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Organ culture of human scalp skin is usually performed with serum-containing medium, which limits its analytical usefulness. Here we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum-free William's E medium for more than 2 weeks. Active hair shaft growth was visible until day 16 and was significantly enhanced compared with minimum essential medium (MEM) + 10% fetal bovine serum (FBS). Moreover, William's E medium protected better against cell death than MEM + 10% FBS before day 12. Using quantitative immunochemistry, proliferating (Ki-67+) cells could still be observed in the epithelium of hair follicles even on day 17 of serum-free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Giemsa stains revealed mature skin mast cells even after 13 days in culture. The percentage of surviving hair follicles (mostly with catagen-or telogen-like morphology) gradually increased over time displaying mostly catagen hair follicles after 17 days of culture. Although epidermis and hair follicle epithelium showed increasing atrophy and degeneration, and their pigmentation decreased gradually over time, some long-term-surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum-free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than 2 weeks under well-defined experimental conditions. This pragmatic assay invites multiple uses, and may become a valuable tool for both skin and hair research.Key words: hair follicle -hair growth -human scalp skinlong-term organ culture -pigmentation Please cite this paper as: Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serumfree conditions. Experimental Dermatology 2007; 16: 37-44.
To date there is ample evidence that patients with vitiligo accumulate millimolar concentrations of hydrogen peroxide (H2O2) in their epidermis as well as in their blood lymphocytes/monocytes. Several enzymes are affected by this H2O2 including catalase, glutathione peroxidase, and 4 alpha-carbinolamine dehydratase. The latter enzyme disrupts the recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) for the aromatic amino acid hydroxylases as well as the nitric oxide synthases. In this report we have elucidated the influence of H2O2 on dihydropteridine reductase (DHPR), the last enzyme in the 6BH4-recycling process. Here we show for the first time that concentrations of less than 30 microM H2O2 increase DHPR activities, whereas levels greater than 30 microM H2O2 deactivate the enzyme based on the oxidation of Met146 and Met151 in the sequence, consequently leading to disruption of the NADH-dependent enzyme active site. This oxidation was confirmed by Fourier transform-Raman spectroscopy yielding the expected SO band at 1025 cm-1 characteristic of methionine sulfoxide. Hence these results unmasked a novel regulatory mechanism for DHPR enzyme activity. Moreover, we also demonstrated that DHPR activities in whole blood of patients with vitiligo are significantly decreased in untreated patients, whereas activities are normalized after removal of epidermal H2O2 with a topical pseudocatalase (PC-KUS). Taken together, these new data add more evidence to a systemic involvement of H2O2 in the pathomechanism of vitiligo.
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