Assessment of the potential skin irritation of lysine-derivative anionic surfactants using mouse fibroblast and human keratinocytes as an alternative to animal testing

Purpose. The aim of this study was to identify new surfactants with low skin irritant properties for use in pharmaceutical and cosmetic formulations, employing cell culture as an alternative method to in vivo testing. In addition, we sought to establish whether potential cytotoxic properties were related to the size of the counterions bound to the surfactants. Methods. Cytotoxicity was assessed in the mouse fibroblast cell line 3T6, and the human keratinocyte cell line NCTC 2544, using the MTT assay and uptake of the vital dye neutral red 24 h after dosing (NRU). Results. Lysine-derivative surfactants showed higher IC50s than did commercial anionic irritant compounds such as sodium dodecyl sulphate, proving to be no more harmful than amphoteric betaines. The aggressiveness of the surfactants depended upon the size of their constituent counterions: surfactants associated with lighter counterions showed a proportionally higher aggressivity than those with heavier ones. Conclusions. Synthetic lysine-derivative anionic surfactants are less irritant than commercial surfactants such as sodium dodecyl sulphate and Hexadecyltrimethylammonium bromide and are similar to Betaines. These surfactants may offer promising applications in pharmaceutical and cosmetic preparations, representing a potential alternative to commercial anionic surfactants as a result of their low irritancy potential.

Dose-dependent effect of atrial natriuretic peptide on blood pressure, heart rate, and skin blood flow of normal volunteers.

Synthetic atrial natriuretic peptide, containing 26 amino acids in the rat sequence, L-364, 343 (Ileu-ANP), was infused intravenously at increasing rates (1-40 micrograms/min) into four normal volunteers. Mean intraarterial blood pressure decreased and heart rate increased in cumulative-dose-dependent fashion. Skin blood flow as measured with a laser Doppler device rose already with a cumulative dose of 55 micrograms Ileu-ANP and further rises were directly related to dose. The only side effects observed were those accompanying symptomatic hypotension at higher doses. These findings provide strong evidence that Ileu-ANP acts as a vasodilator in normal volunteers.

Online teaching of inflammatory skin pathology by a French-speaking international university network

Introduction: Developments in technology, webbased teaching and whole slide imaging have broadened the teaching horizon in anatomic pathology. Creating online learning material including many types of media like radiologic images, videos, clinical and macroscopic photographs and whole slides imaging is now accessible to almost every university. Unfortunately, a major limiting factor to maintain and update the learning material is the amount of work, time and resources needed. In this perspective, a French national university network was initiated in 2011 to build mutualised online teaching pathology modules with clinical cases and tests. This network has been extended to an international level in 2012-2014 (Quebec, Switzerland and Ivory Coast). Method: One of the first steps of the international project was to build a learning module on inflammatory skin pathology intended for interns and residents of pathology and dermatology. A pathology resident from Quebec spent 6 weeks in France and Switzerland to develop the contents and build the module on an e-learning Moodle platform (http: //moodle.sorbonne-paris-cite.fr) under the supervision of two dermatopathologists (BV, MB). The learning module contains text, interactive clinical cases, tests with feedback, whole slides images (WSI), images and clinical photographs. For that module, the virtual slides are decentralized in 2 universities (Bordeaux and Paris 7). Each university is responsible of its own slide scanning, image storage and online display with virtual slide viewers. Results: The module on inflammatory skin pathology includes more than 50 web pages with French original content, tests and clinical cases, links to over 45 WSI and more than 50 micro and clinical photographs. The whole learning module is currently being revised by four dermatopathologists and two senior pathologists. It will be accessible to interns and residents in spring 2014. The experience and knowledge gained from that work will be transferred to the next international fellowship intern whose work will be aimed at creating lung and breast pathology learning modules. Conclusion: The challenges of sustaining a project of this scope are numerous. The technical aspect of whole-slide imaging and storage needs to be developed by each university or group. The content needs to be regularly updated, completed and its use and existence needs to be promoted by the different actors in pathology. Of the great benefits of that kind of project are the international partnerships and connections that have been established between numerous Frenchspeaking universities and pathologists with the common goals of promoting education in pathology and the use of technology including whole slide imaging. * The Moodle website is hosted by PRES Sorbonne Paris Cité, and financial supports for hardware have been obtained from UNF3S (http://www.unf3s.org/) and PRES Sorbonne Paris Cité. Financial support for international fellowships has been obtained from CFQCU (http://www.cfqcu.org/).

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice.

Critical roles of the nuclear receptor PPARbeta (peroxisome-proliferator-activated receptor beta) in skin wound healing.

The PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. While all three receptors are undetectable in adult mouse interfollicular epidermis, PPARbeta expression and activity is strongly re-activated by inflammatory stimuli during epidermal injury. The pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) stimulates transcription of the PPARbeta gene via an activator protein-1 site in its promoter and it also triggers the production of PPARbeta ligands in keratinocytes. This increase of PPARbeta activity in these cells up-regulates the expression of integrin-linked kinase and 3-phosphoinositide-dependent kinase-1, which phosphorylates protein kinase B-alpha (Akt1). The resulting increase in Akt1 activity suppresses apoptosis and ensures the presence of a sufficient number of viable keratinocytes at the wound margin for re-epithelialization. Together, these observations reveal that PPARbeta takes on multiple roles and contributes favourably to the process of wound closure.

Regulation of epithelial-mesenchymal IL-1 signaling by PPARbeta/delta is essential for skin homeostasis and wound healing.

Skin morphogenesis, maintenance, and healing after wounding require complex epithelial-mesenchymal interactions. In this study, we show that for skin homeostasis, interleukin-1 (IL-1) produced by keratinocytes activates peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) expression in underlying fibroblasts, which in turn inhibits the mitotic activity of keratinocytes via inhibition of the IL-1 signaling pathway. In fact, PPARbeta/delta stimulates production of the secreted IL-1 receptor antagonist, which leads to an autocrine decrease in IL-1 signaling pathways and consequently decreases production of secreted mitogenic factors by the fibroblasts. This fibroblast PPARbeta/delta regulation of the IL-1 signaling is required for proper wound healing and can regulate tumor as well as normal human keratinocyte cell proliferation. Together, these findings provide evidence for a novel homeostatic control of keratinocyte proliferation and differentiation mediated via PPARbeta/delta regulation in dermal fibroblasts of IL-1 signaling. Given the ubiquitous expression of PPARbeta/delta, other epithelial-mesenchymal interactions may also be regulated in a similar manner.

Foot skin blood flow following infrainguinal revascularization for critical lower limb ischemia.

INTRODUCTION: The aim of this study was to assess the blood flow in the feet before and after lower limb revascularization using laser Doppler imaging (LDI). METHODS: Ten patients with critical lower limb ischemia were prospectively enrolled from June to October 2004. All patients underwent successful unilateral surgical interventions including above-knee bypass, distal bypass and endarterectomy. Skin blood flow (SBF) over the plantar surface of both forefeet and heels was measured by LDI 24h before and 10 days after revascularization, expressed in perfusion units (PU), and reported as mean+/-SD. RESULTS: Measurements in the forefoot and heel were similar. Before revascularization mean SBF was significantly lower in the ischemic foot (130+/-71 PU) compared to the contralateral foot (212+/-68 PU), p<0.05. After revascularization a significant increase of the SBF in the forefoot (from 135+/-67 to 202+/-86 PU, p=0.001) and hindfoot (from 148+/-58 to 203+/-83, p=0.001) was observed on the treatment side. However, a large decrease of the SBF was seen in forefoot and hindfoot on the untreated side (from 250+/-123 PU to 176+/-83 and from 208+/-116 to 133+/-40, p=0.001, respectively). CONCLUSION: This study confirms the benefits of revascularization in patients with nonhealing foot lesions due to critical limb ischemia. A significant increase of the SBF was observed on the treatment side. However, an unexpected decrease was observed on the untreated side.

The anti-apoptotic role of PPARbeta contributes to efficient skin wound healing.

PPARalpha and PPARbeta are expressed in the mouse epidermis during fetal development, but their expression progressively disappears after birth. However, the expression of PPARbeta is reactivated in adult mice upon proliferative stimuli, such as cutaneous injury. We show here that PPARbeta protects keratinocytes from growth factor deprivation, anoikis and TNF-alpha-induced apoptosis, by modulating both early and late apoptotic events via the Akt1 signaling pathway and DNA fragmentation, respectively. The control mechanisms involve direct transcriptional upregulation of ILK, PDK1, and ICAD-L. In accordance with the anti-apoptotic role of PPARbeta observed in vitro, the balance between proliferation and apoptosis is altered in the epidermis of wounded PPARbeta mutant mice, with increased keratinocyte proliferation and apoptosis. In addition, primary keratinocytes deleted for PPARbeta show defects in both cell-matrix and cell-cell contacts, and impaired cell migration. Together, these results suggest that the delayed wound closure observed in PPARbeta mutant mice involves the alteration of several key processes. Finally, comparison of PPARbeta and Akt1 knock-out mice reveals many similarities, and suggests that the ability of PPARbeta to modulate the Akt1 pathway has significant impact during skin wound healing.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin.

BACKGROUND: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth. METHODOLOGY AND PRINCIPAL FINDINGS: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia. SIGNIFICANCE: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

No implication of thromboxane-prostanoid receptors in reactive hyperemia of skin skeletal muscle in human forearm

L'hyperhémie réactive, définie comme l'augmentation transitoire du flux sanguin après une courte période d'ischémie, pourrait être influencée par des vasoconstricteurs de la famille des prostanoïdes, telle que la thromboxane. Le terutroban (S18886) est un antagoniste spécifique des récepteurs à la thromboxane. L'étude présentée a cherché à déterminer l'effet du terutroban sur l'hyperhémie réactive dans la peau et le muscle squelettique de l'avant-bras de volontaires sains. Vingt volontaires sains ont été randomisés en aveugle pour recevoir oralement 30mg/j de terutroban ou un placebo pendant 5 jours puis réciproquement pendant une deuxième période de 5 jours, selon un schéma cross-over. L'ischémie transitoire a été provoquée par l'occlusion de l'artère brachiale par une manchette gonflée au dessus de la pression systolique. L'hyperhémie réactive était évaluée dans les tissus de l'avant- bras, en mesurant le flux sanguin, pour la peau par une méthode laser Doppler, et pour le muscle au moyen d'une pléthysmographie par jauge de contrainte durant une occlusion veineuse. Au premier et au dernier jour de chaque période de traitement, l'hyperhémie réactive était mesurée avant et 2 heures après l'ingestion du comprimé. Que ce soit dans la peau ou le muscle, le terutroban n'a pas montré d'effet sur le flux de pic post-occlusion ni sur la réponse globale d'hyperhémie, exprimée en aire sous la courbe. En conclusion, dans la peau et le muscle de sujets sains, l'hypérémie réactive n'est pas influencée par les récepteurs spécifiques à la thromboxane.

Peroxisome proliferator activated receptor BETA/DELTA as a central player in the skin response to ultra-violets radiation

Previous studies in the lab of Dr. Liliane Michalik, have shown thai the nuclear hormone receptor Peroxisome Proliferator Activated Receptor beta/delta (PPARß/ö) is an important regulator of skin homeostasis, being involved in the regulation of keratinocyte differentiation, inflammation, apoptosis, arid mouse skin wound healing. Studies of PPARß/ö knock out mice have suggested a possible role for this receptor in cancer. However, contradictory observations of the role for PPARß/ö on tumor growth have been published, depending on cellular contexts and biological models. Given the controversial role of PPARß/ö in skin carcinoma development, the main aim of this PhD work has been to further explore the implication of PPARß/ö in skin response to UV and skin tumor growth. This PhD dissertation is divided in four chapters. The first chapter describes the core part of the project, where I explored the changes in miRNA expression in the skin upon chronic UV irradiation of PPARß/ö wild type and knock-out mice. This analysis shed light on a miRNA- PPARß/ö signature and also predicted thai miR-21-3p (previously named miR-21*) is a key regulator of the PPARß/ö-dependent UV response in the pre-lesiona! skin. Using mice acutely UV-irradiated, ! further demonstrated that miR-21-3p is indirectly regulated by PPARß/ö through activation of Transforming Growth Factor (TGFß)-1 under UV exposure. I also show that miR-21-3p is deregulated in human cutaneous squamous celi carcinoma. In cultured keratinocytes, application of a miR-21 -3p mimic oligonucleotide sequence leads to the regulation of lipid metabolism-related pathway. In the second chapter, I demonstrate that the usage of an mRNA/miRNA combined bioinformatics analysis leads to the discovery of important pathways involved in the PPARß/ö-miRNA response of the skin to chronic UV irradiation, indeed, I validated angiogenesis and lipid metabolism as important functions regulated by PPARß/ö in this context. In the third chapter, we demonstrate that PPARß/5 knockout mice have decreased cutaneous squamous cell carcinomas incidence compared to wild type mice and that PPARß/5 directly activates the cSrc kinase gene. In the last chapter, we review novel insights into PPAR functions in keratinocytes and liver, with emphasis on PPARß/ö but also on PPARa. In summary, this PhD study shows that i) PPARß/5 is able to regulate biological function through regulation of miRNAs, and specifically through miR-21-3p, the passenger miRNA of the oncomiR miR-21, and that ii) the PPARß/5-dependent skin response to UV involves the regulation of angiogenesis and lipid metabolism. Furthermore, the bioinformatics study highlights the relevance of performing integrated mRNA and miRNA genome-wide studies in order to better screen mRNAs and/or miRNAs of interest in the biological context of diseases. - Des études préalables dans le laboratoire du Dr. Liliane Michalik ont démontré que le récepteur nucléaire PPARß/5 est un régulateur important de l'homéostasie de la peau, étant impliqué dans la régulation de la différenciation des keratinocytes, dans l'inflammation, dans l'apoptose et dans la cicatrisation de la peau chez !a souris. L'étude de souris knock-out pour le gène PPARß/5, ont suggérées un rôle possible de ce récepteur dans le cancer. Cependant, des observations opposées ont été publiées suggérant un rôle pro- ou anti- cancer selon le tissue impliqué et le type- cellulaire. En considérant cette controverse autour du rôle de PPARß/5 dans le développement des cancers de la peau, le but principal de mon projet de recherche aura été d'approfondir l'exploration du rôle de PPARß/5 dans la réponse de la peau aux UVs et dans le développement du cancer. Cette dissertation de thèse est divisée en quatre parties. Une première partie, représentant le coeur de mon travail de recherche, décrit la découverte de l'implication des microRNAs (rniRNAs) dans la réponse aux UVs de PPARß/ö et plus spécifiquement l'implication du miRNA miR- 21 -3p (précédemment nommé miR-21*). En étudiant un modèle de souris irradiées de manière aigüe aux UVs, nous montrons que ia régulation de miR-21-3p est PPARß/ö-däpenaante et que cette régulation à lieu par l'intermédiaire du facteur de transcription TGFß-1. Dans des cultures de keratinocytes Humains, la transfecticn d'une séquence oligonucléotidique similaire à celle de miR-21-3p (mimic), montre l'implication de rniR-21-3p dans des fonctions importantes pour le développement des cancers telles que le métabolisme des lipides. Dans un second chapitre, nous montrons que l'usage d'une méthode bioinformatique combinant l'expression des ARN messagers et des miRNAs permet de mettre en évidence des fonctions biologiques importantes lors de ia réponse de PPARß/ö à l'irradiation chronique. L'angiogenèse, le stress oxydatif et le métabolisme des lipides font partie de ces fonctions régulées par PPARß/5 dans la peau irradiée aux UVs. Nous mettons également en évidence la régulation du gène LpcatS par PPARß/5 dans la peau irradiée aux UV ainsi que dans des keratinocytes humains suggérant un rôle pour PPARß/5 dans le remodelage des lipides membranaires. Dans une troisième partie, nous établissons un lien entre la régulation de l'oncogène Src et l'activation de PPARß/5 dans les carcinomes spinocellulaires de la peau. Finalement dans un quatrième chapitre, nous faisons une revue des dernières recherches portées sur le rôle de PPARß/5 et de PPARa dans le foie et ia peau. En résumé ce projet de thèse représente un avancement pour la recherche sur rimplication de PPARß/5 dans la réponse aux UVs de la peau. Pour la première fois, un lien est établi entre ce facteur de transcription et la régulation de microRNAs dans le cadre du carcinome spinocellulare. Jusqu'alors resté dans l'ombre de rniR-21-5p, miR-21-3p est en fait fortement augmenté à la fois dans un modèle de souris d'irradiation aux UVs ainsi que dans ie carcinome spinocellulare chez i'humain. De nouvelles fonctions biologiques pour PPARß/5 ont été également mises en évidence dans ce travail, comme la régulation de l'angiogenèse ou du métabolisme des lipides dans Sa peau. De plus cette dissertation valorise l'intérêt d'une association entre le travail de laboratoire et celui de la bioinformatique.