Cutaneous DNA delivery and gene expression in ex vivo human skin explants via wet-etch microfabricated microneedles

Human skin is constantly exposed to UV light, the most ubiquitous environmental stressor. Here, we investigated the expression and regulation of Nrf1-3, transcription factors crucially involved in protection against oxidative stress in human skin cells in vitro, ex vivo, and in situ. In particular, we examined whether α-MSH, a UV-induced peptide, is capable of modulating Nrf2 and Nrf-dependent gene expression. Nrf1, -2, and -3 were found to be expressed in various cutaneous cell types in vitro. Surprisingly, UVB irradiation at physiological doses (10 mJ/cm2) reduced Nrf2 and Nrf-dependent gene expression in normal keratinocytes and melanocytes in vitro as well as ex vivo in skin organ cultures. α-MSH alone significantly increased Nrf2 as well as Nrf-dependent heme oxygenase-1, γ-glutamylcysteine-synthetase, and glutathione-S-transferase Pi gene expression in both keratinocytes and melanocytes. This effect of α-MSH occurred at physiological doses and was due to transcriptional induction, mimicked by the artificial cAMP inducer forskolin, and blocked by protein kinase A pathway inhibition. In silico promoter analysis of Nrf2 further identified several putative binding sites for activator protein 1 and cAMP response element-binding protein, transcription factors typically activated by α-MSH. Importantly, α-MSH prevented or even overcompensated the UVB-induced suppression of Nrf2 and Nrf-dependent genes not only in normal keratinocytes and melanocytes in vitro but also in skin organ cultures. These findings, for the first time, show regulation of Nrf2 and Nrf-dependent genes by α-MSH. Our data also highlight a novel facet in the cytoprotective and antioxidative effector mechanisms of α-MSH and perhaps of related melanocortin peptides.

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Protective Effect of a Novel Anti-Aging Facial Cream Containing Alteromonas Ferment Extract and Carnosine Against Environmental Pollution in Human Skin Explants in Ex-Vivo Study

SKIN The Journal of Cutaneous Medicine ◽

10.25251/skin.2.supp.38 ◽

2018 ◽

Vol 2 ◽

pp. S38

Author(s):

M Narda ◽

C Granger

Keyword(s):

Protective Effect ◽

Environmental Pollution ◽

Human Skin ◽

Ex Vivo ◽

Skin Explants ◽

Ex Vivo Study

Abstract not available. Disclosures: Study sponosred by ISDIN. Copyright SKIN 2018.

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Characterization of a Topically Testable Model of Burn Injury on Human Skin Explants

International Journal of Molecular Sciences ◽

10.3390/ijms21186956 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6956

Author(s):

Olivia Gross-Amat ◽

Marine Guillen ◽

Damien Salmon ◽

Serge Nataf ◽

Céline Auxenfans

Keyword(s):

Human Skin ◽

Burn Injury ◽

Ex Vivo ◽

Drug Formulation ◽

Burn Injuries ◽

Major Health Problem ◽

Constant Tension ◽

Skin Explants ◽

Set Up ◽

Improved Model

Severe burn injuries remain a major health problem due to high rates of mortality, residual morbidity, and/or aesthetic damages. To find new therapies aimed at promoting a harmonious healing of skin burns, it is important to develop models which take into account the unique properties of the human skin. Based on previously described models of burn injury performed on human skin explants, we hypothesized that maintaining explants under constant tension forces would allow to more closely reproduce the pathophysiological processes of skin remodeling. We thus. Here, we set up and characterized an improved model of deep second-degree burn injury on ex vivo cultured human skin explants at air-liquid interface and maintained under conditions of constant tension forces. A spontaneous re-epithelialization of the lesion was observed 8 to 9 days post burn and was found to rely on the proliferation of basal keratinocytes at the wound edges. Collagen VII at the dermo-epidermal junction reformed along with the progression of re-epithelializatio and a synthesis of procollagen III was observed in the dermis at the wound site. These findings indicate that our model is suitable for the assessment of clinically-relevant therapies aimed at modulating the kinetics of re-epithelialization and/or the activation of fibroblasts following skin burn injuries. In this regard, we evaluated the use of a thermoreversible poloxamer hydrogel as a vehicle for topically-testable therapeutic molecules. Our data showed that, although useful for drug formulation, the p407/p188 poloxamer hydrogel induces a delay of skin re-epithelialization in humans skin explants submitted to experimental burn injury.

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Active neutrophil responses counteract Candida albicans burn wound infection of ex vivo human skin explants

Scientific Reports ◽

10.1038/s41598-020-78387-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Christin von Müller ◽

Fionnuala Bulman ◽

Lysett Wagner ◽

Daniel Rosenberger ◽

Alessandra Marolda ◽

...

Keyword(s):

Human Skin ◽

Ex Vivo ◽

Burn Wound ◽

Local Immunity ◽

Fungal Virulence ◽

Opportunistic Fungi ◽

Burn Wounds ◽

Wound Model ◽

Skin Explants ◽

Bacteria And Fungi

AbstractBurn wounds are highly susceptible sites for colonization and infection by bacteria and fungi. Large wound surface, impaired local immunity, and broad-spectrum antibiotic therapy support growth of opportunistic fungi such as Candida albicans, which may lead to invasive candidiasis. Currently, it remains unknown whether depressed host defenses or fungal virulence drive the progression of burn wound candidiasis. Here we established an ex vivo burn wound model, where wounds were inflicted by applying preheated soldering iron to human skin explants, resulting in highly reproducible deep second-degree burn wounds. Eschar removal by debridement allowed for deeper C. albicans penetration into the burned tissue associated with prominent filamentation. Active migration of resident tissue neutrophils towards the damaged tissue and release of pro-inflammatory cytokine IL-1β accompanied the burn. The neutrophil recruitment was further increased upon supplementation of the model with fresh immune cells. Wound area and depth decreased over time, indicating healing of the damaged tissue. Importantly, prominent neutrophil presence at the infected site correlated to the limited penetration of C. albicans into the burned tissue. Altogether, we established a reproducible burn wound model of candidiasis using ex vivo human skin explants, where immune responses actively control the progression of infection and promote tissue healing.

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The Ex Vivo Skin Model as an Alternative Tool for the Efficacy and Safety Evaluation of Topical Products

Alternatives to Laboratory Animals ◽

10.1177/0261192920914193 ◽

2020 ◽

Vol 48 (1) ◽

pp. 10-22 ◽

Cited By ~ 1

Author(s):

Samara Eberlin ◽

Michelle Sabrina da Silva ◽

Gustavo Facchini ◽

Gustavo Henrique da Silva ◽

Ana Lúcia Tabarini Alves Pinheiro ◽

...

Keyword(s):

Human Skin ◽

Ex Vivo ◽

Safety Evaluation ◽

Test System ◽

Dermal Matrix ◽

Standard Size ◽

Skin Explants ◽

Alternative Approaches ◽

Air Liquid Interface

The development of alternative approaches for safety and efficacy testing that avoid the use of animals is a worldwide trend, which relies on the improvement of current models and tools so that they better reproduce human biology. Human skin from elective plastic surgery is a promising experimental model to test the effects of topically applied products. As the structure of native skin is maintained, including cell population (keratinocytes, melanocytes, Langerhans cells and fibroblasts) and dermal matrix (containing collagen, elastin, glycosaminoglycans, etc.), it most closely matches the effects of substances on in vivo human skin. In this review, we present a collection of results that our group has generated over the last years, involving the use of human skin and scalp explants, demonstrating the feasibility of this model. The development of a test system with ex vivo skin explants, of standard size and thickness, and cultured at the air–liquid interface, can provide an important tool for understanding the mechanisms involved in several cutaneous disorders.

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Staphylococcus aureus-Specific Tissue-Resident Memory CD4+ T Cells Are Abundant in Healthy Human Skin

Frontiers in Immunology ◽

10.3389/fimmu.2021.642711 ◽

2021 ◽

Vol 12 ◽

Author(s):

Astrid Hendriks ◽

Malgorzata Ewa Mnich ◽

Bruna Clemente ◽

Ana Rita Cruz ◽

Simona Tavarini ◽

...

Keyword(s):

Staphylococcus Aureus ◽

T Cells ◽

Human Skin ◽

Ex Vivo ◽

Functional Characterization ◽

T Cell Response ◽

Skin Microbiome ◽

Entry Site ◽

Healthy Human ◽

Skin Explants

The skin is an immunocompetent tissue that harbors several kinds of immune cells and a plethora of commensal microbes constituting the skin microbiome. Staphylococcus aureus is a prominent skin pathogen that colonizes a large proportion of the human population. We currently have an incomplete understanding of the correlates of protection against S. aureus infection, however genetic and experimental evidence has shown that CD4+ T cells play a key role in orchestrating a protective anti-S. aureus immune response. A high S. aureus-specific memory CD4+ T cell response has been reported in the blood of healthy subjects. Since T cells are more abundant in the skin than in blood, we hypothesized that S. aureus-specific CD4+ T cells could be present in the skin of healthy individuals. Indeed, we observed proliferation of tissue-resident memory CD4+ T cells and production of IL-17A, IL-22, IFN-γ and TNF-β by cells isolated from abdominal skin explants in response to heat-killed S. aureus. Remarkably, these cytokines were produced also during an ex vivo epicutaneous S. aureus infection of human skin explants. These findings highlight the importance of tissue-resident memory CD4+ T cells present at barrier sites such as the skin, a primary entry site for S. aureus. Further phenotypical and functional characterization of these cells will ultimately aid in the development of novel vaccine strategies against this elusive pathogen.

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Vitamin C–squalene bioconjugate promotes epidermal thickening and collagen production in human skin

Scientific Reports ◽

10.1038/s41598-020-72704-1 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

R. Gref ◽

C. Deloménie ◽

A. Maksimenko ◽

E. Gouadon ◽

G. Percoco ◽

...

Keyword(s):

Vitamin C ◽

Human Skin ◽

Ex Vivo ◽

Ex Vivo Model ◽

Collagen Formation ◽

Skin Physiology ◽

Collagen Iii ◽

Skin Explants ◽

Natural Lipid ◽

Vit C

Abstract Vitamin C (Vit C) benefits to human skin physiology notably by stimulating the biosynthesis of collagen. The main cutaneous collagens are types I and III, which are less synthesized with aging. Vit C is one of the main promotors of collagen formation but it poorly bypasses the epidermis stratum corneum barrier. To address this challenge, we developed a lipophilic version of Vit C for improving skin diffusion and delivery. Vit C was covalently conjugated to squalene (SQ), a natural lipid of the skin, forming a novel Vit C–SQ derivative suitable for cream formulation. Its biological activity was investigated on human whole skin explants in an ex vivo model, through histology and protein and gene expression analyses. Results were compared to Vit C coupled to the reference lipophilic compound palmitic acid, (Vit C–Palmitate). It was observed that Vit C–SQ significantly increased epidermal thickness and preferentially favored collagen III production in human skin after application for 10 days. It also promoted glycosaminoglycans production in a higher extent comparatively to Vit C–Palmitate and free Vit C. Microdissection of the explants to separate dermis and epidermis allowed to measure higher transcriptional effects either in epidermis or in dermis. Among the formulations studied, the strongest effects were observed with Vit C–SQ.

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