Francis Pruche

Induction of the skin endogenous protective mitochondrial MnSOD by Vitreoscilla filiformis extract

Vitreoscilla filiformis (Vf), a filamentous bacteria living in fresh water is thought to contribute to the observed beneficial effects of Spa water on skin. An active fraction obtained from a Vf biomass was evaluated for its ability to modulate mRNA expression in cultured skin cells. cDNA array analysis was conducted first using a customized membrane including 1176 selected and fully identified genes involved in skin physiology and homeostasis then the newly developed full genome U133 plus 2.0 GeneChip from Affymetrix. The mitochondrial protective manganese superoxide dismutase (MnSOD/SOD‐2) was identified as a preferentially induced mRNA target in both normal human dermal fibroblasts and keratinocytes. Induction at the transcriptional level in both cell types was confirmed using quantitative real time/polymerase chain reaction and a kinetic analysis revealed a maximal increase in mRNA expression 20 h after stimulation with Vf extract (Vfe). Using immunofluorescent (fluorescent cell sorter) analysis, an induction of MnSOD protein in both normal human dermal skin fibroblasts (×1.6; P < 0.01) and epidermal keratinocytes (×1.4; P < 0.01) was confirmed. As MnSOD is a major inducible free‐radical scavenger in skin, these results suggest that the Vfe could induce skin cells to produce their own endogenous protective defences in vivo against both exogenous environmental stressors such as UV irradiation or microflora as well as to combat endogenous sources of deleterious free radicals involved in skin ageing. Finally, in order to confirm the in vivo potential of this original extract in human, we evaluated its protective activity vs. placebo on the generation of sunburn cells in epidermis under UVB stress. As expected from in vitro profiling, Vfe was indeed found to significantly inhibit the appearance of sunburn cells in UVB‐exposed areas, a signature of skin alteration which has been suggested to be linked to a defect in MnSOD protective activity. Altogether, those data suggest that the combination of a suitable protective UV filter together with this bioactive Vfe might improve skin protection through complementary pathways.

Protective effects of taurine on human hair follicle grown in vitro

Taurine is a naturally occurring β‐amino acid produced by methionine and cysteine metabolism. It is involved in a variety of physiological functions, including immunomodulatory and antifibrotic. Taking advantage of the ability of human hair follicle grown in vitro to recapitulate most of the characteristic features of normal hair follicle in vivo, we studied (i) taurine uptake by isolated human hair follicles; (ii) its effects on hair growth and survival rate; and (iii) its protective potential against transforming growth factor (TGF)‐β1, an inhibitor of in vitro hair growth and a master switch of fibrotic program. We showed that taurine was taken up by the connective tissue sheath, proximal outer root sheath and hair bulb, promoted hair survival in vitro and prevented TGF‐β1‐induced deleterious effects on hair follicle.

Evidence for an age-correlated change in glutathione metabolism enzyme activities in human hair follicle

In this investigation, glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-RD), glutathione-S-transferase (GSH-S-T), gamma-glutamyl transpeptidase (gamma-GT) and glucose-6-phosphate dehydrogenase (G6PDH) were measured in human hair follicle obtained by plucking as source of keratinized cells. This non-invasive method was used on 27 men and women volunteers ranging from 19 to 102 years. Our results show that GSSG-RD, GSH-S-T, gamma-GT and G6PDH activities decrease as a function of age, whereas GSH-PX activity does not vary. We discriminated 2 groups: a first one from 19 to 60 years with a large dispersion of the enzymatic activities and a second one corresponding to elderly people (up to 70) with a smaller dispersion of the values. This study suggests the keratinocytes possess an age-correlated enzymatic detoxification response potential.

Changes in glutathione content in human hair follicle keratinocytes as a function of age of donor: relation with glutathione dependent enzymes

Glutathione (GSH) plays an important role in cellular protection during ageing. The hair plucking technique is a non‐invasive method for the direct biochemical study of keratinocytes. Hair was taken from the suboccipital area of 63 volunteers (men and women whose ages ranged from 13 to 103 years). The results show a diminution in the glutathione content as a function of age. We compare two groups of population: group A (less than 80) and group B (more than 80). A remarkable fact is observed: group B displays a weak dispersion of the values as compared to A. The glutathione content (nmol 10−3 mg DNA) is 5.42 ± 0.60 for A and 1.86 ± 0.35 for B. A reduction of 88% was observed in glutathione reductase activity and of 78% in the activity of glutathione‐S‐transferase from group A to group B. The glutathione peroxidase activity remains relatively constant. The decrease in the GSH concentration and the constancy of the glutathione peroxidase suggest that the capacity of the cell to protect itself from peroxides remains unchanged but that the GSH concentration may become the limiting factor.

Direct quantitative digital autoradiography of testosterone metabolites in the pilosebaceous unit: an environmentally advantageous trace radioactive technology

Androgen metabolism is one of the major keys for a better understanding of conditions such as androchronogenetic alopecia, acne, and other androgen-related skin disorders. This paper addresses the process by which testosterone metabolism leads to the following major androgens: androstenedione, dihydrotestosterone, 3 alpha- and 3 beta-androstanediol, androsterone, epiandrosterone, and androstanedione. Our report describes a methodology developed for the direct quantitative measurement from the silica gel plate of these metabolites. After detailing the chromatographic procedures to achieve the complete separation of the seven testosterone metabolites on a single plate, specifications are given for obtaining accurate measurements by 1) calibration of the radiodetectors and 2) internal and external standardization of samples and plates. Analytical criteria are discussed in terms of comparison of the level of sensitivity, reproducibility, and practicability obtained by both the linear analyzer and the direct digital autoradiograph. Signals as weak as 25 dpm were easily detected and calibration curves were obtained for the range of 50-500 dpm. For biological measurements the coefficients of variation do not exceed 10%. Given the difficulty of obtaining large amounts of microdissected subfractions of the pilosebaceous unit and the necessity of evaluating the complete pattern of testosterone transformed into its 3 alpha,3 beta-, 5 alpha-reduced and 17 beta-dehydrogenated secondary derivatives, digital autoradiography appears to be a powerful yet simple tool for studying androgen metabolism. In addition, this methodology offers an important environmental advantage: the high sensitivity of the detectors makes it possible to minimize the quantities of radioactive materials that must be handled or discarded.