Expression Of Caspase-14 Research Articles

Role of syntaxin3 an apical polarity protein in poorly polarized keratinocytes: regulation of asymmetric barrier formations in the skin epidermis.

The skin epidermis exhibits an asymmetric structure composed of multilayered keratinocytes and those in the outer layers form two-way physical barriers, cornified cell envelope (CCE), and tight junctions (TJs). While undifferentiated keratinocytes in the basal layer continuously deliver daughter cells outward, which undergo successive differentiation with losing their polarized characteristics, they retain the expression of several polarity proteins. In the present study, we revealed that the t-SNARE protein syntaxin3, a critical element for the formation of the apical compartment in simple epithelial cells, is required to confer the ability to organize the physical barriers on "poorly polarized" keratinocytes in epidermal outer layers. HaCaT keratinocytes with genetic ablation of syntaxin3 readily succumbed to hydrogen peroxide-induced cell death. Additionally, they lost the ability to organize TJ and CCE structures, accompanied by notable downregulation of transglutaminase1 and caspase14 (a cornification regulator) expression. These syntaxin3-knockout cells appeared to restore oxidative stress tolerance and functional TJ formation ability, in response to the inducible re-expression of exogenous syntaxin3. While plausible mechanisms underlying these phenomena remain unclear, syntaxin3, an apical polarity protein in the simple epithelia, has emerged as a potentially crucial element for barrier formation in poorly polarized keratinocytes in polarized epidermal tissue.

Galactomyces Ferment Filtrate Potentiates an Anti-Inflammaging System in Keratinocytes.

Skincare products play a crucial role in preventing the dry skin induced by various causes. Certain ingredients can help to improve the efficacy of skincare products. Galactomyces ferment filtrate (GFF) is such a functional ingredient. Its use originated from the empirical observation that the hands of sake brewers who deal with yeast fermentation retain a beautiful and youthful appearance. Consequently, skincare products based on GFF are widely used throughout the world. Recent studies have demonstrated that GFF activates an aryl hydrocarbon receptor (AHR) and upregulates the expression of filaggrin, a pivotal endogenous source of natural moisturizing factors, in epidermal keratinocytes. It also activates nuclear factor erythroid-2-related factor 2 (NRF2), the antioxidative master transcription factor, and exhibits potent antioxidative activity against oxidative stress induced by ultraviolet irradiation and proinflammatory cytokines, which also accelerate inflammaging. GFF-mediated NRF2 activation downregulates the expression of CDKN2A, which is known to be overexpressed in senescent keratinocytes. Moreover, GFF enhances epidermal terminal differentiation by upregulating the expression of caspase-14, claudin-1, and claudin-4. It also promotes the synthesis of the antiinflammatory cytokine IL-37 and downregulates the expression of proallergic cytokine IL-33 in keratinocytes. In addition, GFF downregulates the expression of the CXCL14 and IL6R genes, which are involved in inflammaging. These beneficial properties might underpin the potent barrier-protecting and anti-inflammaging effects of GFF-containing skin formulae.

Pep27 Mutant Immunization Inhibits Caspase-14 Expression to Alleviate Inflammatory Bowel Disease via Treg Upregulation.

Inflammatory bowel disease (IBD) is a highly prevalent gut inflammatory disorder. Complicated clinical outcomes prolong the use of conventional therapy and often lead to compromised immunity followed by adverse events and high relapse rates. Thus, a profound medical intervention is required. Previously, intranasal immunization of pneumococcal pep27 mutant (Δpep27) exhibited long-lasting protection against immune-related disorders. System biology analysis has predicted an inverse correlation between Δpep27 immunization and gastroenteritis. Recently, we established that Δpep27-elicited Tregs repressed Wnt5a expression and enhanced barrier integrity, suggesting the restoration of immunological tolerance. Therefore, we evaluated whether Δpep27 can alleviate IBD. Δpep27 dose-dependent response was analyzed in dextran sulfate sodium-induced mice using transcriptome analysis. Pro- and anti-inflammatory signatures were cross-correlated by quantitative PCR and western blot analyses. To address the hierarchy regulating the activity of caspase-14, an undefined marker in IBD, and regulatory T cells (Tregs), antibody-based neutralization studies were conducted. Fecal microbiome profiles were analyzed by 16S rRNA pyrosequencing. Δpep27 significantly attenuated dextran sulfate sodium-induced oxidative stress parameters, proinflammatory cytokines, caspase-14 expression level, and upregulated tight junction, anti-inflammatory genes IL-10 and TGF-β1 via upregulation of Tregs to restore healthy gut microbiota. Neutralization studies unveiled that ∆pep27 had a remedial effect via Treg upregulation. Caspase-14, being an important mediator in the pathogenesis of IBD, can be an alternate therapeutic target in IBD. ∆pep27-increased Tregs repressed caspase-14 expression and reversed gut microbial dysbiosis, aiding to re-establish immunological tolerance.

Peculiarities of epidermal proliferation and terminal differentiation in various histological types of seborrheic keratosis

Aim of the study is to determine the peculiarities of epidermal proliferation and terminal differentiation in various histological types of seborrheic keratosis (SK). Materials and methods. Pathomorphological and immunohistochemical analysis was performed on the skin biopsy material of 60 patients with SK, who constituted the study group, and on the material of 30 healthy skin samples, which were considered a control group. Results. Level of expression of Ki-67 marker in papillomatous and acanthotic types of SK is significantly lower (P < 0.005) compared to healthy skin samples, which indicates a low proliferative potential of tumors. While the expression of the marker of terminal differentiation between these groups of SK and healthy skin is significantly higher (P < 0.005). Comparative characterization of the association of the expression degree of the immunohistochemical marker of proliferation Ki-67 depending on the activity of terminal differentiation processes in acanthotic and papillomatous types of SK indicates a statistically significant inverse correlation of these pathogenetically determined processes. Thus, activation of terminal differentiation with increase of caspase-14 expression level inhibits proliferation in basal epidermal layers that is accompanied by decrease of Ki-67 level. Conclusions. Level of expression of Ki-67 as a marker of proliferative activity in patients with acanthotic and papillomatous types is significantly lower than in the control group, which may indicate no tendency to malignancy. The expression level of caspase-14 in acanthotic and papillomatous types is increased compared to healthy skin, which may indicate a violation of keratinization, which occurs during the terminal differentiation of epidermocytes. Statistical inverse conjugacy between caspase-14 and Ki-67 indicates inhibition of proliferation against the background of increased terminal differentiation activity and may justify the vectors of therapeutic action of topical agents.

Efficacy of Dendrobium candidum polysaccharide extract as a moisturizer.

This study investigated the role of natural polymers as moisturizers with low toxicity and biodegradability in the cosmetic and pharmaceutical industries. We isolated a polysaccharide extract from Dendrobium candidum (D.candidum) and determined its efficacy in skin hydration when used as an active cosmetic ingredient. The molecular weight distribution of D.candidum polysaccharides was analyzed via gel permeation chromatography (GPC). We performed real-time reverse transcription PCR (RT-PCR) and western blotting assays to investigate the physiological mechanism of the polysaccharides extracted from D.candidum (PDC). Based on in vitro data, the efficacy of PDC in improving skin condition was tested on the face of 21 volunteers. The expression of filaggrin (FLG), caspase-14, and bleomycin hydrolase, which are the major components contributing to skin hydration, was significantly increased in the PDC-treated group. Further, the PDC upregulated the mRNA expression of occludin and claudin-1, which play a key role in epidermal barrier function. In addition, a topical application of PDC markedly increased skin hydration and improved trans-epidermal water loss (TEWL) and skin elasticity after 2weeks. It is the first study reporting the efficacy of PDC-mediated FLG mechanism associated with positive skin hydration. PDC can be used as an active ingredient in moisturizers. Long-term application of PDC-based moisturizers may result in significant improvement in elasticity and barrier function.

28140 How a lotion with prebiotic yeast extract regulates the environmental aggressors impact on the skin microbiome

Environmental aggressors cause skin damage and have been conjectured to modulate the skin microbiome. An 8-week clinical study (N = 100) and an ex vivo pollution simulation study were conducted to explore the profile of consumer lifestyle-based aggressor exposure and its impact on skin. The application of a topical lotion with prebiotic yeast extract was tested for the ability to perform as a preventive and restorative solution. Clinical study results indicated that continuous light pollution exposure (PM2.5 <100) significantly correlated with higher TEWL and lower moisture. Ex vivo pollution simulation study showed a decreased expression of caspase-14, a skin barrier biomarker, further indicating that the skin barrier becomes less resilient in harsh city environment. Although the microbiome diversity on healthy subjects’ skin was stable, the harsh city environment resulted in a significantly lower richness (Chao1) compared with baseline, indicating a change in low abundant species. As a solution to the change in microbiome richness, the application of topical lotion with prebiotic yeast extract over 8 weeks can boost growth of skin commensal bacteria and maintain microbiome richness. In addition, dominant bacterium counts were significantly increased, with 10 more dominant bacteria including a detox bacterium that metabolizes polycyclic aromatic hydrocarbons (PAHs), a major pollutant. In conclusion, an individual’s residential environment and lifestyle have a significant impact on the skin microenvironment. The use of a topical lotion with prebiotic yeast extract regulate skin microbiome balance by boosting the growth of beneficial bacteria, thereby preventing and restoring the impact of daily aggressors on the skin.

A Novel Multi-Action Emollient Plus Cream Improves Skin Barrier Function in Patients with Atopic Dermatitis: In vitro and Clinical Evidence

Background: Emollients capable of restoring the skin barrier function would extend their role beyond basic maintenance therapy in atopic dermatitis (AD). Objectives: Investigate the effect of a novel emollient plus cream (EC; Dermoflan®) on the skin barrier in vitro and in patients with mild-to-moderate AD. Methods: The effect of EC on the skin barrier recovery was evaluated using a tape-stripping (TS) model. After TS, organ cultures were treated with EC (undiluted or diluted 1:1 with water) and analyzed at 18–120 h using hematoxylin and eosin, Oil Red O, immunohistochemical, and immunofluorescent techniques. In a double-blind, randomized study, EC or placebo was applied once daily for 2 months to antecubital folds of the upper and lower limbs of patients with mild-to-moderate AD in clinical remission. Epidermal thickness, vascularization, and epidermal hydration were assessed by optical coherence tomography and corneometry, respectively, at baseline, and 1 and 2 months following treatment initiation. Results: Following TS, EC treatment significantly increased epidermal thickness and lipid content versus diluent in the skin organ culture, as well as claudin-1, involucrin, and caspase-14 expression, suggesting skin barrier repair. EC treatment also decreased keratin-16 expression and increased levels of Toll-like receptors 1 and 2 versus diluent, suggesting involvement in regulating the epidermal immune response. In 20 patients randomized 1:1 to EC or placebo, EC treatment at the elbow fold/popliteal fossa significantly decreased epidermal thickness after 2 months, and the number of blood vessels at the elbow fold after 1 and 2 months, versus placebo. EC significantly improved the skin hydration after 2 months versus baseline. Conclusions: This novel multi-action EC may help to restore epidermal homeostasis and improve the skin of patients with AD. Results indicate that this novel multi-action EC could be a valid adjuvant therapy in patients with AD. Key Message: Novel multi-action emollient cream helps to restore epidermal homeostasis and improves the skin affected by AD.

In Vitro Effects of Dehydrotrametenolic Acid on Skin Barrier Function.

Dehydrotrametenolic acid (DTA) is a lanostane-type triterpene acid isolated from Poria cocos Wolf (Polyporaceae). Several studies have reported the anti-inflammatory and antidiabetic effects of DTA; however, its effects on the skin are poorly understood. In this study, we investigated the effects of DTA on skin barrier function in vitro and its regulatory mechanism in human keratinocyte cell line HaCaT cells. DTA increased the microRNA (mRNA) expression of natural moisturizing factor-related genes, such as HAS-2, HAS-3, and AQP3 in HaCaT cells. DTA also upregulated the mRNA expression of various keratinocyte differentiation markers, including TGM-1, involucrin, and caspase-14. Moreover, the protein expression of HAS-2, HAS-3, and TGM-2 were significantly increased by DTA. To examine the regulatory mechanisms of DTA, Western blotting, luciferase-reporter assays, and RT-PCR were conducted. The phosphorylation of mitogen-activated protein kinases (MAPKs) and IκBα were increased in DTA-treated HaCaT cells. In addition, AP-1 and NF-κB transcriptional factors were dose-dependently activated by DTA. Taken together, our in vitro mechanism studies indicate that the regulatory effects of DTA on skin hydration and keratinocyte differentiation are mediated by the MAPK/AP-1 and IκBα/NF-κB pathways. In addition, DTA could be a promising ingredient in cosmetics for moisturizing and increased skin barrier function.

Expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis and effect of ultraviolet B radiation on its mRNA and protein expression in HaCaT cells

Objective To determine the expression of caspase-14 in skin lesions of patients with chronic actinic dermatitis (CAD) , and to explore the effect of ultraviolet B (UVB) radiation on its mRNA and protein expression in HaCaT cells. Methods In 2016, skin samples were collected from lesions of 10 patients with CAD (test group), 10 patients with eczema (positive control group) and from normal skin of 10 healthy controls after cosmetic surgery (negative control group) in the Department of Dermatology, First Affiliated Hospital of Kunming Medical University. Immunohistochemical staining was performed to determine the expression of caspase-14 in the normal skin, CAD and eczema lesions. Cultured HaCaT cells were divided into several groups: UVB groups irradiated with 0, 30, 60, 90 mJ/cm2 UVB separately, and 5-AzaC groups irradiated with 0, 30, 60, 90 mJ/cm2 UVB separately followed by the treatment with the methylase inhibitor 5-AzaC for 24 hours. Then, the cells were collected, and real-time fluorescence-based quantitative PCR (RT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of caspase-14 respectively in HaCaT cells in the UVB groups and 5-AzaC groups. Statistical analysis was carried out with SPSS22.0 software by using chi-square test for the comparison of rates, and t test and two-factor analysis of variance for the comparison of means. Results In the CAD and eczema lesions, caspase-14 was mainly expressed in the spinous and granular layers, but not in the stratum corneum. However, caspase-14 was markedly expressed in the stratum corneum of the normal skin tissues. Of the 10 CAD samples, 5 were positive for caspase-14, and 9 of 10 normal skin samples were positive for caspase-14. The positive rate of caspase-14 significantly differed between the two above groups (χ2=7.30, P < 0.05) . RT-PCR and Western blot analysis showed significant changes in the mRNA and protein expression of caspase-14 in HaCaT cells after irradiation with different doses of UVB (F=87.54, 23.46, both P < 0.05) , which showed a decreasing trend along with the increase in the dose of UVB. After exposure to 0, 30, 60 and 90 mJ/cm2 UVB, the mRNA and protein expression of caspase-14 was significantly higher in the 5-AzaC groups than in the UVB groups (all P < 0.05) . Conclusions In CAD lesions, the expression of caspase-14 markedly decreased, and was absent in the stratum corneum. UVB radiation can down-regulate the mRNA and protein expression of caspase-14 in HaCaT cells. Key words: Photosensitivity disorders; Eczema; Ultraviolet rays; Caspase 14; Keratinocytes; Chronic actinic dermatitis

Impaired Skin Barrier Function and Downregulated Expression of Caspase-14 in Moderate to Severe Chronic Hand Eczema

Aim: To investigate whether the skin barrier function is impaired with regard to the pH value, water content, transepidermal water loss (TEWL), and the integrity of the stratum corneum, and whether the expression of caspase-14 is altered in moderate to severe chronic hand eczema (CHE). Methods: Thirty patients with moderate to severe CHE treated at our institute and 30 healthy volunteers were included in this study. The pH value, water content, TEWL, and the integrity of the stratum corneum were measured in all subjects. Results: Significantly increased pH value, decreased water content, elevated TEWL, and impaired integrity of the stratum corneum were observed in the lesional skin of CHE patients compared with the nonlesional skin of CHE patients and the normal skin of healthy volunteers. The expression of caspase-14 decreased in the lesional and nonlesional skin of CHE patients compared with the normal skin of healthy volunteers, especially prominent in the nonlesional skin. The mean optical density (OD) value of immunohistochemical staining for caspase-14 was significantly lower in the nonlesional skin than in the lesional skin and normal skin (p < 0.01 for both). Although the mean OD value was lower in the lesional skin than in the normal skin, the difference was not statistically significant (p > 0.05). Conclusion: Skin barrier dysfunction indeed occurs in CHE patients, which may be related to mechanisms associated with a downregulated expression of caspase-14.

Brasilianoids A-F, New Meroterpenoids From the Sponge-Associated Fungus Penicillium brasilianum.

3,5-Dimethylorsellinic acid (DMOA) derived meroterpenoids comprise an unique class of natural products with diverse scaffolds and with a broad spectrum of bioactivities. Bioinformatics analysis of the gene clusters in association with the qRT-PCR detection of the amplification of two key genes led to speculate that the sponge associated fungus Penicillium brasilianum WZXY-m122-9 is a potential producer of meroterpenoids. Chromatographic separation of the EtOAc extract of this fungal strain on a large-scale fermentation resulted in the isolation of six new DMOA-related meroterpenoids with trivial names of brasilianoids A–F (1-6), together with preaustinoid D and preaustinoid A2. The structures were determined by extensive analyses of spectroscopic data, including the X-ray diffraction and the ECD data for configurational assignment. Brasilianoids A and F showed an unprecedented skeleton with a γ-lactone in ring A, while brasilianoids B–C featured a 7/6/6/5/5 pentacyclic ring system finding in nature for the first time. The biosynthetic relationship among the isolated compounds was postulated. Compound 1 significantly stimulated the expression of filaggrin and caspase-14 in HaCaT cells in dose-dependent manner, while compounds 2 and 3 showed moderate inhibition against NO production in LPS-induced RAW 264.7 macrophages.

Chitosan-based nanoformulated (-)-epigallocatechin-3-gallate (EGCG) modulates human keratinocyte-induced responses and alleviates imiquimod-induced murine psoriasiform dermatitis.

BackgroundPsoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation, and inflammation, leading to disrupted skin barrier function. The use of natural agents that can abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG) mitigated inflammation and increased the expression of caspase-14 while promoting epidermal differentiation and cornification. However, bioavailability issues have restricted the development of EGCG for the treatment of psoriasis.Materials and methodsTo overcome these limitations, we employed a chitosan-based polymeric nanoparticle formulation of EGCG (CHI-EGCG-NPs, hereafter termed nanoEGCG) suitable for topical delivery for treating psoriasis. We investigated and compared the efficacy of nanoEGCG versus native or free EGCG in vitro and in an in vivo imiquimod (IMQ)-induced murine psoriasis-like dermatitis model. The in vivo relevance and efficacy of nanoEGCG formulation (48 µg/mouse) were assessed in an IMQ-induced mouse psoriasis-like skin lesion model compared to free EGCG (1 mg/mouse).ResultsLike free EGCG, nanoEGCG treatment induced differentiation, and decreased proliferation and inflammatory responses in cultured keratinocytes, but with a 4-fold dose advantage. Topically applied nanoEGCG elicited a significant (p<0.01) amelioration of psoriasiform pathological markers in IMQ-induced mouse skin lesions, including reductions in ear and skin thickness, erythema and scales, proliferation (Ki-67), infiltratory immune cells (mast cells, neutrophils, macrophages, and CD4+ T cells), and angiogenesis (CD31). We also observed increases in the protein expression of caspase-14, early (keratin-10) and late (filaggrin and loricrin) markers of differentiation, and the activator protein-1 factor (JunB). Importantly, a significant modulation of several psoriasis-related inflammatory cytokines and chemokines was observed compared to the high dose of free EGCG (p<0.05). Taken together, topically applied nanoEGCG displayed a >20-fold dose advantage over free EGCG.ConclusionBased on these observations, our nanoEGCG formulation represents a promising drug-delivery strategy for treating psoriasis and possibly other inflammatory skin diseases.

Sphingoid Base-Upregulated Caspase-14 Expression Involves MAPK.

Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Especially, sphingoid base-backbones of sphingolipids (sphingosine, sphinganine, and phytosphingosine) and their metabolites N-acyl-sphingoid bases (ceramides) are highly bioactive. In skin, one of the caspases, caspase-14, is expressed predominantly in cornifying epithelia, and caspase-14 plays an important role in keratinocyte differentiation. As ceramides were surrounding lipids in the keratinocytes and ceramides stimulate keratinocyte differentiation, we therefore examined the upregulation of caspase-14 by various sphingoid bases and ceramide. Sphingosine, sphinganine, phytosphingosine, and C2-ceramide treatment at the doses not damaging cells significantly increased caspase-14 mRNA and protein expression in dose-dependent manner on human keratinocyte HaCaT cells. These results indicated that sphingoid bases and ceramide upregulated caspase-14 mRNA to increase intracellular caspase-14 protein level. We next examined the caspase-14 upregulation mechanism by sphingoid bases. We used the most effective sphingoid base, phytosphingosine, and revealed that specific inhibitors of the mitogen-activated protein kinase, p38 and c-jun N-terminal protein kinase (JNK), blocked caspase-14 expression. This indicates that phytosphingosine upregulation of caspase-14 is involved of p38 and JNK activation. Moreover, phytosphingosine induced caspase-14 upregulation in vivo, suggesting that sphingoid bases were involved in keratinocyte differentiation by affecting caspase-14.

465 (PB-098) - Caspase14 expression is associated with triple negative phenotypes and cancer stem cell marker expression in breast cancer patients

465 (PB-098) - Caspase14 expression is associated with triple negative phenotypes and cancer stem cell marker expression in breast cancer patients

Caspase14 expression is associated with triple negative phenotypes and cancer stem cell marker expression in breast cancer patients.

The Caspase14 (CASP14) was reported that the low expression of CASP14 in ovarian cancer and colon cancer was associated with cancer progression, on the other hand, that the CASP14 expression in breast cancer was higher than that of non-cancerous tissues. The purpose of this study is to determine the clinical significance of CASP14 in breast cancer. We performed immunohistochemistry for CASP14, ER, PgR, HER2, Ki67, EGFR, CK5/6, CD44, CD24, ALDH1, claudins, and androgen receptor in 222 breast cancer patients including 55 TNBC cases, and evaluated the relationship of CASP14, above-mentioned markers, and prognosis. Using public microarray database of breast cancer, the prognostic value of CASP14 was calculated. High CASP14 expression was significantly associated with TNBC subtype (P = 0.015), nuclear grade (P = 0.006), Ki67, EGFR (P < 0.001, P = 0.016), ALDH1, CD44 and CD24 (P < 0.001, P < 0.001, P = 0.001) in 222 breast cancer cases, and the high expression of claudin1 (P = 0.017), and androgen receptor (P = 0.002) in TNBC cases was related to the high CASP14. According to the public database, survival in the high CASP14 breast cancer patients was shorter than low CASP14 patients. High CASP14 expression is a marker of breast cancer aggressiveness in association with proliferation, TNBC phenotype, and cancer stemness.

Increased filaggrin-metabolizing enzyme activity in atopic skin: a pilot study using a canine model of atopic dermatitis.

Filaggrin (FLG) and its metabolites are essential for skin barrier function and hydration of the stratum corneum. Alteration of the FLG metabolism could be the basis for an abnormal skin barrier in allergic dogs. To investigate the expression and distribution of calpain-1, caspase-14, furin and matriptase, four enzymes involved in FLG metabolism, in the skin of atopic and healthy beagles. Skin biopsies were collected from four healthy and four atopic beagles before and after allergen exposure. The dogs were challenged for three consecutive days to mimic an acute exposure, or once weekly to mimic a chronic exposure to allergens. Skin biopsies were taken on days 0 (nonlesional), 3 and 10 in the "acute" model and on days 0 (nonlesional), 14 and 28 in the "chronic" model. Four healthy dogs were used as controls. Indirect immunofluorescence was used to analyse the distribution and the expression of FLG enzymes in a semi-quantitative manner. Five consecutive pictures/section were taken and the intensity analysed tracing the epidermis and using ImageJ on the traced areas. The enzymes' expression was compared between healthy and atopic nonlesional skin (Day 0) and over time in each group. All enzymes were expressed in all layers of the epidermis. A significantly higher expression of calpain-1 (P = 0.028), caspase 14 (P = 0.028) and matriptase (P=0.028) was evident in atopic compared to control dogs on Day 0. No differences over time were seen for any enzyme analysed. This preliminary study suggests an abnormal catabolism of FLG in canine atopic skin.

Skin aging caused by intrinsic or extrinsic processes characterized with functional proteomics.

The skin provides protection against environmental stress. However, intrinsic and extrinsic aging causes significant alteration to skin structure and components, which subsequently impairs molecular characteristics and biochemical processes. Here, we have conducted an immunohistological investigation and established the proteome profiles on nude mice skin to verify the specific responses during aging caused by different factors. Our results showed that UVB-elicited aging results in upregulation of proliferating cell nuclear antigen and strong oxidative damage in DNA, whereas chronological aging abolished epidermal cell growth and increased the expression of caspase-14, as well as protein carbonylation. Network analysis indicated that the programmed skin aging activated the ubiquitin system and triggered obvious downregulation of 14-3-3 sigma, which might accelerate the loss of cell growth capacity. On the other hand, UVB stimulation enhanced inflammation and the risk of skin carcinogenesis. Collectively, functional proteomics could provide large-scale investigation of the potent proteins and molecules that play important roles in skin subjected to both intrinsic and extrinsic aging.

Epigallocatechin-3-gallate (EGCG) inhibits imiquimod-induced psoriasis-like inflammation of BALB/c mice.

BackgroundPsoriasis is a chronic inflammatory immune disease with undefined pathogenesis. It is associated with T cells, and the IL-23/IL17 axis is believed to be crucial in the pathogenesis. The present treatments have side effects that influence the compliance of patients. Tea polyphenol is extracted from tea polyphenols, and its main active ingredient is Epigallocatechin-3-gallate (EGCG), which has been shown to have antioxidant, anti-tumor, and anti-ultraviolet radiation effects. Here, we aim to report that EGCG can inhibit imiquimod (IMQ)-induced psoriasis-like inflammation.MethodsWe used BALB/c mice, which were topically treated with IMQ for 6 consecutive days, as a psoriasis mouse model. Topical application of EGCG and treatment with EGCG were conducted in the experiments. Then observed the effects of the two methods on psoriasis-like mice dermatitis. Statistics are presented as the means ± standard error of mean (SEM) and compared using unpaired two-tailed Student’s t tests or one-way ANOVA.ResultsTopical application of EGCG alleviated psoriasiform dermatitis, improved the skin pathological structure by reduce the expression of epidermal PCNA, promoted the expression of caspase-14. Treatment with EGCG attenuated skin inflammation, accompanied by reduced infiltrations of T cells; reduced percentages of CD11c+ DC in the composition of immunocytes of spleens; reduced levels of interleukin (IL)-17A, IL-17F, IL-22, IL-23 and malondialdehyde (MDA) in plasma; increased percentages of CD4+ T cells in the composition of immunocytes of spleens; and increased bioactivities of superoxide dismutase (SOD) and catalase (CAT) in plasma.ConclusionsAll the results demonstrated that EGCG had anti-inflammatory, immune regulatory and antioxidant effects. It is a promising intervention in psoriasis in the future.

536 In vitro and in vivo efficacy of nitric oxide-releasing antiviral therapeutic agents

536 In vitro and in vivo efficacy of nitric oxide-releasing antiviral therapeutic agents KA McHale, K Balogh, H Wang, S Hollenbach, N Christensen, L Chow, T Broker and N Stasko 1 Novan, Inc, Durham, NC, 2 Department of Experimental Pathology, Pennsylvania State University at Hershey Park, Hershey, PA and 3 Department of Biochemistry & Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL The efficacy of topical nitric oxide-releasing therapeutics to inhibit viral replication was demonstrated in vitro and in vivo. NVN1000 or NVN4000 drug substance, dissolved in phosphate buffered saline, was applied 1 hour/day for 6 days to organotypic cultures of primary human keratinocytes containing HPV-18 genomic replicons. Preliminary results indicate a dose responsive reduction of viral DNA copy number to less than 20% of the untreated cultures. At the highest concentrations applied (1.5 mg or 2 mg/ml), no cytotoxicity was detected, as judged from hematoxylin and eosin staining of normal or HPV-18 containing raft cultures. The NVN1000 drug substance was formulated into SB206 gel and the ability of a daily topical drug product (5 doses/week for 8 weeks) to inhibit papilloma growth in vivo was assessed in the Cottontail Rabbit Papillomavirus (CRPV) model. Dosing of SB206 gel was initiated two weeks after inoculation of CRPV viral DNA into the scarified skin sites. Topical SB206 treatment inhibited papilloma formation in a dose dependent fashion with the highest dose, 10% SB206 gel, achieving 82% inhibition compared to the Vehicle control group. Blinded histological analysis of biopsies from 10% SB206-treated animals exhibited mild hyperplasias, with an absence of viral infection as evidenced by a lack of intra-nuclear basophilic inclusions. Qualitative assessment of inflammation and quantitative cytokine gene expression was similar across all dose groups, suggesting immune activation did not account for the efficacy observed in the CRPV model and supports a direct anti-viral effect of Novan’s nitric oxide releasing therapeutic agents. SB206 Gel is currently being evaluated as a topical therapy for external genital warts in a Phase 2 clinical trial. 537 Topical application of chitosan-based nanoformulated green tea polyphenol EGCG ameliorates imiquimod-induced psoriasis-like skin lesion in mice J Chamcheu, I Siddiqui, V Adhami, S Dodwad, D Bharali, G Wood, S Mousa and H Mukhtar Dermatology, University of Wisconsin Madison, Madison, WI Psoriasis is a chronic and currently incurable inflammatory skin disease characterized by hyperproliferation, aberrant differentiation and inflammation, leading to disrupted skin barrier function. The use of natural agents that possess the ability to abrogate these effects could be useful for the treatment of psoriasis. Earlier studies have shown that treatment of keratinocytes and mouse skin with the green tea polyphenol, epigallocatechin-3-gallate (EGCG), increased the expression of caspase-14 that is involved in epidermal differentiation and cornification. However, dose and bioavailability restrict the therapeutic development and application of EGCG in the treatment of psoriasis. To overcome these limitations, we developed fully characterized, chitosan-based nanocarrier to encapsulate EGCG (hereafter termed as nanoEGCG) suitable for topical delivery. Here, we assessed the efficacy of nanoEGCG (48mg/ animal) using an IMQ-induced mouse psoriasis-like skin model and compared the effects to free EGCG (1mg/amimal). Treatment with nanoEGCG resulted in significant (p<0.01) amelioration of pathological markers of psoriasiform lesions including reduction in i) ear and skin thickness, erytherma and scales, ii) proliferation (Ki-67), iii) infiltratory immune cells (mast cells, neutrophils, macrophages and CD4+ T cells), iv) angiogenesis (CD31), and v) increase in the protein expression of caspase-14, early (K1 and k10) and late (filaggrin and loricrin) differentiation markers and AP-1 factors (JunB and cJun), and (v) modulates several psoriasis-related inflammatory cytokines and chemokines analyzed compared to high dose of free EGCG (p<0.05). Taken together, topical application of nanoEGCG was found to ameliorate IMQ-induced Balb/c mouse psoriasis-like lesions with greater than 25-fold dose advantage over free EGCG. Our observations warrant further in vivo efficacy studies of this unique nanoEGCG formulation in robust inducible and genetic models of psoriasis.

T Helper 1 and T Helper 2 Cytokines Differentially Modulate Expression of Filaggrin and its Processing Proteases in Human Keratinocytes.

Background:Atopic dermatitis (AD) is characterized by defective skin barrier and imbalance in T helper 1/T helper 2 (Th1/Th2) cytokine expression. Filaggrin (FLG) is the key protein to maintaining skin barrier function. Recent studies indicated that Th1/Th2 cytokines influence FLG expression in keratinocytes. However, the role of Th1/Th2 cytokines on FLG processing is not substantially documented. Our aim was to investigate the impact of Th1/Th2 cytokines on FLG processing.Methods:HaCaT cells and normal human keratinocytes were cultured in low and high calcium media and stimulated by either interleukin (IL)-4, 13 or interferon-γ (IFN-γ). FLG, its major processing proteases and key protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) were measured by both real-time quantitative polymerase chain reaction and Western blotting. Their expression was also evaluated in acute and chronic AD lesions by immunohistochemistry.Results:IL-4/13 significantly reduced, while IFN-γ significantly up-regulated FLG expression. IL-4/13 significantly increased, whereas IFN-γ significantly decreased the expression of kallikreins 5 and 7, matriptase and channel-activating serine protease 1. On the contrary, IL-4/13 significantly decreased, while IFN-γ increased the expression of LEKTI and caspase-14. Similar trends were observed in AD lesions.Conclusions:Our results suggested that Th1/Th2 cytokines differentially regulated the expression of major FLG processing enzymes. The imbalance between Th1 and Th2 polarized immune response seems to extend to FLG homeostasis, through the network of FLG processing enzymes.