Canine Epidermal Keratinocytes (CPEK) Grown in Monolayer Are Not Representative of Normal Canine Keratinocytes for Permeability Studies: Pilot Studies

Canine progenitor epidermal keratinocytes (CPEK) are used as canine keratinocyte cell line. Their suitability for skin barrier studies is unknown. Measurement of transepithelial electric resistance (TEER) evaluates epithelial permeability. We compared TEER and tight junction (TJ) expression in CPEKs and normal keratinocytes (NK) harvested from biopsies of normal dogs. CPEKs and NK were grown until confluence (D0) and for 13 additional days. Slides were fixed on D0 and stained with ZO-1 and claudin-1 antibodies. Five images/antibody were taken, randomized and evaluated blindly by three investigators for intensity, staining location, granularity, and continuousness. Cell size and variability were evaluated. TEER increased overtime to 2000 Ohms/cm in NK, while remained around 100–150 Ohms/cm in CPEK. ANOVA showed significant effect of time (p < 0.0001), group (p < 0.0001) and group x time interaction (p < 0.0001) for TEER. Size of CPEKs was significantly (p < 0.0001) smaller and less variable (p = 0.0078) than NK. Intensity of claudin-1 staining was greater in CPEKs (p < 0.0001) while granularity was less in CPEKs (p = 0.0012). For ZO-1, cytoplasmic staining was greater in CPEK (p < 0.0001) while membrane continuousness of staining was greater in NK (p = 0.0002). We conclude that CPEKs grown in monolayer are not representative of NK for permeability studies.

In Vitro Evaluation of the Photoreactivity and Phototoxicity of Natural Polyphenol Antioxidants

Polyphenols are a large family of natural compounds widely used in cosmetic products due to their antioxidant and anti-inflammatory beneficial properties and their ability to prevent UV radiation-induced oxidative stress. Since these compounds present chromophores and are applied directly to the skin, they can react with sunlight and exert phototoxic effects. The available scientific information on the phototoxic potential of these natural compounds is scarce, and thus the aim of this study was to evaluate the photoreactivity and phototoxicity of five phenolic antioxidants with documented use in cosmetic products. A standard ROS assay was validated and applied to screen the photoreactivity of the natural phenolic antioxidants caffeic acid, ferulic acid, p-coumaric acid, 3,4-dihydroxyphenylacetic acid (DOPAC), and rutin. The phototoxicity potential was determined by using a human keratinocyte cell line (HaCaT), based on the 3T3 Neutral Red Uptake phototoxicity test. Although all studied phenolic antioxidants absorbed UV/Vis radiation in the range of 290 to 700 nm, only DOPAC was able to generate singlet oxygen. The generation of reactive oxygen species is an early-stage chemical reaction as part of the phototoxicity mechanism. Yet, none of the studied compounds decreased the viability of keratinocytes after irradiation, leading to the conclusion that they do not have phototoxic potential. The data obtained with this work suggests that these compounds are safe when incorporated in cosmetic products.

Menstrual Fluid Factors Mediate Endometrial Repair

Menstruation is a process whereby the outer functionalis layer of the endometrium is shed each month in response to falling progesterone and estrogen levels in a non-conception cycle. Simultaneously with the tissue breakdown, the surface is re-epithelialized, protecting the wound from infection. Once menstruation is complete and estrogen levels start to rise, regeneration progresses throughout the proliferative phase of the cycle, to fully restore endometrial thickness. Endometrial repair is unique compared to tissue repair elsewhere in the adult, in that it is rapid, scar-free and occurs around 400 times during each modern woman's reproductive life. The shedding tissue and that undergoing repair is bathed in menstrual fluid, which contains live cells, cellular debris, fragments of extracellular matrix, activated leukocytes and their products, soluble cellular components and extracellular vesicles. Proteomic and other analyses have revealed some detail of these components. Menstrual fluid, along with a number of individual proteins enhances epithelial cell migration to cover the wound. This is shown in endometrial epithelial and keratinocyte cell culture models, in an ex vivo decellularized skin model and in pig wounds in vivo. Thus, the microenvironment provided by menstrual fluid, is likely responsible for the unique rapid and scar-free repair of this remarkable tissue. Insight gained from analysis of this fluid is likely to be of value not only for treating endometrial bleeding problems but also in providing potential new therapies for poorly repairing wounds such as those seen in the aged and in diabetics.

Investigation of the interaction of keratinocytes and Candida species

Our skin provides immunological protection against several pathogens. Skin epithelial cells respond to microbial stimuli in various ways, such as through the production of antimicrobial peptides or secretion of cytokines, although phagocytosis of potentially evading microbes was also reported. Relatively little is known about how skin keratinocytes differentiate between the presence of pathogenic and commensal fungi. In this project, we aimed to investigate how human keratinocytes interact with different Candida species, as common colonizers of the skin. While C. albicans is a common cause of cutaneous candidiasis, C. parapsilosisis rarely associated with this disease.For the experimentshuman skin keratinocyte cell lines (HaCaT, HPV-KER)were applied andchallengedwith C. albicans (SC5314 and WO1 strains) and C. parapsilosis (GA1 and CLIB214 strains)strains.We aimedto determine the extent to which C. albicans and C. parapsilosis damage human keratinocytes, their attachment to host cells, the keratinocytes’ ability to internalize these fungi and to examinecytokine production in response to stimuli. Our results suggest that C. albicans causes significantly more damage to human keratinocytes than C. parapsilosis and the HPV-KER cell line was more susceptibleto the infection. In both HaCaT and HPV-KER cells, the production of IL-6, IL-8, and CCL5 increased primarilyafter C. albicans infection. Based on the adhesion studies, there was a low degree of association in case of C. parapsilosis GA1 and CLIB214 compared to C. albicans SC5314 and WO1.

Cosmeceutical Potentials of Grammatophyllum speciosum Extracts: Anti-Inflammations and Anti-Collagenase Activities with Phytochemical Profile Analysis Using an Untargeted Metabolomics Approach

Grammatophyllum speciosum is the largest orchid species and a well-known traditional medicinal plant. Due to skin aging, natural products that inhibit this process can attract the attention of consumers and scientists because radical-scavenging activity, collagenase inhibition, and inflammatory suppression are valuable in dermatological applications. This study investigated the phytochemicals in G. speciosum leaves extracts that have cosmeceutical potentials, including radical-scavenging, anticollagenase, and anti-inflammatory abilities. G. speciosum leaves were extracted using water-based extraction methods. High-resolution mass spectrometry was used to identify the phytochemicals in the extracts. Fibroblast and keratinocyte cell cytotoxicity was determined. Antioxidant abilities were measured using DPPH and ABTS assays. The effect of the extracts on nitric oxide (NO) in macrophage cells was investigated. ELISA of the collagenase enzyme was determined. A total of 721 annotated metabolites were identified in the extracts. Vitexin and orientin were the most abundant metabolites. Cell viability was >80% in both cell lines when the extract concentration was <1 mg/mL. The IC50 values for DPPH and ABTS were 56 and 117 μg/mL, respectively. Furthermore, the extracts revealed that NO and collagenase activity were suppressed by 42% and 23%, respectively. The extracts can suppress ROS, inflammatory, and collagenase activities without causing fibroblast and keratinocyte cell death. Thus, this study provides information on metabolites in G. speciosum leaves, which is promising as cosmeceuticals or pharmaceuticals with anti-inflammatory and anti-collagenase activities.

Multimodal Analyses of Stable Vitiligo Skin Identify Tissue Interactions That Control Disease Persistence

Vitiligo is an autoimmune skin disease that is characterized by the progressive destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells but why white patches in stable disease persist is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. Here, we combine non-invasive multiphoton microscopy (MPM) imaging and single-cell RNA sequencing (scRNA-seq) to identify distinct subpopulations of keratinocytes in lesional skin of stable vitiligo patients. We show that these keratinocytes are enriched in lesional vitiligo skin and differ in metabolism, an observation corroborated by both MPM and scRNA-seq. Systematic investigation of cell-cell communication show that CXCL is the prominent signaling change in this small population of keratinocytes, which secrete CXCL9 and CXCL10 to create local inflammatory cytokine loops with T cells to drive stable vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative keratinocyte differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cellcell communication networks and keratinocyte cell states that perpetuate inflammation and prevent repigmentation.One Sentence SummaryCommunication between keratinocytes, immune cells, and melanocytes maintain depigmented patches in stable vitiligo.

Traditional Turkish Coffee with Medicinal Effect

Traditional Turkish coffee (TTC) is highly associated with caffeine and is known as a mind and heart stimulant as it helps keep tiredness at bay. Daily consumption of TTC naturally benefits human health such as anti-cancer, anti-diabetic, improved energy, anti-depression, reduced risk of heart disease, etc. The TTC was derived from particular types of Arabic coffee beans (ACB), and the preparation method of TTC is unique from other types of coffee. The main objective of the study was to investigate the therapeutic and biological effects of TTC. The ACB powder was characterized physicochemically using UV-Vis spectroscopy, Fourier transforms infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). In vitro analysis using HaCaT (Human keratinocyte cell line) proved the biocompatibility of ACB powder. Case studies which were focusing on healthy individuals as the research populace were conducted using TTC. Consumption of TTC was found beneficially compared to other types of coffee. The TTC was obtained from ACB, which was characterized by spectroscopic techniques and displayed biocompatibility due to the results on HaCaT cell lines. The TTC has beneficial therapeutic effects on individuals. According to statistical analysis, the disease-affected ratio of diabetes, heart disease, and depression was significantly decreased.

α4/α9 Integrins Coordinate Epithelial Cell Migration Through Local Suppression of MAP Kinase Signaling Pathways

Adhesion of basal keratinocytes to the underlying extracellular matrix (ECM) plays a key role in the control of skin homeostasis and response to injury. Integrin receptors indirectly link the ECM to the cell cytoskeleton through large protein complexes called focal adhesions (FA). FA also function as intracellular biochemical signaling platforms to enable cells to respond to changing extracellular cues. The α4β1 and α9β1 integrins are both expressed in basal keratinocytes, share some common ECM ligands, and have been shown to promote wound healing in vitro and in vivo. However, their roles in maintaining epidermal homeostasis and relative contributions to pathological processes in the skin remain unclear. We found that α4β1 and α9β1 occupied distinct regions in monolayers of a basal keratinocyte cell line (NEB-1). During collective cell migration (CCM), α4 and α9 integrins co-localized along the leading edge. Pharmacological inhibition of α4β1 and α9β1 integrins increased keratinocyte proliferation and induced a dramatic change in cytoskeletal remodeling and FA rearrangement, detrimentally affecting CCM. Further analysis revealed that α4β1/α9β1 integrins suppress extracellular signal-regulated kinase (ERK1/2) activity to control migration through the regulation of downstream kinases including Mitogen and Stress Activated Kinase 1 (MSK1). This work demonstrates the roles of α4β1 and α9β1 in regulating migration in response to damage cues.