IntroductionAbnormalities of keratinocyte differentiation and impairment of permeability barrier are features of inflammatory skin diseases driven by Th1/Th17 and Th2 immune response, such as psoriasis and atopic dermatitis. We aimed at identifying the signature of the Th1/Th17 and Th2 environments on keratinocytes, focusing on the expression of genes involved in the lipid metabolism and profiles of abundance of lipid metabolites.MethodsHuman immortalized keratinocytes in prodifferentiative conditions induced by increasing calcium concentration, and 3D epidermal equivalents were treated with mixtures either of TNF-α and IL-17A plus Th1-related cytokines (IL-1α, IL-6) or of Th2 cytokines (IL-4, IL-13). The expression of genes involved in epidermal differentiation and lipid metabolism was evaluated by RT-PCR at 2, 4 and 7 days of treatment. The protein levels of early and late keratinocyte differentiation markers were assessed. The lipid composition was investigated by GCMS and LCMS.ResultsBoth Th1/Th17 and Th2 cytokine mixtures changed the expression of genes involved in the metabolism of fatty acids (FAs), i.e., FAS, FADS2, SCD1, and ALOX12B. Th1/ Th17 downregulated the ELOVL3 gene, which is implicated in the FAs elongation, while the mRNA levels of ABCA12 and HMGCR, genes involved in lipids transport and cholesterol synthesis, respectively, were decreased with both cytokine mixtures. DEGS1 and DEGS2, key enzymes in the ceramide synthesis, were downregulated and upregulated in the Th1/Th17 and Th2 environments, respectively. The mRNA expression of CERS3, which synthesizes ceramides containing long chain FAs, was increased by Th1/Th17 cytokines. Both Th1/ Th17 and Th2 cytokine mixtures lowered the CERS6 mRNA levels in differentiated keratinocytes. Effects specific to Th1/Th17 or Th2 cytokines were observed on freely extractable cell lipids. Th1/Th17 cytokines significantly inhibited the high calcium-induced synthesis of phospholipids (PCs, PEs, SMs), and short-chain ceramides, while the synthesis of ceramides with medium to long carbon chains was upregulated. Th2 cytokines caused a generalized decrement of free FAs, including long-chain ones. In contrast to 2D cultures, the 3D epidermal equivalents allowed the identification of altered profiles of acyland hexosyl-ceramides.ConclusionThe different effects exerted by Th1/Th17 and Th2 cytokines support, at least in part, the features of lipid barrier alterations specific to psoriasis or atopic dermatitis.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

17-β-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-β-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 17-β-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects(n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-β-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-β-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-β-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-β-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-β-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-β-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-α-reductase1 and hydroxysteroid-17β-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-β-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care.

Atopic dermatitis (AD) is a composite disease presenting disruption of the skin permeability barrier (SPB) in the stratum corneum (SC). Recent evidence supports derangement of the sebaceous gland (SG) activity in the AD pathomechanisms. The objective of this study was to delineate profiles of both sebaceous and epidermal lipids and of aminoacids from SG-rich (SGR) and SG-poor (SGP) areas in AD. Both sebum and SC were sampled from SGR areas, while SC was sampled also from SGP areas in 54 adult patients with AD, consisting of 34 and 20 subjects, respectively with and without clinical involvement of face, and in 44 age and sex-matched controls. Skin biophysics were assessed in all sampling sites. Disruption of the SBP was found to be associated with dysregulated lipidome. Abundance of sapienate and lignocerate, representing, respectively, sebum and the SC type lipids, were decreased in sebum and SC from both SGR and SGP areas. Analogously, squalene was significantly diminished in AD, regardless the site. Extent of lipid derangement in SGR areas was correlated with the AD severity. The abundance of aminoacids in the SC from SGR areas was altered more than that determined in SGP areas. Several gender-related differences were found in both controls and AD subgroups. In conclusion, the SG activity was differently compromised in adult females and males with AD, in both SGR and SGP areas. In AD, alterations in the aminoacidome profiles were apparent in the SGR areas. Lipid signatures in association with aminoacidome and skin physical properties may serve the definition of phenotype clusters that associate with AD severity and gender.

Lipids are key constituents of the barrier function in the human stratum corneum (SC), which is the outermost layer of the epidermis and amenable to non-invasive sampling by tape stripping. The three major lipid classes in the SC, i.e., ceramides, fatty acids, and cholesterol, present equimolar concentration. Liquid chromatography coupled with mass spectrometry (LCMS) is elective in profiling lipids in the SC in both positive and negative ion modes. Nevertheless, the latter one allows for the simultaneous detection of the three major epidermal components of the SC. Determination of ceramides in the SC poses analytical challenges due to their wide range of structures and concentrations especially in the case of limited sample amounts. Ammonium formate is a commonly used modifier added to the mobile phase to assist ionization. However, it introduces uncertainty in the identification of ceramides when operating in negative ion mode, even with high resolution MS. We tested the advantages of using fluoride in the lipid profiling of SC and unambiguous identification of ceramides subclasses. The use of fluoride enhanced the ionization of ceramides, regardless the specific substructure, solved misidentification issues, and was successfully applied to the simultaneous detection of all three lipid classes in the human SC.

Antibody-based cancer therapy has achieved significant success, and mAbs targeting ERBB2 (trastuzumab, pertuzumab) and ERBB3 (patritumab, seribantumab, lumretuzumab) are used to treat various types of cancer (Propper et al., 2023Propper D.J. Gao F. Saunders M.P. Sarker D. Hartley J.A. Spanswick V.J. et al.Panther: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer.Br J Cancer. 2023; 128: 245-254Crossref PubMed Scopus (0) Google Scholar). The ERBB2/3 heterodimer, the most active signaling dimer in the ERBB family (Schneider and Yarden, 2016Schneider M.R. Yarden Y. The EGFR-HER2 module: a stem cell approach to understanding a prime target and driver of solid tumors.Oncogene. 2016; 35: 2949-2960Crossref PubMed Scopus (54) Google Scholar), is often hyperactive in tumors, making it a promising target in cancer therapy. Although antibodies targeting EGFR and their toxicity are largely recognized (Lichtenberger et al., 2013Lichtenberger B.M. Gerber P.A. Holcmann M. Buhren B.A. Amberg N. Smolle V. et al.Epidermal EGFR controls cutaneous host defense and prevents inflammation.Sci Transl Med. 2013; 5199ra111Crossref PubMed Scopus (196) Google Scholar), the side effects of targeting the related Y kinase receptors ERBB2 and ERBB3 remain to be determined (Braden and Anadkat, 2016Braden R.L. Anadkat M.J. EGFR inhibitor-induced skin reactions: differentiating acneiform rash from superimposed bacterial infections.Support Care Cancer. 2016; 24: 3943-3950Crossref PubMed Scopus (31) Google Scholar; Hynes and Lane, 2005Hynes N.E. Lane H.A. ERBB receptors and cancer: the complexity of targeted inhibitors.Nat Rev Cancer. 2005; 5: 341-354Crossref PubMed Scopus (2804) Google Scholar). The development of anticancer drugs depends on preclinical research based on reliable animal models able to predict the efficacy and toxicity of candidate drugs or drug combinations. Therefore, we generated a skin-specific double knockout for both receptors (Erbb2/3del) by crossing mice carrying conditional Erbb2 and Erbb3 alleles with transgenic mice expressing keratin (K) 5 promoter–driven cre recombinase. All animal experiments were approved by the governmental body of the state of Upper Bavaria, Germany and by the Austrian Federal Ministry of Education, Science, and Research. Previous studies with skin-specific knockouts of ERBB2 (Dahlhoff et al., 2017bDahlhoff M. Muzumdar S. Schäfer M. Schneider M.R. ERBB2 is essential for the growth of chemically induced skin tumors in mice.J Invest Dermatol. 2017; 137: 921-930Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) or ERBB3 (Dahlhoff et al., 2015Dahlhoff M. Schäfer M. Muzumdar S. Rose C. Schneider M.R. ERBB3 is required for tumor promotion in a mouse model of skin carcinogenesis.Mol Oncol. 2015; 9: 1825-1833Crossref PubMed Google Scholar) revealed that both proteins are dispensable for skin development and homeostasis but are required for the progression of skin tumors. Similarly, Erbb2/3del mice were born at the predicted ratios and showed no abnormalities in coat appearance or behavior but were reduced in body size and weight (Figure 1a). From the age of 3 months, Erbb2/3del mice showed skin changes and often developed a brittle, thickened epidermis on the tail and in the neck and abdomen area, where wounds with scabs occasionally appeared later (Figure 1b). These alterations were never seen in control littermates. Moreover, H&E-stained skin sections from Erbb2/3del mice revealed an inflamed skin compared with those from control mice (Figure 1c). Changes in hair follicle architecture were never detected. The epidermal alterations were accompanied by a significant increase in the inflammatory cytokine IL-1A and a decrease in the anti-inflammatory cytokine IL-10 in the plasma of Erbb2/3del mice compared with those from the control littermates (Figure 1d). Other inflammatory markers such as TNFA, INFG, and IL-23 were not significantly altered (Supplementary Figure S1). The loss of ERBB2 and ERBB3 in the epidermis and the pilosebaceous unit was confirmed by immunofluorescence (Figure 1e). Quantitative morphometry revealed a significantly thicker epidermis in Erbb2/3del mice and significantly enlarged sebaceous glands compared with those in control mice (Figure 1f and g). Accordingly, epidermal and sebaceous gland cell proliferation, evaluated by Ki-67 immunohistochemical staining, was significantly increased in both skin compartments of Erbb2/3del mice compared with those in the control littermates (Figure 1h). Next, we investigated the differentiation of the epidermis by immunofluorescence staining of differentiation markers (Figure 1i). K5 (marking the basal layer), loricrin, FLG, involucrin, and transglutaminase 1 (marking the keratinized epidermal layer) staining showed no differences between Erbb2/3del mice and the control mice. However, Erbb2/3del mice expressed more K10 in the suprabasal layer of the epidermis (Figure 1i), exactly the layer in which ERBB2 and ERBB3 are highly expressed (Figure 1e). Furthermore, we detected K6 expression in the epidermis of Erbb2/3del mice, indicating impaired epidermal differentiation (Figure 1i). Western blot analysis and densitometric quantification revealed an almost complete epidermal loss of ERBB2 and ERBB3 in Erbb2/3del mice (Figure 2a). In contrast, the levels of total and of phosphorylated EGFR remained unchanged in Erbb2/3del epidermis compared with those in control mice (Figure 2a and b). No changes in the phosphorylation or total levels of MAPK1/2 and protein kinase B were detected in ERBB2/3-deficient epidermis (Figure 2a and b). Confirming the histological analyses, the proliferation marker proliferating cell nuclear antigen was significantly increased in the back skin of Erbb2/3del mice compared with those in the controls (Figure 2a and b).Figure 2Epidermal differentiation and sebum composition are altered in Erbb2/3del mice. (a) Western blot and (b) densitometrical analysis of phosphorylated EGFR, ERBB2, and ERBB3 receptors and of phosphorylated MAPK, Akt, and PCNA in skin samples from male mice aged 4 months. GAPDH was used as reference protein. Error bars represent SEM (n = 6 per group). (c) Significantly changed amounts of lipids measured in 1 mg hair of male Erbb2/3del and control mice (n = 5 per group). Data are illustrated as box plots with median. Data were analyzed by Student's t-test. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Akt, protein kinase B; CAR, acylcarnitine; CE, cholesterol ester; Cer, ceramide; DS, desmosterol sulfate; NAE, N-acylethanolamine; NDAE, N,N-diacylethanolamine; PCNA, proliferating cell nuclear antigen; TG, triglyceride.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In light of the changes observed in sebaceous glands, we next extracted hair lipids (which are essentially of sebaceous origin) and assessed their composition by mass spectrometry. Lipidome investigations suggested a significant reduction of acylcarnitines, triglycerides, cholesterol esters, desmosterol sulfate, N-acylethanolamine, N,N-diacylethanolamine, and 2 subclasses of ceramides in the hair lipids of Erbb2/3del mice compared with those in the control littermates (Figure 2c). Thus, loss of ERBB2/3 increases sebaceous gland size but decreases the synthesis of specific lipids, which may influence the skin barrier. Because loss of ERBB2/3 alters epidermal differentiation, we set out to study the role of ERBB2 and ERBB3 receptors in the human HaCaT keratinocyte cell line. By employing CRISPR/Cas9 gene editing technology, we deleted ERBB2, ERBB3, and both receptors in HaCaT cells, thus expanding our previous studies (Dahlhoff et al., 2017aDahlhoff M. Gaborit N. Bultmann S. Leonhardt H. Yarden Y. Schneider M.R. CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.FEBS J. 2017; 284: 3339-3349Crossref PubMed Scopus (0) Google Scholar) (Supplementary Figure S2a). All knockout clones were verified by western blot analysis (Supplementary Figure S2b). ERBB2/3del cells show that significantly increased levels of K5; K6; and K17 and the cornified layer proteins FLG, involucrin, and loricrin were increased but that cytoskeleton proteins ACTB and vinculin were significantly less expressed (Supplementary Figure S3). We in part confirmed these results in primary keratinocytes by small interfering RNA knockdown for ERBB2/3 for 4 (Supplementary Figure S4) and 6 (Supplementary Figure S5) days. In this study, as in HaCaT cells, EGFR phosphorylation is reduced, and loricrin expression is significantly increased. To evaluate the consequences of deleting ERBB2 and ERBB3 on gap closure, confluence formation, and velocity, all cell lines were analyzed with a lens-free live cell-imaging technology. A scratch assay showed significantly increased gap closure of ERBB2del cells and a significantly decreased gap closure behavior for ERBB3del and ERBB2/3del cells compared with those of control cells (Supplementary Figures S6a and S7). All clones showed a significantly decreased confluence formation compared with the control mock cell line (Supplementary Figure S6b). ERBB3del and ERBB2/3del cells were more affected than ERBB2del cells, suggesting that ERBB3 may play a greater role here than ERBB2. The velocity was significantly increased in all knockout cell lines compared with that in the controls (Supplementary Figure S6c). However, the data clearly show that the ERBB2del and the ERBB2/3del cells move much faster than the ERBB3del cells. Notably, only ERBB2/3del cells showed significant changes in all 3 assays, whereas the single knockouts showed no changes in at least 1 assay, indicating a cumulative or synergistic effect in the double knockout cells and resembling the results in triple knockouts for EGFR, ERBB2, and ERBB3 in canine kidney cells (Matsuda et al., 2023Matsuda K. Hirayama D. Hino N. Kuno S. Sakaue-Sawano A. Miyawaki A. et al.Knockout of all ErbB-family genes delineates their roles in proliferation, survival and migration.J Cell Sci. 2023; 136jcs261199Crossref Scopus (0) Google Scholar). The interaction between the EGFR and adhesion proteins such as E-cadherin (CDH1) regulates contact inhibition of proliferation (Rübsam et al., 2017Rübsam M. Mertz A.F. Kubo A. Marg S. Jüngst C. Goranci-Buzhala G. et al.E-cadherin integrates mechanotransduction and EGFR signaling to control junctional tissue polarization and tight junction positioning.Nat Commun. 2017; 8: 1250Crossref PubMed Scopus (138) Google Scholar). To investigate whether the loss of ERBB2 and ERBB3 changes contact inhibition, the phosphorylation of EGFR was analyzed in a confluence experiment. In mock cells as well as in the ERBB2del and ERBB3del cells, EGFR is significantly more phosphorylated in 100% confluent cells than in 50% confluent cells (Supplementary Figure S8a and b). However, in ERBB2/3del cells, EGFR is equally activated in 100% confluent cells and in 50% confluent cells (Supplementary Figure S8a and b). In mock, ERRB2del, and ERBB3del cells, CDH1 is significantly increased in 100% confluent cells compared with that in 50% confluent cells but not in double knockout cells, where the CDH1 signal is equally strong in 100 and 50% confluent cells (Supplementary Figure S8a and c). ERBB2 and ERBB3 appear to regulate the activation of EGFR in nonconfluent cells because the loss of ERBB2/3 leads to increased activation of EGFR, resulting in increased expression of CDH1, which is normally only seen in confluent cells. In summary, our data show that concomitant deletion of ERBB2 and ERBB3 in mice leads to impaired differentiation of the epidermis, inflammation, and altered sebaceous glands and eventually results in skin wound lesions. Although the single loss of ERBB2 or ERBB3 seems to be compensated by other ERBB components, this ability is lost upon a simultaneous deletion of ERBB2 and ERBB3. Inhibition of both ERBB2 and ERBB3 is thus an attractive approach for tumor therapies because both receptors are often active together in tumors, but their inhibition, similarly to the inhibition of EGFR, leads to cutaneous side effects. All animal experiments were approved by the governmental body of the state of Upper Bavaria, Germany (Regierung von Oberbayern, 55.2-1-54-2532-206-2012) and by the Austrian Federal Ministry of Education, Science, and Research (BMBWF-68.205/106-V/3b/2019) and were performed in strict compliance with the European Communities Council Directive (86/609/EEC) recommendations for the care and use of laboratory animals. All data generated or analyzed during this study are included in this published article and its supplementary information materials. Theresa Hommel: http://orcid.org/0009-0009-0922-9948 Paula F. Meisel: http://orcid.org/0009-0009-4657-2358 Emanuela Camera: http://orcid.org/0000-0001-6633-0449 Grazia Bottillo: http://orcid.org/0000-0002-6671-7384 Andrea R. Teufelberger: http://orcid.org/0000-0002-1142-949X Theresa H. Benezeder: http://orcid.org/0000-0001-6218-2792 Peter Wolf: http://orcid.org/0000-0001-7777-9444 Lisa Kleissl: http://orcid.org/0000-0001-8011-796X Georg Stary: http://orcid.org/0000-0003-1746-4250 Christian Posch: http://orcid.org/0000-0003-0296-3567 Marlon R. Schneider: http://orcid.org/0000-0002-9570-3491 Maik Dahlhoff: http://orcid.org/0000-0001-9189-7631 The authors state no conflict of interest. We thank Ingrid Renner-Müller and Petra Renner (Gene Center, Ludwig Maximilian University of Munich, Munich, Germany) for excellent animal care, Franziska Kress for assistance with western blot analysis, and Josef Millauer for mouse genotyping. We thank Angel Ramirez and Jose Jorcano (CIEMAT, Madrid, Spain) for providing K5-Cre mice and Carmen Birchmeier (Max-Delbrück-Center for Molecular Medicine, Berlin, Germany) for providing Erbb2-floxed mice. No funding was available for this study. Conceptualization: MD, MRS, TH; Data Curation: MD, TH, PFM, EC, THB, GB, ART, LK; Formal Analysis: MD, TH, PFM, EC, THB, GB, ART, CB, LK; Investigation: MD, TH, PFM, EC, GB, THB, CB, LK; Methodology: MD, TH, PW, EC, GS, CP; Project Administration: MD, MRS; Resources: MD, PW, GC; Visualization: MD, TH, MRS, EC, PW, GS; Writing – Original Draft Preparation: MD, TH, MRS, PW; Writing – Review and Editing: MD, TH, MRS, EC, GS, CP, PW Supplementary Table S1Antibodies Employed for Western Blot Analysis and Immunofluorescence StainingAntigenAntibodyHostDilutionEGFRCell Signaling Technology, number 2232Rabbit1:1000ERBB2Cell Signaling Technology, number 4290Rabbit1:1000ERBB2R&D System, AF5176Sheep1:1000ERBB3Cell Signaling Technology, number 12708Rabbit1:1000ERBB3R&D System, AF4518Sheep1:1000p-EGFR (Tyr 1068)Cell Signaling Technology, number 2236Mouse1:1000p-ERBB2 (Tyr 1221/1222)Cell Signaling Technology, number 2243Rabbit1:1000p-ERBB3 (Tyr 1289)Cell Signaling Technology, number 4791Rabbit1:1000CDH1Cell Signaling Technology, number 14472Mouse1:1000PCNACell Signaling Technology, number 13110Rabbit1:1000Caspase 3Cell Signaling Technology, number 9662Rabbit1:1000pAKT (Ser473)Cell Signaling Technology, number 4060Rabbit1:1000AKTCell Signaling Technology, number 4691Rabbit1:1000p-P44/42 MAPK (Thr202/Tyr204)Cell Signaling Technology, number 13110Rabbit1:1000P44/42 MAPKCell Signaling Technology, number 9102Rabbit1:1000K1Proteintech, 16848-1-APRabbit1:1000K5BioLegend, number 905504Rabbit1:1000K6BioLegend, number 905701Rabbit1:1000K10BioLegend, number 905401Rabbit1:1000K14BioLegend, number 905304Rabbit1:1000K17Abcam, ab109725Rabbit1:1000LORBioLegend, number 905104Rabbit1:1000IVLBioLegend, number 924401Rabbit1:1000FLGBioLegend, number 905804Rabbit1:1000TGM1Proteintech, 12912-3-APRabbit1:1000ACTBCell Signaling Technology, number 4970Rabbit1:1000VCLSanta Cruz Biotechnology, sc-25336Mouse1:500GAPDHCell Signaling Technology, number 2118Rabbit1:5000Goat α RabbitCell Signaling Technology, number 7074Goat1:5000Horse α MouseCell Signaling Technology, number 7076Horse1:5000Donkey α Sheep AF594Dianova, 713-586-147Donkey1:1000Donkey α Rabbit AF594Dianova, 711-585-152Donkey1:1000Abbreviations: AKT, protein kinase B; IVL, involucrin; K, keratin; LOR, loricrin; p-AKT, phosphorylated protein kinase B; PCNA, proliferating cell nuclear antigen; p-ERBB, phosphorylated ERBB; p-EGFR, phosphorylated EGFR; p-P44/42 MAPK, phosphorylated P44/42 MAPK; VCL, vinculin. Open table in a new tab Supplementary Table S2List of Metabolites Determined by GC-MS in the Sebum Extract from Mouse HairSystematic NameSupplierSynonymsFormulaMWTMSQuantifier ionRT AvgTypeLabelDodecanoic acidLARODANC12:0C12H24O2200.21257.216.2EveneFATetradecanoic acidLARODANC14:0C14H28O2228.21285.219.7EveneFAHexadecanoic acidLARODANC16:0C16H32O2256.21313.222.9EveneFAOctadecanoic acidLARODANC18:0C18H36O2284.31341.325.9EveneFAHeptadecanoic acidLARODANC17:0C17H34O2270.31327.324.5OddoFANonadecanoic acidLARODANC19:0C19H38O2298.31355.326.9OddoFA9Z-Hexadecenoic acidLARODANC16:1n-7C16H30O2254.21311.222.6MonounsaturatedMUFA10Z-octadecenoic acidTentatively assignedC18:1C18H34O2282.31339.325.6MonounsaturatedMUFA9Z-octadecenoic acidLARODANC18:1n-9C18H34O2282.31339.325.5MonounsaturatedMUFA9Z,12Z-Octadecadienoic acidLARODANC18:2C18H32O2280.21337.225.4PolyunsaturatedPUFACholest-5-en-3β-olTRCCholesterolC27H46O386.61458.136.6SterolCholCholest-5,24-dien-3β-olTRCDesmosterolC27H44O384.61456.137.0SterolSterold17PalmitateC/D/N Isotopesd17C16:0C16H15D17O2273.51330.522.7EveneFAd6CholesterolC/D/N Isotopesd6CholesterolC27H40D6O392.71464.436.5SterolChold6DesmosterolTRCd6DesmosterolC27H38D6O390.71462.236.9SterolSterold6SqualeneTRCd6SqualeneC30H44D6416.8075.433.2PrenolSqn-hexadecyl-1,1,2,2-d4 hexadecanoate-16,16,16-d3C/D/N Isotopesd7WE 32:0C32H57D7O2487.90487.938.8Wax esterWE1,2,3-PropanetriolTentatively assignedGlycerolC3H8O392.13205.08.6GlycerolGly(2S)-5-oxopyrrolidine-2-carboxylic acidTentatively assigned5-OxoprolineC5H7NO3129.12156.113.6Amino acidAAβ-D-Fructofuranosyl α-D-glucopyranosideTentatively assignedSucroseC12H22O11342.38361.331.1SugarSUnknown193Tentatively assignedUnknown193UnUnUn193.116.0UnUnUnknown296Tentatively assignedUnknown296UnUnUn296.115.9UnUnlanosta-8,24-dien-3β-olTentatively assignedLanosterolC30H50O426.71393.238.5SterolSterolcholest-7-en-3β-olTentatively assignedLathosterolC27H46O386.61458.137.2SterolSterol5-α-Cholesta-8,24-dien-3-β-olTentatively assignedZymosterolC27H44O384.61456.137.6SterolSterolCarbamideTentatively assignedUreaCH4N2O60.02189.28.3AmideUAbbreviations: AA, amino acid; Avg, average; d7WE, n-hexadecyl-1,1,2,2-d4 hexadecanoate-16,16,16-d3; GC-MS, gas chromatography–mass spectrometry; FA, fatty acid, MUFA, monounsaturated fatty acid; MW, molecular weight; PUFA, polyunsaturated fatty acid; RT, retention time (min); TMS, trimethyl silyl group; TRC, Toronto Research Chemicals; Un, unknown; WE, wax ester.The m/z ratio in bold were taken into account for the quantitative assessments. Open table in a new tab Abbreviations: AKT, protein kinase B; IVL, involucrin; K, keratin; LOR, loricrin; p-AKT, phosphorylated protein kinase B; PCNA, proliferating cell nuclear antigen; p-ERBB, phosphorylated ERBB; p-EGFR, phosphorylated EGFR; p-P44/42 MAPK, phosphorylated P44/42 MAPK; VCL, vinculin. Abbreviations: AA, amino acid; Avg, average; d7WE, n-hexadecyl-1,1,2,2-d4 hexadecanoate-16,16,16-d3; GC-MS, gas chromatography–mass spectrometry; FA, fatty acid, MUFA, monounsaturated fatty acid; MW, molecular weight; PUFA, polyunsaturated fatty acid; RT, retention time (min); TMS, trimethyl silyl group; TRC, Toronto Research Chemicals; Un, unknown; WE, wax ester. The m/z ratio in bold were taken into account for the quantitative assessments. Mice carrying floxed Erbb2 alleles or Erbb3 alleles or expressing cre recombinase under the keratin 5 promoter (a courtesy of A. Ramirez and J. Jorcano, CIEMAT, Madrid, Spain) have been described previously (Garratt et al., 2000Garratt A.N. Voiculescu O. Topilko P. Charnay P. Birchmeier C. A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool.J Cell Biol. 2000; 148: 1035-1046Crossref PubMed Scopus (231) Google Scholar; Lee et al., 2009Lee D. Yu M. Lee E. Kim H. Yang Y. Kim K. et al.Tumor-specific apoptosis caused by deletion of the ERBB3 pseudo-kinase in mouse intestinal epithelium.J Clin Invest. 2009; 119: 2702-2713Crossref PubMed Scopus (78) Google Scholar; Ramirez et al., 2004Ramirez A. Page A. Gandarillas A. Zanet J. Pibre S. Vidal M. et al.A keratin K5Cre transgenic line appropriate for tissue-specific or generalized Cre-mediated recombination.Genesis. 2004; 39: 52-57Crossref PubMed Scopus (166) Google Scholar). Mice strains were maintained in the C57BL/6N background under specific pathogen-free conditions and had access to water and standard rodent diet (V1534, Ssniff) ad libitum. All animal experiments were approved by the governmental body of the state of Upper Bavaria, Germany (Regierung von Oberbayern, 55.2-1-54-2532-206-2012) and by the Austrian Federal Ministry of Education, Science, and Research (BMBWF-68.205/106-V/3b/2019) and were performed in strict compliance with the European Communities Council Directive (86/609/EEC) recommendations for the care and use of laboratory animals. Genotyping of mouse lines was performed according to the original publications. Both females and males of E2/E3del double knockouts show the same phenotype, but all experiments published in this article were performed with males. HaCaT cells were originally obtained from CLS Cell Lines Service. For the generation of ERBB2 and ERBB3 double-knockout clones, we used the already established ERBB2-knockout cell line HaCaT 2/F6 and the ERBB3-knockout cell line HaCaT 3/B8 (Dahlhoff et al., 2017Dahlhoff M. Gaborit N. Bultmann S. Leonhardt H. Yarden Y. Schneider M.R. CRISPR-assisted receptor deletion reveals distinct roles for ERBB2 and ERBB3 in skin keratinocytes.FEBS J. 2017; 284: 3339-3349Crossref PubMed Scopus (0) Google Scholar). Cells were cultured in DMEM high glucose (Merck) supplemented with 10% fetal calf serum (Merck) and 1% streptomycin/penicillin (Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% carbon dioxide. The following plasmids were used for the CRISPR/Cas9-based deletion of ERBB2 and ERBB3: Cas9 plasmid (Mali et al., 2013Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. et al.RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (7279) Google Scholar), surrogate plasmid, and guide RNA (gRNA) plasmid (Mulholland et al., 2015Mulholland C.B. Smets M. Schmidtmann E. Leidescher S. Markaki Y. Hofweber M. et al.A modular open platform for systematic functional studies under physiological conditions.Nucleic Acids Res. 2015; 43: e112Crossref PubMed Scopus (0) Google Scholar) with the gRNA sequences 5′-CTGGACATGCTCCGCCACCTCTACCA-3′ for ERBB2 gRNA and 5′-TACGAGAGGTGTGAGGTGGTGATG-3′ for ERBB3 gRNA. For transfection, we used Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Selection of cells with Cas9 activity was accomplished by single-cell sorting for GFP and mCherry-positive cells 48 hours after transfection. The cells were dissociated with 0.25% Trypsin-EDTA (Merck), resuspended in DMEM containing 2% fetal calf serum, and sorted using CytoFLEX SRT (Beckman Coulter) into 96-well plates (Falcon) containing 100 μl DMEM complete medium. After expansion of the clones, the sequence variation was analyzed by PCR amplification of the gRNA-binding region using the following primers: 5′-TCTCCCTGTCTGAGGTGGC-3′ for hERBB2_s, 5′-GGGACATGATCATGCTGGC-3′ for hERBB2_as, 5′-CTACAGCTTCTGCCTATCGC-3′ for hERBB3_s, and 5′-TAGGTCCCAGATGACAGCC-3′ for hERBB3_as. PCR products were purified by gel extraction (New England Biolabs) and cloned using the StrataClone Blunt PCR Cloning Kit (Agilent Technologies). Six different clones were sequenced using T7 primer 5′-TAATACGACTCACTATAGGG-3′. Identified knockouts were confirmed by western blot analysis. Normal human epidermal keratinocytes were isolated from healthy human skin biopsies. The dermis was removed as good as possible, and the remaining tissue was digested overnight at 4 °C using Dispase II (2.2 IU/ml, Roche). Subsequently, the epidermis was separated and incubated with Trypsin/EDTA (Lonza) for 5 minutes at 37 °C to isolate primary keratinocytes. Normal human epidermal keratinocytes were cultured in Keratinocyte Growth Medium 2 (PromoCell) supplemented with 1% streptomycin/penicillin (Thermo Fisher Scientific) in a humidified incubator at 37 °C with 5% carbon dioxide. Cells were passaged approximately twice a week depending on confluency. Lipofectamine RNAiMAX (Invitrogen) was used according to the manufacturer's instructions to transfect normal human epidermal keratinocytes at a confluence of 50–60% in a 6-well plate with 30 pmol pooled small interfering RNAs for ERBB2 and ERBB3 or with a negative control small interfering RNA (Silencer Select, Ambion). After 96 or 144 hours, cells were harvested with ice-cold PBS for protein isolation. Protein was extracted using RIPA lysis buffer with protease inhibitor (Merck) and PhosSTOP (Roche). The concentration of the protein lysates was estimated by bicinchoninic acid assay (Thermo Fisher Scientific). A total of 20 μg of HaCaT total protein or 10 μg of normal human epidermal keratinocyte total protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (GE Healthcare) by semidry blotting for 1 hour at 14 V. The membranes were stained with Ponceau S solution to confirm successful blotting and then blocked in 5% w/v skim milk for 1 hour at room temperature. After washing in Tris-buffered saline (TBS) solution with 0.05% Tween 20 (Merck), membranes were incubated overnight at 4 °C in 5% w/v BSA (Sigma-Aldrich) with the appropriate primary antibody. Antibodies and their dilutions are listed in Supplementary Table S1. Membranes were washed and incubated in 5% w/v skim milk powder with the corresponding horseradish peroxidase–labeled secondary antibody. Signals were detected using an enhanced chemiluminescence detection reagent (Bio-Rad Laboratories). After detection, membranes were stripped by incubation with an appropriate buffer (2% SDS, 62.5 mM Tris/hydrogen chloride, pH 6.8, and 100 mM β-mercaptoethanol) for 40 minutes at 60 °C and incubated with a second primary antibody recognizing the total protein of a phosphorylated protein or a housekeeper protein. Band density measurement was performed using ImageJ (http://rsb.info.nih.gov/ij), and values were normalized to GAPDH. Cell proliferation and migration were monitored in 6-well plates using a PHIO Cellwatcher M microscope (PHIO Scientific, Munich, Germany) placed inside the incubator, imaging each well every 30 minutes (over a period of x [to y] hours). The proliferation, motility, and wound healing assays were analysed using PHIOme Data Management and Analysis Platform with the Software Add-Ons Proliferation, Motility, and Wound Healing, version 1.4.2, from PHIO Scientific. For the proliferation assay, 8 × 104 HaCaT cells, either ERBB2 or ERBB3 single- or double-knockout cells and a mock-transfected control cell line, were seeded in a 6-well plate. The cells were allowed to attach for 24 hours, and then they were washed twice with PBS and covered with 3 ml culture medium, and then the plate was transferred to the PHIO Cellwatcher. Migration was assessed by wound healing assays. Therefore, cells were seeded on a 6-well plate and allowed to attach and grow until they were 100% confluent. The cell monolayer was scraped with a 200-μl pipet tip. To remove the debris, the scratch was washed twice with PBS before adding 3 ml of culture medium and starting the measurement. A confluency experiment was performed to study the ERBB receptors under conditions of high and low cell–cell contact. Briefly, the cells were seeded and cultured up to 50 or 100% confluency. Then, they were harvested using RIPA lysis buffer. Skin samples were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. For target retrieval, the sections were boiled in a microwave for 20 minutes in 10 mM citrate buffer (pH 6.0). Tissue sections were incubated with an anti–Ki-67 antibody (rat anti-mouse Ki-67, TEC-3, DakoCytomation, 1:200) over night at 4 °C, followed by incubation with a horseradish peroxidase–conjugated secondary antibody rabbit anti-rat antibody (DakoCytomation, E0468, 1:200) for 1 hour at room temperature. The compound 3,3'-diaminobenzidine (KemEnTec, Copenhagen, Denmark) was used as a chromogen. Tumor cell proliferation rate was measured on Ki-67–stained sections by counting 10 visual fields for each mouse. Both Ki-67–positive and –negative nuclei were counted on pictures taken with a 200-magnification lens and a Leica DFC425C digital camera (Leica Microsystems) covering an area of 1.3 mm2. Skin samples were fixed in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin. Paraformaldehyde-fixed sections were microwaved for 22 minutes in 10 mM citrate buffer (pH 6.0). Tissue sections were blocked with 5% normal donkey serum in TBS for 1 hour at room temperature and incubated over night at 4 °C with the designated primary antibodies in 1% BSA in TBS. Afterward, the slides were washed with TBS with 0.1% Tween and incubated with the appropriate fluorescent secondary antibody in 1% BSA in TBS for 1 hour at temperature. After washing with TBS, the sections were mounted with Vectashield with DAPI (Vectorlabs). Images were acquired using Zeiss LSM 880 Airyscan confocal laser-scanning microscope (Carl Zeiss MicroImaging) with a ×40/1.4

17-8-estradiol, involved in mesothelioma pathogenesis, and its precursors were explored as potential biomarkers for the early diagnosis of mesothelioma. Using enzyme-linked immunosorbent assay(ELISA) for 17-8-estradiol and ultra-high performance liquid chromatography/tandem mass spectrometry(UHPLC-MS/MS) for 19 178-estradiol precursors, a comprehensive analysis of 20steroid hormones was conducted in the serum of mesothelioma patients(n=67), asbestos-exposed healthy subjects(n=39), and non-asbestos-exposed healthy subjects (n=35). Bioinformatics analysis explored three potential serum biomarkers: 17-8-estradiol, DHEA-S, and androstenedione. The results revealed significant differences in 17-8-estradiol levels between mesothelioma patients and both non-asbestos-exposed and asbestos-exposed healthy subjects. No significant variations in serum 17-8-estradiol levels were observed among mesothelioma patients at different stages, suggesting its potential as an early diagnostic marker. 17-8-estradiol levels were similar in mesothelioma patients with environmental and occupational asbestos exposure, while males with occupational asbestos exposure exhibited significantly higher levels of 17-8-estradiol compared to females. Significant reduction in androstenedione and an increase in DHEA-S were observed in asbestos-exposed individuals compared to non-asbestos-exposed individuals. The analysis of DHEA-S-androstenedione-17-8-estradiol signature score showed an increase in asbestos-exposed individuals and mesothelioma patients compared to non-asbestos-exposed individuals, and this score effectively distinguished between the groups. The Cancer Genome Atlas data was utilized to analyze the expression of 5-a-reductase1 and hydroxysteroid-178-dehydrogenase2 genes. The findings indicated that mesothelioma patients with elevated gene values for 5-a-reductase1 and hydroxysteroid-178-dehydrogenase2 have a worse or better prognosis on overall survival, respectively. In conclusion, this study suggests 17-8-estradiol, DHEA-S, and androstenedione as biomarkers for mesothelioma risk and early diagnosis of mesothelioma in asbestos-exposed individuals, aiding timely intervention and improved care.

BACKGROUND:Data on sexual function in patients with adrenal insufficiency are scarce and largely controversial. OBJECTIVES:To investigate sexual dysfunction in patients with primary and secondary adrenal insufficiency and the effects of switching to once-daily dual-release hydrocortisone on sexual function in outcome assessors blinded, randomized, multicenter, active comparator clinical trial. MATERIALS AND METHODS:Eighty-nine adrenal insufficiency patients on conventional, multiple daily doses of glucocorticoid replacement, enrolled in the Dual RElease hydrocortisone versus conventionAl glucocorticoid replaceMent in hypocortisolism (DREAM) trial, were randomly assigned to continue their therapy or to switch to an equivalent dose of dual-release hydrocortisone. Sixty-three patients (34 women) consented to sex steroid measurements and questionnaires completion for quality of life (Addison's disease-specific quality-of-life questionnaire) and sexual function evaluation (female sexual function index for women, International Index of Erectile Function-Erectile Function for men) at baseline and 24 weeks after randomization. RESULTS:At baseline, sexual dysfunction was observed in 41% of women and 59% of men with adrenal insufficiency. In both sexes, no associations were found between sexual function and hormone levels, whereas Addison's disease-specific quality-of-life questionnaire total and fatigue domain scores positively correlated with total female sexual function index and International Index of Erectile Function-Erectile Function scores. At 24 weeks, there was no significant difference either in sexual function or sex steroid levels between study groups. In the dual-release hydrocortisone group, the variation in the female sexual function index desire domain score was positively associated with the change in Addison's disease-specific quality-of-life questionnaire's symptom domain score (ρ = 0.478, p = 0.045). DISCUSSION:Sexual dysfunction is common in adrenal insufficiency patients and is likely explained by multiple factors. dual-release hydrocortisone treatment is not directly associated with sexual function improvement, but an indirect effect mediated by quality-of-life amelioration cannot be excluded.

The immune response in atopic dermatitis (AD) is driven predominantly by T helper (Th2) cells. The Th2-type cytokines interleukin (IL)-4, and IL-13 activate the Janus kinase/signal transduction and activator of transcription (JAK-STAT) signaling pathway, which is essential for their pro-inflammatory action and detrimental effects on the permeability barrier in the epidermis. The lipid abnormalities driven by Th2-type cytokines have not been completely elucidated in AD. The objective of this study was to investigate the role of JAK/STAT in the lipid perturbations in the 3D-epidermal equivalent model of AD conditioned by Th2-type cytokines. We used the small molecule JAK inhibitor tofacitinib to delineate effects involving the JAK/STAT signaling pathway in the lipid perturbation. Th2-type cytokines caused decrement in the elongation process of fatty acids and in the ceramide synthesis, as demonstrated by the reduced levels of ELOVLs 1, 3, and 6, and SPT mRNAs. In contrast, mRNAs of the lipid metabolism-related enzymes DEGS2 and CA2 were increased. Expression levels of PPARG mRNA were also significantly decreased. Altogether, these effects were abrogated by tofacitinib. Th2 signals caused a significant depletion of triglycerides (TGs) paralleled by a lower content of intracellular lipid droplets and increased levels of phosphatidylcholines (PCs). The depletion of TGs was partly associated with the inhibition of the monounsaturated palmitoleic acid formation. Likely, the observed lipid abnormalities occurred prior to changes in ceramide profiles, which were minimally perturbed. Lipid changes induced by Th2-type cytokines were abrogated upon co-treatment with tofacitinib. In conclusion, inhibition of JAK/STAT activation counteracts the unbalance in the lipid metabolism caused by Th2-type cytokines.

Abnormalities of the permeability barrier and impaired epidermal differentiation characterize both psoriasis and atopic dermatitis, which are driven by immune responses mediated by Th1 and Th2 cytokines. The spectrum of interference of the Th1 and Th2 type cytokines with normal terminal differentiation of keratinocytes is not fully characterized. Our aim was to identify specific and common features of epidermal perturbation associated with each group of cytokines. Immortalized human keratinocytes (Ker-CT) were treated with Th1 and Th2 cytokines in pro-differentiative culture conditions with high calcium concentration. The expression of genes involved in epidermal differentiation and lipid metabolism was evaluated by RT-PCR at 2, 4 and 7 days of treatment. In parallel, Western blot analyses were performed to assess the expression levels of keratinocyte differentiation markers. Lipid composition was investigated by GCMS and LCMS at 7 days. Treatment with Th1 and Th2 cytokines showed decreased and increased expression of K10 and K6, respectively. Both Th1 and Th2 cytokines increased the expression of CCL26, CerS3, ALOX12B, FAS, FADS2 and SCD1. Conversely, LOR and CASP14, CerS6, ABCA12, ELOVL3, HMG-CoA reductase, and HMGB1 displayed low expression with both cytokine types. Specific effects of Th1 and Th2 cytokines were observed on the lipid components. Th1 cytokines inhibited significantly the calcium-induced levels of phospholipids (PCs, PEs, SMs), and free fatty acids (FFAs). Th1 and Th2 cytokines elevated and decreased, respectively, ceramides with medium to long carbon chain. Th2 cytokines promoted the accumulation of short chain ceramides and of cholesterol sulfate, as opposed to the generalized decrement of FFAs, which included long chain ones. Overall, our data indicated that the two families of cytokines feature perturbation of the lipid pathways that underlie the barrier derangement associated with psoriasis and atopic dermatitis.

Bed bugs (Cimex lectularius) have proliferated globally and have become one of the most challenging pests to control indoors. They are nocturnal and use multiple sensory cues to detect and orient towards their human hosts. After feeding, usually on a sleeping human, they return to a shelter on or around the sleeping surface, but not directly on the host. We hypothesized that although human skin odors attract hungry bed bugs, human skin compounds may also prevent arrestment on hosts. We used arrestment assays to test human skin swabs, extracts from human skin swabs, and pure compounds identified from human skin swabs. When given a choice, bed bugs preferred to arrest on substrates not previously conditioned by humans. These responses were consistent among laboratory-reared and apartment-collected bed bugs. The compounds responsible for this behavior were found to be extractable in hexane, and bed bugs responded to such extracts in a dose-dependent manner. Bioassay-guided fractionation paired with thin-layer chromatography, GC-MS, and LC-MS analyses suggested that triglycerides (TAGs), common compounds found on human skin, were preventing arrestment on shelters. Bed bugs universally avoided sheltering in TAG-treated shelters, which was independent of the number of carbons or the number of double bonds in the TAG. These results provide strong evidence that the complex of human skin compounds serve as multifunctional semiochemicals for bed bugs, with some odorants attracting host-seeking stages, and others (TAGs and possibly other compounds) preventing bed bug arrestment. Host chemistry, environmental conditions and the physiological state of bed bugs likely influence the dual nature behavioral responses of bed bugs to human skin compounds.

Background Atopic dermatitis (AD) is a composite disease characterized by derangement of the skin permeability barrier (SPB), altered immune defence, and dysbiosis. Little is known on the role played by the sebaceous gland (SG) activity in the SPB integrity and in the AD pathomechanisms. Objectives To investigate profiles of sebaceous and epidermal free fatty acids (FFAs), squalene, cholesterol, triglycerides (TGs), and wax esters (WEs) in sebum and stratum corneum (SC) from seborrheic and non-seborrheic areas, in healthy subjects and patients with AD. To simultaneously acquire aminoacidome in SC. Methods In healthy controls and patients with AD, sebum and SC were sampled consecutively from facial areas (forehead, and cheeks). SC was sampled also from non sebaceous areas (arm) in healthy controls and from the non lesional and lesional areas on the arm in AD. Sampling was preceded by assessments of skin biophysics, i.e. TEWL and corneometry. Results Disruption of the SBP was associated with decreased levels of lipids of both sebaceous and epidermal type. Extent of lipid derangement in the SG and the SC was correlated with the AD severity. Relative composition of natural moisturizing factors was altered in the SC of patients with AD. Conclusions The SG activity is compromised in adult AD. Aminoacidome is deranged in the facial areas in AD. Lipid signatures in association with aminoacidome, and skin physical properties may serve the definition of phenotype clusters that associate with AD severity.

Derangement of ceramide levels in the skin permeability barrier (SPB) is widely demonstrated in atopic dermatitis (AD). By GCMS chemometric analysis we have observed significant deficiency of sebum-specific free fatty acids (FFAs), i.e. species with odd carbon number and terminal branching, in association to sebostasis in AD sebum. To investigate the interplay between sebum and epidermal lipids, we conducted a targeted LCMS study on the sphingolipidome of the stratum corneum (SC) in sebaceous and non sebaceous areas. The study involved 44 adult controls and 54 age and gender balanced AD patients, which included 20 with uninvolved face and 34 with involved face. SC was sampled from forehead, cheek, and non lesional arm. We profiled 111 all-classes ceramides, i.e. arising from the binding of P, S, DS, and H long chain bases (LCBs) with hydroxylated and non hydroxylated FAs, indicated as A and N, respectively. SC from facial sites presented higher levels (pmol/mg protein) of species belonging to the CerAH, CerAS and CerNS classes compared to arm, in controls and both AD subgroups. Medium-to-short and odd chained species (C≤44) were largely responsible for the discrimination between sebaceous and non-sebaceous areas. Facial sphingolipid signature was more effective than arm sphingolipidome in discriminating controls from both AD groups, being 60 and 40 ceramide species significantly different in face and arm SC, respectively. Modification of CerAH, CerAP, and CerNS level was more pronounced at sebum-rich areas than arm. Odd-chained ceramides accounted for half of the total discriminating species in all cases. These findings support a role played by the sebaceous secretion in the plasticity of the SPB. Ceramide bio-signature in sebaceous areas may empower the characterization of AD patients.