Facial skin photo-aging and development of hyperpigmented spots from children to middle-aged Japanese woman.

BACKGROUND/PURPOSE: Facial skin hyperpigmention caused by chronic sun exposure is a major skin complaint, however, its characteristics and influential factors are still limitedly known. METHODS: A cross-sectional survey in healthy Japanese women aged from 6 to 62 years (n=169) was conducted using a facial image analyzer VISIA™ for knowing onset age of hyperpigmented spot formation, its chronological changes, and influence of environmental factors. RESULTS: UV Pigmented Spot (PS) Score was positively correlated with age (R=.487, P=.000). Hyperpigmented spots appeared first around 18 years old in most subjects, and PS score remarkably increased at 20s then gradually increased by ages. The subjects with Skin Type I, one of the three grades of Japanese Skin Type (JST), whose melanin formation is genetically lower, showed higher PS score. A woman aged 31 years was subjected a weekly VISIA measurement for 2 years, and found no changes in the number, place, size and intensity of the pigment spots in this duration. CONCLUSION: Hyperpigmented spots developed in women over 20 years of age due to chronic sun exposure without sun protection during childhood and adolescent and it was stable afterwards, whose intensity was influenced by age and skin type.

Mechanisms of inhibitory effects of CoQ10 on UVB-induced wrinkle formation in vitro and in vivo.

Photodamaged skin exhibits wrinkles, pigmented spots, dryness and tumors. Solar UV radiation induces cyclobutane pyrimidine dimers (CPD) and further produces base oxidation by reactive oxygen species (ROS). ROS are thought to be a major factor to initiate the up-regulation of matrix metalloproteinases (MMPs) in keratinocytes and fibroblasts via activation of receptor proteins on the cell membrane of keratinocytes and fibroblasts, and to degrade fiber components in dermis, leading to wrinkle formation. Coenzyme Q10 (CoQ10) was reported to reduce ROS production and DNA damage triggered by UVA irradiation in human keratinocytes in vitro. Further, CoQ10 was shown to reduce UVA-induced MMPs in cultured human dermal fibroblasts. We speculated that UVB radiation-induced cytokine production in keratinocytes may be inhibited by CoQ10, resulting in the reduction of MMPs in fibroblasts leading to wrinkle reduction. Our in vitro studies showed that UVB-induced IL-6 production of normal human keratinocyte (NHKC) decreased in the presence of CoQ10. Furthermore, MMP-1 production of fibroblasts cultured with the medium containing CoQ10 collected from UVB-irradiated NHKC significantly decreased during 24 h culture. In the clinical trial study, we found that the use of 1% CoQ10 cream for five months reduced wrinkle score grade observed by a dermatologist. Taken together, our results indicate that CoQ10 may inhibit the production of IL-6 which stimulate fibroblasts in dermis by paracrine manner to up-regulate MMPs production, and contribute to protecting dermal fiber components from degradation, leading to rejuvenation of wrinkled skin.

A Zn(II)-glycine complex suppresses UVB-induced melanin production by stimulating metallothionein expression.

Oxidative stress caused by ultraviolet (UV) radiation generates reactive oxygen species (ROS) in the skin, induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyper-pigmentation. Thus, increasing the anti-oxidative ability of skin cells is expected to be a good strategy for skin-lightening cosmetics. Metallothionein (MT) is one of the stress-induced proteins and is known to exhibit a strong anti-oxidative property. We previously reported that a zinc(II) complex with glycine (Zn(II)(Gly)(2)) effectively induces MT expression in cultured human keratinocytes. To determine its potential as a new skin lightening active, we examined whether Zn(II)(Gly)(2) regulates the release of melanocyte-activating factors from UVB-irradiated keratinocytes and affects melanin production in a reconstructed human epidermal equivalent. Conditioned medium from UVB-irradiated keratinocytes accelerated melanocyte proliferation to 110%, and that increase could be prevented by pre-treatment with Zn(II)(Gly)(2). In addition, Zn(II)(Gly)(2) significantly reduced both the production of prostaglandin E(2) and proopiomelanocortin expression in UVB-irradiated keratinocytes. Zn(II)(Gly)(2) also decreased melanin production in a reconstructed human epidermal equivalent. These results indicate that MT-induction in the epidermis effectively up-regulates tolerance against oxidative stress and inhibits the secretion of melanocyte growth and activating factors from keratinocytes. Thus, Zn(II)(Gly)(2) is a good candidate as a new skin-lightening active.

N-retinoyl-D-glucosamine, a new retinoic acid agonist, mediates topical retinoid efficacy with no irritation on photoaged skin.

BACKGROUND: Chronic ultraviolet (UV) radiation from sunlight induces wrinkle formation. Retinoic acid (RA) can markedly improve wrinkles, although RA does have some side-effects, such as skin irritation. As the efficacy and cytotoxicity of RA has been traced to its free carboxylic acid, we synthesized a new molecule, N-retinoyl-D-glucosamine (GRA), in which a glucosamine has been attached to the polar end group of all-trans retinoic acid. OBJECTIVES: To analyse the effect of topical GRA in wrinkle repair and anti-irritation in photoaged mice compared with topical RA, as well as to determine retinoic acid receptor (RAR) and retinoid X receptor (RXR) transactivation activity in vitro. METHODS: Hairless mice were irradiated with 60 mJ cm-2 of UVB for 10 weeks, and then topically treated with 0.05% GRA or 0.05% RA for 8 weeks. An in vitro transcriptional assay was performed and the activity of GRA in 293 cells transfected with RAR-alpha or RXR-alpha expression plasmid and luciferase reporter plasmid then determined. RESULTS: Topical GRA and RA brought about almost complete disappearance of the wrinkles caused by UVB irradiation. The two ligands promoted both a wide repair zone histologically, and the expression of type 1 collagen in the skin. In contrast, topical GRA treatment did not produce irritation such as erythema or roughness, or alteration of transepidermal water loss values, compared with RA. In the in vitro luciferase assay, GRA resulted in significant dose-dependent RAR transactivation activity in a 100 times higher concentration range than RA. GRA did not mediate RXR transactivation activity at all. CONCLUSIONS: Topical GRA appears to be able to repair photoaged skin damage without any of the irritation caused by topical RA, probably via RAR transactivation activity.

Bowen's carcinoma of the scrotal skin associated with human papillomavirus type 82.

We have previously cloned human papillomavirus type 82 (HPV-82) from a vaginal intraepithelial neoplasia, but it is not known whether HPV-82 can induce a cutaneous lesion. A large erosive nodule developed on the scrotum of a 50-year-old Japanese patient. Histopathologically, the lesion was composed of two distinct parts; one part showing changes characteristic of Bowen's disease in the epidermis, and the other showing elongated rete ridges and proliferation of atypical basaloid cells in the dermis. These parts were partially connected, giving the diagnosis of Bowen's carcinoma. Immunohistochemically, HPV capsid antigen was detected only in the nuclei of a few cells on the upper part of the epidermis. HPV-82 was identified in the lesion by blot hybridization and viral DNA was demonstrated in the lesion by in situ hybridization. HPV-82 has tropism for both the skin and the genital regions.

Effects of glycolic acid on desquamation-regulating proteinases in human stratum corneum.

In order to investigate the mechanism of glycolic acid (GA) function in human stratum corneum, we monitored changes in cathepsin D-like (CD) and chymotrypsin-like (SCCE) proteinases for 3 weeks following topical GA application (50% w/v, pH 0.9) for 30 min to human skin. In the early phase, weakened stratum corneum cohesion in the lower layers was observed on day 2 and the amount of active CD in the upper layer of the stratum corneum was significantly decreased from 30 min until day 2, whereas that in the lower layer remained normal. In contrast, the amount of active SCCE showed no change during the experimental period. The surface pH of the stratum corneum drastically decreased to pH 2 at 30 min and slightly recovered to around pH 3 until 1 day after treatment. From 9 to 19 days, a decrease in corneocyte cell area and a remarkable long-term increase in the amount of active CD in the upper layer were observed. In an in vitro study, the activities of desquamation-regulating proteinases were shown to have remarkably increased at around pH 3, due to activation of CD at its optimal pH. These results suggest that GA functions via at least two different mechanisms, acute activation of CD in the lower layer by acidification around pH 3, along with inactivation of CD in the upper layer, and long-term enhancement of de novo CD production in the few weeks following GA treatment.

Observation of magnetic multilayers by electron holography.

Magnetic structures of Co/Cu multilayers in cross section are observed by two kinds of electron holography: a Fourier method and a phase-shifting method, which is introduced briefly. The Fourier method can easily reconstruct wave functions and is applied to many specimens, whereas the phase-shifting method requires longer time for processing, but has a higher spatial resolution that permits us to discuss fine structures. Magnetization vectors in Co layers aligning parallel and separating into two blocks with antiparallel alignment are observed. Magnetic blurring on the boundary between Co and Cu in the reconstructed phase images is larger than the estimated atomic roughness.

Boron neutron capture therapy (BNCT) for malignant melanoma with special reference to absorbed doses to the normal skin and tumor.

Twenty-two patients with malignant melanoma were treated with boron neutron capture therapy (BNCT) using 10B-p-boronophenylalanine (BPA). The estimation of absorbed dose and optimization of treatment dose based on the pharmacokinetics of BPA in melanoma patients is described. The doses of gamma-rays were measured using small TLDs of Mg2SiO4 (Tb) and thermal neutron fluence was measured using gold foil and wire. The total absorbed dose to the tissue from BNCT was obtained by summing the primary and capture gamma-ray doses and the high LET radiation doses from 10B(n, alpha)7Li and 14N(n,p)14C reactions. The key point of the dose optimization is that the skin surrounding the tumour is always irradiated to 18 Gy-Eq, which is the maximum tolerable dose to the skin, regardless of the 10B-concentration in the tumor. The neutron fluence was optimized as follows. (1) The 10B concentration in the blood was measured 15-40 min after the start of neutron irradiation. (2) The 10B-concentration in the skin was estimated by multiplying the blood 10B value by a factor of 1.3. (3) The neutron fluence was calculated. Absorbed doses to the skin ranged from 15.7 to 37.1 Gy-Eq. Among the patients, 16 out of 22 patients exhibited tolerable skin damage. Although six patients showed skin damage that exceeded the tolerance level, three of them could be cured within a few months after BNCT and the remaining three developed severe skin damage requiring skin grafts. The absorbed doses to the tumor ranged from 15.7 to 68.5 Gy-Eq and the percentage of complete response was 73% (16/22). When BNCT is used in the treatment of malignant melanoma, based on the pharmacokinetics of BPA and radiobiological considerations, promising clinical results have been obtained, although many problems and issues remain to be solved.

Involvement of EGF receptor activation in the induction of cyclooxygenase-2 in HaCaT keratinocytes after UVB.

Because selective inhibition of cyclooxygenase-2 (COX-2) suppressed the induction of skin tumors in mice by UV and as UV has been shown to induce expression of COX-2 in skin and cells, COX-2 may be crucial for photocarcinogenesis of the skin. We studied the mechanism of UVB-induced expression of COX-2 focusing on the signal transduction pathway involved. Hydrogen peroxide (H2O2) treatment of HaCaT cells induced expression of COX-2 and pretreatment with the antioxidant N-acetylcysteine (NAC) partly inhibited the UVB-induced expression of COX-2 protein in HaCaT cells, suggesting that oxidative stress contributes to COX-2 induction. To examine the signaling pathways involved in the UVB-induced expression of COX-2 in HaCaT cells, we analysed the expression of COX-2 protein after treatment with various inhibitors of signaling molecules. Inhibition of EGFR by a specific inhibitor and by a neutralizing antibody suppressed the induction of COX-2 expression by UV. Although a neutralizing antibody to transforming growth factor-alpha (TGF-alpha) suppressed COX-2 expression induced by TGF-alpha, it did not suppress COX-2 expression by UV, indicating that a direct activation of EGFR is involved. Treatment of cells at low temperature (4 degrees C) inhibited UVB-induced JNK activation, but it did not inhibit COX-2 expression by UV. Inhibitors of MEK, p38 MAP kinase and PI3-kinase, suppressed the induction of COX-2 expression by UV. In contrast, an erbB-2 inhibitor augmented the UVB-induced increase of COX-2 protein. These data indicate that oxidative stress in association with activation of EGFR, ERK, p38 MAP kinase, and PI3-kinase plays crucial roles in the UVB induction of expression of COX-2.

UV-induced skin damage.

Solar radiation induces acute and chronic reactions in human and animal skin. Chronic repeated exposures are the primary cause of benign and malignant skin tumors, including malignant melanoma. Among types of solar radiation, ultraviolet B (290-320 nm) radiation is highly mutagenic and carcinogenic in animal experiments compared to ultraviolet A (320-400 nm) radiation. Epidemiological studies suggest that solar UV radiation is responsible for skin tumor development via gene mutations and immunosuppression, and possibly for photoaging. In this review, recent understanding of DNA damage caused by direct UV radiation and by indirect stress via reactive oxygen species (ROS) and DNA repair mechanisms, particularly nucleotide excision repair of human cells, are discussed. In addition, mutations induced by solar UV radiation in p53, ras and patched genes of non-melanoma skin cancer cells, and the role of ROS as both a promoter in UV-carcinogenesis and an inducer of UV-apoptosis, are described based primarily on the findings reported during the last decade. Furthermore, the effect of UV on immunological reaction in the skin is discussed. Finally, possible prevention of UV-induced skin cancer by feeding or topical use of antioxidants, such as polyphenols, vitamin C, and vitamin E, is discussed.

Widespread expression of parathyroid hormone-related peptide in melanocytic cells.

BACKGROUND: Parathyroid hormone-related peptide (PTH-rP) was associated with the syndrome of hypercalcaemia of malignancy. An increased serum level of PTH-rP could occur in patients with advanced melanoma. OBJECTIVES: We examined PTH-rP expression in cultured melanocytic cell lines and in lesions of melanocytic origin for associations with clinicopathological variables of disease progression. We measured the supernatant and cell lysate level of PTH-rP in cultured melanoma cells to clarify whether melanoma cells secrete PTH-rP. METHODS: PTH-rP expression was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in cultured melanocytic cell lines and by immunoperoxidase staining in 18 melanocytic naevi, 40 primary melanoma and 19 metastatic melanoma lesions. The supernatant level of PTH-rP was measured with an immunoradiometric assay. RESULTS: RT-PCR products of PTH-rP mRNA were detected in six of eight melanoma cell lines; however, neither naevus cells nor melanocytes showed positive products. On the other hand, immunohistochemical analysis showed that PTH-rP was widely expressed both in benign and malignant melanocytic lesions. In addition, PTH-rP expression was not associated with any clinicopathological variables. Cell lysate but not the supernatant of melanoma cells showed high PTH-rP levels. CONCLUSIONS: These results suggest that PTH-rP was widely expressed in melanocytic cells; however, the cells did not secrete PTH-rP.

Localized heat urticaria in a patient is associated with a wealing response to heated autologous serum.

We report a case of localized heat urticaria in a 71-year-old woman who developed weals and loss of consciousness after taking a bath. Exposing her skin to heat at 40 degrees C or immersing her hands in water at 40 degrees C produced urticarial lesions and increased her plasma histamine level. Desensitization with hot water improved her symptoms and normalized her plasma histamine level after heat challenge. An intracutaneous injection of her serum produced no reaction, while an injection of her serum that had been heated at 40 degrees C for 15 min induced a weal flare response. Further examination revealed that the weal-inducing activity of her heated serum remained for at least for 6 h and that treatment of her serum at 60 degrees C for 2 h did not abrogate its weal-inducing activity. These findings indicate that certain materials in her serum that are activated by heat are responsible for the development of her anaphylactic and urticarial reactions and that these reactions may be mediated by histamine.

Protean manifestations of human papillomavirus type 60 infection on the extremities.

BACKGROUND: Human papillomavirus type 60 (HPV-60) induces a ridged wart or an epidermal cyst on the sole of the foot, exhibiting identical pathological changes, with a single refractile eosinophilic intracytoplasmic inclusion body in infected cells. However, there is no information on the role of HPV-60 in the development of cutaneous lesions on other anatomical sites. OBJECTIVES: To perform the clinicopathological analysis of various cutaneous lesions of a patient in relation to HPV genotype. PATIENT: A 50-year-old male patient developed multiple papules, plaques and nodules on his hand, arm and legs. RESULTS: Clinicopathologically, the lesions were classified into three categories. A common wart on the finger showed papillomatosis and acanthosis characterized by numerous keratohyalin granules. Plane warts on the arm showed perinuclear vacuolization of the cells in the upper Malpighian layer. On the other hand, a pigmented papillomatous nodule on the finger, and the other lesions on the hands and legs exhibited similar histological features with a unique cytoplasmic eosinophilic inclusion body. All the three categorized lesions were equally positive for HPV capsid antigen by immunohistochemistry. By blot hybridization analysis for HPV sequences, it was revealed that a common wart on the finger and plane warts on the arm harboured HPV-27 and HPV-3, respectively, while all the other lesions harboured HPV-60. The histological localization of each viral DNA was confirmed in the corresponding lesions by in situ hybridization. CONCLUSIONS: HPV-60 is able to induce papular and nodular lesions on the extremities.

Fusion hybrids with macrophage and melanoma cells up-regulate N-acetylglucosaminyltransferase V, beta1-6 branching, and metastasis.

It was shown previously that a majority of hybrids produced by in vitro fusion of normal macrophages with Cloudman S91 melanoma cells displayed enhanced metastatic potential in vivo, increased motility in vitro, increased ability to produce melanin, and responsiveness to melanocyte stimulating hormone compared with the parental Cloudman S91 melanoma cells. These hybrids also showed altered N-glycosylation consistent with a slower migration pattern of lysosome-associated membrane protein (LAMP-1) on electrophoretic gels. Because LAMP-1 is the major carrier of polylactosamine sugar structures, and synthesis of this complex sugar moiety indicates the extent of beta1,6 branch formation by beta1,6-N-acetyl-glucosaminyltransferase V (GnT-V), we analyzed the expression of GnT-V and beta1,6 branching in highly metastatic macrophage-fusion hybrids and compared with poorly metastatic ones. GnT-V was up-regulated in regard to both mRNA levels and enzymatic activity specifically in metastatic hybrids as well as parental macrophages compared with weakly metastatic hybrids and parental melanoma cells. Macrophages and metastatic hybrids also showed increased binding of the lectin L-phytohemagglutinin, which specifically binds to the beta1,6-branched sugar moiety. In addition, in metastatic hybrids there was increased cell surface expression of LAMP-1 and beta1 integrin, two prominent substrates for GnT-V also known to be associated with metastasis. Finally, exposure of metastatic hybrids in vitro to L-phytohemagglutinin or LAMP-1 completely eliminated melanocyte stimulating hormone/ isobutylmethyl xanthine-induced motility, suggesting a role for GnT-V in the motility of these cells. In summary, macrophage fusion with melanoma cells often increased metastatic potential, which was associated with enhanced expression of GnT-V and beta1,6-branching in glycoproteins. It is suggested that the known correlation with elevated GnT-V in both human and animal metastasis could, at least in some cases, reflect previous fusion of tumor cells with tumor-infiltrating macrophages, which, similar to malignant cells, show elevated expression of GnT-V and beta1,6-branched polylactosamines.

UV-induced tyrosine phosphorylation of PKC delta and promotion of apoptosis in the HaCaT cell line.

Protein kinase C delta (PKC delta) is activated through tyrosine phosphorylation and is involved in apoptosis induction in the H(2)O(2)-treated fibroblasts. In the human keratinocyte HaCaT cell line, ultraviolet radiation, which induces apoptosis, promoted tyrosine phosphorylation and activation of PKC delta, but neither enhanced threonine phosphorylation in the activation loop nor generated the catalytic fragment of the PKC isoform. Tyrosine phosphorylation of PKC delta was prevented by a radical scavenger, N-acetyl-l-cysteine, and by a tyrosine kinase inhibitor, genistein, in the ultraviolet-irradiated keratinocyte cell line. Ultraviolet radiation-induced apoptosis was attenuated by N-acetyl-l-cysteine and genistein as well as by a PKC inhibitor, bisindolylmaleimide I. These results indicate that reactive oxygen species generated by ultraviolet radiation enhance tyrosine phosphorylation of PKC delta, and the PKC isoform thus activated by the modification reaction contributes to induction of apoptotic cell death in keratinocytes.

Regulation of Ras and Rho small G proteins by SHP-2.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor, c-Met. We have previously shown that SHP-2, a protein tyrosine phosphatase, positively regulates the HGF/SF-induced cell scattering through modulating the activity of Rho to form stress fibres and focal adhesions. To further investigate the role of SHP-2 in HGF/SF-induced cell scattering, we have now examined the effect of a dominant active mutant of SHP-2 (SHP-2-DA). RESULTS: Expression of SHP-2-DA markedly increased the formation of lamellipodia with ruffles, while it decreased the accumulation of E-cadherin and beta-catenin at cell-cell adhesion sites in MDCK cells. In addition, expression of SHP-2-DA markedly enhanced cell scattering of MDCK cells in response to HGF/SF. Expression of SHP-2-DA induced the activation of MAP kinase without HGF/SF stimulation, whereas an inhibitor of MEK partly reversed the SHP-2-DA-induced morphological phenotypes. Furthermore, expression of either a dominant-active mutant of Rho or Vav2 also reversed the SHP-2-DA-induced morphological phenotypes. CONCLUSION: These results indicate that SHP-2 plays a crucial role in the HGF/SF-induced cell scattering through the regulation of two distinct small G proteins, Ras and Rho.