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The aims of the study were to write an image analysis (IA) program allowing the stereological quantification of human epidermal melanocyte melanization at the ultrastructural level and to specify the suitable preparative methods, in keeping with IA limits and stereological principles. Micrographs of cultured human melanocytes obtained in transmission electron microscopy were digitized with a scanner. The key step of the designed IA program is a thresholding based on the gray levels. Hence, gray level histograms (pixel frequency as a function of gray level) of melanocyte images exhibit a peak specific to melanin. The gray level thresholding used consists in isolating the melanin pixels that form profiles on a binary image and in storing the numerical data produced for a given melanocyte profile. These primary data are used to calculate numerous parameters via stereology with melanocyte cytoplasm and melanized melanosome as main reference spaces. The most important stereological parameters obtained are v(mi,cy) (melanin volume per average cell), v(mi,m) (melanin volume per average melanized melanosome), and nm (number of melanized melanosomes per average cell), and their validity is discussed. Melanocytes embedded in situ were abandoned for stereological reasons but pelleted melanocytes were found suitable. Using this computerized tool and stereology, we are able to perform quantitative studies producing varied data even from small cell samples. To our knowledge, this is the first stereological approach for quantifying intracellular melanization. A quantitative comparison of spectrophotometrical results (melanin assay) with stereological results obtained in ultraviolet B-irradiated Caucasian epidermal melanocytes will be performed in order to appraise this method.

No abstract

The aims of the study were to write an image analysis (IA) program allowing the stereological quantification of human epidermal melanocyte melanization at the ultrastructural level and to specify the suitable preparative methods, in keeping with IA limits and stereological principles. Micrographs of cultured human melanocytes obtained in transmission electron microscopy were digitized with a scanner. The key step of the designed IA program is a thresholding based on the gray levels. Hence, gray level histograms (pixel frequency as a function of gray level) of melanocyte images exhibit a peak specific to melanin. The gray level thresholding used consists in isolating the melanin pixels that form profiles on a binary image and in storing the numerical data produced for a given melanocyte profile. These primary data are used to calculate numerous parameters via stereology with melanocyte cytoplasm and melanized melanosome as main reference spaces. The most important stereological parameters obtained are v (mi,cy) (melanin volume per average cell), v (mi,m) (melanin volume per average melanized melanosome), and n m (number of melanized melanosomes per average cell), and their validity is discussed. Melanocytes embedded in situ were abandoned for stereological reasons but pelleted melanocytes were found suitable. Using this computerized tool and stereology, we are able to perform quantitative studies producing varied data even from small cell samples. To our knowledge, this is the first stereological approach for quantifying intracellular melanization. A quantitative comparison of spectrophotometrical results (melanin assay) with stereological results obtained in ultraviolet B‐irradiated Caucasian epidermal melanocytes will be performed in order to appraise this method. Microsc. Res. Tech. 36:188–200, 1997. © 1997 Wiley‐Liss, Inc.

Encapsulation of DNA in a new non-cationic multilamellar vector (Spherulites), composed of phosphatidylcholine, cholesterol and polyoxyethylene alcohol, is described here for the first time. Spherulites entrapping DNA were prepared by shearing a phospholipid lyotropic lamellar phase, using a recently discovered method. The average diameter of these vesicles ranges around 300 nm, and can be adjusted depending on the conditions of the process. The formulation did not result in cytotoxicity for the human cells and could be used as a DNA delivery system. More emphasis is brought to the role of condensing agents like histones on the encapsulation yield, which has been studied using radiolabelled DNA. It is shown that use of histones (histone to DNA ratio of 0.4) can increase significantly the encapsulation of DNA, thus improving the transfection efficiency. Transfection experiments were done with success using the beta-galactoside reporter gene on human primary cells (human skin fibroblasts and human bone marrow stromal cells). The results suggest that the spherulites have to be considered as a new and promising tool for gene transfection.