Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration

2020 ◽  
pp. 1-12
Author(s):  
Irene Castellano-Pellicena ◽  
M. Julie Thornton
Keyword(s):  
Human Skin ◽  
Epidermal Keratinocytes ◽  
Epidermal Keratinocyte ◽  
Keratinocyte Migration ◽  
Scratch Wound ◽  
Scratch Wound Assay

Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model

2011 ◽  
Vol 51 (4) ◽  
pp. 884-892 ◽  
Author(s):  
Alvin Eng Kiat Loo ◽  
Rongjian Ho ◽  
Barry Halliwell
Keyword(s):  
Hydrogen Peroxide ◽  
Keratinocyte Migration ◽  
Wound Model ◽  
Scratch Wound

Alpha pinene modulates UVA-induced oxidative stress, DNA damage and apoptosis in human skin epidermal keratinocytes

Life Sciences ◽  
2018 ◽  
Vol 212 ◽  
pp. 150-158 ◽  
Author(s):  
Ramasamy Karthikeyan ◽  
Govindasamy Kanimozhi ◽  
Nagarajan Rajendra Prasad ◽  
Balupillai Agilan ◽  
Muthusamy Ganesan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Human Skin ◽  
Epidermal Keratinocytes ◽  
Alpha Pinene

Auswirkungen Konservierungsmittel-freier Fluorchinolon-Augenlösungen auf menschliche Hornhaut-Epithelzellen in vitro

2015 ◽  
Vol 1 (1) ◽  
pp. 26-27
Keyword(s):  
Scratch Wound ◽  
Scratch Wound Assay

Ziele: Die Beurteilung der biologischen Wirkungen Konservierungsmittel-freier Fluorchinolon-Augenlösungen auf Zellkulturen menschlichen Hornhaut-Epithels in vitro.Methoden: Wir untersuchten die Auswirkung von topischen Fluorchinolonen verschiedener Generationen, wie Ofloxacin 0,3%, Levofloxacin 0,5%, Tosufloxacin 0,3%, Moxifloxacin 0,5% und Gatifloxacin 0,3%, auf gezüchtete menschliche Hornhaut-Epithelzellen. Die Untersuchung erfolgte mittels MTT-basiertem kalorimetrischem Assay, Laktatdehydrogenase(LDH)-Leakage-Assay und Scratch-Wound-Assay. Die Morphologie der Hornhaut-Epithelzellen wurde mittels inverser Lichtmikroskopie und Transmissionselektronenmikroskopie untersucht.Ergebnisse: Bei allen topischen Fluorchinolonen ging die metabolische Aktivität der Hornhaut-Epithelzellen zeitabhängig zurück, und der LDH-Titer stieg mit zunehmender Dauer der Wirkstoffexposition. Insbesondere nach einer Exposition gegenüber Moxifloxacin 0,5% und Gatifloxacin 0,3% war ein signifikanter Anstieg der LDH-Titer im Vergleich zu den Kontrollen festzustellen. Die Migrationsraten der Hornhaut-Epithelzellen waren bei Ofloxacin 0,3% und Levofloxacin 0,5% höher als bei den anderen Fluorchinolonen. Nach einer Exposition gegenüber Moxifloxacin 0,5% und Gatifloxacin 0,3% waren schwere morphologische Schäden an den Zellen zu beobachten.Schlussfolgerung: Da Moxifloxacin 0,5% und Gatifloxacin 0,3% eine stärkere toxische Wirkung auf die Hornhaut-Epithelzellen ausübten als die anderen Fluorchinolone, sind diese Fluorchinolon-Augenlösungen der 4. Generation nur nach sorgfältiger Abwägung im Hinblick auf die mögliche Schädigung des Hornhaut-Epithels bei langer Behandlungsdauer oder zu hoher Dosierung anzuwenden.Übersetzung aus Ophthalmic Res 2014;51:216-223 (DOI: 10.1159/000357976)

scholarly journals Fisetin, a 3,7,3′,4′-Tetrahydroxyflavone Inhibits the PI3K/Akt/mTOR and MAPK Pathways and Ameliorates Psoriasis Pathology in 2D and 3D Organotypic Human Inflammatory Skin Models

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1089 ◽  
Author(s):  
Jean Christopher Chamcheu ◽  
Stephane Esnault ◽  
Vaqar M. Adhami ◽  
Andrea L. Noll ◽  
Sergette Banang-Mbeumi ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Human Skin ◽  
Mononuclear Cells ◽  
Psoriatic Skin ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Peripheral Blood Mononuclear ◽  
Inflammatory Skin ◽  
2D And 3D

Psoriasis is a chronic immune-mediated skin disease that involves the interaction of immune and skin cells, and is characterized by cytokine-driven epidermal hyperplasia, deviant differentiation, inflammation, and angiogenesis. Because the available treatments for psoriasis have significant limitations, dietary products are potential natural sources of therapeutic molecules, which can repair the molecular defects associated with psoriasis and could possibly be developed for its management. Fisetin (3,7,3′,4′-tetrahydroxyflavone), a phytochemical naturally found in pigmented fruits and vegetables, has demonstrated proapoptotic and antioxidant effects in several malignancies. This study utilized biochemical, cellular, pharmacological, and tissue engineering tools to characterize the effects of fisetin on normal human epidermal keratinocytes (NHEKs), peripheral blood mononuclear cells (PBMC), and CD4+ T lymphocytes in 2D and 3D psoriasis-like disease models. Fisetin treatment of NHEKs dose- and time-dependently induced differentiation and inhibited interleukin-22-induced proliferation, as well as activation of the PI3K/Akt/mTOR pathway. Fisetin treatment of TNF-α stimulated NHEKs also significantly inhibited the activation of p38 and JNK, but had enhanced effect on ERK1/2 (MAPK). In addition, fisetin treatment significantly decreased the secretion of Th1/Th-17 pro-inflammatory cytokines, particularly IFN-γ and IL-17A by 12-O-tetradecanolylphorbol 13-acetate (TPA)-stimulated NHEKs and anti-CD3/CD28-activated human PBMCs. Furthermore, we established the in vivo relevance of fisetin functions, using a 3D full-thickness human skin model of psoriasis (FTRHSP) that closely mimics in vivo human psoriatic skin lesions. Herein, fisetin significantly ameliorated psoriasis-like disease features, and decreased the production of IL-17 by CD4+ T lymphocytes co-cultured with FTRHSP. Collectively, our data identify the prodifferentiative, antiproliferative, and anti-inflammatory effects of fisetin, via modulation of the PI3K-Akt-mTOR and p38/JNK pathways and the production of cytokines in 2D and 3D human skin models of psoriasis. These results suggest that fisetin has a great potential to be developed as an effective and inexpensive agent for the treatment of psoriasis and other related inflammatory skin disorders.

scholarly journals Microtubule-associated protein 4 phosphorylation regulates epidermal keratinocyte migration and proliferation

2019 ◽  
Vol 15 (9) ◽  
pp. 1962-1976 ◽  
Author(s):  
Junhui Zhang ◽  
Lingfei Li ◽  
Qiong Zhang ◽  
Wensheng Wang ◽  
Dongxia Zhang ◽  
...  
Keyword(s):  
Epidermal Keratinocyte ◽  
Keratinocyte Migration

scholarly journals Murine Cutaneous Mastocytosis and Epidermal Melanocytosis Induced by Keratinocyte Expression of Transgenic Stem Cell Factor

1998 ◽  
Vol 187 (10) ◽  
pp. 1565-1573 ◽  
Author(s):  
Takahiro Kunisada ◽  
Shu-Zhuang Lu ◽  
Hisahiro Yoshida ◽  
Satomi Nishikawa ◽  
Shin-ichi Nishikawa ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Skin ◽  
Stem Cell Factor ◽  
Epidermal Keratinocytes ◽  
Melanin Production ◽  
Human Melanocyte ◽  
Kit Receptor ◽  
Membrane Bound ◽  
Epidermal Melanocyte ◽  
Cutaneous Mastocytosis

The growth and differentiation of mast cells and melanocytes require stem cell factor (SCF), the ligand for the kit receptor tyrosine kinase. SCF may exist as a membrane-bound or soluble molecule. Abnormalities of the SCF-kit signaling pathway, with increased local concentrations of soluble SCF, have been implicated in the pathogenesis of the human disease cutaneous mastocytosis, but have not yet been shown to play a causal role. To investigate both the potential of SCF to cause mastocytosis and its role in epidermal melanocyte homeostasis, we targeted the expression of SCF to epidermal keratinocytes in mice with two different transgenes controlled by the human keratin 14 promoter. The transgenes contained cDNAs that either produced SCF, which can exist in both membrane-bound and soluble forms, or SCF, which remains essentially membrane bound. Murine epidermal keratinocyte expression of membrane-bound/ soluble SCF reproduced the phenotype of human cutaneous mastocytosis, with dermal mast cell infiltrates and epidermal hyperpigmentation, and caused the maintenance of a population of melanocytes in the interadnexal epidermis, an area where melanocytes and melanin are found in human skin but where they are not typically found in murine skin. Expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. We conclude, first, that a phenotype matching that of human mastocytosis can be produced in mice by keratinocyte overproduction of soluble SCF, suggesting a potential cause of this disease. Second, we conclude that keratinocyte expression of membrane-bound SCF results in the postnatal maintenance of epidermal melanocytes in mice. Since the resulting animals have skin that more closely approximates human skin than do normal mice, their study may be more relevant to human melanocyte biology than the study of skin of normal mice.

scholarly journals Epidermis instructs skin homing receptor expression in human T cells

Blood ◽  
2012 ◽  
Vol 120 (23) ◽  
pp. 4591-4598 ◽  
Author(s):  
Michelle L. McCully ◽  
Kristin Ladell ◽  
Svetlana Hakobyan ◽  
Robert E. Mansel ◽  
David A. Price ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Human Skin ◽  
Memory T Cells ◽  
Immune Surveillance ◽  
Receptor Expression ◽  
Epidermal Keratinocytes ◽  
Specific Factor ◽  
Peripheral Tissues ◽  
Preferential Expression

Abstract The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. Although the mechanisms controlling memory T-cell migration to peripheral tissues are poorly understood, the current paradigm includes the localized secretion of “imprinting” signals from tissue-resident dendritic cells in the draining lymph nodes. Here we show that CCR8 expression by newly activated naive T cells is regulated by skin-specific factor(s) derived primarily from epidermal keratinocytes, thereby providing a mechanism for the preferential expression of CCR8 by skin-resident memory T cells. Importantly, no such effects were observed after coculture with primary cells from skin-unrelated epithelia, including mesothelium and small intestine. The keratinocyte-derived CCR8-inducing factor(s) were soluble, and independent of vitamins A and D. Furthermore, the induction of CCR8 under these conditions correlated with an increase in cutaneous lymphocyte-associated antigen expression. Our findings challenge current tissue homing paradigms, especially those involving CCR10, and emphasize the importance of steady-state epidermis rather than tissue-resident dendritic cells in controlling the localization of memory T cells within human skin.

scholarly journals The expression of a gamma interferon-induced protein (IP-10) in delayed immune responses in human skin.

1987 ◽  
Vol 166 (4) ◽  
pp. 1098-1108 ◽  
Author(s):  
G Kaplan ◽  
A D Luster ◽  
G Hancock ◽  
Z A Cohn
Keyword(s):  
Endothelial Cells ◽  
Human Skin ◽  
Basal Layer ◽  
Lepromatous Leprosy ◽  
Immunocytochemical Staining ◽  
Epidermal Keratinocytes ◽  
Ifn Gamma ◽  
Intense Staining ◽  
Effector Cells ◽  
Dermal Infiltrate

Our knowledge of the induction of new molecules by IFN-gamma has led to the characterization of IP-10 and the preparation of a monospecific, polyclonal antibody. Using this reagent we have now examined inflammatory states occurring in human skin and used immunocytochemical staining for the expression of both Ia and IP-10 determinants. After evoking a delayed-type response to purified protein derivative of tuberculin (PPD), we noted the presence of IP-10 in dermal macrophages and endothelial cells. Intense staining of the basal layer of epidermal keratinocytes was prominent at 41 h, and by 1 wk the entire epidermis was staining. The comparison of the amount of IP-10 secreted by keratinocytes vs. macrophages, fibroblasts, and endothelial cells revealed that keratinocytes were by far the major producers of this molecule. The expression of Ia occurred in conjunction with IP-10. The injection of rIFN-gamma mimicked many of the features of the PPD response, including the expression of both Ia and IP-10 by epidermal keratinocytes. Coexpression was also found in the natural lesions of tuberculoid leprosy and cutaneous Leishmaniasis. However, it was absent in lepromatous leprosy, a state where activated T lymphocytes are not present. We suggest that the local production of IFN-gamma by T cells of the dermal infiltrate induces IP-10 formation in both the dermis and epidermis. IP-10 and Ia then serve as specific markers of immune IFN and its possible influence on effector cells of the cell mediated immune response.

Dissecting the invasive potential of esophageal cancer by siRNA library screening.

2015 ◽  
Vol 33 (3_suppl) ◽  
pp. 86-86
Author(s):  
Shane P Duggan ◽  
Sinead Phipps ◽  
Fiona Behan ◽  
Catherine Garry ◽  
Dermot P Kelleher
Keyword(s):  
Cell Proliferation ◽  
Esophageal Cancer ◽  
Cell Viability ◽  
Metastatic Potential ◽  
Software Systems ◽  
Invasive Potential ◽  
Significant Difference ◽  
Scratch Wound ◽  
Scratch Wound Assay ◽  
Reverse Transfection

86 Background: The low survival for esophageal cancer is in part attributed to its high invasive potential and distant metastasis. In cancer, abnormal cell migration is an essential component of metastasis, and it is reasonable to consider that the conversion between different forms of morphology permits tumor cells to metastasise. Discovering regulators of esophageal cancer morphogenesis may aid in the development of novel targeted therapies that limit metastatic potential. Methods: GOhTRT cells were seeded and treated with siRNA (Human Druggable Genome, Dharmacon) by reverse transfection. Cells were fixed, immunostained for DNA, tubulin and actin and imaged with automated microscope. Cell images were processed using the InCell Analyzer software and the R statistical software systems CellHTS2 and RNAither. The effect of RNAi knockdown on cell viability and cell motility were assessed using MTT cell proliferation assay and scratch wound assay. Results: 127 gene candidates greatly exhibited effects on F-actin and α-tubulin area staining. This list was refined to six high quality candidates (RRM2, ITGB8, GPS1, SPRY1, NOL1 and MYO9B). Silencing of GPS1, MYO9B and SPRY1 increased the rate of migration in a scratch wound assay, with 86.98% ± 3.097%, 75.78% ±5.454% and 72.97% ± 5.463% (p =0.0022) respectively. There was no significant difference in cell viability absorbance values for siGPS1 (0.9037 ± 0.06575; p =0.1905) and siSPRY1 (0.9088 ± 0.09849; p =0.2985), which suggests that the increased rate of wound closure previously seen is by virtue of migratory signalling as oppose to an increase in cell proliferation. Cell viability was decreased considerably in siRRM2 cells (0.2492 ± 0.02798; p <0.0001) and siMYO9B (0.4048 ± 0.04663; p<0.0001) in comparison to siNT cells (1.046 ± 0.07712). Conclusions: In summary, this screen successfully identified high confidence hits associated with cytoskeletal remodelling, some of which are already associated with metastasis in literature and database searches. Further mechanistic studies and gene pathway analysis of candidate genes will provide novel therapeutic targets which can be utilised to block the spread of cancer in patients.

Alterations in cell-adhesive and migratory properties of proximal tubule and collecting duct cells from bcl-2 −/− mice

2004 ◽  
Vol 287 (6) ◽  
pp. F1154-F1163 ◽  
Author(s):  
Jacqueline Ziehr ◽  
Nader Sheibani ◽  
Christine M. Sorenson
Keyword(s):  
Proximal Tubule ◽  
Collecting Duct ◽  
Proximal Tubule Cells ◽  
Duct Cells ◽  
Scratch Wound ◽  
Cell Adhesive ◽  
Scratch Wound Assay ◽  
Collecting Duct Cells ◽  
Tubule Cells ◽  
Sb 203580

Bcl-2 protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Bcl-2 −/− mice develop renal hypoplastic/cystic dysplasia with renal cyst formation coinciding with renal maturation in normal mice. To gain a better understanding of the role cell-adhesive mechanisms play during renal maturation, we generated proximal tubule and collecting duct cell lines from postnatal day 10 ( P10) and P20 bcl-2 +/+ and bcl-2 −/− mice. Very little is known about the role cell-adhesive and migratory mechanisms play during renal maturation. We observed that modulation of cell-adhesive properties, which normally occur in a nephron segment-specific manner during renal maturation, and cell migration were altered in cells from bcl-2 −/− mice. Enhanced migration of bcl-2 −/− proximal tubule cells in a scratch wound assay was completely inhibited by incubation with PP1 (Src inhibitor) and moderately affected by incubation with SB-203580 (p38 inhibitor). These cells expressed increased levels of fibronectin and had numerous central focal adhesions. P20 bcl-2 −/− proximal tubule cells adhered to fibronectin but adhered poorly to collagen, vitronectin, or laminin. Collecting duct cells, similar to proximal tubule cells from bcl-2 −/− mice, demonstrated enhanced migration in a scratch wound assay that was inhibited by incubation with PP1. Migration of these cells was moderately affected by incubation with PD-98059 (MEK inhibitor) or LY-294002 (PI3 kinase inhibitor), whereas incubation with SB-203580 had no effect. P10 bcl-2 −/− collecting duct cells also expressed increased levels of fibronectin but decreased levels of thrombospondin-1 and demonstrated precocious binding to fibronectin and vitronectin compared with bcl-2 +/+ cells. The ability of P20 bcl-2 +/+ collecting duct cells to adhere to fibronectin and vitronectin corresponded with a decline in thrombospondin-1 expression. Therefore, alterations in cell-adhesive and migratory characteristics may be an early indicator of aberrant renal epithelial cell differentiation.

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