Mi Yeung Sohn

Topical ALA-Photodynamic Therapy for Acne Can Induce Apoptosis of Sebocytes and Down-regulate Their TLR-2 and TLR-4 Expression

BACKGROUND Although photodynamic therapy (PDT) is widely performed for acne, little is known about its exact therapeutic mechanism. OBJECTIVE We aimed to estimate the efficacy and safety of PDT on acne and to discover its mode of action. METHODS We performed PDT on 12 patients with mild to moderate acne. The clinical efficacy was assessed by counting the acne lesions and measuring the sebum secretion before and after PDT. In addition, we took biopsy samples from the peri-lesional areas before and after 3-session of PDT. To examine the degree of apoptosis of the sebaceous follicles, TUNEL assay was performed. To investigate the changes of toll-like receptor (TLR)-2 and TLR-4 expression after PDT, immunohistochemical stainings were also carried out. Finally, we performed TUNEL assay using the cultured sebocytes to confirm the apoptosis of sebocytes in vitro after PDT. RESULTS There was a significant reduction in the number of inflammatory acne lesions after PDT, compared to baseline (p<0.05). Sebum excretion significantly decreased 2 weeks after the first PDT session except for one patient (p<0.05). The TUNEL positive cells in the peri-lesional sebaceous glands after PDT markedly increased, compared with those of before PDT. A decrease in TLR-2 and TLR-4 expression by sebaceous glands and epidermis after PDT was 50% and 30%, respectively. CONCLUSION Our results demonstrate that apoptosis of the sebaceous glands is associated with improvement of acne by PDT. PDT has shown to down-regulate TLR-2 and TLR-4 expression in the sebaceous glands and epidermis of acne patients.

Expression of inflammatory biomarkers from cultured sebocytes was influenced by treatment with vitamin D

Background: Inflammatory cytokines are the key factor in the pathophysiology of acne. It is well known that keratinocytes synthesize many kinds of inflammatory cytokines. In addition, it is reported that inflammatory cytokines are also expressed from sebocytes, which originate from the same stem cells with keratinocytes. Aim: To clarify changes in the expression of inflammatory biomarkers from cultured sebocytes after treatment with vitamin D. Materials and Methods: Reverse transcription-polymerase chain reaction (RT-PCR) was done to measure changes in the expression of inflammatory biomarkers, including interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α), and several subtypes of matrix metalloproteinases (MMPs) after treatment of a group of cultured sebocytes with vitamin D. Vitamin D receptor (VDR) small interfering RNA (siRNA) was added in the other group of cultured sebocytes to assure the role of vitamin D on the expression of inflammatory biomarkers. Enzyme-linked immunosorbent assay (ELISA) was also performed in the vitamin D-treated sebocytes. Results: Cultured sebocytes showed non-significant changes in the gene expression of inflammatory biomarkers after treatment with vitamin D. In cultured sebocytes treated with a VDR siRNA, the expression of inflammatory biomarkers was not blocked after treatment with vitamin D. ELISA showed a significant decrease in the expression of IL-6, IL-8, and MMP-9, but a significant increase in the expression of MMP-1 and MMP-3, after treatment with vitamin D (10-6 M). Conclusion: Expression of inflammatory biomarkers is influenced by treatment with vitamin D in cultured sebocytes, but not through VDR.

Ultraviolet B irradiation increases the expression of inflammatory cytokines in cultured sebocytes.

Sebaceous gland hyperplasia and increased sebum secretion after irradiation of ultraviolet (UV)‐B has been widely accepted. This study was performed to clarify expression of inflammatory cytokines after irradiating UV‐B in cultured sebocytes. Reverse transcription polymerase chain reaction was performed to measure gene expression of inflammatory cytokines, including interleukin (IL)‐1β, IL‐6, IL‐8 and tumor necrosis factor‐α, in cultured sebocytes after exposure to 40 and 70 mJ/cm2 UV‐B. Protein expression of inflammatory cytokines and lipid production in cultured sebocytes after exposure to UV‐B were measured using enzyme‐linked immunoassay and lipid analysis kit. The expression of inflammatory cytokines, especially IL‐1β and IL‐8, was significantly increased in cultured sebocytes after treatment with UV‐B. Many more studies on the effect of UV‐B on sebaceous glands should be performed to reveal the pathogenic mechanism of acne.

Insulin-Like Growth Factor-1 Increases the Expression of Inflammatory Biomarkers and Sebum Production in Cultured Sebocytes

Background Acne vulgaris has been linked to the Western diet. Hyperglycemic diet increases insulin and insulin-like growth factor (IGF)-1. Deeper insights into IGF-1-mediated signal pathway are critical importance to understand the impact of Western diet. Objective We investigated the effect of IGF-1 on the expression of inflammatory biomarkers and sebum production in cultured sebocytes. Methods Polymerase chain reaction and enzyme-linked immunosorbent assay were performed to measure changes in the expression of inflammatory biomarkers including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), IGF1R, IGFBP2, sterol response element-binding protein (SREBP), and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PI3KCA) after the treatment of cultured sebocytes with 10−7 M or 10−5 M IGF-1. Sebum production was evaluated after the treatment of cultured sebocytes with 10−7 M or 10−5 M IGF-1 using lipid analysis. Results The expression levels of IL-1β, IL-6, IL-8, and TNF-α in cultured sebocytes after treatment with 10−7 M or 10−5 M IGF-1 were increased. Increased gene expression levels of NF-κB in cultured sebocytes were also shown after 10−7 M or 10−5 M IGF-1 treatments. Gene expression of these inflammatory biomarkers was decreased after 10−7 M or 10−5 M IGF-1 treatment in the presence of 100 nM NF-κB inhibitor. Treatment with 10−7 M or 10−5 M IGF-1 increased the gene expression levels of IGF1R, IGFBP2, SREBP and PI3KCA in cultured sebocytes. Sebum production from cultured sebocytes treated with 10−7 M or 10−5 M IGF-1 was also increased. Conclusion It is suggestive that IGF-1 might be involved in the pathogenesis of acne by increasing both expression of inflammatory biomarkers and also sebum production in sebocytes.

Ear Keloids as a Primary Candidate for the Application of Mitomycin C after Shave Excision: In Vivo and In Vitro Study

BACKGROUND Although many methods have been developed to treat ear keloids, new therapeutic options are still needed. OBJECTIVE To determine the effects of topical mitomycin C (MC) on shave‐removed wounds and fibroblasts of ear keloids. METHODS Fourteen ear keloids in 12 patients were shaved, and MC (1 mg/mL) was applied to the resected bed for 5 minutes. The application was repeated 3 weeks later. All patients were assessed 2, 4, and 6 months after the procedure to evaluate the cosmetic results, recurrence, and postsurgical complications. An in vitro study to determine the effects of MC on fibroblasts of the excised keloids was conducted using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, the measurement of total cell counts, and immunoassay of DNA synthesis. RESULTS Only one recurrence occurred (on the ear helix), and the patients were satisfied with the cosmetic outcomes. The results of the MTT assay, total cell counts, and DNA synthesis immunoassay confirmed the suppressive effects of MC on the keloid fibroblasts. CONCLUSION The application of topical MC to the resected bed of shave‐removed ear keloids was successful in preventing recurrences and providing an acceptable cosmetic outcome. The authors have indicated no significant interest with commercial supporters.

The Expression of Involucrin, Loricrin, and Filaggrin in Cultured Sebocytes

Dear Editor: It is well established that a complex interplay of corneocytes and intercellular lipids in the stratum corneum of the skin is responsible for the skin barrier against environmental factors. A cornified cell envelope, which is composed of involucrin, loricrin, filaggrin, and other proteins, is a component of fully differentiated epidermal keratinocytes and corneocytes, and it is important in the skin barrier1. Although sebaceous gland sebocytes are considered to originate from the same stem cells as epidermal keratinocyte, it is not clear whether the keratinocyte differentiation markers are expressed in the sebocytes or not2. Because the transfollicular route has been known as a major route for drug delivery, more information is required to investigate the expression of the markers in the sebaceous gland sebocytes. A primary culture of human scalp sebocytes was performed with Dulbeccos modified Eagles medium (DMEM; Gibco BRL, Grand Island, NY, USA) and Epilife (MEPI500CA; Gibco BRL) according to a method described previously3. Sebocytes were cultured at various concentrations of calcium (0.25, 0.5, 1, and 1.2 mM), or treated with vitamin D (10-10, 10-9, 10-8, 10-7, and 10-6 M). Reverse transcription polymerase chain reaction (RT-PCR) for involucrin, loricrin, and filaggrin was conducted in triplicate using the first strand cDNA synthesis kit (Promega, Madison, WI, USA) and oligonucleotide primers (Genotech, Daejeon, Korea). RT-PCR amplification was conducted using the GoTaq Flexi DNA Polymerase: involucrin for 34 cycles at 59℃, loricrin 34 cycles at 69℃, and filaggrin for 30 cycles at 58℃. Immunocytofluorescence for involucrin, loricrin, and filaggrin (Sigma-Aldrich, St. Louis, MO, USA) was also performed on cultured sebocytes. In this study, it was revealed that the expression of involucrin, loricrin, and filaggrin in cultured sebocytes was very weak (Fig. 1, ​,2).2). In addition, their expression in cultured sebocytes was not changed according to calcium concentration or after treatment with vitamin D (Fig. 2). Fig. 1 The expression of involucrin (A), loricrin (B), and filaggrin (C) was very weak in the cytoplasm of cultured sebocytes in immunocytofluorescence. (D) Control (A~D: ×200). Fig. 2 Gene expression of involucrin (A), loricrin (B), and filaggrin (C) in cultured sebocytes after treatment with calcium and VitD (vitamin D), respectively. Cont: control. Sebocytes are highly specialized, lipid-producing epithelial cells that release their contents by a rupture of the cell membrane and cellular degradation during differentiation4. Keratinocyte differentiating markers, including involucrin, loricrin, and filaggrin, provide structural support to the cell. Although sebocytes originate from the same stem cells as keratinocytes, it is thought that there are many differences in the expression of keratinocyte differentiating markers in sebocytes. In addition, it is recognized by our previous study (not published) that sebaceous gland sebocytes in vivo, both basal proliferating cells and central differentiating cells, show little expression of involucrin, loricrin, and filaggrin. Doran et al.5 reported that sebocytes did not produce cornified envelopes in vitro and could only be induced to produce small quantities (less than 5%) of envelopes with a calcium ionophore. However, Lo Celso et al.6 observed the presence of cornifin and involucrin positive immortalized sebocytes. This study showed very weak expression of involucrin, loricrin, and filaggrin in the cytoplasm of cultured sebocytes. Like cultured epidermal keratinocytes, proliferation and differentiation of cultured sebocytes are influenced by extracellular calcium concentration. Vitamin D also induces time- and dose-dependent modulation of cell proliferation and lipid content in cultured sebocytes through binding to vitamin D receptors. Nevertheless, this study showed that both extracellular calcium concentration and treatment with vitamin D did not affect the expression of involucrin, loricrin, and filaggrin in cultured sebocytes. In conclusion, cultured sebocytes showed little expression of keratinocyte differentiation markers. Weak physical barrier in the sebaceous gland may cause much better transfollicular drug delivery.

Some Becker's Nevus Melanocytes Remain Alive after Treatment with Q-Switched Alexandrite Laser

352 Ann Dermatol Received March 2, 2016, Revised May 30, 2016, Accepted for publication June 2, 2016 Corresponding author: Weon Ju Lee, Department of Dermatology, Kyungpook National University Hospital, 130 Dongdeok-ro, Jung-gu, Daegu 41944, Korea. Tel: 82-53-420-5838, Fax: 82-53-426-0770, E-mail: weonju@knu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright

Effect of Vitamin D on the Expression of Inflammatory Biomarkers in Cultured Sebocytes Treated with Propionibacterium acnes or Ultraviolet B Irradiation.

Vol. 28, No. 5, 2016 665 Received July 15, 2015, Revised September 25, 2015, Accepted for publication October 5, 2015 Corresponding author: Weon Ju Lee, Department of Dermatology, Kyungpook National University Hospital, 130 Dongdeok-ro, Jung-gu, Daegu 41944, Korea. Tel: 82-53-420-5838, Fax: 82-53-426-0770, E-mail: weonju@knu.ac.kr This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright