A re-innervated in vitro skin model of non-histaminergic itch and skin neurogenic inflammation: PAR2-, TRPV1- and TRPA1-agonist induced functionality.

Background: Skin, and epidermis, is innervated by sensory nerve fibres. Interactions between them and signal transduction are only partially elucidated in physiological/pathological conditions, especially in pruritus. Objectives: To study the mechanisms involved in pruritus in vitro, we developed a skin explant model re-innervated by sensory neurons. Methods: This model is based on the co-culture of human skin explants and sensory neurons from dorsal root ganglia of rats. Innervation and the expression of protease activated receptor 2 (PAR2), transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin one (TRPA1) was analysed by immunostaining. The response of the model to TRPV1, PAR2 and TRPA1 agonists was analysed by patch-clamp, qPCR and enzyme-linked immunosorbent assay. Results: After 5 days of re-innervating nerve fibres was evidenced in the epidermis. Re-innervation was correlated with decrease of epidermal thickness and the number of apoptotic cells in the tissue. The major actors of non-histaminergic itch (PAR-2, thymic stromal lymphopoietin [TSLP], TSLP-R, TRPA1 and TRPV1) were expressed in neurons and/or epidermal cells of skin explants. After topical exposure of TRPV1-(Capsaicin), TRPA1-(Polygodial) and PAR2-agonist (SLIGKV-NH2) activation of reinnervating neurons could be shown in patch-clamp analysis. The release of TSLP was increased with capsaicin or SLIGKV but decreased with polygodial. Release of CGRP was increased by capsaicin and polygodial but decreased with SLIGKV. Activation by SLIGKV showed a decrease of VEGF; polygodial induced an increase of TSLP, Tumour necrosis factor (TNF) and nerve growth factor and capsaicin lead to a decrease of sema3 and TNF expression. Conclusion: The present model is suitable for studying itch and neurogenic inflammation pathways in vitro. We observed that activation of TRPV1, TRPA1 and PAR-2 leads to different response profiles in re-innervated skin explants.

4-Hexyl-1,3-phenylenediol, a nuclear factor-κB inhibitor, improves photodamaged skin and clinical signs of ageing in a double-blinded, randomized controlled trial.

BACKGROUND: The nuclear factor-κB (NF-κB) pathway is a key mediator of inflammation; however, few studies have examined the direct effects of NF-κB inhibition on the skin. OBJECTIVES: To investigate NF-κB activity in cultured human fibroblasts and to investigate the effects of 4-hexyl-1,3-phenylenediol (an NF-κB inhibitor) on elastin and collagen gene expression in vitro and on the clinical appearance of photodamaged skin. METHODS: The amount and activity of NF-κB in human fibroblasts obtained from donors (17-78 years old) was measured after transfection with a NF-κB reporter and a luciferase promoter system. The expression of extracellular matrix (ECM) genes was determined using quantitative polymerase chain reaction. Women with moderate skin photodamage were randomized to daily treatment with a topical lotion containing 4-hexyl-1,3-phenylenediol (n = 30) or vehicle (n = 29) for 8 weeks, with clinical assessments at baseline and weeks 2, 4 and 8. RESULTS: Fibroblasts obtained from donors older than 50 years had higher NF-κB activity compared with cells from younger donors; inhibition of the NF-κB pathway with 4-hexyl-1,3-phenylenediol enhanced the expression of ECM genes. In women, treatment for 8 weeks with 4-hexyl-1,3-phenylenediol significantly improved crow's feet fine lines, cheek wrinkles, age spots, mottled pigmentation and radiance compared with both the vehicle and baseline. Furthermore, treatment with 4-hexyl-1,3-phenylenediol resulted in a twofold greater clinical improvement in overall photodamage compared with the vehicle group. CONCLUSIONS: Inhibition of the proinflammatory NF-κB pathway resulted in increased expression of ECM proteins in vitro and significant clinical improvement in photodamaged skin.

Evaluation of the efficacy of a topical cosmetic slimming product combining tetrahydroxypropyl ethylenediamine, caffeine, carnitine, forskolin and retinol, In vitro, ex vivo and in vivo studies.

Three studies were performed to investigate the mechanism of action and evaluate the efficacy of a topical cosmetic slimming product combining tetrahydroxypropyl ethylenediamine, caffeine, carnitine, forskolin and retinol. The Ex vivo study on skin explants showed that caffeine and forskolin both stimulated glycerol release and demonstrates for the first time that retinol and carnitine in combination synergistically stimulated keratinocyte proliferation, which leads to an increase epidermal thickness. The double-blind, randomized, placebo-controlled clinical study associating circumference measurements on five selected parts of the body, cutaneous hydration measurements as well as blinded expert grading of skin aspect was conducted on 78 women who applied the product or placebo twice daily for 12 consecutive weeks. After 4 weeks of twice-daily application of the product, significant reductions in circumference of abdomen, hips-buttocks and waist were already observed. Improvements concerned all the measured body parts after 12 weeks. Orange peel and stubborn cellulite decreased significantly from 4 weeks of treatment and tonicity improved from 8 weeks, demonstrating that the product improved skin aspect. At the end of the study, eight parameters of the thirteen evaluated were significantly improved in the active group and compared with placebo.

A novel anti-ageing mechanism for retinol: induction of dermal elastin synthesis and elastin fibre formation.

Dermal elastic fibres are extracellular matrix protein complexes produced by fibroblasts and involved in skin elasticity. Elastin fibres decrease with age as a result of reduced synthesis and increased degradation, resulting in skin sagging and reduced skin elasticity. In this study, we show that retinol (ROL), known to enhance dermal collagen production, is also enhancing elastin fibre formation. ROL induced elastin gene expression and elastin fibre formation in cultured human dermal fibroblasts. Topical treatment of cultured human skin explants with a low dose (0.04%) of ROL increased mRNA and protein levels of tropoelastin and of fibrillin-1, an elastin accessory protein, as documented by QPCR and immunohistochemistry staining. Luna staining confirmed the increased elastin fibre network in the ROL-treated skin explants, as compared with untreated controls. These data demonstrate that ROL exerts its anti-ageing benefits not only via enhanced epidermal proliferation and increased collagen production, but also through an increase in elastin production and assembly.

Antiaging action of retinol: from molecular to clinical.

The antiaging efficacy of retinol (ROL) has been explored mainly clinically in photoprotected skin sites and for high doses of ROL (0.4-1.6%). The objective of the study was to demonstrate the antiaging action of a low and tolerable dose of ROL (0.1%) ex vivo by measuring the expression of cellular retinoic-acid-binding protein II (CRABP2) and heparin-binding epidermal growth factor (HBEGF) by a histological evaluation of the epidermis and in vivo by assessing major aging signs and performing three-dimensional profilometry and digital imaging during a 9-month double-blind placebo-controlled study involving 48 volunteers. Finally, epidermal cell proliferation was evaluated using tryptophan fluorescence spectroscopy. Our results demonstrate that 0.1% ROL induced CRABP2 and HBEGF gene expression and increased keratinocyte proliferation and epidermal thickness. In human volunteers, topical application of a ROL-containing product improved all major aging signs assessed in our study (wrinkles under the eyes, fine lines and tone evenness). Moreover, tryptophan fluorescence increased in the active-agent-treated group and not in the placebo-treated group, indicating that cell proliferation was accelerated in vivo. These data demonstrate that a product containing a low dose (0.1%) of ROL promotes keratinocyte proliferation ex vivo and in vivo, induces epidermal thickening ex vivo and alleviates skin aging signs, without any significant adverse reaction.

A microarray to measure repair of damaged plasmids by cell lysates.

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Expression studies of key adipogenic transcriptional factors reveal that the anti-adipogenic properties of retinol in primary cultured human preadipocytes are due to retinol per se.

The aim of this study was to investigate the mechanism(s) underlying the antiadipogenic effect of retinol that we recently reported in primary cultured human preadipocytes. Exposure of human preadipocytes to the potent alcohol dehydrogenase inhibitor, 4-methyl-pyrazole, failed to alter the antiadipogenic effect of retinol (3.5 microm), suggesting that the latter effect is due to retinol per se rather than to its oxidation product, retinoic acid (RA). Moreover, retinol, in contrast to what is generally observed with RA, did not alter the expression of the major adipogenic transcriptional factors PPARgamma and C/EBPalpha but, like RA, reduced transcription of an adipospecific gene controlled in part by C/EBP, the ob gene. These results indicate that retinol per se inhibits the adipo-conversion of human preadipocytes and suggest that the mechanisms of this antiadipogenic action implies at least in part inhibition of C/EBP transcriptional activity.

Erythropoiesis in long term cultures of foetal liver cells is transiently obtained on adult but not on foetal adherent cell layers.

We have previously reported long term erythroid differentiation of adult bone marrow cells seeded onto adherent cells derived from adult bone marrow. In this paper, we show that the adherent cells obtained from foetal liver do not support the erythroid differentiation of either adult bone marrow cells or foetal liver cells. Adherent layers derived from bone marrow of adult W/Wv mice supported differentiation of adult bone marrow precursors, but foetal liver progenitors only produced erythrocytes for a few weeks and the foetal origin of these red cells was confirmed by haemoglobin typing. The duration and extent of erythropoiesis was generally inversely proportional to the cell dose. Foetal progenitors were as sensitive to erythropoietin as adult cells, but were optimally stimulated at a lower plateau concentration. These results suggest that inhibitory cells present in foetal liver may block erythropoiesis and their growing importance with age may provide an explanation for the arrest of erythropoiesis in the liver at late developmental stages.

Kidney cell lysates contain an activity that stimulates mature erythroid burst-forming-unit (mBFU-E) proliferation.

This study reports the detection of an activity that stimulates the development of a subclass of burst-forming unit-erythroid (BFU-E) progenitors giving rise to small bursts in semi-solid cultures established in the presence of saturating concentrations of erythropoietin. These progenitors are considered to be mature BFU-E. The activity is found in extracts from kidney cells and appears to be physiologically regulated as it was respectively enhanced and decreased in kidneys from anemic and polycythemic mice. The disappearance of activity in kidney-cell extracts during long-term polycythemia correlated with an accumulation of mature BFU-E in the spleen and bone marrow of polycythemic mice. Using specific neutralizing antibodies and in vitro tests, we also show that this activity is different from hemopoietins known to share burst promoting activity (Interleukin-3 [IL-3], granulocyte-macrophage colony-stimulating factor [GM-CSF], Interleukin-4 [IL-4], erythropoietin [EPO], human interleukin for DA cells [HILDA]) and that it can stimulate erythroid differentiation in long term bone marrow cell cultures.

Erythropoiesis in murine long-term bone-marrow cell cultures: dependence on erythropoietin and endogenous production of an erythropoietic stimulating activity.

Erythropoiesis was obtained in murine long-term bone-marrow cell cultures (LTBMCs) in the presence of erythropoietin (Epo) when the medium was frequently renewed. The level of the erythropoietic differentiation was shown to be a function of the erythropoietin concentration. In response to Epo addition, an activity which stimulates CFU-E proliferation in semisolid cultures of fresh bone marrow cells was detected in the LTBMC supernatants. These results suggest that another factor, whose synthesis may be under Epo control, participates in the stimulation of erythropoiesis in vitro.