Daphnane Diterpenes from Daphne genkwa Activate Nurr1 and Have a Neuroprotective Effect in an Animal Model of Parkinson's Disease.

Nurr1 is an orphan nuclear receptor that is essential for the differentiation and maintenance of dopaminergic neurons in the brain, and it is a therapeutic target for Parkinson's disease (PD). During the screening for Nurr1 activators from natural sources using cell-based assay systems, a methanol extract of the combined stems and roots of Daphne genkwa was found to activate the transcriptional function of Nurr1 at a concentration of 3 µg/mL. The active components were isolated and identified as genkwanine N (1) and yuanhuacin (2). Both compounds 1 and 2 significantly enhanced the function of Nurr1 at 0.3 µM. Nurr1-specific siRNA abolished the activity of 1 and 2, strongly suggesting that transcriptional activation by 1 and 2 occurred through the modulation of Nurr1 function. Additionally, treatment with 1 and 2 inhibited 6-hydroxydopamine (6-OHDA)-induced neuronal cell death and lipopolysaccharide (LPS)-induced neuroinflammation. Moreover, in a 6-OHDA-lesioned rat model of PD, intraperitoneal administration of 2 (0.5 mg/kg/day) for 2 weeks significantly improved behavioral deficits and reduced tyrosine hydroxylase (TH)-positive dopaminergic neuron death induced by 6-OHDA injection and had a beneficial effect on the inflammatory response in the brain. Accordingly, compounds 1 and 2, the first reported Nurr1 activators of natural origin, are potential lead compounds for the treatment of PD.

Prevention of thyroidectomy scars in Korean patients using a new combination of intralesional injection of low-dose steroid and pulsed dye laser starting within 4 weeks of suture removal.

BACKGROUND: Regulation of inflammation during the wound healing process reduces scar formation at the injury site. OBJECTIVE: To evaluate the effect of intralesional injection of low-dose steroid with pulsed dye laser on healing of early postoperative thyroidectomy scars. MATERIALS AND METHODS: Twenty Korean women with thyroidectomy scars were enrolled. All were treated with an intralesional injection of low-dose steroid (2 mg/mL) and 595-nm pulsed dye laser starting within 4 weeks of suture removal. The Vancouver Scar Scale (VSS), Global Assessment Score (GAS), and Patient Satisfaction Score were used in this evaluation. RESULTS: Average VSS scores were significantly lower after treatment. The GAS also indicated better cosmetic outcomes after steroid injection in the laser treatment group than after laser treatment only. CONCLUSION: Early postoperative intralesional injection of low-dose steroid and pulsed dye laser treatment is effective and safe.

Clinical improvement of striae distensae in Korean patients using a combination of fractionated microneedle radiofrequency and fractional carbon dioxide laser.

BACKGROUND: Striae distensae are dermal scars with flattening and atrophy of the epidermis. OBJECTIVE: To evaluate the efficacy and safety of combination therapy with fractionated microneedle radiofrequency (RF) and fractional carbon dioxide (CO2) laser in the treatment of striae distensae. MATERIALS AND METHODS: Thirty patients (30 female; mean age 33, range 21-51, Fitzpatrick skin type IV) with moderate to severe striae distensae were enrolled in this study. Patients were divided into three groups: fractional CO2 laser only (n = 10), microneedle RF only (n = 10), and combination (n = 10). RESULTS: Improvement was evaluated using a visual analogue scale (range 1-4). Mean clinical improvement score of the dermatologist was 2.2 in the fractional CO2 laser-treated group, 1.8 in the microneedle RF-treated group, and 3.4 in the combination group. Through skin biopsy, we observed thickened epidermis and a clear increase in the number of collagen fibers in the microneedle RF- and fractional CO2 combination-treated sites. Consistent with these results, greater expression of transforming growth factor-ß1 and stratifin was observed in treated sites. CONCLUSION: Combination therapy of fractionated microneedle RF and fractional CO2 laser is a safe treatment protocol with a positive therapeutic effect on striae distensae.

Tinea incognito in Korea and its risk factors: nine-year multicenter survey.

Tinea incognito (TI) is a dermatophytic infection which has lost its typical clinical appearance because of improper use of steroids or calcineurin inhibitors. The incidence of TI is increasing nowadays. We conducted retrospective review on 283 patients with TI from 25 dermatology training hospitals in Korea from 2002-2010 to investigate the demographical, clinical, and mycological characteristics of TI, and to determine the associated risk factors. More than half (59.3%) patients were previously treated by non-dermatologists or self-treated. The mean duration of TI was 15.0 ± 25.3 months. The most common clinical manifestations were eczema-like lesion, psoriasis-like, and lupus erythematosus-like lesion. The trunk and face were frequently involved, and 91 patients (32.2%) also had coexisting fungal infections. Among 67 isolated strains, Trichophyton rubrum was the most frequently detected (73.1%). This is the largest study of TI reported to date and the first investigational report concerning TI in Korea. We suggest that doctors should consider TI when a patient has intractable eczema-like lesions accompanied by tinea pedis/unguium. Furthermore, there should be a policy change, which would make over-the-counter high-potency topical steroids less accessible in some countries, including Korea.

Roles of SEA-expressing Staphylococcus aureus, isolated from an atopic dermatitis patient, on expressions of human beta-defensin-2 and inflammatory cytokines in HaCaT cells.

Atopic dermatitis (AD) shows an increased susceptibility to Staphylococcus aureus infection partly due to decreased expression of human beta-defensin-2 (HBD-2). Interestingly, it was reported that the nasal carrier S. aureus down-regulates the expression of HBD-2 and -3, thereby the carrier strains of S. aureus retain an advantage to epithelial colonization and infection. In this study, we tried to isolate and characterize S. aureus from an AD patient, with recurrent oozing on his face. We studied the increased expression of inflammatory cytokines, such as IL-1beta, -6, -8, and TNF-alpha in S. aureus treated-HaCaT cells, which are mediated by secreting superantigens (SAgs), structural component, or both. In addition, we investigated whether the SAgs from S. aureus can down-regulate the expression of HBD-2 in HaCaT cells making favorable conditions for colonization on skin. Our data showed that the isolated S. aureus has the exotoxin gene, sea exotoxin. The SEA producing-S. aureus induced the expression of IL-1beta, -6, -8 cytokines, and TNF-alpha in HaCaT cells. The expression of HBD-2 was increased in S. aureus-treated HaCaT cells. Furthermore IL-8 was also induced by the structure component of S. aureus. Taken together, the SEA producing S. aureus induced the up-regulation of pro-inflammatory cytokines as well as HBD-2, thereby resulting in induction of the persistent eczematous skin lesions in AD. Thus, our data may give insight into understanding the pathogenesis by which S. aureus induces and aggravates eczematous skin lesions in AD.

Type VII collagen gene mutations (c.8569G>T and c.4879G>A) result in the moderately severe phenotype of recessive dystrophic epidermolysis bullosa in a Korean patient.

Dystrophic epidermolysis bullosa (DEB) are caused by mutations in the COL7A1 gene, which encodes type VII collagen. Even though more than 500 different COL7A1 mutations have been identified in DEB, it still remains to be under-investigated. To investigate the mutation of COL7A1 in moderately severe phenotype of recessive DEB (RDEB) in a Korean patient, the mutation detection strategy was consisted of polymerase chain reaction (PCR) amplification of genomic DNA, followed by heteroduplex analysis, nucleotide sequencing of the PCR products demonstrating altered mobility. In this study, we found that one mutation (c.8569G>T) was detected within exon 116. The mutation of c.8569G>T in exon 116 changed the GAG (Glu) to TAG, eventually resulted in premature termination of type VII collagen polypeptide. Furthermore the mother did not have the mutation c.8569G>T in exon 116. The other novel mutation (c.4879G>A) was detected within exon 51 of both patient and mother, thereby resulting in changing valine (Val) to isoleucine (Ile) in type VII collagen polypeptide. Taken together, in this study we identified compound heterozygosity for COL7A1 mutations (c.8569G>T and c.4879G>A) in moderately severe RDEB in a Korean patient. We hope that this data contribute to the expanding database on COL7A1 mutations in DEB.

AP-1 transcription factor decoy reduces the TGF-beta1-induced cell growth in scleroderma fibroblasts through inhibition of cyclin E.

The transforming growth factor-beta (TGF-beta) signaling pathway plays a key role in the abnormal accumulation of type I and III collagen of scleroderma. Activator protein-1 (AP-1) is a key regulatory protein in TGF-beta1-induced type I collagen synthesis. However, it is largely unknown whether AP-1 is involved in the cell proliferation of fibroblasts in scleroderma. In this study, we investigated the effects of the AP-1 oligo-deoxynucleotide (ODN) decoy on TGF-beta1-induced cell growth in scleroderma fibroblasts. To investigate the inhibition of AP-1 ODN decoy on the growth rates of scleroderma fibroblasts through the regulation of cell cycle regulatory proteins, we transfected the AP-1 ODN decoy on scleroderma fibroblasts and analyzed the cell cycle regulatory proteins by RT-PCR and Western blot analysis. We found that the growth rates of normal fibroblasts and scleroderma fibroblasts showed similar rates. It is noteworthy that the scleroderma fibroblasts grew more rapidly than normal fibroblasts in the presence of TGF-beta1. Moreover, the transfection of AP-1 decoy ODN into scleroderma fibroblasts resulted in the down-regulation of the growth rates by the down-regulation of cyclin E. These results collectively suggest that AP-1 ODN decoy can down-regulate the growth rates of scleroderma fibroblasts, thus implying that AP-1 ODN decoy is a promising therapeutic tool for overcoming scleroderma.

Modification of gene expression by melatonin in UVB-irradiated HaCaT keratinocyte cell lines using a cDNA microarray.

Excessive ultraviolet B (UVB) irradiation causes apoptotic cell death or induction of tumors in skin. Melatonin is a promising antioxidant and direct radical scavenger. Recently, it was reported that melatonin increases the survival of ultraviolet-B (UVB)-irradiated HaCaT keratinocyte cell lines. However, the precise molecular mechanisms underlying protective effect of melatonin on UVB damage are largely unknown. In this study, to gain more insight into the molecular mechanisms involved in melatonin-induced cell survival on UVB-irradiated HaCaT keratinocytes, we performed cDNA microarray analysis. HaCaT keratinocytes were incubated without or with melatonin at 100 nm for 30 min prior to UVB irradiation at 100 mJ/cm(2), and total RNA was isolated. Our data showed that the expression of apoptosis regulator genes (apoptosis related protein-3, apoptotic chromatin condensation inducer in the nucleus), cancer related genes (tumor suppressor deleted in oral cancer-related 1), cell cycle regulator (cyclin-dependent kinase 2 interacting protein), enzymes (glutathione peroxidase 1, ubiquitin-conjugating enzyme E2M), and signal transducer genes [fibroblast growth factor (acidic) intracellular binding protein, transforming growth factor beta-stimulated protein TSC-22] were decreased by melatonin treatment in the UVB-irradiated HaCaT keratinocyte cell lines, compared to that of UVB-irradiated HaCaT cells without melatonin. Thus, findings of the present study demonstrate that melatonin modulates the expression of apoptosis related genes in UVB-irradiated HaCaT cells, resulting in increasing cell survival, thereby suggesting that melatonin may be used as a promising sunscreen substance to reduce cell death of keratinocytes after excessive UVB irradiation.

Curcumin attenuates the expression of IL-1beta, IL-6, and TNF-alpha as well as cyclin E in TNF-alpha-treated HaCaT cells; NF-kappaB and MAPKs as potential upstream targets.

TNF-alpha induces some proinflammatory cytokines including IL-1beta, IL-6, IL-8, and itself by activation of NF-kappaB or MAPKs (p38, JNK, ERK). These cytokines play important roles in various inflammatory skin diseases, such as psoriasis. Recently it was also reported that expression of cyclin E is up-regulated by ERK pathway after TNF-alpha treatment. However, it was unknown whether curcumin, showing inhibitory effects on NF-kappaB and MAPKs, attenuates the expression of TNF-alpha-induced IL-1beta, IL-6, IL-8, and TNF-alpha as well as cyclin E expression in HaCaT cells. In this study, we investigated the inhibitory effect of curcumin on expression of proinflammatory cytokines and cyclin E in TNF-alpha-treated HaCaT cells. We found that curcumin inhibited the expression of TNF-alpha-induced IL-1beta, IL-6, and TNF-alpha, but not IL-8, in TNF-alpha-treated HaCaT cells as well as the TNF-alpha-induced cyclin E expression. In addition, curcumin inhibited the activation of MAPKs (JNK, p38 MAPK, and ERK) and NF-kappaB in TNF-alpha-treated HaCaT cells. Taken together, curcumin exerts anti-inflammatory and growth inhibitory effects in TNF-alpha-treated HaCaT cells through inhibition of NF-kappaB and MAPK pathways.

Profiling analysis of protein tyrosine phosphatases during neuronal differentiation.

During neuronal differentiation, it is generally accepted that many kinases and phosphatases fulfill different roles. In this study, phospho-tyrosine phosphatases were focused on and their expression profiling was evaluated during neuronal differentiation of mouse J1 embryonic stem cells. Among 83 phospho-tyrosine phosphatases, expressions of 21 PTPs were increased but mRNA expressions of 10 PTPs decreased depending on the differentiation. We checked the protein expression patterns for the cases where PTPs mRNA expressions changed. Some of them showed consistent results with the mRNA expressions. In particular, it was found that dual-specific phosphatase23 (DUSP23) affected neuronal differentiation. The knock-down of DUSP23 decreased neuronal differentiation in terms of neuronal outgrowth and the expression of neuronal marker proteins and mRNAs. Taken together, the obtained results show that many PTPs play specific roles during neuronal differentiation and manipulating their activities by activators or inhibitors could adjust neuronal differentiation.

Curcumin inhibits the expression of COX-2 in UVB-irradiated human keratinocytes (HaCaT) by inhibiting activation of AP-1: p38 MAP kinase and JNK as potential upstream targets.

Ultraviolet B (UVB) irradiation of skin induces an acute inflammation. Cyclooxygenase-2 (COX-2) protein plays key roles in acute inflammation in UVB-irradiated keratinocyte cell line HaCaT. Recently, curcumin has been regarded as a promising anti-inflammatory agent due to its ability to inhibit COX-2 expression. However, it remains largely unknown whether curcumin inhibits the UVB-induced COX-2 expression in HaCaT cells. This study was undertaken to clarify the effect of curcumin on the expression of COX-2 in UVB- irradiated HaCaT cells and further determined the molecular mechanisms associated with this process. In this study, we have found that the expression of COX-2 mRNA and protein were up-regulated in UVB-irradiated HaCaT cells in a dose- and time-dependent manner. Interestingly, treatment with curcumin strongly inhibited COX-2 mRNA and protein expressions in UVB-irradiated HaCaT cells. Notably, there was effective inhibition by curcumin on UVB-induced activations of p38 MAPK and JNK in HaCaT cells. The DNA binding activity of AP-1 transcription factor was also markedly decreased with curcumin treatment in UVB-irradiated HaCaT cells. These results collectively suggest that curcumin may inhibit COX- 2 expression by suppressing p38 MAPK and JNK activities in UVB-irradiated HaCaT cells. We propose that curcumin may be applied as an effective and novel sunscreen drug for the protection of photoinflammation.

Melatonin reduces ultraviolet-B induced cell damages and polyamine levels in human skin fibroblasts in culture.

UV radiation is known to cause photoaging of the skin and is considered one of the leading cause of developing skin carcinogenesis. Melatonin which has a highly lipophilic molecular structure facilitating penetration of cell membranes and serving as an extra- and intracellular free radical scavenger has been demonstrated to protect photodamage of skin affected by UV exposure. In this study, we have examined the role of melatonin in response to UVB induced photodamaging process, using human skin fibroblasts in vitro. Cell survival curves after UVB irradiation showed dose-dependent decrease. Only 60% of fibroblasts were survived at 140 mJ/cm(2) UVB irradiation. By pre-cultivation of cells with melatonin (100 nM), a significant number of cells remained unaffected. After UVB irradiation with 70 mJ/cm(2), the level of putrescine was 1.7+/-0.3 fold increased compared to melatonin pre-treated group. In Northern analyses, the transcriptional level of ornithine decarboxylase (ODC) gene expression was increased by UVB irradiation and prohibited by melatonin. These results indicated that melatonin was effectively able to neutralize membrane peroxidation when present in relevant concentration during UVB irradiation and diminishes the UVB-induced increase of polyamine synthesis and ODC gene expression. Collectively, ODC response to UVB induced changes are possibly involves a melatonin or antioxidant sensitive regulatory pathway in normal human skin fibroblast.

Weber-Cockayne Type Epidermolysis Bullosa Simplex Resulting from a Novel Mutation (c. 608T>C) in the Keratin 5 Gene.

Epidermolysis bullosa simplex (EBS), an inherited genetic disorder, is most often caused by a dominant-negative mutation in either the keratin 5 (KRT5) or the keratin 14 (KRT14) gene. These keratin mutants result in a weakened cytoskeleton and cause extensive cytolysis. It is important to analyze the KRT5 or KRT14 genes of the patient and their family members by mutational analysis in order to identify genetic defects as well as the need for genetic counseling. In this study, we present a 5-year-old Korean boy who had been developing blisters and erosions on the palms of his hands and soles of his feet since infancy. In addition, while his younger sister and father showed similar clinical manifestation, his mother did not. The patient was diagnosed with EBS based on clinical manifestation, which is characterized by the presence of blisters restricted to the palms and soles, histological findings, and mutational analysis. Mutational analysis of the patient's DNA revealed a thymine-to-cytosine transition at codon 608 in the KRT-5 gene, resulting in a leucine-to-proline substitution in the keratin 5 protein. The same mutation was identified in the paternal, but not maternal, DNA. Here, we report a case of Weber-Cockayne type EBS with vesicles and bullae restricted to the palms and soles with a novel, paternally inherited mutation in KRT5 gene (exon2, c.608T>C).

Downregulation of type I collagen expression in silibinin-treated human skin fibroblasts by blocking the activation of Smad2/3-dependent signaling pathways: potential therapeutic use in the chemoprevention of keloids.

The inhibition of the Smad2/3 pathway is a key step involved in the downregulation of type I collagen synthesis, thus preventing keloid formation in tissue. In this study, we investigated the effect of silibinin on the proliferation of human skin fibroblasts (HSFs), as well as its effect on the expression of type I collagen, matrix metalloproteinase (MMP)-1, Smad2 and Smad3. Our results showed that the proliferation rates of the fibroblasts were not markedly decreased in a dose- and time-dependent manner following treatment with silibinin. Even though silibinin did not exert any cytotoxic effects on HSFs, the expression of type I collagen was markedly decreased in a dose- and time-dependent manner in the silibinin-treated HSFs. Consistent with this finding, the decreased promoter activity of type I collagen was observed in the HSFs following treatment with silibinin. The MMP-1 and MMP-2 expression levels were increased in the silibinin-treated HSFs. Moreover, the silibinin-induced downregulation of type I collagen was associated with the inhibition of Smad2/3 activation in the transforming growth factor­ß1 (TGF-ß1)-treated HSFs. We further demonstrated that silibinin attenuated the translocation of Smad2 and Smad3 to the nucleus in the TGF-ß1-treated HSFs. Taken together, our data indicate that silibinin has the potential to prevent fibrotic skin changes by inducing the downregulation of type I collagen expression; this effect was partly mediated by the inhibition of the Smad2/3-dependent signaling pathway in HSFs.

Photodynamic therapy combined with cryotherapy for the treatment of nodular basal cell carcinoma.

Carbon dioxide (CO2) laser ablation in combination with photodynamic therapy (PDT) has previously been successfully used to treat superficial basal cell carcinoma (BCC). However, the efficacy of this treatment modality is limited in the treatment of deeper lesions and the more aggressive subtypes of BCC. In order to improve the outcome of PDT, 8 BCC lesions of variable depths (4 lesions ≤2 mm and 4 lesions >2 mm) and subtypes (1 superficial, 6 nodular and 1 infiltrative) were treated with CO2 laser ablation in combination with PDT, followed by modified cryotherapy. The mean number of treatment sessions was 1.5 and the follow-up period was 22 months. All of the patients demonstrated a complete response and no recurrence of disease, while the majority of patients were satisfied with the cosmetic results upon follow-up examination. The combination therapy of CO2 laser ablation with PDT followed by modified cryotherapy demonstrated a good efficacy and satisfactory cosmetic outcomes in the treatment of nodular BCC.

Application of platelet-rich plasma accelerates the wound healing process in acute and chronic ulcers through rapid migration and upregulation of cyclin A and CDK4 in HaCaT cells.

Application of autologous platelet-rich plasma (PRP) has been used for chronic wound healing. The aim of this study was to evaluate the effect of PRP on the wound healing processes of both acute and chronic ulcers and the underlying molecular mechanisms involved. We treated 16 patients affected by various acute and chronic ulcers with PRP. We performed molecular studies of cell proliferation, migration assays, immunoblotting and chloramphenicol acetyltransferase (CAT) assays in PRP-treated HaCaT keratinocyte cells. PRP treatment induced increased rates of cell proliferation and cell migration of HaCaT cells. In addition, the expression of cyclin A and cyclin dependent kinase (CDK) 4 proteins was markedly increased with a low concentration (0.5%) of PRP treatment in HaCaT cells. In 11 patients with chronic ulcers, including stasis ulcers, diabetic ulcers, venous leg ulcers, livedoid vasculitis, claw foot and traumatic ulcers, 9 patients showed 90-100% epithelization after 15.18 days. In 5 patients with acute ulcers, such as dehiscence, open wound and burn wound, 80-100% epithelization was achieved between 4 to 20 days. Topical application of PRP to acute and chronic skin ulcers significantly accelerated the epithelization process, likely through upregulation of the cell cycle regulatory proteins cyclin A and CDK4.

Platelet-rich plasma induces increased expression of G1 cell cycle regulators, type I collagen, and matrix metalloproteinase-1 in human skin fibroblasts.

Platelet-rich plasma (PRP) is derived from fresh whole blood, which contains a high concentration of platelets. Recently, PRP has been used for skin wound healing and rejuvenation. However, the molecular mechanisms underlying PRP-inducing wound healing processes are still largely unknown. The aim of this study is to evaluate the effect of PRP on the expression of G1 cell cycle regulatory proteins, type I collagen, matrix metalloproteinase-1 (MMP-1), and MMP-2 in human skin fibroblasts (HSF). We performed a cell proliferation and a migration assay, immunoblotting, and a chloramphenicol acetyltransferase (CAT) assay in PRP-treated human skin fibroblasts. PRP treatment induced increased rates of cell proliferation and cell migration. Expression of cyclin A protein was increased by a low concentration (0.5%) of PRP-treated HSF. In addition, expression of Rb, cyclin E, and cyclin-dependent kinase 4 proteins was increased by a high concentration (5%) of PRP-treated HSF. High concentration of PRP induced an up-regulation of type I collagen, MMP-1, and MMP-2 expression in HSF. Taken together, PRP treatment induced an increase in expression of G1 cell cycle regulators, type I collagen and MMP-1, thereby accelerating the wound healing process.

Pityriasis versicolor on penile shaft in a renal transplant recipient.

Pityriasis versicolor is a superficial infection of the stratum corneum, which is caused by the Malassezia species. Tge Malassezia species consist of 12 subspecies, including M. furfur, M. pachydermatis, M. symphodialis and M. globasa. The Malassezia species are classified as a normal flora, particularly in the sebum rich areas of the skin, and they convert from saprophytic yeast to parasitic mycelial morpholgic form to cause clinical disease. But majorities of their distributions are in the upper back, the neck, the thighs, and the forearm, and not in the penis. It is well known that the renal transplant patients, who take immunosuppressive agents, have impairment in the protective cell mediated immunity. Thus, they are more susceptible to infectious diseases, such as a fungal infection. Therefore, clinical manifestations show higher incidence of disease, but they mostly occur in an expected distribution. We here report a case of pityriasis versicolor in a renal transplant recipient on penile shaft, which is an unusual area.