the condition gradually improved by extensive treatments with antibiotics and other medications. Membranous desquamation of skin was seen on the trunk, soles and palms. PCR assays for toxin detection proved positive for staphylococcal enterotoxin B. T cell receptor analysis demonstrated a marked accumulation of Vb14+ and Vb20+CD4+T

230 CMV ulcers mimicking Degos’ disease in drug-induced hypersensitivity syndrome as another manifestation of immune reconstitution syndrome

Drug-induced hypersensitivity syndrome (DIHS) is a severe drug eruption associated with reactivation of human herpesvirus-6 (HHV-6) However, some questions remain unsolved why paradoxical worsening of clinical symptoms occurs after withdrawal of the causative drug; and why no virus genome is detected at onset. In view of common immunosuppressive properties of the causative drugs, clinical symptoms of DIHS might be mediated by restored immune responses rapidly occurring on withdrawal of the causative drug: indeed, clinical manifestations of DIHS are similar to those of immune reconstitution syndrome (IRS) developing in HIVpositive patients, in which recovery of immune responses against previously unrecognized viruses reduces viral load on the one hand but cause tissue damage at sites of subclinical infection on the other. We present such a unique case of 74-year-old man with DIHS who developed cutaneous cytomegalovirus (CMV) ulcers mimicking Degos’ disease shortly after reduction of oral prednisone for the treatment of DIHS, while this therapy appeared to be efficacious in ameliorately dramatic paradoxical worsening observed after withdrawal of the causative drug. Importantly, multiple CMV ulcers involved the trunk, where CMV ulcers rarely occur. The CMV infection in the ulcers was confirmed by immunohistochemistry and PCR. The patient subsequently developed fatal CMV enterocolitis and died of respiratory failure 7 weeks after onset of the CMV ulcers. The CMV disease that had been dormant before onset of DIHS appeared to have been reactivated upon withdrawal of the causative drug followed by rapid reduction of oral prednisone. We propose that CMV ulceration involving the trunk in patients with DIHS is another manifestation of IRS.