Skin Explant Culture: A Reliable Method for Detecting Pemphigoid Antibodies in Pemphigoid Sera that Are Negative by Standard Immunofluorescence and Immunoblotting

Mutasim, Diya F.; Vaughan, Amy.; Supapannachart, Nantapat; Farooqui, Jamal

doi:10.1111/1523-1747.ep12366084

Cited by 8 publications

(8 citation statements)

References 18 publications

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“…We demonstrated the effects of TNF␣, namely the induction of K6b expression and the activation of NFB transcription factor, both in cultured keratinocytes and in explants of human skin, a new experimental system designed to emulate in vivo conditions (9,(71)(72)(73)(74).…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we decided to determine whether TNF␣ could induce, in the absence of EGF/TGF␣, the expression of K6b keratin in human epidermis. Because convenient systems for analysis of the effects of TNF␣ and other growth factors and cytokines in human skin in vivo have not been described, we developed a new and elegant nearly in vivo experimental system that uses organ culture of human skin samples otherwise discarded during surgery (71)(72)(73)(74). We obtained 5-mm diameter biopsies of human skin and placed them in culture medium to which we added TNF␣.…”

Section: Tnf␣ Activates Nfb and Induces Expression Of Keratin K6b Promentioning

confidence: 99%

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Inflammatory Versus Proliferative Processes in Epidermis

Komine¹,

Rao²,

Kaneko³

et al. 2000

Journal of Biological Chemistry

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Epidermal keratinocytes respond to injury by becoming activated, i.e. hyperproliferative, migratory, and proinflammatory. These processes are regulated by growth factors and cytokines. One of the markers of activated keratinocytes is keratin K6. We used a novel organ culture system to show that tumor necrosis factor ␣ (TNF␣) induces the expression of K6 protein and mRNA in human skin. Multiple isoforms of K6 are encoded by distinct genes and have distinct patterns of expression. By having shown previously that proliferative signals, such as epidermal growth factor (EGF), induce expression of the cytoskeletal protein keratin K6b, we here demonstrate that the same isoform, K6b, is also induced by TNF␣, a proinflammatory cytokine. Specifically, TNF␣ induces the transcription of the K6b gene promoter. By using co-transfection, specific inhibitors, and antisense oligonucleotides, we have identified NFB and C/EBP␤ as the transcription factors that convey the TNF␣ signal. Both transcription factors are necessary for the induction of K6b by TNF␣ and act as a complex, although only C/EBP␤ binds the K6b promoter DNA. By using transfection, site-directed mutagenesis, and footprinting, we have mapped the site that responds to TNF␣, NFB, and C/EBP␤. This site is separate from the one responsive to EGF and AP1. Our results show that the proinflammatory (TNF␣) and the proliferative (EGF) signals in epidermis separately and independently regulate the expression of the same K6b keratin isoform. Thus, the cytoskeletal responses in epidermal cells can be precisely tuned by separate proliferative and inflammatory signals to fit the nature of the injuries that caused them.

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Section: Discussionmentioning

confidence: 99%

Section: Tnf␣ Activates Nfb and Induces Expression Of Keratin K6b Promentioning

confidence: 99%

Inflammatory Versus Proliferative Processes in Epidermis

Komine¹,

Rao²,

Kaneko³

et al. 2000

Journal of Biological Chemistry

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“…Frozen sections of 4 to 6 m were cut (Fung Frigocut 2,800 E Cryostat) and then collected onto gelatin-coated slides. The sections were stained according to a standard immunofluorescent staining protocol (40). The primary antibody used was polyclonal rabbit anti-human GR antibody (Affinity Bioreagents).…”

mentioning

confidence: 99%

Novel Mechanism of Steroid Action in Skin through Glucocorticoid Receptor Monomers

Radoja

Komine

Jho³

et al. 2000

Molecular and Cellular Biology

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Glucocorticoids (GCs), important regulators of epidermal growth, differentiation, and homeostasis, are used extensively in the treatment of skin diseases. Using keratin gene expression as a paradigm of epidermal physiology and pathology, we have developed a model system to study the molecular mechanism of GCs action in skin. Here we describe a novel mechanism of suppression of transcription by the glucocorticoid receptor (GR) that represents an example of customizing a device for transcriptional regulation to target a specific group of genes within the target tissue, in our case, epidermis. We have shown that GCs repress the expression of the basal-cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17 but not the differentiation-specific keratins K3 and K10 or the simple epithelium-specific keratins K8, K18, and K19. We have identified the negative recognition elements (nGREs) in all five regulated keratin gene promoters. Detailed footprinting revealed that the function of nGREs is to instruct the GR to bind as four monomers. Furthermore, using cotransfection and antisense technology we have found that, unlike SRC-1 and GRIP-1, which are not involved in the GR complex that suppresses keratin genes, histone acetyltransferase and CBP are. In addition, we have found that GR, independently from GREs, blocks the induction of keratin gene expression by AP1. We conclude that GR suppresses keratin gene expression through two independent mechanisms: directly, through interactions of keratin nGREs with four GR monomers, as well as indirectly, by blocking the AP1 induction of keratin gene expression.

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“…This method has long been employed as a model of wound healing or adult skin epidermal outgrowth rather than a source of keratinocytes for clinical or experimental purposes (19)(20)(21)(22)(23)(24). The major limitation of the explant method is due to the fact that fibroblasts also grow out from the same explants and will eventually outgrow the keratinocytes.…”

Section: Discussionmentioning

confidence: 99%

Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

et al. 2015

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Abstract. Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2-3 mm pieces and placed in trypsin at 4˚C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7-10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2-3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing. IntroductionThe skin is the largest organ in the body that is divided into two anatomically distinct regions, the dermis and epidermis. The normal structure and function of this organ is dependent on the intact epidermis anchored to its vascular, elastic dermis (1,2). Fibroblasts are the most prevalent cell types in the dermis, which produce different growth factors that induce proliferation of keratinocytes in vivo and in vitro (3). The principal cell type of the epidermis is the keratinocyte (1,4), which is a small epithelial cell, located at the top of the epidermal basal membrane and characterized by a low division rate (5,6).Thus far, various enzymatic methods for dermal-epidermal separation have been applied (6-8). For instance, trypsin separates suprabasal hemidesmosomes that causes the basal layer cells to attach to the dermal layer (6). Thermolysin is another enzyme that selectively separates desmosomes (5,9) and is able to separate the epidermis at the basal membrane zone level (5,10).Although the dissociation method of keratinocytes in primary culture is well-established, attempts to acquire purified adult stem-cell like⁄progenitor keratinocytes from whole human skin are still ongoing. In particular, different techniques are currently being applied to achieve high purity or homogeneous primary cultures enriched in keratinocyte progenitor⁄precursor cells. These include filtration, density gradient centrifugation and fluorescence-activated cell sorting using cell surface antibodies, as well as differential adhesion to enrich the cells that rapidly attach to particular substrates (11).A previous study showed that when keratinocytes/precursor cells...

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Skin Explant Culture: A Reliable Method for Detecting Pemphigoid Antibodies in Pemphigoid Sera that Are Negative by Standard Immunofluorescence and Immunoblotting

Cited by 8 publications

References 18 publications

Inflammatory Versus Proliferative Processes in Epidermis

Inflammatory Versus Proliferative Processes in Epidermis

Novel Mechanism of Steroid Action in Skin through Glucocorticoid Receptor Monomers

Comparison of the enzymatic and explant methods for the culture of keratinocytes isolated from human foreskin

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