Background: The etiology and different inflammatory steps associated with the development of an acne nodule remain unsolved. Objectives: This study aimed to investigate the main biological processes involved in acne nodules and compare them to those of papules. Methods: Nodules, papules, and non-involved skin of the back (control) were biopsied to perform proteomic analysis using mass spectrometry, Luminex assay, and elastase staining on skin sections. Results: Many factors involved in the migration and function of immune cells, particularly those impacting leukocytes and neutrophils, were strongly and significantly higher in nodules than in papules and non-involved skin, while several enzymes involved in lipid metabolism were lower. Elastase staining confirmed strong neutrophil infiltration within and around the nodules. Conclusions: Our results highlight the role of neutrophils during nodule formation in severe nodular acne of the back.

Onychomycosis is mainly caused by two dermatophyte species, Trichophyton rubrum and Trichophyton interdigitale. A study of nail invasion mechanisms revealed that the secreted subtilisin Sub6, which has never been detected under in vitro growth conditions, was the main protease secreted by T. rubrum and T. interdigitale during infection. In contrast, most of the proteases secreted during the digestion of keratin in vitro were not detected in infected nails. The hypothesis that proteases isolated from dermatophytes grown in a keratin medium are virulence factors is no longer supported. Non-dermatophyte fungi can also be infectious agents in nails. It is necessary to identify the infectious fungus in onychomycosis to prescribe adequate treatment, as moulds such as Fusarium spp. and Aspergillus spp. are insensitive to standard treatments with terbinafine or itraconazole, which are usually applied for dermatophytes. In these refractory cases, topical amphotericin B treatment has shown to be effective. Terbinafine treatment failure against dermatophytes is also possible, and is usually due to resistance caused by a missense mutation in the squalene epoxidase enzyme targeted by the drug. Trichophyton resistance to terbinafine treatment is an emerging problem, and a switch to azole-based treatment may be necessary to cure such cases of onychomycosis.

Background Onychomycosis is the most prevalent nail disease and is mainly caused by two dermatophyte species Trichophyton rubrum and Trichophyton interdigitale with a frequency in the range of 80% and 20%, respectively. The secreted protease Sub6 of the subtilisin family, which was never detected in vitro growth conditions, was found to be a robust marker of onychomycosis. Objective The aim of this work was to detect tinea unguium using anti-Sub6 monoclonal antibodies in proteins extracted from clinical nail samples. Methods We produced monoclonal antibodies in mice using recombinant Sub6 as an antigen. Selected monoclonal antibodies were tested by Western blot analysis and ELISA on protein extracts from onychomycosis samples. Results Several monoclonal antibodies used to quantify Sub6 in proteins extracted from clinical nail samples were produced and characterised. We showed that these antibodies were very specific and allowed the detection of T. rubrum and T. interdigitale in onychomycosis. Sub6 was detected in clinical samples infected by T. rubrum and not detected in nails with trauma and other diseases. Conclusion Anti-Sub6 monoclonal antibodies could be useful for a rapid diagnosis of tinea unguium and/or therapeutic survey of dermatophyte in onychomycosis by ELISA or an immunochromatography device such as a strip test.

Acne vulgaris is a chronic inflammatory skin condition. Skin biopsies are used for detecting inflammation at the cellular and molecular level by different OMICs technologies. However, biopsies are invasive procedures that can be associated with scarring, pain and risk of infection; furthermore, they are not suitable for the analysis of facial skin areas for evident ethical reasons. Recently tape stripping has emerged as a new possible technique for biomarker analysis and inflammatory profiling in several skin diseases including atopic dermatitis and psoriasis [1-3]. This article is protected by copyright. All rights reserved.

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.

Background Rosacea is a chronic inflammatory skin disease. Characteristic vascular changes in rosacea skin include enlarged, dilated vessels of the upper dermis and blood flow increase. Brimonidine is approved for symptomatic relief of the erythema of rosacea. It acts by selectively binding to alpha 2-adrenergic receptors present on smooth muscle in the peripheral vasculature, resulting in transient local vasoconstriction. Objectives Methods To provide further evidence of the anti-inflammatory potential of brimonidine across preclinical models of skin inflammation and its ability to decrease the neutrophil infiltration in human skin after ultraviolet light exposure. The anti-inflammatory properties of brimonidine through modulation of the vascular barrier function were assessed using in vivo neurogenic vasodilation and acute inflammatory models and a well-described in vitro transmigration assay. A clinical study assessed the neutrophil infiltration in human skin after exposure to UV in 37 healthy Caucasian male subjects. Results Conclusion In vitro, brimonidine affects the transmigration of human neutrophils through the endothelial barrier by modulating adhesion molecules. In vivo, in the mouse, topical treatment with brimonidine, used at a vasoconstrictive dose, confirmed its anti-inflammatory properties and prevented leucocyte recruitment (rolling and adhesion) mediated by endothelial cells. Topical pretreatment with brimonidine tartrate 0.33% gel once a day for 4 days significantly prevented neutrophil infiltration by 53.9% in human skin after exposure to UV light. Results from in vitro, in vivo and from a clinical study indicate that brimonidine impacts acute inflammation of the skin by interfering with neurogenic activation and/or recruitment of neutrophils.

Histone deacetylases (HDACs) are key enzymes involved in epigenetic modulation and were targeted by HDAC inhibitors (HDACis) for cancer treatment. The action of HDACis is not restricted to histones and also prevents deacetylation of other proteins, supporting their wide biological actions. The HuT78 cell line is recognized as a key tool to support and understand cutaneous T-cell lymphoma (CTCL) biology and was used as a predictive model since HDACi such as Vorinostat and Panobinostat have both demonstrated apoptotic activities in HuT78 cells and in primary blood CTCL cells. In this study, Quisinostat (JNJ-26481585) a novel second generation HDACi with highest potency for HDAC1, was tested on HuT78 cell line. Quantitative mass spectrometry (MS)-based proteomics after acetylated-lysine peptide enrichment and a targeted antibody -based immunoassay (DigiWest) were used as complementary technologies to assess the modifications of the acetylated proteome. As expected, several acetylated lysines of histones were increased by the HDACi. Additional acetylated non-histone proteins were modulated after treatment with Quisinostat including the nucleolin (a major nucleolar protein), the replication protein A 70 kDa DNA-binding subunit, the phosphoglycerate kinase 1, the stress-70 protein, the proto-oncogene Myc and the serine hydroxymethyltransferase. A better knowledge of histone and non-histone acetylated protein profile after Quisinostat treatment can strongly support the understanding of non-clinical and clinical results of this HDACi. These technological tools can also help in designing new HDACis in a pharmaceutical drug discovery program. Significance: A better knowledge of histone and non-histone acetylated protein profile after HDAC inhibitors (HDACis) treatment can strongly support the understanding of non-clinical and clinical investigations in a pharmaceutical drug discovery program. Relative quantification using mass spectrometry -based proteomics after acetylated-lysine peptide enrichment and a targeted antibody -based immunoassay (DigiWest) are proposed as complementary technologies to assess the modifications of the acetylated proteome. Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1 was better characterized in vitro in HuT78 cells to support and understand cutaneous T-cell lymphoma (CTCL) therapeutic research program.

BACKGROUND:Protein expression is disturbed in the psoriatic stratum corneum (SC). Noninvasive methods for the description of pathophysiological changes and drug profiling in psoriasis are desirable.OBJECTIVES:Undertake large-scale noninvasive protein expression studies in psoriatic SC to identify biomarkers of pathophysiological processes and use them for drug profiling.METHODS:Psoriatic SC was harvested through repetitive tape-stripping. Nonlesional and lesional SC, as well as vehicle-treated and drug-treated lesional SC samples were collected. Protein extracts from nonlesional and lesional skin biopsies were used for comparison. Calcipotriol-betamethasone (CB) was used as a reference medication. Proteins extracted from pooled tape strips were quantified using mass spectrometry (MS), Western blotting, enzyme-linked immunosorbent assay and Luminex technologies.RESULTS:MS-based methods identified 140 proteins differentially expressed in psoriatic SC. Epidermis development, glycolysis, regulation of apoptosis, cytoskeleton organization and peptide cross-linking were modulated, all reflecting perturbed epidermal differentiation. Using antibody-based techniques, increased levels of sICAM1, of CXCL1- and CXCL8-attracting neutrophils, of CXCL10- and CCL4-attracting T helper (Th) 1 cells, and of CCL2- and CCL4-attracting monocytes and dendritic cells were observed. Quantification of the Th1 and Th17 markers tumour necrosis factor, interleukin (IL) 12B, IL17A and IL17F in lesional SC was successful, while the Th2 cytokines IL4, IL5 and IL13, not involved in the disease process, were not detected. The pruritic cytokine IL31 was detected in lesional SC. CXCL1, CXCL8, CXCL10 and sICAM were used to investigate disease remission, ranking three topical treatments according to their known clinical efficacy.CONCLUSIONS:Protein biomarker quantification in psoriatic SC detects key pathophysiological mechanisms and enables noninvasive drug profiling in translational medicine settings.

Onychomycosis, the most prevalent nail disease, is mainly caused by two dermatophyte species, Trichophyton rubrum and Trichophyton interdigitale, with a frequency in the range of 80% and 20%, respectively (Monod et al., 2002Monod M. Jaccoud S. Zaugg C. Léchenne B. Baudraz F. Panizzon R. Survey of dermatophyte infections in the Lausanne area Switzerland.Dermatology. 2002; 205: 201-203Crossref PubMed Scopus (54) Google Scholar). To determine if the proteases secreted by T. rubrum in vitro during keratin digestion were involved in nail degradation, we investigated the fungus secretome in onychomycosis by proteomics analysis. In a first experiment, mass spectrometry analyses were performed using a pool of extracts from 12 donors infected by T. rubrum as described in the Supplementary Materials and Methods online. Patient consent for experiments was not required because French laws consider human tissue leftover from surgery as discarded material. Proteins were extracted from each sample using a nonionic acid labile surfactant (ALS-400). The secretion of the following four secreted proteases of T. rubrum proteins extracted from nail beds was identified after subsequent SDS-PAGE separation and in-gel digestion coupled to mass spectrometry analysis: subtilisin-like protease 6 (Sub6, Q9UW97), subtilisin-like protease 7 (Sub7, Q8NID9), dipeptidyl-peptidase 5 (DppV, Q9UW98), and leucine aminopeptidase 2 (Lap2, Q5QHG6) (Table 1 and Supplementary Table S1 online). In particular, 12 unique peptides were found for Sub6, suggesting that this protease was abundantly secreted during nail infection. No T. rubrum proteins were detected in the collected samples from abnormal nails with trauma but without fungal infection (data not shown). No additional proteins were identified from a sequential second extract, suggesting that all soluble secreted proteins were already extracted in the first extraction (Supplementary Materials online). The high amount of Sub6 secreted by T. rubrum in onychomycosis and the presence of DppV were confirmed by Western blot analysis and by a shotgun protein identification experiment in SDS-PAGE gels using the same pooled extract for mass spectrometry analysis and specific antisera (Supplementary Table S2 and Figure S1 online). Surprisingly, most proteases secreted by the fungus during its in vitro growth in a keratin medium including subtilisin-like protease 3 (Sub3, B8XGQ6), subtilisin-like protease 4 (Sub4, A7UKV6), leucine aminopeptidase 1 (Lap1, Q5QHG5), dipeptidyl peptidase 4 (DppIV, Q5J6J3), and metallocarboxypeptidase (M14A, A6XGK3) (Giddey et al., 2007Giddey K. Monod M. Barblan J. et al.Comprehensive analysis of proteins secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro conditions.J Proteome Res. 2007; 6: 3081-3092Crossref PubMed Scopus (46) Google Scholar, Zaugg et al., 2008Zaugg C. Jousson O. Léchenne B. Staib P. Monod M. Trichophyton rubrum secreted and membrane-associated carboxypeptidases.Int J Med Microbiol. 2008; 298: 669-682Crossref PubMed Scopus (46) Google Scholar) were not detected either by mass spectrometry or by Western blot analysis (Supplementary Table S3 and Figure S1 online).Table 1Trichophyton rubrum proteins identified by LC-MS/MS in infected nail beds samplesAccessionDescriptionScoreCoverageNo. of proteinsNo. of unique peptidesBandQ9UW97Subtilisin-like protease 6205.9646.121121, 2, 3, 4, 5, 6, 7F2SX42Polyubiquitin119.9270.7410F2SEJ4Ubiquitin62.2226.810Q6QUM1Actin29.2718.410Q8NID9Subtilisin-like protease 718.2222145F2SH28Enolase8.3412.5612Q9UW98Dipeptidyl-peptidase 56.487.16131F2SUU6Glucan 1,3-beta-glucosidase6.2418.1613F2SLY0Putative uncharacterized protein6.1334.2513F2SZI9Beta-glucosidase4.597.7912F2SN23Superoxide dismutase [Cu-Zn]3.9525.3212F2SU38Hsp70-like protein2.266.8811Q5QHG6Leucine aminopeptidase 2133Proteins were extracted from infected nail beds samples using nonionic surfactant ALS-400. Proteins were separated by SDS-PAGE, and then digested using trypsin before peptide identification by mass spectrometry (Supplementary Table S1). Proteins of T. rubrum were identified in the protein grouping mode. Similarly proteases of T. rubrum proteins were also identified by mass spectrometry analysis after SDS-PAGE from pieces of gel corresponding to positive immunostaining observed in Western blot analysis as indicated in Supplementary Figure S1. In the right column, the numbers of bands in which the proteases were identified are indicated. Lap2 was identified by SDS-PAGE/LC/MS/MS in the second analysis coupled to Western blot. Twelve peptides of Sub6 could be identified by LC/MS/MS after separation of proteins using SDS-PAGE with an Orbitrap velos (Thermo Scientific, Bremen, Germany) as a mass spectrometer. In crude extracts from infected nail samples, only four Sub6 peptides could be identified with high confidence without extensive sample preparation and SDS-PAGE using TripleTOF 5600 System (ABSciex, Redwood Shores, Canada), Orbitrap EliteTM (Thermo Scientific, Bremen, Germany), and MaXis impact (Bruker, Bremen, Germany) mass spectrometers (data not shown). No additional proteins of T. rubrum were identified from crude extracts without SDS-PAGE separation.Abbreviations: LC, liquid chromatography; MS, mass spectrometry. Open table in a new tab Proteins were extracted from infected nail beds samples using nonionic surfactant ALS-400. Proteins were separated by SDS-PAGE, and then digested using trypsin before peptide identification by mass spectrometry (Supplementary Table S1). Proteins of T. rubrum were identified in the protein grouping mode. Similarly proteases of T. rubrum proteins were also identified by mass spectrometry analysis after SDS-PAGE from pieces of gel corresponding to positive immunostaining observed in Western blot analysis as indicated in Supplementary Figure S1. In the right column, the numbers of bands in which the proteases were identified are indicated. Lap2 was identified by SDS-PAGE/LC/MS/MS in the second analysis coupled to Western blot. Twelve peptides of Sub6 could be identified by LC/MS/MS after separation of proteins using SDS-PAGE with an Orbitrap velos (Thermo Scientific, Bremen, Germany) as a mass spectrometer. In crude extracts from infected nail samples, only four Sub6 peptides could be identified with high confidence without extensive sample preparation and SDS-PAGE using TripleTOF 5600 System (ABSciex, Redwood Shores, Canada), Orbitrap EliteTM (Thermo Scientific, Bremen, Germany), and MaXis impact (Bruker, Bremen, Germany) mass spectrometers (data not shown). No additional proteins of T. rubrum were identified from crude extracts without SDS-PAGE separation. Abbreviations: LC, liquid chromatography; MS, mass spectrometry. We developed multiple reaction monitoring (MRM) assays to quantify T. rubrum Sub3, Sub4, Sub6, Sub7, DppIV, DppV, Lap1, Lap2, and M14A directly from nail crude protein extracts as described in the Supplementary Material. MRM is a powerful method for sensitive quantitative measurement of target proteins (Castro-Gamero et al., 2014Castro-Gamero A.M. Izumi C. Rosa J.C. Biomarker verification using selected reaction monitoring and shotgun proteomics.Methods Mol Biol. 2014; 1156: 295-306Crossref PubMed Scopus (6) Google Scholar, Keshishian et al., 2007Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol Cell Proteomics. 2007; 6: 2212-2229Crossref PubMed Scopus (576) Google Scholar). Sub6 was detected in all infected nail bed samples but not in the control samples (trauma) or in the blank sample (heavy peptides only). Sub7 and DppV were detected in few samples (Supplementary Table S4 online). This may be related to the abundance and/or the stability of these proteins. Lap2 was detected rarely and at very low intensity in agreement with the faint detection of Lap2 using Western blot analysis, suggesting that Lap2 is discreetly produced in onychomycoses (Supplementary Table S2 and Figure S1). No other proteases secreted by the fungus during its growth in vitro in a keratin medium (i.e., Sub3, Sub4, Mep3, Mep4, Lap1, DppIV, and M14A) could be detected in the protein extracts from infected human nails despite the sensitivity of the method and therefore did not appear to be involved during the establishment of onychomycosis. Quantifications of Sub6, Sub7, and DppV were subsequently performed across 218 clinical samples using MRM assay including negative controls and other nail diseases (Supplementary Table S5 online). Sub6 was detected in almost all samples infected by T. rubrum (105 of 118 samples) with one or more peptides, but not in the other samples including nail trauma, psoriasis, and nail beds infected by other pathogens (molds and yeasts) (Table 2). However, a restricted number of samples (13%) identified as positive after mycology culture and/or direct observation were not identified as positive in the MRM assay. Four of 118 of these samples after extraction from nail beds showed a very low concentration of proteins—less than 0.08 mg/ml—which may explain the negative response in the MRM assay. In addition, three samples (mycological culture negative) were identified as positive by MRM and direct mycological examination. The comparison between the clinical evaluation from direct mycological examination and culture results and our mass-spectrometry-based assay showed a good correspondence, as indicated by the high scores of the sensitivity (0.89) and the specificity (0.99) of the assay. Positive and negative predictive values showed important scores: 0.99 and 0.88, respectively. From these results, Sub6 can be considered as a marker of T. rubrum nail infection. The identification and quantification of Sub6-specific peptides using MRM were restricted to T. rubrum (Table 2). Putative T. interdigitale Sub6 could not be identified using this MRM assay because the amino acid sequences of the peptides used for MRM are different in the T. interdigitale corresponding peptides (Supplementary Table S6 online).Table 2Detection of Trichophyton rubrum Sub6 in clinical samples by multiple reaction monitoring (MRM)Mycology positiveSub6 positive (total sample)Trichophyton rubrum98 (108)Trichophyton rubrum (+ melanine, black pigmentation)3 (3)Trichophyton rubrum (africanis)0 (1)Trichophyton rubrum (+ Scopulariopsis brevicaulis)0 (1)Trichophyton rubrum (+ pyocyanique)1 (1)Trichophyton rubrum (Mycelium observed with negative mycology)3 (4)105 (118)Trichophyton mentagrophytes var. interdigitalis0 (19)Trichophyton soudanense0 (1)Epidermophyton floccosum0 (1)Mycology positive (molds and yeasts)Scytalidium dimidiatum0 (6)Scytalidium hyalinum0 (2)Fusarium0 (4)Aspergillus0 (6)Scopulariopsis brevicaulis0 (2)Acremonium sp0 (1)Candida albicans0 (3)0 (24)Mycology negativePsoriasis Onychopathy0 (2)Onychogryphosis0 (1)Trauma0 (10)Others1 (42)1 (55)Quantifications of Sub6 across 218 clinical samples using an MRM assay including samples infected by T. rubrum, negative controls and other nail diseases (for details see Supplementary Table S5). Open table in a new tab Quantifications of Sub6 across 218 clinical samples using an MRM assay including samples infected by T. rubrum, negative controls and other nail diseases (for details see Supplementary Table S5). The gene encoding Sub6 was previously revealed by microarray analysis to be the most upregulated protease gene during skin infection in guinea pigs with the dermatophyte Arthroderma benhamiae (Staib et al., 2010Staib P. Zaugg C. Mignon B. et al.Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.Microbiology. 2010; 156: 884-895Crossref PubMed Scopus (69) Google Scholar). As in the present investigations on onychomycosis, none of the A. benhamiae genes encoding specific endo- and exoproteases involved in keratin digestion in vitro was found to be upregulated during skin infection. In contrast to T. rubrum, which is anthropophilic, A. benhamiae is zoophilic and causes highly inflammatory cutaneous infections in humans and rodents. As a general conclusion, the proteases secreted in vitro during protein degradation and in vivo during infection are different whatever the dermatophyte species and the tinea. Sub6 and DppV were first described as the major allergens Tri r 2 and Tri r 4, respectively, in T. rubrum (Woodfolk, 2005Woodfolk J.A. Allergy and dermatophytes.Clin Microbiol. 2005; 18: 30-43Crossref PubMed Scopus (131) Google Scholar, Woodfolk et al., 1998Woodfolk J.A. Wheatley L.M. Piyasena R.V. Benjamin D.C. Platts-Mills T.A. Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity: sequence homology to two families of serine proteinases.J Biol Chem. 1998; 273: 29489-29496Crossref PubMed Scopus (89) Google Scholar). These antigens were found to induce dual immune responses and elicit either immediate hypersensitivity or delayed-type hypersensitivity skin test reactions in different individuals. Exposure to Trichophyton proteins may result in bronchial sensitization and symptomatic asthma that can be controlled with systemic antifungal therapy (Ward et al., 1989Ward Jr., G.W. Karlsson G. Rose G. Platts-Mills T.A. Trichophyton asthma: sensitisation of bronchi and upper airways to dermatophyte antigen.Lancet. 1989; 22: 859-862Abstract Scopus (98) Google Scholar, Ward et al., 1999Ward Jr., G.W. Woodfolk J.A. Hayden M.L. Jackson S. Platts-Mills T.A. Treatment of late-onset asthma with fluconazole.J Allergy Clin Immunol. 1999; 104: 541-546Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, Woodfolk, 2005Woodfolk J.A. Allergy and dermatophytes.Clin Microbiol. 2005; 18: 30-43Crossref PubMed Scopus (131) Google Scholar, Woodfolk et al., 1998Woodfolk J.A. Wheatley L.M. Piyasena R.V. Benjamin D.C. Platts-Mills T.A. Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity: sequence homology to two families of serine proteinases.J Biol Chem. 1998; 273: 29489-29496Crossref PubMed Scopus (89) Google Scholar). There are also eczematous skin reactions to dermatophytes that are distant of the area of the dermatophytes. These skin reactions, called dermatophytids, are generally reported in patients with tinea pedis, most often with T. interdigitale but also with T. rubrum (Ilkit et al., 2012Ilkit M. Durdu M. Karakaş M. Cutaneous id reactions: a comprehensive review of clinical manifestations, epidemiology, etiology, and management.Crit Rev Microbiol. 2012; 38: 191-202Crossref PubMed Scopus (44) Google Scholar, Veien et al., 1994Veien N.K. Hattel T. Laurberg G. Plantar Trichophyton rubrum infections may cause dermatophytids on the hands.Acta Derm Venereol. 1994; 74: 403-404PubMed Google Scholar). The dermatophytids go away once the dermatophyte infection has been cured. In cases of eczematous skin reaction of unknown origin, it could be relevant to perform skin test reactions using both Sub6 and DppV antigens. A positive reaction could be indicative of a nondetected dermatophyte infection and suggest further clinical investigations for antifungal treatment. The authors state no conflict of interest. Nail samples were collected from patients by medical doctors in the Dermatology Department at Saint Louis Hospital (Paris). The samples were analyzed as a part of an internal quality control program according to hospital internal practice and French regulation. Mass spectrometric analyses and database searching for protein were performed (by Jean-William Dupuy) at Plateforme Proteome, Functional Genomic Centre of Bordeaux, University of Bordeaux, Bordeaux, France. MRM assays were developed by Biognosis AG, Wagistrasse, Schlieren, Switzerland. We thank Alexandre Genette and Alexia Seraidaris for their technical assistance, Galderma R&D, Les Templiers, Sophia Antipolis, France. Download .pdf (.91 MB) Help with pdf files Supplementary Data

Le vitiligo touche environ 1 % de la population mondiale. Stopper l'évolutivité de la maladie et repigmenter les zones lésionnelles sont les deux challenges thérapeutiques. La physiopathologie du vitiligo est complexe et fait intervenir de nombreux acteurs cellulaires. Nous avons réalisé une étude transcriptomique des peaux de patients ayant un vitiligo actif. Étude transcriptomique chez 10 patients ayant un vitiligo actif avec biopsies en zone lésionnelle dépigmentée, périlésionnelle et saine et chez 10 sujets témoins. Le profil d'expression des cytokines et chémokines présentes dans la couche cornée a également été analysé. Dans un second temps, une confirmation par qRT-PCR puis des études fonctionnelles ont été réalisées in vitro et sur un modèle ex vivo de peau vitiligo que nous avons développé. Les résultats montrent une augmentation de CXCL10 dans les zones « saines » et péri-lésionnelles de peaux de patients vitiligo comparée aux peaux témoins. Cependant, ni CXCL10, ni aucun autre facteur immunitaire ne sont dérégulés dans les peaux de vitiligo déjà dépigmentées. L'analyse des données du transcriptome montre aussi que la voie de WNT, fortement impliquée dans la différenciation mélanocytaire, est moins active dans les peaux vitiligo. Nous avons ensuite montré que le stress oxydatif était capable de diminuer l'activité de la voie de WNT dans les cultures de mélanocytes et de kératinocytes. Nous avons développé un modèle de peau ex vivo qui reste viable et fonctionnel pendant 15 jours et nous avons montré que le stress oxydatif réduisait aussi l'activité de la voie WNT dans ce modèle. Enfin, nous avons traité des peaux de patients vitiligo dans notre modèle ex vivo avec des agents stimulant la voie de WNT (agonistes WNT et inhibiteurs de GSK3b) et nous avons montré que ces agents permettaient d'obtenir la différenciation des cellules souches présentes dans ces peaux en pré-mélanocytes. Nos Résultats confirment l'implication de CXCL10 dans la physiopathologie du vitiligo avec une activité à bas bruit dans les peaux encore non lésionnelles. Ils montrent par contre qu'il n'y a plus d'activation immunitaire lorsque les peaux sont dépigmentées. Enfin, ils montrent pour la première fois une diminution d'activité de la voie de WNT probablement secondaire au stress oxydatif. Nos résultats montrent que le système immunitaire n'agit plus lorsque les peaux de patients vitiligos sont dépigmentées mais qu'il existe alors une diminution de l'activité de la voie de WNT qui freine la différenciation et la prolifération mélanocytaire. Ils montrent aussi que des activateurs de WNT sont capables d'induire sans UV une différenciation des mélanocytes au sein de la peau vitiligineuse offrant ainsi de nouvelles perspectives thérapeutiques.

Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.

The calmodulin-like skin protein (CLSP) or so-called calmodulin-like protein 5, a recently discovered skin-specific calcium-binding protein, is closely related to keratinocyte differentiation. The 16-kDa protein is proteolytically degraded in the upper layers of the stratum corneum (SC) of healthy skin. With the use of specific new monoclonal antibodies to CLSP, we were able to demonstrate that the abnormal elevated levels of CLSP, characteristic of psoriatic epidermis, were probably not due to an overexpression of the protein, but most likely the result of its non-degradation. Further in vitro experiments using recombinant CLSP and in situ data clearly showed that calcium protected and chelator accelerated CLSP degradation. These data indicate that CLSP degradation in the SC of psoriatic skin might be hindered by the abnormally elevated calcium concentration. No degradation of CLSP in psoriatic epidermis keeping its ability to bind protein as transglutaminase 3 may have a physiological role in skin diseases such as psoriasis.

Proteases play a pivotal role in epidermal differentiation and desquamation. Separation of a total protein extract from human reconstructed epidermis by two-dimensional gel electrophoresis and subsequent peptide analysis of a specific protein spot identified a new protein exhibiting similarities with the retroviral aspartic protease family. Cloning of the corresponding full-length cDNA revealed an open reading frame encoding for a new protease of 343 amino acids, containing a putative aspartic protease catalytic domain. We named this protein Skin ASpartic Protease (SASPase). RT-PCR and northern blot analysis of various human tissues revealed that SASPase was specifically expressed within the epidermis. Immunohistochemical analysis showed a particularly intense expression restricted to the granular layers, whereas in diseased skin, its expression was changed. Western blot analysis, using a monoclonal antibody, revealed the expression of two forms of the enzyme: a 28 kDa putative proform and the active 14 kDa form. Recombinant truncated SASPase (SASP28) was generated from a prokaryotic expression system in Escherichia coli as a fusion protein with GST. SASP28 degraded insulin and to a lesser extent casein with a pH optimum of 5. As seen for retroviral proteases, an auto-activation processing was evidenced, generating a 14 kDa protein (SASP14). Site-directed mutagenesis inhibited auto-activation of the enzyme. Indinavir, a potent HIV protease inhibitor used in AIDS therapy, had a significant inhibitory effect on rSASPase auto-activation, which could explain its side effects on skin.

The gene expression profiles of three different models of reconstructed human epidermis were analyzed in a comparative study using cDNA array technology. The study also included normal human subconfluent keratinocytes cultured on plastic. Arrays were custom-made and comprised 504 known genes related to cutaneous biology. The gene expression profiles of the three reconstructed epidermis models shared 86% similarity; only 22 of the 504 examined genes showed a different expression level. A comparison of the 3D models with keratinocyte cultures on plastic dishes revealed a set of six genes with a considerably higher expression in the 3D models. These genes were keratin 1, corneodesmosin, filaggrin, loricrin, calmodulin-like skin protein and caspase 14, all related to keratinocyte terminal differentiation. The reported data may contribute to a better understanding and characterization of reconstructed epidermal models and may also serve as established references for investigations related to epidermal differentiation and proliferation.