Stan Pavel

Melanin content of cultured human melanocytes and UV-induced cytotoxicity.

Cultured melanocytes originating from persons with different skin phototypes were utilized for measurement of endonuclease sensitive sites induced by UVB and the determination of cell survival after UVA or UVB irradiation. During culture, the melanocytes largely maintained their phenotypic characteristics according to their original skin phototype. Total melanin concentrations were 4.9 times higher in the darker skin phototype (IV-VI) melanocytes when compared to the cells from lighter skin phototypes (I-III). Also phaeomelanin contents were higher (2.5 times) in the skin phototype (IV-VI) melanocytes which implies that the cells from light skin types contain less melanin, but a relatively high proportion of phaeomelanin. After UVB irradiation a stronger induction of endonuclease sensitive sites was found for melanocytes with a lower level of total melanin and a high content of pheomelanin. By measuring the clone forming ability in different melanocyte cultures after UVB irradiation, significant better survival was found in case of the cells with the higher melanin content. Despite the large variations in melanin content, no significant difference in survival after UVA irradiation could be demonstrated in this way. Our results suggest a protective effect of melanin for UVB and indicate the importance of the measurements of melanin content and composition when different parameters of UV-induced damage are studied in melanin producing cells.

Polymorphic light eruption occurs in 18% of Europeans and does not show higher prevalence with increasing latitude: multicenter survey of 6,895 individuals residing from the Mediterranean to Scandinavia.

Schneider MR, Antsiferova M, Feldmeyer L, Dahlhoff M, Bugnon P, Hasse S et al. (2008a) Betacellulin regulates hair follicle development and hair cycle induction and enhances angiogenesis in wounded skin. Journal of Investigative Dermatology 128:1256–65 Schneider MR, Werner S, Paus R, Wolf E (2008b) Beyond wavy hairs: the epidermal growth factor receptor and its ligands in skin biology and pathology. Am J Pathol 173:14–24

Variations in melanin formation by cultured melanocytes from different skin types

Abstract In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin typeu2002VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher l -tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated.

Normalized ultraviolet (UV) induction of Langerhans cell depletion and neutrophil infiltrates after artificial UVB hardening of patients with polymorphic light eruption

Backgroundu2002 Ultraviolet (UV) B hardening has been widely used as a prophylactic treatment in patients with polymorphic light eruption (PLE). Recent investigations have shown that in patients with PLE Langerhans cells (LCs) and neutrophils display less migration from and to the epidermis after an intense UVB irradiation compared with controls.

Catechol-O-methyltransferase in vitiligo

Catechol-O-methyltransferase (COMT) is involved in the metabolism of neurotransmitters such as epinephrine, norepinephrine and dopamine. For melanocytes, the enzyme is of particular importance in preventing the formation of toxic o-quinones during melanin synthesis. It has been suggested that COMT plays a regulatory role in melanin synthesis. Indeed, when the melanin precursor molecule DHI(2C) is methylated by COMT it is no longer available for incorporation into melanin. Autodestruction by intermediates of melanin metabolism has been implicated in the aetiology of vitiligo. Therefore enzyme activities in vitiligo patients and in healthy controls were compared. Systemic COMT activities were measured using red blood cells (RBC) as starting material. However, as local alterations in COMT activity may be specifically involved in vitiligo, the enzyme activity was also measured in epidermal homogenates. Finally, to ascribe epidermal COMT activity to the responsible cell type(s), enzyme activity was measured in cultured vitiligo non-lesional melanocytes and melanocytes from healthy controls as well as in cultured keratinocytes from lesional skin and in purified keratinocytes from control skin. It was found that epidermal homogenates from vitiligo patients expressed higher levels of COMT activity than homogenates from healthy controls. Such differences were not found at the systemic level (i.e. in RBC) nor could they be explained by measurements on separately cultured epidermal cell types, indicating that the COMT activity was induced at the tissue level by extracellular factors. It is possible that elevated levels of catecholamines secreted by keratinocytes or by nerve endings in vitiliginous skin in close proximity to the epidermis cause damage to all epidermal cells, an effect which is insufficiently neutralized by elevated levels of COMT activity. Catecholamines may well be more damaging to the melanocytes than to the keratinocytes because of their slower turnover rate.

Morphological changes of the dermatophyte Trichophyton rubrum after photodynamic treatment: a scanning electron microscopy study.

Treatment strategies for superficial mycosis caused by the dermatophyte Trichophyton rubrum consist of the use of topical or oral antifungal preparations. We have recently discovered that T. rubrum is susceptible to photodynamic treatment (PDT), with 5,10,15-tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride (Sylsens B) as a photosensitizer. The susceptibility appeared to depend on the fungal growth stage, with PDT efficacy higher with microconidia when compared to mycelia. The aim of this study was to investigate, with the use of scanning electron microscopy, the morphological changes caused by a lethal PDT dose to T. rubrum when grown on isolated human stratum corneum. Corresponding dark treatment and light treatment without photosensitizer were used as controls. A sub-lethal PDT dose was also included in this investigation The morphologic changes were followed at various time points after the treatment of different fungal growth stages. Normal fungal growth was characterized by a fiber-like appearance of the surface of the hyphae and microconidia with the exception of the hyphal tips in full mycelia and the microconidia shortly after attachment to the stratum corneum. Here, densely packed globular structures were observed. The light dose (108 J/cm2) in the absence of Sylsens B, or the application of the photosensitizer in the absence of light, caused reversible fungal wall deformations and bulge formation. However, after a lethal PDT, a sequence of severe disruptions and deformations of both microconidia and the mycelium were observed leading to extrusion of cell material and emptied fungal elements. In case of a non-lethal PDT, fungal re-growth started on the remnants of the treated mycelium.

Preclinical studies with 5,10,15-Tris(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine trichloride for the photodynamic treatment of superficial mycoses caused by Trichophyton rubrum.

Dermatophytes are fungi that cause infections of keratinized tissues. We have recently demonstrated the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment (PDT) with 5,10,15‐Tris(4‐methylpyridinium)‐20‐phenyl‐[21H,23H]‐porphine trichloride (Sylsens B) in 5u2003mm citric acid/sodium citrate buffer (pH 5.2, formulation I). In this work, we examined the penetration of Sylsens B in healthy and with T. rubrum infected skin and we investigated the susceptibility of T. rubrum to PDT using formulation I and UVA‐1 radiation (340–550u2003nm). Skin penetration studies were performed with formulations I and II (Sylsens B in PBS, pH 7.4) applied on dermatomed skin, human stratum corneum (SC), disrupted SC by T. rubrum growth and SC pretreated with a detergent. No penetration was observed in healthy skin. Disruption of SC by preceding fungal growth caused Sylsens B penetration at pH 7.4, but not at pH 5.2. However, chemically damaged SC allowed Sylsens B to penetrate also at pH 5.2. UVA‐1 PDT was applied ex vivo during two fungal growth stages of two T. rubrum strains (CBS 304.60 and a clinical isolate). Both strains could be killed by UVA‐1 alone (40u2003J/cm2). Combined with formulation I (1 and 10u2003μm Sylsens B for, respectively, CBS 304.60 and the clinical isolate), only 18u2003J/cm2 UVA‐1 was required for fungal kill. Therefore, PDT with 10u2003μm Sylsens B (formulation I) and 18u2003J/cm2 UVA‐1 could be considered as effective and safe. This offers the possibility to perform clinical studies in future.

Hydrophilic and lipophilic moisturizers have similar penetration profiles but different effects on SC water distribution in vivo

Abstract:u2002 Dry skin is often treated with hydrophilic and/or lipophilic moisturizers. Hydrophilic moisturizers must penetrate the stratum corneum (SC) deeply to function properly, whereas lipophilic moisturizers should remain in the upper SC layers. In this study, both types of moisturizers were applied on volunteers for 3u2003h, after which the relative amount of moisturizer and the water distribution in the SC were determined using attenuated total reflectance‐Fourier transform infrared (ATR‐FTIR) spectroscopy in combination with tape‐stripping. The results show that while hydrophilic moisturizers penetrate much more readily than lipophilic moisturizers, the latter are abundantly present in the upper regions of the SC. It was also observed that a 3‐h treatment with lipophilic moisturizer did not result in increased water levels in the SC, whereas hydrophilic moisturizers retained water where they are located. The results suggest that upon prolonged application, adequate amounts of moisturizer can be obtained in those regions where they may cause moisturization in the central part of the SC. However, a single application of 3u2003h is probably too short to exert increased hydration as measured with ATR‐FTIR.

Prevention and treatment of vitamin D deficiency in Dutch psychogeriatric nursing home residents by weekly half-body UVB exposure after showering: a pilot study

BACKGROUNDnin older people, induction of cutaneous vitamin D production by ultraviolet B (UVB) exposure may be preferable to oral supplementation: it cannot cause toxic levels, it helps to prevent polypharmacy and, moreover, there are indications that UVB exposure has beneficial effects on health and well being by mechanisms other than the vitamin D pathway alone.nnnOBJECTIVEnthe aim of this pilot study is to investigate whether weekly, half-body, UVB irradiation after showering can increase serum 25-hydroxyvitamin D (25(OH)D) to sufficient levels, in a Dutch psychogeriatric nursing home population.nnnMETHODnsubjects were eight psychogeriatric nursing home patients, mean age: 79 ± 8. Exclusion criteria were going outdoors into the sun more than once a week, the presence of actinic or cancer skin lesions and known resistance to body contact. The intervention consisted of weekly half-body UVB irradiation, after showering, over 8 weeks, with 0.5 minimal erythemal dose (MED). Main outcome measures were change in fasting serum levels of 25(OH)D and parathyroid hormone (PTH) at 0, 2, 4 and 8 weeks.nnnRESULTSnat baseline, mean serum 25(OH)D was 28.5 nmol/l. Mean serum 25(OH)D levels increased to 46.5 nmol/l. Median serum PTH levels decreased by 20% after 8 weeks of treatment.nnnCONCLUSIONnan 8 week course of weekly, frontal half-body irradiation with UVB, at 0.5 MED, leads to an significant increase in 25(OH)D serum levels, but this period is too short to reach vitamin D sufficiency.

Melanogenesis in transfected fibroblasts induces lysosomal activation

Normal melanosome biogenesis requires the association of structural proteins with tyrosinase. 3T3 Swiss fibroblasts transfected with mouse tyrosinase cDNA (line 13.4, clone c) are a unique system in which melanogenesis takes place in the absence of melanosomal structural proteins. Our study confirmed that transfected fibroblasts displayed tyrosinase activity and some of them produced pigment granules. In the absence of melanosomal structural proteins the granules failed both to show a typical ultrastructure and to undergo the usual melanosome ontogenesis. The differentiating agent – dimethyl sulfoxide- increased phaeomelanin production. Pigment was localized in membrane-bound vesicles which were identified as lysosomes by means of immunogold electron microscopy. Cell line 13.4 had higher levels of lysosomal enzymes (β-hexosaminidase, α-mannosidase) than both parental 3T3 cells and clone pKG4 (fibroblasts transfected with the G418 resistance plasmid). Melanosomal proteins act as scavengers of toxic products of melanogenesis, and our results suggest that in their absence cells may employ an alternative mechanism to sequester injurious products.