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The Japanese Dermatological Association established an advisory committee in 1995 to develop a severity scoring system for atopic dermatitis (AD). Its interim and concluding reports were published in Japanese in the Japanese Journal of Dermatology (: 1491-1496, 1998 and : 2023-2033, 2001). Because of the strong demand for an English version, we have decided to publish the reports in English. This manuscript is the English version of the concluding report. The interim report suggested that eruption components such as erythema, papule, erosion, crust, excoriation and lichenification with extent of involved areas in five body regions, including the head and neck, anterior and posterior trunks, and upper and lower limbs, were important items for assessing AD severity. Additionally, it was recommended that streamlining of eruption components was mandatory for improving the statistical validity and reliability. The committee members subsequently concentrated their efforts on this task, and finally proposed an Atopic Dermatitis Severity Classification Criteria of the Japanese Dermatological Association. [ABSTRACT FROM AUTHOR]

Skin hyperpigmentation, and the reactions that precipitate it, have been linked to free radicals by the fact that free radical scavengers or antioxidants can slow that hyperpigmentation. We have screened several hundred plant extracts for antioxidants and discovered one that is both a strong antioxidant and can reduce skin hyperpigmentation. Extracts of Dianella ensifolia contain 1-(2,4-dihydrophenyl)-3-(2,4-dimethoxy-3-methylphenyl) propane (DP), which was found to inhibit the free radical 1-1-diphenyl-2-picryl-hydrazyl (DPPH) with an EC50 value of 78 μm. DP was also found to inhibit Ultraviolet (UV)C-induced lipid oxidation with an EC50 of about 30 μm. We next investigated the effects of this antioxidant on skin hyperpigmentation. The reduction of discoloration by different topical treatments has been assessed in human volunteers using an in vivo assay for the rate of fading of UVB-induced tan. Two pharmaceutical formulas containing 4% hydroquinone (HQ) were used as positive controls, and we tested the ability of DP, a plant-derived amphoteric antioxidant, to increase performance of non-HQ cosmetic formulations. We found that the cosmetic formula containing DP produced an increase in the rate of fading compared to the two pharmaceutical treatments containing HQ. [ABSTRACT FROM AUTHOR]

There are many techniques for evaluating melanosome transfer to keratinocytes but the spectrophotometric quantification of melanosomes incorporated by keratinocyte phagocytosis has not been previously reported. Here we describe a new method that allows the spectrophotometric visualization of melanosome uptake by normal human keratinocytes in culture. Fontana-Masson staining of keratinocytes incubated with isolated melanosomes showed the accumulation of incorporated melanosomes in the perinuclear areas of keratinocytes within 48 h. Electron microscopic observations of melanosomes ingested by keratinocytes revealed that many phagosomes containing clusters of melanosomes or their fragments were localized in the perinuclear area. A known inhibitor of keratinocyte phagocytosis which inhibits protease-activated receptor-2, i.e., soybean trypsin inhibitor, decreased melanosome uptake by keratinocytes in a dose-dependent manner. These data suggest that our method is a useful model to quantitate keratinocyte phagocytosis of melanosomes visually in vitro. [ABSTRACT FROM AUTHOR]

Background: It has been reported that patients with cholinergic urticaria have a type 1 allergy to autologous sweat; however, the pathogenesis of that disorder has not been fully elucidated. Objective: We investigated the responsiveness to autologous sweat and serum in patients with cholinergic urticaria in relation to their clinical characteristics. We further classified the clinical subtypes that are clearly characterized by responsiveness to in vivo and in vitro tests as well as their clinical features. Methods: Intradermal tests with autologous sweat and serum were performed in 18 patients with cholinergic urticaria. Histamine release from peripheral blood basophils induced by autologous sweat was measured. Results: Eleven of 17 patients with cholinergic urticaria showed positive reactions in skin tests with their own diluted sweat. Substantial amounts of sweat-induced histamine release from autologous basophils were observed in 10 of 17 patients. Eight of 15 patients with cholinergic urticaria showed positive reactions in the autologous serum skin tests. All 6 patients who developed satellite wheals after the acetylcholine test showed hypersensitivity to sweat. Further, patients whose eruptions were coincident with hair follicles showed positive responses to the skin test with autologous serum, whereas patients whose eruptions were not coincident with hair follicles did not. Conclusion: On the basis of these findings, we propose that cholinergic urticaria should be classified into 2 distinct subtypes. The first (nonfollicular) subtype shows strong positive reactions to autologous sweat and negative reactions to autologous serum. The second (follicular) subtype shows weak reactions to autologous sweat and positive reactions to autologous serum. [Copyright &y& Elsevier]

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease-activating factor-1 (Apaf-1) is a cell death effector that acts with cytochrome c and caspase-9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf-1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf-1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf-1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf-1 expression was observed when comparing nevi and melanomas (chi-square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf-1 (chi-square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf-1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf-1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf-1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy. [ABSTRACT FROM AUTHOR]

UV-sensitive syndrome (UVsS) is a rare autosomal recessive disorder characterized by photosensitivity and mild freckling but with- out neurological abnormalities or skin tumors. UVsS cells show UV hypersensitivity and defective transcription-coupled DNA repair of UV damage. It was suggested that UVsS does not belong to any complementation groups of known photosensitive disorders such as xeroderma pigmentosum and Cockayne syndrome (CS). To identify the gene responsible for UVsS, we performed a microcell- mediated chromosome transfer based on the functional complementation of UV hypersensitivity. We found that one of the UVsS cell lines, UV9 KO, acquired UV resistance when human chromosome 10 was transferred. Because the gene responsible for CS group B (CSB), which involves neurological abnormalities and photosensitivity as well as a defect in transcription-coupled DNA repair of UV damage, is located on chromosome 10, we sequenced the CSB gene from UVs1KO and detected a homozygous null mutation. Our results indicate that previous complementation analysis of UVs1KO was erroneous. This finding was surprising because a null mutation of the CSB gene would be expected to result in CS features such as severe developmental and neurological abnormalities. On the other hand, no mutation in the CSB cDNA and a normal amount of CSB protein was detected in Kps3, a UVsS cell line obtained from an unrelated patient, indicating genetic heterogeneity in UVsS. Possible explanations for the discrepancy in the genotype-phenotype relationship in UVs1KO are presented. [ABSTRACT FROM AUTHOR]

CD4+CD25+ T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25+ regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAbad ministration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8+ T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8+ T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4+ T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25+ T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25+ T cells did not induce vitiligo-like coat color changes, though that of CD4+ T cells induced the change in 60%of mice. Furthermore, we confirmed that elimination of CD25+ T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4+ T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25+ regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25+ T-cell depletion is mediated through CD8+ T cells and may differ from the enhancing mechanism caused by CD4+ T-cell depletion. [ABSTRACT FROM AUTHOR]

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion. [ABSTRACT FROM AUTHOR]

The regulation of phospholipase D1 (PLD1) by protein kinase C (PKC) isoforms was analyzed in human melanoma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced PLD1 activation was suppressed by the introduction of PKCδ as well as its kinase-negative mutant in MeWo cells, which contain PKCα but lack PKCβ. PLD activity was not affected by PKCδ in G361 cells, which have PKCβ but are deficient in PKCα. In MeWo cells introduced by PKCα and PLD1, the association of these proteins was observed, which was enhanced by the TPA treatment. In cells overexpressing PKCδ in addition to PKCα and PLD1, TPA treatment increased the association of PKCδ and PLD1, while it attenuated the association of PKCα and PLD1. These results indicate that PKCδ inhibits TPA-induced PLD1 activation mediated by PKCα through the association with PLD1. [Copyright &y& Elsevier]

Genistein, a naturally occurring isoflavone found chiefly in soybeans, has been reported to be a potent antitumor agent. Genistein is presumed to exert multiple effects related to the inhibition of cancer growth. Metastatic melanoma is a chemotherapy-refractory neoplasm. The present study was designed to explore the possible activity of genistein to inhibit the aberrant proliferation and to induce apoptosis of human malignant melanoma cells in cooperation with cisplatin treatment. Five human melanoma cell lines were utilized for these experiments. Genistein at physiologic concentrations (20 μ M) did not induce apoptosis by itself but did enhance cisplatin-induced apoptosis in all five human melanoma cell lines tested. The enhanced susceptibility among the cell lines was diverse. Changes in the expression of two anti-apoptotic proteins, bcl-2 and bcl-xL, and one pro-apoptotic protein, apoptotic protease activating factor-1 (Apaf-1), were examined. Genistein alone or cisplatin alone generally did not alter bcl-2 expression or bcl-xL expression, but slightly increased Apaf-1 in some cell lines. The combined treatment with genistein and cisplatin significantly reduced bcl-2 and bcl-xL protein and increased Apaf-1 protein expression. These data suggest that genistein therapy may enhance the chemosensitivity of melanoma patients. [ABSTRACT FROM AUTHOR]

Thymus and activation‐regulated chemokine (TARC)/CCL17 and macrophage‐derived chemokine (MDC)/CCL22 are a pair of CC chemokines known to selectively attract Th2 type memory T cells via CCR4. Here we examined circulating levels of TARC and MDC in patients with atopic dermatitis (AD) and control subjects by using plasma samples, which reflect blood contents of chemokines more accurately than serum samples. The plasma levels of TARC and MDC were significantly elevated in AD patients. These values also strongly correlated with disease severity and serum lactate dehydrogenase levels, and weakly correlated with serum total IgE levels and blood eosinophilia. Previous studies demonstrated TARC immunoreactivity in the epidermal layer of AD lesional skin and production of TARC by a human keratinocytic cell line HaCaT upon stimulation with IFN‐γ. Here we demonstrated MDC immunoreactivity in the epidermal layer of AD skin at levels stronger than that of TARC. Furthermore, primary epidermal keratinocytes expressed both TARC and MDC mRNA upon stimulation with IFN‐γ, but efficiently secreted only MDC. These results suggest a post‐transcriptional regulation in TARC production. IFN‐γ also induced TARC and MDC mRNA in mouse skin. Collectively, both TARC and MDC play important roles in the local accumulation of Th2 cells in AD lesional skin. Production of Th2‐attracting chemokines by epidermal keratinocytes upon treatment with IFN‐γ, which is also the potent inducer of Th1‐attracting chemokines, may underline the pivotal role of IFN‐γ in the chronic phase of AD where both Th1 and Th2 responses are mixed. [ABSTRACT FROM PUBLISHER]

The effect of plant flavonoids on intercellular adhesion molecule-1 (ICAM-1) expression in human keratinocyte was investigated. ICAM-1 is known to mediate skin inflammation. Among the flavonoids tested, taxifolin was the most potent in inhibiting interferon γ (IFNγ)-induced ICAM-1 protein as well as mRNA expression in human keratinocytes. Much smaller dosages of taxifolin were required in primary keratinocytes compared to HaCaT (immortalized cell) to achieve similar levels of inhibition in the inducible ICAM-1 expression. Regulation of inducible ICAM-1 expression by taxifolin was at transcriptional level by inhibiting the activation of signal transducers and activators of transcription (STAT)1 and protein tyrosine phosphorylation of Janus kinase (JAK)1 suggesting that the JAK–STAT pathway may be the molecular site of action of taxifolin. Finally, taxifolin pre-treatment also potently inhibited IFNγ-induced ICAM-1 expression in a reconstructed human skin equivalent suggesting therapeutic potential of taxifolin in skin pathological conditions related to increased cell adhesion and inflammation. [Copyright &y& Elsevier]

Tissue factor (TF) is involved in tumor progression and metastatic potency in some malignant tumors and its function is regulated by tissue factor pathway inhibitor (TFPI) therefore the interaction of both molecules is crucial for their functional role. We evaluated the clinical relevance of TF and TFPI expression in benign and malignant melanocytic lesions. Expression of both was examined by immunoperoxidase staining using serial tissue sections in 16 nevi, 34 primary and 15 metastatic melanoma lesions. TF and TFPI were ubiquitously expressed in benign and malignant melanocytic lesions. This finding was confirmed by Western blot analysis using cultured human melanocytes, nevi cells (NCN) and melanoma cell lines. Although TF expression was not associated with malignant transformation and disease progression, TFPI expression in primary and metastatic melanoma lesions was significantly lower and weaker than that in nevi lesions in terms of intensity and percentage of stained cells. In addition, TFPI expression in metastatic lesions was significantly lower and weaker than that of TF. These results suggest that the relative expression of TF to TFPI may play a crucial role in the malignant transformation and metastatic potency in melanocytic cells. [ABSTRACT FROM AUTHOR]

We previously demonstrated that advanced melanoma cells express high amounts of proopiomelanocortin (POMC) that correlate with tumor progression. We now investigated whether the high expression of POMC derives from increased expression of corticotropin-releasing hormone (CRH) and the possible role of CRH as a melanoma growth factor. Forty-five cases of melanoma [25 primary malignant melanoma; 20 metastatic melanoma (MetM)] were immunohistochemically analysed for coexpression of POMC and CRH peptides. The ability of CRH to induce POMC expression in cultured melanoma cells was examined using CRH and a CRH antagonist. In CRH positive melanomas, seven out of nine cases (78%) of primary melanoma, and 7 out of 12 cases (58%) of MetM showed colocalization of CRH and POMC peptides. CRH induced POMC mRNA expression, an effect that was inhibited by a CRH antagonist. These results provide evidence for the existence of the CRH/POMC axis in pigmented lesions. [ABSTRACT FROM AUTHOR]