Morphological characterization of solar lentigines by in vivo reflectance confocal microscopy: a longitudinal approach.

Solar lentigines are benign hyperpigmented skin lesions. Despite their widespread distribution, knowledge on the mechanisms of development is largely unknown. A clinical study was designed in which solar lentigines were characterized using various non-invasive clinical techniques. A subset of solar lentigines was followed over a 5-year time period. One hundred and twenty-eight solar lentigines were evaluated using in vivo reflectance confocal microscopy (RCM) for the evaluation of the length and density of their dermal papillae as well as the deformation of the alignment pattern of hyperrefractive basal cells. Skin colour, colour contrast, the size of the solar lentigo, epidermal proliferation rate, melanin and haemoglobin content were quantified. RCM imaging of solar lentigines revealed a profound structural deformation of the dermal papillae, as the alignment pattern of hyperrefractive basal cells shifted from a circle in non-lesional skin to an irregular non-circular shape in solar lentigines. There was a rise in the number of dermal papillae, and these dermal papillae were significantly longer. Solar lentigines had increased melanin and haemoglobin levels and a higher rate of epidermal proliferation. For a subset of nineteen solar lentigines, a longitudinal study was set-up in which these measurements were repeated 5 years after the first evaluation. The deformation and the number of the hyperrefractive dermal papillary rings increased significantly over the 5-year time span. The size of the lesion increased, and the skin colour became darker. RCM is a useful non-invasive clinical tool for the characterization of solar lentigines, in particular the compressive deformation of the dermal papillae. This deformation became more severe over a time period of 5 years. To our knowledge, this is the first time that the in vivo time-dependent progression of solar lentigines was supported by RCM images, contributing to an improved understanding of the formation and progression of solar lentigines.

Arginase is overactive in psoriatic skin.

BACKGROUND: Psoriatic keratinocytes are poorly differentiated and hyperproliferative. Low concentrations of nitric oxide (NO) induce keratinocyte proliferation, while high concentrations induce differentiation. The NO-producing enzyme inducible NO synthase is overexpressed in psoriatic skin, but so is arginase. The overexpressed arginase competes for arginine, the common substrate for both enzymes, and may reduce NO production. OBJECTIVES: To determine whether arginase activity is elevated in psoriatic skin and whether exogenous NO will improve psoriatic plaques. METHODS: Tape strips were taken from healthy skin of eight control subjects and nonlesional skin of eight patients with psoriasis and L-arginine, L-citrulline and L-ornithine concentrations measured by high-performance liquid chromatography. In a second study, four psoriatic patients with a pair of similar symmetrical plaques were treated with an NO donor and vehicle control. Plaques were scored for size, erythema, induration and scaling at the start and after 6 weeks of treatment. RESULTS: Ornithine, the end-product of arginase, was at higher concentrations in nonlesional psoriatic than in healthy skin (mean +/- SEM 2.08 +/- 0.98 vs. 1.13 +/- 0.44 microg mg(-1) protein; P = 0.0002). Arginine, its substrate, was at lower concentrations. Topical application of an NO donor improved psoriatic plaques clinically [mean +/- SD reduction in severity from baseline score (100%) to 35% +/- 16% in active NO donor and to 93% +/- 10% in control]. CONCLUSIONS: Arginase is overactive in psoriatic skin, leading to a relative increase in the consumption of arginine. We therefore hypothesize a relative decrease in NO synthase-derived NO production. NO donors may be effective topical treatments for psoriasis.

Glycation associated skin autofluorescence and skin elasticity are related to chronological age and body mass index of healthy subjects.

Glycation is the non-enzymatic reaction between reducing sugars and proteins that leads to the formation of advanced glycation end products (AGEs). In vivo skin autofluorescence (lambda(ex)/lambda(em)=370/440 nm) was used as a non-invasive clinical tool to study skin AGE accumulation in healthy panellists. Using multiple linear regression analysis, it was shown that for panellists below the age of 40, glycation associated in vivo skin fluorescence intensity increased as a function of chronological age and body mass index (BMI). Above the age of 40, the fluorescence was associated to age but not to BMI, suggesting that the effect of age became dominant over BMI. Since the accumulation of AGEs is expected to affect the biomechanical properties of the skin, in vivo skin elasticity data were gathered on a second panel. It was found that skin elasticity depended on age and BMI in a similar fashion as to what we observed for the skin fluorescence data. It is hypothesised that skin AGE accumulation contributes to the loss of skin elasticity in aged and/or overweight people.

Prevention of oxidative damage that contributes to the loss of bioenergetic capacity in ageing skin.

Skin ageing is a complex biological process related to a decline in physiological and biochemical performance. A decline in the mitochondrial energy production is a feature of ageing at the cellular level. This is partially attributed to excessive production of reactive oxygen species such as superoxide and hydrogen peroxide in aged individuals. We have investigated the effect of (glyc)oxidative stress on two biochemical targets relevant for the energy metabolism of the skin. First, we showed an age dependent decline in the activity of the hydrogen peroxide detoxifying antioxidant catalase in stratum corneum on a chronically sun-exposed site. Furthermore catalase was sensitive to peroxynitrite-induced in vitro inactivation. Catalase mimetics as well as peroxynitrite scavengers are proposed to maintain hydrogen peroxide detoxification pathways. The second target was creatine kinase, an enzyme that controls the creatine-creatine phosphate shuttle. Creatine kinase lost activity after in vitro glycation by methylglyoxal. This activity loss could be prevented by antiglycation actives. These data suggest that biomolecules involved in energy homeostasis become damaged by different sources of stress. Actives specifically selected for optimal protection against these stress situations will decrease skin vulnerability and prevent the premature loss of skin function.

Adaptive response of the skin to UVB damage: role of the p53 protein.

Different adaptation mechanisms like heat shock response, cell cycle arrest and DNA repair, melanin pigmentation and thickening of the epidermis are present in the human skin to protect against the adverse effects of solar UV irradiation. When DNA damage is beyond repair, cells undergo apoptosis to prevent their replication. We discuss the current knowledge on these different adaptation mechanisms to UVB damage, the most energetic fraction of solar UV that reaches the skin. As p53 protein, the guardian of the genome, plays a key role in protective response to genotoxic damage, its role in this adaptive response of the skin to UV will be further discussed.

Human keratinocytes respond to osmotic stress by p38 map kinase regulated induction of HSP70 and HSP27.

Human skin is exposed to an environment that varies in humidity from 100 to 0%, leading to seasonal variations in the condition of the skin. Exposure to a low humidity environment creates an osmotic gradient across the stratum corneum, which is known to modulate cutaneous barrier function. Heat shock proteins protect against stress-induced destabilization of proteins. We investigated whether osmotic shock (sorbitol) induced a heat shock protein response in normal human keratinocytes, and used heat shock as a positive control. Both heat shock and osmotic stress (200 and 300 mM sorbitol) clearly induced heat shock proteins 70 and 27 mRNA levels. The induction of heat shock protein 70 mRNA levels by osmotic stress peaked at 16 h and persisted until 24 h, whereas upregulation of heat shock protein 70 mRNA levels by heat peaked at 2 h and returned to baseline levels by 6 h. Sorbitol also increased heat shock protein 70 levels in a concentration-dependent manner. The kinetics of heat shock protein 27 mRNA induction by osmotic stress and heat were similar with peak induction at 6 h. The mitogen activated protein kinase family of proteins plays an important part in the coordination of gene responses to various stress conditions. We have demonstrated that the p38 mitogen activated protein kinase was strongly activated by 200 mM and 300 mM sorbitol. The specific p38 mitogen activated protein kinase inhibitor PD169316 almost completely blocked heat shock protein 70 mRNA induction by 200 mM and 300 mM sorbitol and completely suppressed heat shock protein 27 mRNA induction with 200 mM sorbitol. PD169316 also counteracted upregulation of heat shock protein 70 levels by sorbitol. These data indicate that keratinocytes respond to osmotic stress by p38 mitogen activated protein kinase regulated induction of heat shock proteins. This molecular pathway may be relevant for the mechanisms regulating the response of human skin to variations in environmental humidity.

A summary report of the COLIPA international validation study on alternatives to the draize rabbit eye irritation test.

The principal goal of this study was to determine whether the results from a set of selected currently available alternative methods as used by cosmetics companies are valid for predicting the eye irritation potential of cosmetics formulations and ingredients and, as a consequence, could be valid replacements for the Draize eye irritation test. For the first time in a validation study, prediction models (PMs) that convert the in vitro data from an assay to a prediction of eye irritation were developed for each alternative method before the study began. The PM is an unequivocal description of the relationship between the in vitro and the in vivo data and allows an objective assessment of the reliability and relevance of the alternative methods. In this study, 10 alternative methods were evaluated using 55 test substances selected as representative of substances commonly used in the cosmetics industry (23 ingredients and 32 formulations). Twenty of the single ingredients were common to the European Commission/British Home Office (EC/HO) eye irritation validation study (Balls et al., 1995b). The test substances were coded and supplied to the participating laboratories. The results were collected centrally and analysed independently, using statistical methods that had been agreed before the testing phase began. Each alternative method was then evaluated for reliability and relevance in assessing eye irritation potential. Using the criteria of both reliability and relevance as defined in the study, the preliminary results indicate that none of the alternative methods evaluated could be confirmed as a valid replacement for the Draize eye irritation test across the full irritation scale. However, three alternative methods-the fluorescein leakage test, the red blood cell assay (classification model) and the tissue equivalent assay-each satisfied one criterion of reliability or relevance. Further investigation of the decoded data from this study to explore more fully the relationship between the in vitro data and the in vivo data is recommended. Such a review may allow the development of new prediction models to be tested in a subsequent validation study.

Use of the synthetic superoxide dismutase/catalase mimetic EUK-134 to compensate for seasonal antioxidant deficiency by reducing pre-existing lipid peroxides at the human skin surface.

The generation of reactive oxygen species (ROS) in UV-exposed skin is believed to contribute to the photoaging process. The stratum corneum (SC) contains a variety of enzymatic and non-enzymatic antioxidants to protect against various environmental sources of free radicals. We have previously shown a seasonal variation in SC catalase activity with strong deactivation in sun-exposed skin in the summer, whereas SC superoxide dismutase (SOD) activity remained intact in those conditions. This potentially leads to the local overproduction of H(2)O(2). The oxidized lipid squalene hydroperoxide accumulates at the surface of sun-exposed skin in the summer and upon exposure to ultravoilet A (UVA) doses as low as 0.1 J cm(-2) and adequate protection against excessive lipid peroxidation at times of UV exposure should be aimed for. We have been using the induction of lipid hydroperoxides at the skin surface by a single dose of UVA (1 J cm(-2)) as a model system to evaluate the protective effect of antioxidants in vivo. Topical treatment with the synthetic SOD/catalase mimetic molecule (EUK-134) 1 h before UVA exposure reduced the level of lipid peroxides at the surface of UVA-exposed skin but also baseline peroxide levels on non-irradiated skin were reduced in a dose-dependent fashion. In contrast to alpha-tocopherol, EUK-134 even reduced the level of lipid peroxides at the surface of UVA-exposed skin when it was applied after irradiation. We confirmed that this salen-manganese complex was able to reduce squalene hydroperoxide levels in vitro, suggesting peroxidase-like activity towards organic peroxides. These data support the concept that the synthetic SOD/catalase mimetic EUK-134 might be able to compensate for seasonal deficiencies in antioxidant defense capacity at the skin surface, thereby contributing to an optimal protection of the skin against the accumulation of oxidative damage.

Aging of human skin: review of a mechanistic model and first experimental data.

The physical, chemical, and biochemical factors that accelerate skin aging have been proposed to activate a self-maintained microinflammatory process, one of the expected end results of which is an imbalance in the turnover of macromolecules in the dermis. Surface peroxides are recognized as controllable factors of skin aging, and their accumulation is attributed to environmentally induced impairment of defense enzymes. Topical application of antioxidants decreases the rate at which skin elasticity and skin thickness are modified.

The diagnostic significance of the antiperinuclear factor for rheumatoid arthritis.

Sensitivity and specificity of antiperinuclear factor (APF) determination were assessed in 127 patients with classical or definite rheumatoid arthritis (RA). The control population consisted of 262 patients with rheumatic complaints other than RA. Our results showed a sensitivity of 86.6%, a specificity of 96.2% and a Youden index of 82.8% for APF in RA. In 14.2% of the total population with RA the rheumatoid factor (RF) became negative in the course of the disease; all but one were found to be actually APF positive. Of the population with RA analyzed, 8.6% were consistently RF negative but APF positive. In view of its strong sensitivity and specificity for RA, APF determination is a valuable serological tool for the diagnosis of the disease. In APF positive, RF negative patients with RA the prognosis seems to be poor.