Healing effect of a spray containing Rhealba oat colloidal extract in an in vitro reconstitution model of skin.

By using a reconstituted skin model, we aimed to evaluate the efficacy of a spray containing Rhealba oat extract on the rapidity of healing and the extension of the newly formed epithelium. A dermal equivalent was first made in a petri dish by combining skin fibroblasts with collagen type I. Then a punch biopsy as a source of epidermal cells was implanted on this dermal equivalent, where a multilayered epidermis developed. The spray containing Rhealba oat extract was added to the culture medium to evaluate epidermal growth by immunohistochemical analysis of mitotic activity (5-bromo-2'-deoxyuridine [BrDu] incorporation). The extension of the neoepithelium in comparison with untreated reconstituted skin over 22 days was evaluated histologically. On day 12, 16% of positive BrDu basal cells was detected after spray treatment in comparison with 4.2% positive cells in untreated reconstituted skin (p < 0.05). During epidermal differentiation between days 12 and 22, we observed a significant increase in the number of cellular epithelial layers after 16 and 18 days of spray treatment. Moreover the extension of re-epithelialization was also significantly increased after spray treatment on days 16 and 18. In conclusion, our results demonstrate a positive modulation of re-epithelialization on a newly formed epithelium by a spray containing Rhealba oat extract displaying a healing effect.

Identification of clustered cells in human hair follicle responsible for MMP-9 gelatinolytic activity: consequences for the regulation of hair growth.

BACKGROUND: The control of human hair follicle growth and differentiation is dependent upon several well-identified factors, including androgens, cytokines, and growth factors. In humans, alopecia androgenetica is a common aging process thought to be regulated through complex genetic imbalances, which also involve several of these crucial identified factors (and probably others not yet characterized), alone or in combination. Among these factors, epidermal growth factor (EGF), as well as pro-inflammatory cytokines, play a pivotal role, as evidenced by their direct inhibitory effects on hair growth both in vitro and in vivo. Following such treatments, the in vitro growth of hair follicles was rapidly arrested and deleterious modifications of hair morphology were also observed. AIM: Because these cytokines act, at least partly, through the induction of matrix metalloproteinases (MMP), and because tissue remodeling occurs during the hair cycle, we attempted to identify and localize MMP in the human pilosebaceous unit. METHOD: We used zymography to observe human hair follicles in culture in vitro. RESULTS: We observed that human hair follicles in culture in vitro mainly and almost exclusively produce MMP-2 and MMP-9 gelatinolytic activities. Furthermore, after stimulation with EGF, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1alpha (IL-1alpha), MMP-9 production was strongly increased. Using immunohistochemistry, we then precisely localized MMP-9 in the lower part of the inner root sheath (Henle's layer) of control human anagen hair follicles. CONCLUSIONS: Cytokine- and EGF-induced upregulation of MMP-9 in the lower epithelial compartment of the human hair bulb is a major mechanism through which hair follicle involution, observed in alopecia, may occur.

Alteration of cytokeratin expression in oral lichen planus.

The purpose of this investigation is to examine the possible biochemical and topographic cytokeratin alterations in lichen planus of oral mucosa. Biopsy samples of clinically normal buccal mucosa (n = 5), normal gingiva (n = 5), lichen planus from buccal mucosa (n = 5), and lichen planus from gingiva (n = 5) were obtained from patients of both sexes. Cytokeratin expression was determined by means of immunohistochemical labeling with use of a battery of monoclonal antibodies against cytokeratins and filaggrin and two-dimensional gel electrophoresis. In buccal mucosa, which is not keratinized cytokeratins 4 and 13 are expressed in the majority. In buccal mucosa lichen planus, the appearance of cytokeratins 1, 2, 10, and 11 coincides with a decrease in cytokeratins 4 and 13 and a moderate increase in cytokeratins 6, 16, 17, and 19. In normal gingiva, which is normally keratinized, the main cytokeratins are 1, 2, 10, and 11. In gingival lichen planus, a slight decrease in these cytokeratins and in cytokeratin 13 expression was noted. Finally, alterations in cytokeratins 5 and 14, explained by marked alterations of basal cells, were observed. The battery of antibodies used in this study, in correlation with two-dimensional gel electrophoresis, could represent useful diagnostic tools that enable the distinction between inflammatory keratosis and so-called quiescent lichen planus. Moreover, this work showed that cytokeratins 1, 2, 10, and 11 and filaggrin are sensitive tools that may help detect early relapse before clinical exacerbation. Finally, these biochemical techniques may be useful to follow the evolution of lichen planus under treatment.

[Topical tretinoin in the treatment of lichen planus and leukoplakia of the oral mucosa. A biochemical evaluation of the keratinization]. / Trétinoïne topique dans le traitement des lichens plans et des leucoplasies de la muqueuse buccale. Evaluation biochimique de la kératinisation.

In earlier work, we demonstrated that 0.1 p. 100 topical tretinoin is clinically effective and well tolerated compared with placebo for the treatment of oral leukoplakia and oral keratosic or erythematous lichen planus. Here we aimed to complete this clinical protocol with histological and biochemical analyses comparing the biopsy specimens collected at inclusion and those collected after 4 months of treatment. Histological results were based on changes in keratinization observed between onset of treatment and 4 months treatment. Biochemical studies included the use of antibodies (anti-cytokeratins 10-11, anti-filaggrine) for the immunohistochemical evaluation of keratinization and 2-dimensional gel electrophoresis for measuring cytokeratins. In patients with lichen planus, histological changes during treatment showed that, in the 10 patients in the tretinoin group, keratinization disappeared in 6 and decreased significantly in 3. Immunohistochemistry revealed that cytokeratins 10-11 and filaggrin disappeared in 57 p. 100 of the patients treated with tretinoin versus 25 p. 100 in the patients given placebo. Bidimensional gel electrophoresis showed that cytokeratins 1, 2, 10 and 11 disappeared only in the tretinoin group (60 p. 100 of the cases). In patients with leukoplakia, histological changes during treatment showed that, in the tretinoin group, keratinization disappeared in 5 cases and decreased in 5 others. Immunohistochemistry revealed that cytokeratins 10-11 disappeared in 30 p. 100 of the patients treated with tretinoin versus 25 p. 100 in the placebo group. Bidimensional electrophoresis demonstrated that cytokeratins 1, 2, 10 and 11 disappeared in 43 p. 100 of the patients treated with tretinoin.

[Topical tretinoin in the treatment of lichen planus and leukoplakia of the mouth mucosa. A clinical evaluation]. / Trétinoïne topique dans le traitement des lichens plans et des leucoplasies de la muqueuse buccale. Evaluation clinique.

A randomized study was conducted to evaluate the effect of tretinoin and patient tolerance to treatment with topical applications in series of 20 cases of smoking-related or traumatic oral keratoses leukoplakia and of 20 cases of lichen planus. In each group, patients applied the topical ointment containing tretinoin (10 patients) or placebo (10 patients) twice daily. Clinical outcome was evaluated on the basis of the surface area of the lesion, measured monthly during treatment, as compared with the area observed at treatment onset. After 4 months treatment, there was a significant decrease in the surface area of the lesion in the patients with lichen planus (p < 0.02): 94 p. 100 in the tretinoin group versus 21.4 p. 100 in the placebo group. In patients with leukoplakias, there was also a very significant reduction in the surface area of the lesion after 4 months of treatment (p < 0.001): 80 p. 100 in the tretinoin group and 16 p. 100 in the placebo group. Tolerance to treatment was generally good despite a few complaints of quite temporary burning sensation at application rapidly resolutive.

Expression of HLA class II antigens on skin fibroblasts in scleroderma.

Skin biopsies were taken from 11 patients with morphoea, nine with acrosclerosis and 10 with diffuse systemic sclerosis and processed for immunohistochemical studies using a panel of monoclonal antibodies including antibodies to MHC class II antigens. A significantly higher percentage of HLA-DR positive dermal cells were observed in the reticular dermis in biopsies from patients with morphoea (44.1 +/- 16.2%), acrosclerosis (15.9 +/- 5.4%) and systemic sclerosis (39.5 +/- 2.3%) when compared with the controls (6.6 +/- 2%). A smaller percentage of dermal cells also expressed HLA-DP and -DQ. The degree of monocnuclear cell infiltrate in the biopsies, however, did not correlate with the percentage of HLA class II positive fibroblasts. In organ culture, the expression of the HLA class II antigens was almost totally lost after 3 days and was no longer detected on fibroblasts after 3 weeks of culture.

Changes in the elastic tissue of the non-sun-exposed skin of cigarette smokers.

Biopsies were taken from the upper and inner arm of 10 60-year-old male cigarette smokers and compared with 10 age-matched controls who were non-smokers. The mean relative area, number and thickness of the elastic fibres were significantly increased in the cigarette smokers compared to the controls. These results were confirmed using antibodies to elastin or the microfibrillar component of elastic tissue. In the smokers the broader and more fragmented elastic fibres in the skin were not as intensely stained as those of the non-smokers and the ultrastructural alterations of the elastic fibres were similar to those in solar elastosis.

Morphometric analysis of dermal collagen fibers in normal human skin as a function of age.

A quantitative study of dermal collagen as a function of age was carried out by computerized digital image analysis. Fast Green-Syrius Red stained sections were obtained of skin biopsies taken from the upper inner arm of 33 healthy women and 38 healthy men. The Leitz texture Analysis System (Leitz-TAS) and mathematical morphology (Serra, 1982) were used for the evaluation of the data. Collagen was studied in the superficial dermis and also in the reticular dermis using the same program. There were significant correlations, firstly between the percentage of collagen measured by the morphometric method and the concentration of collagen analysed biochemically (microg/mm(2) of tissue section) (r = 0.79, p < 0.001) and secondly between the decreased concentration of collagen and age (r = 0.58, p < 0.05). The morphometrical measurements have shown that the relative percentage of collagen bundles (surface of collagen fibers as a function of the dermal area analyzed) was 93.35% in the superficial dermis and 89.2% in the reticular dermis. Although this value is higher than the chemically determined ratio of collagen to other proteins (over 70%), this may be due to the relatively uniform distribution pattern of (type I and III) collagen through the dermis covering most other components of the skin. As the collagen fiber density per unit dermal surface did not change with age, the decrease in collagen content of the skin may be ascribed to the loss of about 6% of dermal mass per decade (Branchet, 1990), although large individual variations exist. The histogram of the diameter distribution of collagen fiber bundles of the reticular dermis showed thinner diameters in persons between 20 and 40 years of age than in older persons. The histogram of the distribution of interfiber spaces did not show any variation with age in the superficial dermis, while in the reticular dermis there was a predominance of smaller interspaces in persons older than 50 years.

Immunohistochemical study of oral lesions of lichen planus: diagnostic and pathophysiologic aspects.

The immunophenotype of lymphoid cells in the epithelium and lamina propria of the oral mucosa were examined in patients with lichen planus, nondysplastic leukoplakia, leukoplakia with lichen planus, and other unrelated lesions. In all groups T lymphocytes were predominant; however, the T4/T8 lymphocyte ratio was higher with lichen planus than with other groups. This may be of diagnostic value in the histologic evaluation of oral lesions not typical of lichen planus. Finally, a higher percentage of Langerhans cells were observed in lichen planus. An immunologic pathogenesis of lichen planus is proposed.

Skin thickness changes in normal aging skin.

The age-dependent decrease of skin thickness was studied with a morphometric procedure on upper inner arm skin biopsies. Epidermal thickness decreased somewhat faster in men (7.2% of the original value/decade) than in women (5.7%). The total dermal thickness decreased at about the same rate in men and women (6%/decade). The thickness of the superficial layer of the dermis exhibited a biphasic evolution with age and these variations were not significantly different between men and women because of the large individual variations. This may be due partially to the difficulties of delineating with precision the limit between superficial and reticular dermis. These results are somewhat lower than those obtained by physical measurements of skin thickness. This may be due to fixation artifacts and also to the overestimation of skin thickness by physical measurements.

Elastic fibers in normal human skin. Variations with age: a morphometric analysis.

A quantitative study of dermal elastic fibers was carried out by computerized digital image analysis. Sections of skin biopsies taken from the upper inner arm of 33 healthy women and 38 healthy men were stained by a selective procedure for elastic fibers. The Leitz texture analysis system (Leitz-Tas) and mathematical morphology were used for the evaluation of the data. Distinct programs were used for the vertical superficial elastic fibers and for the mature elastic fibers of the reticular dermis. For elastic fibers of the superficial dermis there was no significant variation with age or sex for total area occupied by the fibers or for the total number of fibers per unit skin area or total fiber length. The distribution of the relative fiber number as a function of their length showed that about 80% of the fibers had a length of 0-20 microm, with no detectable age- or sex-dependent variation. The distribution of fiber number frequency as a function of fiber diameter did not show any significant variation with sex or age, either. The relative area of mature elastic fibers increased significantly with age after the sixth decade of life. The total number (Nt) of these fibers did not show however any significant variation with age or sex: Nt per mm(2) of skin surface was 3220+/-660 (S.D.). We found for all age groups a significant increase of total fiber length by comparing persons between 20 and 30 years with those above 60 years. There was, however, no significant variations according to age or sex for the frequency distribution of mature elastic fiber length and diameter. As a significant decrease with age of the dermal thickness was also found in males and females, the loss of elastic fibers with age is mainly due to a loss of skin thickness (skin volume) with age. This loss is about 30% at 50 years and nearly 50% at 80 years. Loss of skin elasticity with age can be due to loss of cells (fibroblasts), to a decrease of their biosynthetic activity and to qualitative modifications of extracellular matrix macromolecules.

[Morphometric study of collagen and bone tissue in fibrous dysplasia and ossifying fibroma]. / Etude morphométrique du collagène et du tissu osseux dans la dysplasie fibreuse et le fibrome ossifiant.

Parallel morphometric studies were conducted in the same cases of ossifying fibroma and fibrous dysplasia to confirm differences suggested by the previous study between the two bone diseases.