Basal Layer Of Epidermis Research Articles

Capsaicin attenuates the effect of inflammatory cytokines in a HaCaT cell model for basal keratinocytes.

The resolution of the skin's inflammatory response is only possible if its barrier function is restored. TRPV1 channel activation plays an important role during inflammation but the effect of this activation on the skin barrier under inflammatory conditions has not been clarified. We hypothesize that it could potentially aid the keratinocyte barrier by reducing inflammatory cytokine release and promoting tight junction development. To explore the role of TRPV1 activation in inflammation, we designed and optimized an in vitro model of keratinocytes with basal epidermal layer characteristics using HaCaT cells and TNFα to induce inflammation. TNFα increased the gene expression of tight junction protein claudin 1 (CLDN1) by at least 2.60 ± 0.16-fold, in a concentration-dependent manner, over a 48h period. The administration of a capsaicin pre-treatment reduced the CLDN1 expression to 1.51 ± 0.16-fold during the first 6h after TNFα induction, whereas IL-8 cytokine release was reduced 0.64 ± 0.17-fold. After 48h, CLDN1 protein levels increased by a factor of 6.57 ± 1.39 compared to cells only treated with TNFα. These results suggest that activation of TRPV1 by capsaicin can potentiate the increase in CLDN1 expression and CLDN1 protein synthesis induced by TNFα in cultured keratinocytes, while reducing the release of IL-8.

RUNX2 as a novel biomarker for early identification of patients progressing to advanced-stage mycosis fungoides.

The majority of patients with mycosis fungoides (MF) have an indolent disease course, but a substantial fraction (20-30%) of patients progress to advanced stages - usually with a grave prognosis. Early differentiation between indolent and aggressive types of MF is important for the choice of treatment regimen and monitoring of the individual patient. Good biomarkers are therefore desired. Here, we used spatial transcriptomics on skin samples at time-of-diagnosis to enable prediction of patients who later progressed to advanced stages of MF. Formalin-fixed, paraffin-embedded skin biopsies at time of diagnosis from six patients with MF who progressed to advanced stages of disease within 4 months to 12 years after diagnosis, and nine patients who remained in early-stage disease over 9 to 27 years were analyzed using the GeoMx Digital Spatial Profiler to capture spatially resolved high-plex RNA gene expression data. Five different regions of interest (the epidermis, the basal layer of epidermis, CD4+ T-cells and neighboring cells, and Pautrier's microabscesses) were profiled for further assessment. Interestingly, RUNX2, SHMT2, and MCM7 were upregulated in the enriched population of malignant T-cells in Pautrier's microabscesses in patients who later developed advanced stages of disease. Expression of RUNX2, SHMT2 and MCM7 in malignant T-cells was confirmed in a subset of patients in MF skin using scRNA-seq datasets across multiple studies and correlating with stage of disease. Taken together, we provide first evidence that RUNX2 has potential as a biomarker to identify MF patients progressing to advanced stage disease. As RUNX2 has not previously been linked to MF, our data also shows the analytical strength of combining spatial transcriptomics with scRNA-seq analysis.

Analysis of Immunohistochemical Expression of Bcl-2 in Cases of Psoriasis and Psoriasiform Dermatitis.

Introduction Psoriasis involves rapid cell growth and abnormal differentiation of skin cells. Dysregulation of apoptosis has been proposed in the pathogenesis of psoriasis. The role of Bcl-2 as an anti-apoptotic proteinin pathogenesis has not been studied in detail in these cases.Our study aims to evaluate the expression of Bcl-2 in different skin compartments in psoriasis and psoriasiform dermatitis cases. Materials and methodology An analytical cross-sectional study was performed using paraffin blocks of psoriasis and psoriasiform dermatitis patients between 2022 and 2023. For light microscopy screening, sections were initially stained with hematoxylin and eosin stains. In further sections, a primary antibody against Bcl-2 was applied. The level of Bcl-2 expression in keratinocytes of supra-basal and basalepidermal layers and dermal lymphocytes was evaluated using a scoring system. Results Sixty patients, 30 with psoriasis and 30 with psoriasiform dermatitis were included. Among the histopathological features, parakeratosis (p - 0.038), Munro's microabscesses (p - 0.001), supra-papillary plate thinning (p - 0.004), and increased dermal vasculature (p - 0.001) were significantly associated with psoriasis when compared with psoriasiform dermatitis. Most psoriatic lesions had hypogranulosis whereas it was alternating hypo and hypergranulosis in psoriasiform dermatitis (p - 0.001). Acanthosis was regular in psoriasis lesions (p - 0.036). Bcl-2 expression was seen in dermal lymphocytes in all psoriasis and psoriasiform lesions with significantly stronger positive expression compared to basal and supra-basal layers. Psoriasis cases demonstrated more significant Bcl-2 positivity in dermal lymphocytes and basal layers than psoriasiform dermatitis cases. Conclusion Bcl-2 has stronger expression in dermal lymphocytes and basal layers than supra-basal layers, which comply with the proposed pathogenesis. Increased Bcl-2 expression in psoriasis cases compared with other psoriasiform dermatitis lesions implies the more chronic and recurrent nature of the disease.

Cutaneous Basal Cell Carcinoma In Situ: A Review of the World Literature.

Cutaneous basal cell carcinoma (BCC) in situ is a recently recognized subtype of the skin neoplasm in which the abnormal cells are confined to the epidermis. BCC in situ of the skin was previously referred to as a superficial BCC. A review of the world literature has revealed 10 cutaneous BCCs in situ that have been described in nine patients but likely reflect a more general phenomenon. The neoplasm typically presents as an asymptomatic red plaque on the abdomen, upper extremity, back, and chest. Pathologic changes frequently show confluent tumor cells along the epidermal basal layer or superficial aggregates of neoplastic cells that are contiguous with the epidermis and extend into the dermis. Genomic evaluation has been performed in neoplasms from one individual with cutaneous BCC in situ and metastatic BCC; like other variants of BCC, an aberration of the PTCH1 gene was observed. In contrast to his liver metastasis, the in situ carcinoma had a lower tumor mutational burden, lacked programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) amplification and had a distinct PTCH1 mutation, suggesting that the in situ BCC of his skin and the metastatic BCC of his liver were derived from different clones of cells.

Histopathologic Analysis of Chronic Cutaneous Graft-Versus-Host Disease.

Chronic graft-versus-host disease (cGVHD) is the leading cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation. Skin biopsy of cGVHD is recommended when clinical features are not diagnostic, yet the histopathologic features of skin cGVHD are not well described. The objective of this study is to describe the histopathologic features of skin cGVHD in epidermal, sclerotic, and combination cGVHD. Of 49 patients with skin cGVHD, 30 of 49 (61.2%) were male, and mean age was 55 years (SD 11.1). Clinically, 33 of 49 (67.3%) had epidermal cGVHD (E-cGVHD), 1 of 49 (2.1%) had sclerotic cGVHD (S-cGVHD), and 15 of 49 (30.6%) had combination disease. The 49 patients corresponded to 83 unique pathologic specimens with 67 of 83 (80.7%) taken from E-cGVHD, and 16 of 83 (19.3%) from S-cGVHD lesions. Nearly all biopsy specimens from E-cGVHD showed minimal features of active GVHD, including apoptosis in the epidermal basal layer (n = 63, 94.0%), vacuolar change (n = 62, 92.5%), and lymphocyte satellitosis (n = 57, 85.1%). The predominant histologic pattern of E-cGVHD was lichen planus/interface dermatitis (n = 31, 47.0%). S-cGVHD specimens also showed minimal features of active GVHD with apoptosis of the epidermal basal layer (n = 11, 68.8%) and vacuolar change (n = 8, 50.0%). In addition, S-cGVHD showed sclerosis of the papillary and reticular dermis and subcutaneous septae (n = 8, 50.0%; n = 11, 68.8%; n = 5, 31.2%, respectively). The predominant histologic pattern of S-cGVHD was lichen sclerosus/morphea-like pattern (n = 10, 62.5%). Although minimal pathologic features of active GVHD are common, the majority of cGVHD biopsies share features with the inflammatory skin diseases that they clinically resemble. Complete histologic reporting is recommended with implications for disease endotyping and personalized therapy.

Identification of a new human senescent skin cell marker ribonucleoside-diphosphate reductase subunit M2 B.

In skin aging, it has been hypothesized that aging fibroblasts accumulate within the epidermal basal layer, dermis, and subcutaneous fat, causing abnormal tissue remodeling and extracellular matrix dysfunction, thereby inducing an aging-related secretory phenotype (SASP). A new treatment for skin aging involves the specific elimination of senescent skin cells, especially fibroblasts within the dermis and keratinocytes in the basal layer. This requires the identification of specific protein markers of senescent cells, such as ribonucleoside-diphosphate reductase subunit M2 B (RRM2B), which is upregulated in various malignancies in response to DNA stress damage. However, the behavior and role of RRM2B in skin aging remain unclear. Therefore, we examined whether RRM2B functions as a senescence marker using a human dermal fibroblast model of aging. In a model of cellular senescence induced by replicative aging and exposure to ionizing radiation or UVB, RRM2B was upregulated at the gene and protein levels. This was correlated with decreased uptake of the senescence-associated β-galactosidase activity and proliferation marker bromodeoxyuridine. RRM2B upregulation was concurrent with the increased expression of SASP factor genes. Furthermore, using fluorescence flow cytometry, RRM2B-positive cells were recovered more frequently in the aging cell population. In aging human skin, RRM2B was also found to be more abundant in the dermis and epidermal basal layer than other proteins. Therefore, RRM2B may serve as a clinical marker to identify senescent skin cells.

The people behind the papers - Sangeeta Ghuwalewala, David Shalloway and Tudorita (Doina) Tumbar.

Skin renewal is characterised by progenitor cells at the epidermal basal layer dividing and differentiating, yet the identity and properties of these progenitors remain poorly understood. In a new study, Tudorita Tumbar and colleagues identify, for the first time in vivo in the mouse epidermis, a distinct population of non-self-renewing progenitor cells that exhibit biphasic transit-amplifying behaviour. To find out more about the story behind the paper, we caught up with first author Sangeeta Ghuwalewala, penultimate author David Shalloway and corresponding author Tudorita (Doina) Tumbar, Professor of Molecular Biology and Genetics at Cornell University, USA.

P05 The aryl hydrocarbon receptor pathway is dysfunctional in atopic dermatitis

Abstract Introduction and aims Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by epidermal barrier disruption and type-2 immune dysregulation. While treatment has improved with targeted biologics, clinical response varies, highlighting the need for additional therapeutic strategies. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and environmental sensor, which maintains skin barrier integrity and homeostasis by reducing inflammation. Upon activation, AHR induces the expression of CYP1A1, a biomarker of pathway activation and critical for AHR ligand metabolism. Tapinarof, a topical AHR agonist, is approved for psoriasis and is effective in phase III clinical trials in AD, highlighting the importance of this pathway in inflammatory skin disease. Nevertheless, AHR expression is increased in AD skin, leading to the hypothesis that the pathway is dysregulated, thus not exerting its anti-inflammatory effect. Methods Here we begin to perform a global investigation of the AHR pathway in AD. We measured AHR and CYP1A1 mRNA expression by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in healthy and AD skin, with spatial mRNA expression assessed by RNAscope. We also investigated AHR mRNA expression in interleukin (IL)-4 stimulated (100 ng mL−1) normal human epidermal keratinocytes (NHEKs) using qRT-PCR. Results We found that AHR mRNA is significantly upregulated in lesional AD (n = 6) compared with healthy skin (n = 9; P < 0.05), whereas CYP1A1 mRNA expression shows a downward trend in lesional AD compared with healthy skin (P = 0.063). The AHR expression pattern differs in lesional AD (n = 7) compared with healthy skin (n = 8), with AHR localizing in the epidermal basal layer. Finally, IL-4 significantly upregulated AHR mRNA (n = 4 wells; P < 0.001) expression in NHEKs compared with unstimulated cells. Conclusions Overall, our data have uncovered a dysfunctional AHR pathway in AD, with increased AHR expression, possibly driven by the AD inflammatory milieu, but reduced pathway activation. Further investigations are required to fully dissect this pathway and maximize its potential as a therapeutic target for AD.

Basal Cell Carcinoma: Diagnosis, Management and Prevention

Basal cell carcinoma (BCC) is a slow-growing, locally aggressive, rarely metastasizing, low-grade cutaneous neoplasm that arises from the epidermal basal layer and invades the adjoining tissues. It is the most common skin cancer. It is fairly common in fair Caucasians and quite uncommon in dark-skinned populations. It contributes to 65–75% of cutaneous malignancies in whites and 20–30% in Asian Indians. The most important causal factors appear to be radiation exposure and genetic predisposition. It may present as a nonhealing lesion that occasionally bleeds or as a pruritic lesion with no symptoms. Tumours rarely spread to regional lymph nodes. The clinical appearances and morphology of BCC are diverse. Clinical types include nodular, cystic, superficial, pigmented, morphoeaform, (sclerosing), keratotic and fibroepithelioma of Pinkus. Most of the lesions appear on the head and neck, usually above the line joining the tragus and the angle of the mouth. A biopsy should be performed on all lesions suspected of BCC. The primary aim of treatment is the complete excision of the tumour tissue. Other treatment modalities include cryotherapy, immunomodulatory drugs, laser treatment or locally applicable chemotherapeutic agents. Prevention consists of lifestyle changes such as avoiding sunburn, tanning beds and prolonged direct sun exposure, shade seeking, sunscreen application on the skin, and physical barrier methods such as protective clothing, hats and sunglasses. Regular sunscreen use in childhood and adolescence seems more beneficial than in adulthood.

Epidermal growth factor receptor inhibition leads to cellular phenotype correction of DSP-mutated keratinocytes.

Desmoplakin (DSP) is a desmosomal component expressed in skin and heart, essential for desmosome stability and intermediate filament connection. Pathogenic variants in the DSP gene encoding DSP, lead to heterogeneous skin, adnexa and heart-related phenotypes, including skin fragility, woolly hair (WH), palmoplantar keratoderma (PPK) and arrhythmogenic/dilated cardiomyopathy (ACM/DCM). The ambiguity of computer-based prediction analysis of pathogenicity and effect of DSP variants, indicates a necessity for functional analysis. Here, we report a heterozygous DSP variant that was not previously described, NM_004415.4:c.3337C>T (NM_004415.4(NP_004406.2):p.(Arg1113*)) in a patient with PPK, WH and ACM. RNA and protein analysis revealed ~50% reduction of DSP mRNA and protein expression. Patient's keratinocytes showed fragile cell-cell connections and perinuclear retracted intermediate filaments. Epidermal growth factor receptor (EGFR) is a transmembrane protein expressed in the basal epidermal layer involved in proliferation and differentiation, processes that are disrupted in the development of PPK, and in the regulation of the desmosome. In skin of the abovementioned patient, evident EGFR upregulation was observed. EGFR inhibition in patient's keratinocytes strongly increased DSP expression at the plasma membrane, improved intermediate filament connection with the membrane edges and reduced the cell-cell fragility. This cell phenotypic recovery was due to a translocation of DSP to the plasma membrane together with an increased number of desmosomes. These results indicate a therapeutic potential of EGFR inhibitors for disorders caused by DSP haploinsufficiency.

The Mechanism of 5-Fluorouracil-Induced Hyperpigmentation in HRM-2 Hairless Mice: Focus on the Increase of Blood Vessels.

5-Fluorouracil (5-FU), an effective chemotherapeutic agent for many solid tumors, has long been reported to cause pigmentation in patients treated intravenously, which occurs with increasing frequency of administration and decreases the QOL of the patients. Although melanin accumulation is thought to be the cause, the mechanism of pigmentation induced by 5-FU administration remains unclear, and there is no effective treatment for this problem. In this study, we investigated the mechanism of pigmentation induced by continuous 5-FU administration in 9-week-old male HRM-2 hairless mice for 8 weeks by focusing on the blood vessels for basic verification. In the auricular skin of 5-FU-administered mice, hyperpigmentation caused by melanin accumulation was observed macroscopically and by Fontana-Masson Staining. In addition, the expression of tyrosinase, melanin synthase, and blood vessel markers in the auricular skin was increased by 5-FU-administration in mice auricular skin. Other anticancer agents, cytarabine (Ara-C) and irinotecan (CPT-11), were also administered, and the differences between them and 5-FU were investigated; these changes were not observed in the auricles of these mice. These results suggest that tyrosinase is associated with 5-FU-induced melanin production and that an increase in blood vessels may be involved. Furthermore, pigmentation with melanin accumulation in the basal epidermal layer is a characteristic finding of 5-FU compared with Ara-C and CPT-11. In conclusion, this study indicates that 5-FU causes hyperpigmentation by melanin accumulation in a characteristic manner, including an increase in blood vessels.

Hesperetin activates CISD2 to attenuate senescence in human keratinocytes from an older person and rejuvenates naturally aged skin in mice

BackgroundCDGSH iron-sulfur domain-containing protein 2 (CISD2), a pro-longevity gene, mediates healthspan in mammals. CISD2 is down-regulated during aging. Furthermore, a persistently high level of CISD2 promotes longevity and ameliorates an age-related skin phenotype in transgenic mice. Here we translate the genetic evidence into a pharmaceutical application using a potent CISD2 activator, hesperetin, which enhances CISD2 expression in HEK001 human keratinocytes from an older person. We also treated naturally aged mice in order to study the activator’s anti-aging efficacy.MethodsWe studied the biological effects of hesperetin on aging skin using, firstly, a cell-based platform, namely a HEK001 human keratinocyte cell line established from an older person. Secondly, we used a mouse model, namely old mice at 21-month old. In the latter case, we investigate the anti-aging efficacy of hesperetin on ultraviolet B (UVB)-induced photoaging and naturally aged skin. Furthermore, to identify the underlying mechanisms and potential biological pathways involved in this process we carried out transcriptomic analysis. Finally, CISD2 knockdown HEK001 keratinocytes and Cisd2 knockout mice were used to study the Cisd2-dependent effects of hesperetin on skin aging.ResultsFour findings are pinpointed. Firstly, in human skin, CISD2 is mainly expressed in proliferating keratinocytes from the epidermal basal layer and, furthermore, CISD2 is down-regulated in the sun-exposed epidermis. Secondly, in HEK001 human keratinocytes from an older person, hesperetin enhances mitochondrial function and protects against reactive oxygen species-induced oxidative stress via increased CISD2 expression; this enhancement is CISD2-dependent. Additionally, hesperetin alleviates UVB-induced damage and suppresses matrix metalloproteinase-1 expression, the latter being a major indicator of UVB-induced damage in keratinocytes. Thirdly, transcriptomic analysis revealed that hesperetin modulates a panel of differentially expressed genes that are associated with mitochondrial function, redox homeostasis, keratinocyte function, and inflammation in order to attenuate senescence. Intriguingly, hesperetin activates two known longevity-associated regulators, namely FOXO3a and FOXM1, in order to suppress the senescence-associated secretory phenotype. Finally, in mouse skin, hesperetin enhances CISD2 expression to ameliorate UVB-induced photoaging and this occurs via a mechanism involving CISD2. Most strikingly, late-life treatment with hesperetin started at 21-month old and lasting for 5 months, is able to retard skin aging and rejuvenate naturally aged skin in mice.ConclusionsOur results reveal that a pharmacological elevation of CISD2 expression at a late-life stage using hesperetin treatment is a feasible approach to effectively mitigating both intrinsic and extrinsic skin aging and that hesperetin could act as a functional food or as a skincare product for fighting skin aging.

Skin-protective biological activities of bio-fermented Aframomum angustifolium extract by a consortium of microorganisms.

Background: Aframomum sp. is a genus of plants in the Zingiberaceae family. It includes several species, some of which are used in cosmetics for their various properties, making them useful in skincare products, particularly for anti-aging, moisturizing, and brightening the skin. However, to date, there is no experimental evidence on its natural extracts obtained or modified using microorganisms (bio-fermentation) as an anti-aging agent. Objective: The present study aimed to evaluate the antiaging effect of a Bio-fermented Aframomum angustifolium (BAA) extract on 3D bioprinted skin equivalent. Methods: The consortium of microorganisms contained Komagataeibacter, Gluconobacter, Acetobacter, Saccharomyces, Torulaspora, Brettanomyces, Hanseniaspora, Leuconostoc, Lactobacillus, Schizosaccharomyces. It was developed on a media containing water, sugar, and infused black tea leaves. The seeds of Aframomum angustifolium previously grounded were mixed with the culture medium, and the ferments in growth; this fermentation step lasted 10 days. Then, the medium was collected and filtered (0.22µm) to obtain the BAA extract. To enhance our comprehension of the impact of BAA extract on skin aging, we developed skin equivalents using bio-printing methods with the presence or absence of keratinocyte stem cells (KSC). These skin equivalents were derived from keratinocytes obtained from both a middle-aged donor, with and without KSC. Moreover, we examined the effects of treating the KSC-depleted skin equivalents with Bio-fermented Aframomum angustifolium (BAA) extract for 5days. Skin equivalents containing KSC-depleted keratinocytes exhibited histological characteristics typical of aged skin and were compared to skin equivalents derived from young donors. Results: The BAA extract contained specific organic acids such as lactic, gluconic, succinic acid and polyphenols. KSC-depleted skin equivalents that were treated with BAA extract exhibited higher specular reflection, indicating better hydration of the stratum corneum, higher mitotic activity in the epidermis basal layer, improved dermal-epidermal connectivity, and increased rigidity of the dermal-epidermal junction compared to non-treated KSC-depleted equivalents. BAA extract treatments also resulted in changes at the dermis level, with an increase in total collagen and a decrease in global laxity, suggesting that this extract could help maintain youthful-looking skin. Conclusion: In summary, our findings indicated that BAA extract treatments have pleiotropic beneficial effects on skin equivalents and that the bio-fermentation provides new biological activities to this plant.

Role of interleukin-6 and endothelin-1 receptors in enhanced melanocyte dendricity of facial spots and suppression of their ligands by niacinamide and tranexamic acid.

Hyperpigmented spots are common issues in all ethnicities with a hallmark characteristic of increased melanocyte dendricity. To determine (1) potential receptors and/or cytokines that are involved in increased melanocyte dendricity in multiple facial spot types; (2) treatment effects of skin-lightening compounds on identified cytokine release from keratinocytes and on dendricity in melanocytes. Facial spots (melasma, solar lentigo, acne-induced post-inflammatory hyperpigmentation) and adjacent non-spot skin biopsies were collected from Chinese women (age 20-70). The epidermal supra and basal layers were laser dissected to enrich keratinocyte or melanocyte biology respectively for transcriptome analysis. Melanocyte dendricity was assessed histologically by immunofluorescent staining. Effect of interleukin-6 (IL-6) and endothelin-1 (ET-1) on melanocyte dendricity and melanosome transfer were assessed in human melanocytes or melanocyte-keratinocyte co-culture models. Treatment effects of skin-lightening compounds (niacinamide, tranexamic acid [TxA], sucrose laurate/dilaurate mixture [SDL]) were assessed on IL-6 or ET-1 release from keratinocytes and on dendricity in melanocytes. Transcriptome analysis revealed IL-6 receptor and ET-1 receptor were significantly upregulated compared to the adjacent normal skin, visually confirmed at the protein level through immunostaining. Melanocytes in spot areas are more dendritic than melanocytes in adjacent non-spot skin. The addition of IL-6 and ET-1 to cell culture models increased melanocyte dendricity and melanosome transfer. IL-6 release was significantly suppressed by niacinamide and its combination, while ET-1 release was significantly reduced by both niacinamide and TxA. In contrast, SDL acted directly upon melanocytes to reduce dendricity. Interleukin-6 and ET-1 receptors are significantly upregulated in multiple facial spot types. The invitro testing demonstrated their respective ligands increased melanocyte dendricity. Tested skin-lightening compounds showed reduction in release of IL-6/ET-1 from epidermal keratinocytes and/or inhibition of melanocyte dendricity. This work sheds light on pathophysiological mechanism of facial spots and potential new mechanisms of these skin-lightening compounds which warrant further human clinical validation.

Single-cell RNA sequencing of human epidermis identifies Lunatic fringe as a novel regulator of the stem cell compartment

Single-cell RNA sequencing (scRNA-seq) of human skin provides a tool for validating observations from invitro experimental models. By analyzing a published dataset of healthy adult epidermis, we confirm that the basal epidermal layer is heterogeneous, and three subpopulations of non-dividing cells can be distinguished. We show that Delta-like ligand 1 (DLL1) is expressed in a subset of basal cells previously identified as stem cells in cultured human keratinocytes and map the distribution of other Notch ligands and receptors to specific epidermal cell compartments. Although DLL1 is expressed at low levels, it is expressed in the same cell state as the Notch regulator, Lunatic -fringe (LFNG, O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase). Overexpression of LFNG amplifies the effects of DLL1 in cultured keratinocytes, increasing proliferation and colony-forming ability. We conclude that using scRNA-seq resources from healthy human skin not only validates previous experimental data but allows formulation of testable new hypotheses.

Immunolocalization of Some Epidermal Proteins and Glycoproteins in the Growing Skin of the Australian Lungfish (Neoceratodus forsteri).

Here we report the immunolocalization of mucin, nestin, elastin and three glycoproteins involved in tissue mineralization in small and large juveniles of Neoceratodus forsteri. Both small and larger juvenile epidermis are mucogenic and contain a diffuse immunolabeling for nestin. Sparse PCNA-labeled cells, indicating proliferation, are found in basal and suprabasal epidermal layers. No scales are formed in small juveniles but are present in a 5 cm long juvenile and in larger juveniles. Elastin and a mineralizing matrix are localized underneath the basement membrane of the tail epidermis where lepidotriches are forming. The latter appears as "circular bodies" in cross sections and are made of elongated cells surrounding a central amorphous area containing collagen and elastin-like proteins that undergo calcification as evidenced using the von Kossa staining. However, the first calcification sites are the coniform teeth of the small juveniles of 2-3 cm in length. In the superficial dermis of juveniles (16-26 cm in length) where scales are formed, the spinulated outer bony layer (squamulin) of the elasmoid scales contains osteonectin, alkaline phosphatase, osteopontin, and calcium deposits that are instead absent in the underlying layer of elasmodin. In particular, these glycoproteins are localized along the scale margin in juveniles where scales grow, as indicated by the presence of PCNA-labeled cells (proliferating). These observations suggest a continuous deposition of new bone during the growth of the scales, possibly under the action of these mineralizing glycoproteins, like in the endoskeleton of terrestrial vertebrates.

Post-injury hydraulic fracturing drives fissure formation in the zebrafish basal epidermal cell layer

The skin epithelium acts as the barrier between an organism's internal and external environments. In zebrafish and other freshwater organisms, this barrier function requires withstanding a large osmotic gradient across the epidermis. Wounds breach this epithelium, causing a large disruption to the tissue microenvironment due to the mixing of isotonic interstitial fluid with the external hypotonic fresh water. Here, we show that, following acute injury, the larval zebrafish epidermis undergoes a dramatic fissuring process that resembles hydraulic fracturing, driven by the influx of external fluid. After the wound has sealed-preventing efflux of this external fluid-fissuring starts in the basal epidermal layer at the location nearest to the wound and then propagates at a constant rate through the tissue, spanning over 100μm. During this process, the outermost superficial epidermal layer remains intact. Fissuring is completely inhibited when larvae are wounded in isotonic external media, suggesting that osmotic gradients are required for fissure formation. Additionally, fissuring partially depends on myosin II activity, as myosin II inhibition reduces the distance of fissure propagation away from the wound. During and after fissuring, the basal layer forms large macropinosomes (with cross-sectional areas ranging from 1 to 10μm2). We conclude that excess external fluid entry through the wound and subsequent closure of the wound through actomyosin purse-string contraction in the superficial cell layer causes fluid pressure buildup in the extracellular space of the zebrafish epidermis. This excess fluid pressure causes tissue to fissure, and eventually the fluid is cleared through macropinocytosis.

Expression in skin biopsies supports genetic evidence linking CAMKK2, P2X7R and P2X4R with HIV-associated sensory neuropathy

HIV-associated sensory neuropathy (HIV-SN) affects 14–38% of HIV+ individuals stable on therapy with no neurotoxic drugs. Polymorphisms in CAMKK2, P2X7R and P2X4R associated with altered risk of HIV-SN in Indonesian and South African patients. The role of CaMKK2 in neuronal repair makes this an attractive candidate, but a direct role for any protein is predicated on expression in affected tissues. Here, we describe expression of CaMKK2, P2X7R and P2X4R proteins in skin biopsies from the lower legs of HIV+ Indonesians with and without HIV-SN, and healthy controls (HC). HIV-SN was diagnosed using the Brief Peripheral Neuropathy Screen. Biopsies were stained to detect protein gene product 9.5 on nerve fibres and CaMKK2, P2X7R or P2X4R, and were examined using 3-colour sequential scanning confocal microscopy. Intraepidermal nerve fibre densities (IENFD) were lower in HIV+ donors than HC and correlated directly with nadir CD4 T-cell counts (r = 0.69, p = 0.004). However, IENFD counts were similar in HIV-SN+ and HIV-SN− donors (p = 0.19) and so did not define neuropathy. CaMKK2+ cells were located close to dermal and epidermal nerve fibres and were rare in HC and HIV-SN− donors, consistent with a role for the protein in nerve damage and/or repair. P2X7R was expressed by cells in blood vessels of HIV-SN− donors, but rarely in HC or HIV-SN+ donors. P2X4R expression by cells in the epidermal basal layer appeared greatest in HIV-SN+ donors. Overall, the differential expression of CaMKK2, P2X7R and P2X4R supports the genetic evidence of a role for these proteins in HIV-SN.

Enlarged rete pegs with excessive accumulation of melanosomes leading to darker aging spots revealed by histomorphological measurements of internal structures of the epidermis.

Various histological studies of facial pigmented spot sites such as solar lentigo have been reported, but few studies have used quantitative indices by histomorphometric analysis of the internal structure of pigmented spot sites using non-invasive methods. In the present study, to quantitatively elucidate morphological changes in the epidermis in male, darker-pigmented spots and female, light-pigmented spots, indices that characterize the internal structure of the epidermis in pigmented spot sites were measured using in vivo confocal laser scanning microscopy (CLSM). The darkness of pigmented spots on the cheeks of 69 women and 43 men was analysed using image analysis software. The L* value was calculated from RGB values obtained from facial images. The internal structures of pigmented spots on the cheeks of 13 subjects were observed by CLSM. Various parameters were measured using CLSM images from the surface of the stratum corneum to the bottom of the dermal papillae, including the thickness of the epidermis, melanosome content, and shape of the dermal papillae. Mean ΔL* values between pigmented spots and non-pigmented areas of male subjects were significantly increased in the 40s and 50s compared with those of female subjects. Conspicuous pigmented spots increased in the 40s in male subjects and the 50s in female subjects. In CLSM observations, significant increases in the thickness of the epidermis and melanosome content were confirmed in pigmented spots compared with surrounding non-pigmented areas. In particular, melanosome content in the male subject group with dark-coloured pigmented spots increased significantly to about eight times that of non-pigmented areas, and more than double that of the male subject group with light-coloured pigmented spots. From the measurements of quantitative parameters, morphological changes in the epidermis were clearly related to the surface colour tone of pigmented spots. Darker pigmented spot sites tended to show longer rete pegs in the epidermis. Accumulation of melanosomes in epidermal basal cells could be considered to increase with the degree of elongation of rete pegs at pigmented spot sites and, thus, induce darker pigmented spots.

PRAME immunohistochemistry can distinguish melanocytic pseudonests of lichenoid reactions from melanoma in situ.

Distinguishing melanocytic pseudonests encountered in lichenoid dermatoses or lichenoid keratoses from melanoma in situ (MIS) with brisk lichenoid inflammation can prove challenging. We designed a case-control study to evaluate the accuracy metrics of PRAME immunohistochemistry to distinguish melanocytic pseudonests in lichenoid dermatoses or keratoses from inflamed MIS. Immunostaining for PRAME was performed on paraffin-embedded formalin-fixed diagnostic tissue using a rabbit monoclonal antibody to PRAME (Abcam), with a 1:3200 dilution on a Leica Bond detection system. Our search identified 21 cases of melanocytic pseudonests (n=21, 46%) encountered in lichenoid dermatoses and 24 cases of inflamed MIS (n=24, 53%). Each method of evaluating PRAME immunohistochemistry (PRAME+ clusters, PRAME % of melanocytes by four categories and PRAME+ melanocyte counts per linear mm of epidermal basal layer) showed statistically significant differences between the MIS and the pseudonest cohorts (respectively, p < 0.001; p < 0.001; and p < 0.001). Receiver operating characteristics analysis for PRAME+ melanocyte counts per linear mm of epidermal basal layer revealed an area under the curve of 0.9 ± 0.05 (95% confidence interval 0.9-1.0). When determining an optimal cut-off point for the best Youden index [sensitivity (%) + specificity (%) - 100], the cut-off of 1.0 PRAME+ melanocytes per linear mm showed a sensitivity of 79.2% and specificity of 85.7% (Youden index 0.65) to distinguish MIS from pseudonests. PRAME immunohistochemistry may constitute an additional tool for this challenging differential diagnosis.