Formation Of Stratum Corneum Research Articles

Epidermal keratinocytes regulate hyaluronan metabolism via extracellularly secreted hyaluronidase 1 and hyaluronan synthase 3

Hyaluronan (HA) is a high-molecular-weight (HMW) glycosaminoglycan, which is a fundamental component of the extracellular matrix that is involved in a variety of biological processes. We previously showed that the HYBID/KIAA1199/CEMIP axis plays a key role in the depolymerization of HMW-HA in normal human dermal fibroblasts (NHDFs). However, its roles in normal human epidermal keratinocytes (NHEKs) remained unclear. HYBID mRNA expression in NHEKs was lower than that in NHDFs, and NHEKs showed no depolymerization of extracellular HMW-HA in culture, indicating that HYBID does not contribute to extracellular HA degradation. In this study, we found that the cell-free conditioned medium of NHEKs degraded HMW-HA under weakly acidic conditions (pH 4.8). This degrading activity was abolished by HYAL1 knockdown, but not by HYAL2 knockdown. Newly synthesized HYAL1 was mainly secreted extracellularly, and the secretion of HYAL1 was increased during differentiation, suggesting that epidermal interspace HA is physiologically degraded by HYAL1 according to pH decrease during stratum corneum formation. In HA synthesis, HAS3 knockdown reduced HA production by NHEKs, and interferon-γ-dependent HA synthesis was correlated to increased HAS3 expression. Furthermore, HA production was increased by TMEM2 knockdown through enhanced HAS3 expression. These results indicate that NHEKs regulate HA metabolism via HYAL1 and HAS3, and TMEM2 is a regulator of HAS3-dependent HA production.

Qinzhuliangxue mixture ameliorates psoriasis by restraining apoptosis in psoriasis via downregulating the MDA-5 pathway

Ethnopharmacological relevancePsoriasis is characterized by hyperkeratosis that produces the classic silvery scales, and the pathogenesis of psoriasis involves abnormal proliferation of keratinocytes. Emerging evidence supports that apoptosis regulates keratinocyte proliferation and formation of stratum corneum, which maintains the homeostasis of the skin. Qinzhuliangxue mixture (QZLX) is a representative formula for the treatment of psoriasis, which was earliest recorded in the classic Chinese medicine book Xia's Surgery. In our previous clinical studies, QZLX demonstrated 83.33% efficacy with few side effects in the treatment of psoriasis. Furthermore, our published basic research has also proved that the QZLX mixture effectively inhibits the hyperproliferation of keratinocytes, thus exerting therapeutic effects on psoriasis. However, whether QZLX mixture can regulate keratinocytes apoptosis requires further clarification. Objective of the studyTo investigate the mechanism of QZLX in the treatment of psoriasis from the perspective of keratinocyte apoptosis. Materials and methodsFirst, psoriasis-like mice with imiquimod (IMQ)-induced were given QZLX intragastric administration and Psoriasis Area Severity Index (PASI) scores were recored for 11 consecutive days to appraise the efficacy. Then, tissue samples were collected for transcriptome analysis. The DEseq2 method detected significantly differentially expressed genes (DEGs), Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway databases were used to analyze the functions and pathway enrichment of DEGs. After that, the therapeutic mechanisms of QZLX in intervening with psoriasis were explored using TUNEL, immunohistochemical staining, and western blotting. ResultsQZLX ameliorated the symptoms and pathological characteristics of IMQ-induced psoriasis in mice. The epidermal cell hyperplasia in the skin was inhibited, in accordance with the suppressed expression of PCNA and Ki67 after treatment. Transcriptome sequencing showed that melanoma differentiation associated gene-5 (MDA-5) was downregulated. GO and KEGG enrichment analysis of the signaling pathways indicated that the differentially expressed genes were significantly enriched in apoptosis pathways. Besides, QZLX treatment decreased the apoptosis of keratinocyte as shown by reduced TUNEL-positive cells. As MDA-5 protein levels decreased, so did the expression of the downstream protein Caspase-8, which indicates that the apoptotic pathway was triggered. Furthermore, QZLX therapy might also help to balance the apoptotic Bcl-2 family expression. ConclusionQZLX restrains the apoptosis of keratinocyte in psoriasis-like mice by downregulating the MDA-5 pathway. The restoration of the balance between cell apoptosis and proliferation in the skin may lead to considerable psoriasis relief. Our study reveals the possible molecular processes behind the effects of QZLX therapy on the skin lesions of psoriasis, and lends support to its clinical efficacy.

Are the Cutaneous Microbiota a Guardian of the Skin's Physical Barrier? The Intricate Relationship between Skin Microbes and Barrier Integrity.

The skin is a tightly regulated, balanced interface that maintains our integrity through a complex barrier comprising physical or mechanical, chemical, microbiological, and immunological components. The skin's microbiota affect various properties, one of which is the establishment and maintenance of the physical barrier. This is achieved by influencing multiple processes, including keratinocyte differentiation, stratum corneum formation, and regulation of intercellular contacts. In this review, we summarize the potential contribution of Cutibacterium acnes to these events and outline the contribution of bacterially induced barrier defects to the pathogenesis of acne vulgaris. With the combined effects of a Westernized lifestyle, microbial dysbiosis, epithelial barrier defects, and inflammation, the development of acne is very similar to that of several other multifactorial diseases of barrier organs (e.g., inflammatory bowel disease, celiac disease, asthma, atopic dermatitis, and chronic rhinosinusitis). Therefore, the management of acne requires a complex approach, which should be taken into account when designing novel treatments that address not only the inflammatory and microbial components but also the maintenance and strengthening of the cutaneous physical barrier.

Investigations into the FLG Null Phenotype: Showcasing the Methodology for CRISPR/Cas9 Editing of Human Keratinocytes

Ever since the association between filaggrin (FLG) loss-of-function mutations and ichthyosis vulgaris and atopic dermatitis disease onset was identified, filaggrins function has been under investigation. Intra-individual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG knockout (ΔFLG) N/TERT-2G keratinocytes. Filaggrin deficiency was demonstrated by immunohistochemistry of human epidermal equivalent (HEE) cultures. Next to (partial) loss of structural proteins (IVL, HRNR, KRT2, and TGM1), the stratum corneum was more dense and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG-HEEs. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, filaggrin protein expression, and expression of aforementioned proteins. The beneficial effects on stratum corneum formation were reflected by normalization of EIS and TEWL. This study demonstrates the causal phenotypical and functional consequences of filaggrin deficiency, indicating filaggrin is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of filaggrin in skin biology and disease.

Ethnobotanical Survey of Medicinal Plants Used against Infertility in the Nyong and So’o Division (Cameroon) and Pro-fertilizing Activities of Mammea africana (Clusiaceae) Aqueous Extract Rats

Background: World-renowned as primary healthcare, traditional medicine often represents the only therapeutic resource for many communities with very low incomes, although it shows valuable benefits. The objective of this investigation was to evaluate the medicinal plants used to treat infertility in the Nyong & So’o Division (Cameroon) and the effect of Mammea africana, the most commonly used medicinal plant extract, on reproductive parameters in female rats.
 Methods: First, a transversal and analytic investigation using semi-structured interviews was conducted on a sample of 22 traditional healers. Secondly, two pharmacological assays were carried out to evaluate M. africana aqueous extract activities in 8-10-week-old female Wistar rats. During 14 days, non-ovariectomized (Novx) mice were given distilled water (DW) at 10 mL/kg and M. africana extract at 40, 80, and 160 mg/kg. In the end, cervical smears were realized, animals were sacrificed, and extract activities were evaluated on reproductive parameters. The estrogenic activities of M. africana extract were assessed on estrogenic-dependent tissues per os in ovariectomized (Ovx) rats in the second assay. They received respectively distilled water (DW), estradiol valerate (E2V) at 1 mg/kg, and the extract at the aforesaid doses, and 3 groups were co-treated with E2V (0.75 mg/kg) and the extract. A sham-operated group received DW
 Results: Ethnobotanical investigation revealed that 20 species of plant belonging to 13 different families are used to treat infertility. Mammea africana was the most used plant and its extract at 80 mg/kg aqueous extract induced a significant increase (p <0.001) of FSH, LH, and estradiol in Novx rats compared to control. Furthermore, the extract induced the maturation of ovarian follicles. M. africana extract exhibited also estrogenic activities in Ovx rats. Indeed, M. africana aqueous extract induced the formation of stratum corneum in the vagina of Ovx rats. E2V activities at 0.75 mg/kg were maximized by M. africana extract.
 Conclusion: To sum up, many species are used in the Nyong & So’o Division to manage reproductive failure. Among them, Mammea africana is the most commonly used and possesses estrogenic-like activities.

Novel filagrinol-containing emollient

Background. In the last decade, there has been a significant increase in interest in the study of the role of epidermal barrier structural integrity in order to determine the prospects for its pathogenetic correction. It is now known that patients with filaggrin gene mutations have increased risk of developing atopic dermatitis, xerosis, ichthyosis, eczema. Filaggrin participates in the formation of a full-fledged stratum corneum, provides formation of a natural moisturizing factor, stabilizing the water balance of the stratum corneum. In Russia, the innovative filaggrin-containing Admera has been registered, which opens up prospects in the management impaired skin barrier function diseases.
 Aims. Efficacy and safety of "Admera" emollient in patients with primary and secondary xerosis.
 Materials and methods. A single-center prospective interventional study December 2021 March 2022 at the Department of Skin and Venereal Diseases of the Sechenov University. The study included 32 patients (22 main group, 10 control group) aged 18 to 60 years (29 9.78), 14 men and 18 women diagnosed with atopic dermatitis, skin xerosis. Patients applied moisturizers 3 times a day for 21 days. Performance was evaluated using vIGA-AD, POEM, EASI, 5D Elman scale, SKINDEX-29, HADS, on Capricorn AI, ANTERA 3D machines.
 Results. A steady regression of clinical symptoms of xerosis was noted during the study by the dynamics of the study scales. The mean EASI score decreased by 67% from 25.25 18.78 screening to 7.8 4.41 control visit (p 0.001). Assessment of POEM index dynamics showed a significant decrease in total score from 18.65 to 6.6 (p 0.001). There was good tolerability of the product and absence of adverse events.
 Conclusions. The filagrinol-containing emollient Admera (Dr. Reddy's Laboratories Ltd., India) demonstrated high efficacy and safety in all patients. This emollient can be recommended as a basic care product in the treatment schemes and prevention of primary, secondary xerosis, relapses of atopic dermatitis.

254 Characterization of keratinocyte lipid composition of bioengineered human epidermis

In the three past decades, tissue engineering allowed to develop skin models with characteristics close to ex vivo skin, particularly at the level of the epidermis. The main function of the epidermis is to protect the body against environmental damage (UVs, pollution, microbial pathogens…) and to prevent water loss. Keratinocytes, the major cellular component of the epidermis, proliferate and differentiate to form in fine a physical barrier sustaining the epidermal barrier function. This cutaneous impermeability is ensured by the formation of a well-defined stratum corneum constituted by corneocytes and a lipidic matrix.

409 Corneoptosis, functional keratinocyte cell death, is tightly associated with spaciotemporal dynamics of epidermal tight junctions

Epidermis has two physical barrier components, stratum corneum (SC) and tight junction (TJ). Formation of SC or cornification is initiated by the cell death of the top layer (SG1) of stratum granulosum, termed ‘corneoptosis’ after prolonged intracellular Ca2+([Ca2+]i) elevation (∼60min), followed by rapid intracellular acidification. However, the exact timing of the onset of corneoptorsis remains to be clarified. In this study, we aimed to determine the spatiotemporal relationship between the turnover of TJs and corneoptosis. We performed intravital confocal microscopic imaging of mouse ear co-expressing ZO-1-Venus (TJ strand marker) and GCaMP3 (intracellular Ca2+ marker). Hoechst 33342 was intradermally injected to monitor the change of nuclear pH. Consistent with our previous observation, ZO-1-Venus signals showed two TJ strand-pattens in upper SG. According to the TJ turnover, three phases are observed; TJ-phase 1 which shows single old TJ strand formed in SG2 cells; TJ-phase 2 which shows double TJ strands; TJ-phase 3 which shows new single TJ strand. Time lapse imaging analysis revealed that the elevation of GCaMP3 signal was observed only in TJ-phase 2 which shows double TJs, but not in other TJ-phases. The elevation of [Ca2+]i lasted for approximately 60min. Subsequently, intracellular acidification was induced in these SG1 cells as detected by both the decrease of GCaMP3 signal and the increase of Hoechst 33342 signals. Simultaneously, ‘upper larger TJ strand’ of double-TJ strand in SG1 cells disappeared, resulting in change from TJ-phase 2 to TJ-phase 3. These findings indicated that the onset of corneoptosis is tightly associated with the spaciotemporal dynamics of TJs.

T Helper 17/T Helper 22‒Skewed Inflammation with Epidermal Barrier Dysfunction in Nonmajor Inherited Ichthyosis Subtypes

Ichthyoses are heterogeneous genetic skin diseases that are characterized by thickened and desquamating skin. Causative genes are associated with stratum corneum (SC) formation. The two most common ichthyoses are ichthyosis vulgaris and recessive X-linked ichthyosis. Ichthyosis vulgaris is caused by gene variants in FLG encoding FLG (Oji et al., 2010), and it is known to increase the risk of developing atopic dermatitis (AD). Recessive X-linked ichthyosis results from variants in STS. STS encodes steroid sulfatase, and STS abnormalities lead to the accumulation of cholesterol sulfate and result in corneocyte retention (Oji et al., 2010).

Epigenetic Mechanisms of Epidermal Differentiation.

Keratinocyte differentiation is an essential process for epidermal stratification and stratum corneum formation. Keratinocytes proliferate in the basal layer of the epidermis and start their differentiation by changing their functional or phenotypical type; this process is regulated via induction or repression of epidermal differentiation complex (EDC) genes that play a pivotal role in epidermal development. Epidermal development and the keratinocyte differentiation program are orchestrated by several transcription factors, signaling pathways, and epigenetic regulators. The latter exhibits both activating and repressive effects on chromatin in keratinocytes via the ATP-dependent chromatin remodelers, histone demethylases, and genome organizers that promote terminal keratinocyte differentiation, and the DNA methyltransferases, histone deacetylases, and Polycomb components that stimulate proliferation of progenitor cells and inhibit premature activation of terminal differentiation-associated genes. In addition, microRNAs are involved in different processes between proliferation and differentiation during the program of epidermal development. Here, we bring together current knowledge of the mechanisms controlling gene expression during keratinocyte differentiation. An awareness of epigenetic mechanisms and their alterations in health and disease will help to bridge the gap between our current knowledge and potential applications for epigenetic regulators in clinical practice to pave the way for promising target therapies.

Simple and robust 3D bioprinting of full-thickness human skin tissue

ABSTRACT Artificial skins have been used as skin substitutes for wound healing in the clinic, and as in vitro models for safety assessment in cosmetic and pharmaceutical industries. The three-dimensional (3D) bioprinting technique provides a promising strategy in the fabrication of artificial skins. Despite the technological advances, many challenges remain to be conquered, such as the complicated preparation conditions for bio-printed skin and the unavailability of stability and robustness of skin bioprinting. Here, we formulated a novel bio-ink composed of gelatin, sodium alginate and fibrinogen. By optimizing the ratio of components in the bio-ink, the design of the 3D model and the printing conditions, a fibroblasts-containing dermal layer construct was firstly fabricated, on the top of which laminin and keratinocytes were sequentially placed. Through air-liquid interface (ALI) culture by virtue of sterile wire mesh, a full-thickness skin tissue was thus prepared. HE and immunofluorescence staining showed that the bio-printed skin was not only morphologically representative of the human skin, but also expressed the specific markers related to epidermal differentiation and stratum corneum formation. The presented easy and robust preparation of full-thickness skin constructs provides a powerful tool for the establishment of artificial skins, holding critical academic significance and application value.

Microwave-Assisted Physically Cross-Linked Chitosan-Sodium Alginate Hydrogel Membrane Doped with Curcumin as a Novel Wound Healing Platform.

This project purposes to develop chitosan and sodium alginate-based hydrogel membranes loaded with curcumin through microwave-based physical cross-linking technique and its evaluation for wound healing potential. For the purpose, curcumin-loaded chitosan and sodium alginate membranes were developed using microwave at fixed frequency of 2450 MHz, power 350 W for 60 s, and tested for their physicochemical attributes like swelling, erosion, surface morphology, drug content, and in vitro drug release. The membranes were also subjected to tensile strength and vibrational and thermal analysis followed by testing in vivo on animals. The results indicated that microwave treatment significantly enhanced the swelling ability, reduced the erosion, and ensured smooth surface texture with optimal drug content. The drug was released in a slow fashion releasing total of 41 ± 4.2% within 24-h period with a higher tensile strength of 16.4 ± 5.3 Mpa. The vibrational analysis results revealed significant fluidization of hydrophilic domains and defluidization of hydrophobic domains which translated into a significant rise in the melting temperature and corresponding enthalpy which were found to be 285.2 ± 3.2 °C and 4.89 ± 1.4 J/g. The in vivo testing revealed higher percent re-epithelialization (75 ± 2.3%) within 14 days of the treatment application in comparison to only gauze and other treatments applied, with higher extent of collagen deposition having well-defined epidermis and stratum corneum formation. The microwave-treated chitosan-sodium alginate hydrogel membranes loaded with curcumin may prove to be another alternative to treat skin injuries. Graphical Abstract.

Evolution of innate defense in human skin.

More often as compared to other barrier systems (gastrointestinal, urogenital, and respiratory linings) human skin over millions of years has been subject to fundamental changes in structure and function. When life on land started, the first changes consisted in the formation of a coherent impermeable stratum corneum. Two-legged locomotion was followed by loss of body hair and formation of sweat glands. Major changes took place after the agricultural revolution, investigating settlements with domestication of animals and plants. Living together after giving up nomadic life, hairless skin became a battlefield for pathogens, members of the skin microbiome, and arthropod visits. Human skin became exceptional in showing a boosted, highly developed immune system which is much more complex as compared to the "skins" of other species. A recently found skin disinfection system ("Cationic Intrinsically Disordered Antimicrobial Peptides, CIDAMPs") dates back to the origins of life and still is active in present-day integuments. As a skin-restricted and effective principle, keratinocyte- myeloid synergy (KMS) is recognized. As a consequence of such highly developed immune defense, the basic contributions of KMS - cells (keratinocytes, neutrophils, macrophages) in regulating innate immunity is emphasized. Antimicrobial peptides and chemokines became major keratinocyte products. The formation of impermeable str. corneum membrane has enabled KMS - cells to accumulate within upper skin levels and cause a special group of human skin diseases, pustular dermatoses.

The aryl hydrocarbon receptor at the forefront of host-microbe interactions in the skin: A perspective on current knowledge gaps and directions for future research and therapeutic applications.

The skin is home to a community of skin microbiota including bacteria, viruses and fungi, which are widely accepted to be of importance for skin homeostasis but also associated with skin diseases. Detailed knowledge on the skin microbiota composition and its changes in a number of skin diseases is available. Yet, specific interactions between microbes and the host skin cells or how they communicate with each other are less well understood. To identify, understand and eventually therapeutically exploit causal relationships of microbial dysbiosis with disease, studies are required that address the receptors and mediators involved in host‐microbe interactions. In this perspective article, we provide an outlook on one of such receptors, namely the aryl hydrocarbon receptor (AHR). The AHR is well known for being a ligand‐activated transcription factor regulating the proliferation, differentiation and function of many cell types present in the skin. Its targeting by anti‐inflammatory therapeutics such as coal tar and Tapinarof is effective in atopic dermatitis and psoriasis. AHR signalling is activated upon binding of wide variety of small chemicals or ligands, including microbiota‐derived metabolites. New evidence has emerged pointing towards a key role for epidermal AHR signalling through skin microbiota‐derived metabolites. In response, AHR‐driven expression of antimicrobial peptides and stratum corneum formation may alter the skin microbiota composition. This a self‐perpetuating feedback loop calls for novel therapeutic intervention strategies for which we herein discuss the requirements in future mechanistic studies.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect.In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

Comprehensive stratum corneum ceramide profiling reveals reduced acylceramides in ichthyosis patient with CERS3 mutations.

The stratum corneum (SC) of the epidermis acts as a skin permeability barrier, and abnormalities in SC formation lead to several skin disorders. Lipids, especially the epidermis-specific ceramide classes ω-O-acylceramides (acylceramides) and protein-bound ceramides, are essential for skin barrier formation. Ceramide synthase 3 (CERS3) is involved in the synthesis of acylceramides and protein-bound ceramides, and CERS3 mutations cause autosomal recessive congenital ichthyosis. In the present study, we measured ceramide synthase activity and performed comprehensive SC ceramide profiling in an ichthyosis patient with compound heterozygous CERS3 mutations: nonsense mutation p.Arg75* and missense mutation p.Arg229His. The activity of p.Arg75* and p.Arg229His mutant CERS3 proteins was reduced to 4% and 56%, respectively, of the wild-type protein. In the patient's SC, acylceramide levels were greatly reduced, but the levels of protein-bound ceramides remained almost unchanged. Non-acylated ceramide levels were also affected in the patient; in particular, the levels of ceramides composed of sphingosine and non-hydroxy or α-hydroxy fatty acid were substantially higher than in healthy controls. These results suggest that a reduction in acylceramide levels alone leads to ichthyosis. Although protein-bound ceramides are synthesized from acylceramides, levels of acylceramides and protein-bound ceramides are not necessarily correlated.

Analysis of BCL-2 protein expression in keratinocytic hyperproliferative lesions

Apoptosis regulates keratinocytic proliferation and formation of stra-tum corneum in skin and is regulated by proteins like BCL-2 which con-trols cell survival and p53 which regulates cell death. Dysfunctional apoptosis plays an important role in development of hyperproliferative skin disorders including non-tumorigenic, pre-tumorigenic and tumor-igenic conditions. Therefore, we conducted a study to analyze the ex-pression pattern of apoptosis regulating protein BCL-2 in keratinocytic hyperproliferative lesions. The aim of this study was to evaluate and compare BCL-2 expression in various non-tumorigenic, pre-tumorigenic and tumorigenic keratinocytic hyperproliferative lesions. Skin biopsies of hyperproliferative keratinocytic lesions were studied from October 2018 to September 2020. The cases were categorized into 3 groups based on histological diagnosis (non-tumorigenic, pre-tumorigenic and tumorigenic). Following this, IHC for BCL-2 was performed on all cases using standard protocol. Tumorigenic lesions included 7 cases of SCC, pre-tumorigenic included 6 cases of seboherric keratosis, 2 cas-es of condyloma acuminata, 1 case of epidermodysplasia verruciform-is and 1 case of actinic keratosis, while the remaining 35 cases were non-tumorigenic, including psoriasis (6 cases), verruca vulgaris (4 cas-es), lichen planus (12 cases), chronic dermatitis (9 cases) and lichen simplex chronicus (4 cases). BCL-2 average weighted score in non-tumorigenic lesions was (1.25±1.13) as compared with pre-tumorigenic and tumorigenic lesions (5.30±3.32) and thus showing a significant p value (p<0.001). To conclude, significantly increased BCL-2 expression in neoplastic lesions shows that increased cell survival may contribute to neoplastic transformation.

Addressing Differentiation in Live Human Keratinocytes by Assessment of Membrane Packing Order.

Differentiation of keratinocytes is critical for epidermal stratification and formation of a protective stratum corneum. It involves a series of complex processes leading through gradual changes in characteristics and functions of keratinocytes up to their programmed cell death via cornification. The stratum corneum is a relatively impermeable barrier, comprised of dead cell remnants (corneocytes) embedded in lipid matrix. Corneocyte membranes are comprised of specialized lipids linked to late differentiation proteins, contributing to the formation of a stiff and mechanically strengthened layer. To date, the assessment of the progression of keratinocyte differentiation is only possible through determination of specific differentiation markers, e.g., by using proteomics-based approaches. Unfortunately, this requires fixation or cell lysis, and currently there is no robust methodology available to study keratinocyte differentiation in living cells in real-time. Here, we explore new live-cell based approaches for screening differentiation advancement in keratinocytes, in a “calcium switch” model. We employ a polarity-sensitive dye, Laurdan, and Laurdan general polarization function (GP) as a reporter of the degree of membrane lateral packing order or condensation, as an adequate marker of differentiation. We show that the assay is straightforward and can be conducted either on a single cell level using confocal spectral imaging or on the ensemble level using a fluorescence plate reader. Such systematic quantification may become useful for understanding mechanisms of keratinocyte differentiation, such as the role of membrane in homogeneities in stiffness, and for future therapeutic development.

Genomic and physiological mechanisms underlying skin plasticity during water to air transition in an amphibious fish.

The terrestrial radiation of vertebrates required changes in skin that resolved the dual demands of maintaining a mechanical and physiological barrier while also facilitating ion and gas transport. Using the amphibious killifish Kryptolebias marmoratus, we found that transcriptional regulation of skin morphogenesis was quickly activated upon air exposure (1 h). Rapid regulation of cell-cell adhesion complexes and pathways that regulate stratum corneum formation was consistent with barrier function and mechanical reinforcement. Unique blood vessel architecture and regulation of angiogenesis likely supported cutaneous respiration. Differences in ionoregulatory transcripts and ionocyte morphology were correlated with differences in salinity acclimation and resilience to air exposure. Evolutionary analyses reinforced the adaptive importance of these mechanisms. We conclude that rapid plasticity of barrier, respiratory and ionoregulatory functions in skin evolved to support the amphibious lifestyle of K. marmoratus; similar processes may have facilitated the terrestrial radiation of other contemporary and ancient fishes.

Basket-weave structure in the stratum corneum is an important factor for maintaining the physiological properties of human skin as studied using reconstructed human epidermis and tape stripping of human cheek skin.

The normal stratum corneum (SC) has an upper basket-weave (BW) pattern layer and a lower compact layer. The transition from compact to BW SC is well associated with a transition from diffuse to peripheral distributions of corneodesmosomes (CDs). The loss of transition from compact SC to BW SC appears to cause structural and barrier-function impairments. To show the involvement of the BW SC in maintaining the physiological properties of the skin. Reconstructed human epidermis (RHE) with a complete BW structure was created by treatment with prepared emulsion-A, an oil-in-water emulsion. The RHE tissues were subjected to histological analysis, and the distribution of CDs on the SC with or without BW SC was analysed by anti-desmoglein (Dsg)1 antibody immunofluorescence and ultrastructural and Western blotting analyses. Ultrastructural analysis of intercellular lipids was performed. The mechanical properties of the RHE were evaluated. Emulsion-A successfully generated the BW SC in the RHE in which the degradation of CDs was promoted. The intercellular space of the BW SC generated by emulsion-A was filled with multilamellar lipid sheets. The softness of the SC with a BW structure formed with emulsion-A was higher than that of the compact SC in RHE. The outermost SC Dsg1 degradation (formation of the BW SC as determined with Dsg1 pixels) was correlated with water-barrier functions and the SC softness of healthy human cheek, which varied widely. Emulsion-A successfully generated the BW SC in RHE for the first time. This method is suggested to be a useful tool for investigating the physiological significance of the BW SC invitro. Determination of Dsg1 content in the SC obtained by tape stripping from human skin allows study of the effects of external stimulants, such as creams and ointments, including cosmetics, on the completeness of the BW SC insitu without biopsy. What's already known about this topic? The normal stratum corneum (SC) has two layers, an upper basket-weave (BW) pattern layer and a lower compact layer. Epidermal diseases such as ichthyosis vulgaris and X-linked ichthyosis have an incomplete or no BW SC and impaired SC barrier functions, in which corneodesmosome (CD) degradation in a peripheral distribution is impaired. The roles of the BW SC in the physiological properties of human skin have not been clearly elucidated. What does this study add? Reconstructed human epidermis (RHE) with a complete BW structure was generated for the first time by treatment with oil-in-water emulsion-A. The formation of the BW SC was associated with a decrease in Dsg1 content, which represents the CD number in the SC. The intercellular space of the BW SC generated by emulsion-A, but not compact SC, was filled with multilamellar lipid sheets. The softness of the SC with a BW structure formed by emulsion-A treatment was higher than that of the compact SC in RHE. What is the translational message? RHE with a complete BW SC generated by emulsion-A treatment is suggested to be a useful tool for investigating effects on the physiological functions of the BW SC, as in treatments with creams and ointments including cosmetics. Determination of desmoglein 1 content in the SC obtained by tape stripping from human skin can make it possible to study the effects of external stimulants, such as creams and ointments, including cosmetics, on the completeness of the BW SC insitu without biopsy.