Isolation of Bovine Milk Exosome Using Electrophoretic Oscillation Assisted Tangential Flow Filtration with Antifouling of Micro-ultrafiltration Membrane Filters.

Tangent flow-driven ultrafiltration (TF-UF) is an efficient isolation process of milk exosomes without morphological deformation. However, the TF-UF approach with micro-ultrafiltration SiNx membrane filters suffers from the clogging and fouling of micro-ultrafiltration membrane filter pores with large bioparticles. Thus, it is limited in the long term, continuous isolation of large quantities of exosomes. In this work, we introduced electrophoretic oscillation (EPO) in the TF-UF approach to remove pore clogging and fouling of with micro-ultrafiltration SiNx membrane filters by large bioparticles. As a result, the combined EPO-assisted TF (EPOTF) filtration can isolate large quantities of bovine milk exosomes without deformation. Furthermore, several morphological and biological analyses confirmed that the EPOTF filtration approach could isolate the milk exosomes in high concentrations with high purity and intact morphology. In addition, the uptake test of fluorescent-labeled exosomes by the keratinocyte cells visualized the biological function of purified exosomes. Hence, compared to the TF-UF process, the EPOTF filtration produced a higher yield of bovine milk exosomes without stopping the filtering process for over 200 h. Therefore, this isolation process enables scalable and continuous production of morphologically intact exosomes from bovine milk, suggesting that high-quality exosome purification is possible for future applications such as drug nanocarriers, diagnosis, and treatments.

Induction of autophagy improves skin and hair conditions in dogs with underlying diseases.

Background: Autophagy was reported to play a crucial role in maintaining general and skin health. Methods: The study used a synthesized autophagy inducer (AI) (Aquatide™ cospharm Inc.; Daejeon, Korea), for evaluating the effects of autophagy on skin and hair in dogs. Twenty-two dogs with poor skin and hair which were diagnosed with canine atopic dermatitis (CAD) or pituitary-dependent hyperadrenocorticism (PDH) were included. Clinical scores using Canine Atopic Dermatitis Extent and Severity Index-04 (CADESI-04), Pruritus Visual Analog Scale (PVAS) and skin barrier function using measurement of transepidermal water loss (TEWL) were evaluated and canine keratinocytes were also used in vitro investigation of pro-inflammatory cytokines after AI treatment. Results: In the AI group, clinical scores and skin barrier function were improved at week 8 significantly compared to in the other groups. In particular, the AI significantly improved the hair surface damage at 8 weeks compared to the baseline. In vitro, the AI reduced pro-inflammatory cytokines by activating the 78-kDa glucose-regulated protein (GRP78). Conclusion: AI improve skin barrier function and hair damage and reduce pro-inflammatory cytokines by inhibiting reactive oxygen species (ROS) production in dogs.

Enhanced Large-Scale Production of Hahella chejuensis-Derived Prodigiosin and Evaluation of Its Bioactivity.

Prodigiosin as a high-valued compound, which is a microbial secondary metabolite, has the potential for antioxidant and anticancer effects. However, the large-scale production of functionally active Hahella chejuensis-derived prodigiosin by fermentation in a cost-effective manner has yet to be achieved. In the present study, we established carbon source-optimized medium conditions, as well as a procedure for producing prodigiosin by fermentation by culturing H. chejuensis using 10 L and 200 L bioreactors. Our results showed that prodigiosin productivity using 250 ml flasks was higher in the presence of glucose than other carbon sources, including mannose, sucrose, galactose, and fructose, and could be scaled up to 10 L and 200 L batches. Productivity in the glucose (2.5 g/l) culture while maintaining the medium at pH 6.89 during 10 days of cultivation in the 200 L bioreactor was measured and increased more than productivity in the basal culture medium in the absence of glucose. Prodigiosin production from 10 L and 200 L fermentation cultures of H. chejuensis was confirmed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) analyses for more accurate identification. Finally, the anticancer activity of crude extracted prodigiosin against human cancerous leukemia THP-1 cells was evaluated and confirmed at various concentrations. Conclusively, we demonstrate that culture conditions for H. chejuensis using a bioreactor with various parameters and ethanol-based extraction procedures were optimized to mass-produce the marine bacterium-derived high purity prodigiosin associated with anti-cancer activity.

Phenotypic overlap between atopic dermatitis and autism.

BACKGROUND: Autism, a childhood behavioral disorder, belongs to a large suite of diseases, collectively referred to as autism spectrum disorders (ASD). Though multifactorial in etiology, approximately 10% of ASD are associated with atopic dermatitis (AD). Moreover, ASD prevalence increases further as AD severity worsens, though these disorders share no common causative mutations. We assessed here the link between these two disorders in the standard, valproic acid mouse model of ASD. In prior studies, there was no evidence of skin involvement, but we hypothesized that cutaneous involvement could be detected in experiments conducted in BALB/c mice. BALB/c is an albino, laboratory-bred strain of the house mouse and is among the most widely used inbred strains used in animal experimentation. METHODS: We performed our studies in valproic acid (VPA)-treated BALB/c hairless mice, a standard mouse model of ASD. Mid-trimester pregnant mice received a single intraperitoneal injection of either valproic acid sodium salt dissolved in saline or saline alone on embryonic day 12.5 and were housed individually until postnatal day 21. Only the brain and epidermis appeared to be affected, while other tissues remain unchanged. At various postnatal time points, brain, skin and blood samples were obtained for histology and for quantitation of tissue sphingolipid content and cytokine levels. RESULTS: AD-like changes in ceramide content occurred by day one postpartum in both VPA-treated mouse skin and brain. The temporal co-emergence of AD and ASD, and the AD phenotype-dependent increase in ASD prevalence correlated with early appearance of cytokine markers (i.e., interleukin [IL]-4, 5, and 13), as well as mast cells in skin and brain. The high levels of interferon (IFN)γ not only in skin, but also in brain likely account for a significant decline in esterified very-long-chain N-acyl fatty acids in brain ceramides, again mimicking known IFNγ-induced changes in AD. CONCLUSION: Baseline involvement of both AD and ASD could reflect concurrent neuro- and epidermal toxicity, possibly because both epidermis and neural tissues originate from the embryonic neuroectoderm. These studies illuminate the shared susceptibility of the brain and epidermis to a known neurotoxin, suggesting that the atopic diathesis could be extended to include ASD.

Topical application of autophagy-activating peptide improved skin barrier function and reduced acne symptoms in acne-prone skin.

BACKGROUND: Recent studies about the important roles of autophagy signaling in sebaceous lipogenesis and epidermal differentiation suggest potential benefits of autophagy activation in acne. AIMS: To investigate the effects of an autophagy activator on acne-prone skin. METHODS: Autophagy signaling in human immortalized SZ95 sebocytes, normal human epidermal keratinocytes, and 3D reconstituted skin was examined. Effects of an autophagy-activating peptide on sebaceous lipogenesis were measured by fluorescence microscopic analysis. The clinical efficacy in acne-prone skin was evaluated through an eight-week, double-blind, randomized, vehicle-controlled study. Changes in skin surface lipid compositions were further analyzed. RESULTS: In cultured sebocytes and keratinocytes, the investigated autophagy-activating peptide increased LC3-II expression, indicating a stimulation of autophagy signaling. Testosterone and linoleic acid treatment induced lipogenesis in cultured sebocytes and is further inhibited by the autophagy activator peptide treatment. Increased expression of differentiation marker proteins in cultured keratinocytes was also observed by autophagy-activating peptide. In clinical study, reduction of closed comedones and the amount of skin surface lipids as well as of trans-epidermal water loss (TEWL) were observed in acne-prone skin after autophagy-activating peptide application. In addition, reduction of squalene and increase in cholesterol were observed after an 8-week application. CONCLUSIONS: Topical application of an autophagy activator downregulated sebaceous lipogenesis and improved the skin barrier function. Considering the important roles of sebum and skin barrier function in acne pathogenesis, autophagy activation might represent a new therapeutic option in early forms of acne.

Anti-Wrinkle Benefits of Peptides Complex Stimulating Skin Basement Membrane Proteins Expression.

The dermal-epidermal junction (DEJ) provides a physical and biological interface between the epidermis and the dermis. In addition to providing a structural integrity, the DEJ also acts as a passageway for molecular transport. Based on the recently reported importance of the DEJ in skin aging, novel peptide derivatives have been tested for their effects on basement membrane (BM) protein expressions in cultured human epidermal keratinocytes. As a result, protein expressions of collagen XVII, laminin and nidogen were stimulated by the test peptide and peptides complex. Further ex vivo evaluation using excised human skin, confirmed that the topical application of the peptides complex significantly increased dermal collagen expression, as well as expressions of collagen XVII and laminin. Interestingly, while the origin of the laminin protein is epidermal keratinocytes, the immunohistochemical staining of skin showed that laminin was only detected in the uppermost layer of the dermis, which suggests a tight assembly of laminin protein onto the dermal side of the DEJ. These results suggest that a peptide complex could improve the structural properties of the DEJ through its ability to stimulate BM proteins. In order to evaluate the anti-wrinkle benefits of the peptide complex in vivo, a clinical study was performed on 22 healthy Asian female volunteers older than 40 years. As a result, significant improvements in skin wrinkles for all of the five sites were observed after two weeks, as assessed by skin topographic measurements. Collectively, these results demonstrate the anti-aging efficacy of the peptides complex.

Protease-Activated Receptors 2-Antagonist Suppresses Asthma by Inhibiting Reactive Oxygen Species-Thymic Stromal Lymphopoietin Inflammation and Epithelial Tight Junction Degradation.

PURPOSE: Protease-activated receptor 2 (PAR2) reportedly triggers the immune response in allergic asthma. We aimed to investigate the mechanism on allergic inflammation mediated by PAR2. METHODS: Human lung epithelial cells (A549 cells) were used for in vitro, and the German cockroach extract (GCE)-induced mouse model was developed for in vivo studies. RESULTS: In A549 cells, the levels of reactive oxygen species (ROS) and thymic stromal lymphopoietin (TSLP) were significantly increased by GCE treatment, but were suppressed by PAR2-antagonist (PAR2-ant) or N-acetylcysteine (NAC) treatment. Claudin-1 was degraded by GCE, and was restored by PAR2-ant or NAC in the cells. In the mouse model, the clinical appearance including bronchial hyperresponsiveness, bronchoalveolar lavage fluid analysis and total immunoglobulin E were significantly suppressed by PAR2-ant or NAC. Moreover, TSLP levels in the lung were suppressed by the same treatments in the lung. Claudin-1 was also degraded by GCE, and was restored by PAR2-ant or NAC. CONCLUSIONS: ROS generation and epidermal tight junction degradation are triggered by protease, followed by the induction of TSLP in allergic asthma. Our findings could suggest that PAR2-ant or anti-oxidants could be considered for allergic diseases as preventive alternatives.

Autophagy Activation by Crepidiastrum Denticulatum Extract Attenuates Environmental Pollutant-Induced Damage in Dermal Fibroblasts.

Pollution-induced skin damage results in oxidative stress; cellular toxicity; inflammation; and, ultimately, premature skin aging. Previous studies suggest that the activation of autophagy can protect oxidation-induced cellular damage and aging-like changes in skin. In order to develop new anti-pollution ingredients, this study screened various kinds of natural extracts to measure their autophagy activation efficacy in cultured dermal fibroblast. The stimulation of autophagy flux by the selected extracts was further confirmed both by the expression of proteins associated with the autophagy signals and by electron microscope. Crepidiastrum denticulatum (CD) extract treated cells showed the highest autophagic vacuole formation in the non-cytotoxic range. The phosphorylation of adenosine monophosphate kinase (AMPK), but not the inhibition of mammalian target of rapamycin (mTOR), was observed by CD-extract treatment. Its anti-pollution effects were further evaluated with model compounds, benzo[a]pyrene (BaP) and cadmium chloride (CdCl2), and a CD extract treatment resulted in both the protection of cytotoxicity and a reduction of proinflammatory cytokines. These results suggest that the autophagy activators can be a new protection regimen for anti-pollution. Therefore, CD extract can be used for anti-inflammatory and anti-pollution cosmetic ingredients.

Antiaging and antioxidant effects of topical autophagy activator: A randomized, placebo-controlled, double-blinded study.

BACKGROUND: Recently, potential roles of autophagy in skin homeostasis received many interests. But, little has been reported for the potential antiaging effects of autophagy activator. OBJECTIVE: With the newly synthesized autophagy activator, heptasodium hexacarboxymethyl dipeptide-12 (Aquatide™) in vitro and clinical efficacy of the topical autophagy activator as an antiaging cosmeceutical ingredient was evaluated. METHODS: Antioxidant effect of Aquatide™ was evaluated by radical scavenging assay. In vitro effect was assessed by measuring the cytotoxicity of hydrogen peroxide in cultured normal human epidermal keratinocytes. Clinical evaluation was performed by a randomized, placebo-controlled, double-blinded study. Antioxidant efficacy was observed by measuring the carbonylated proteins in stratum corneum (SC) by fluorescein-5-thiosemicarbazide (FTZ) staining. RESULTS: Radical scavenging effects of Aquatide were observed with the ABTS assay, and significant reduction in hydrogen peroxide-induced cytotoxicity was observed in Aquatide™-treated cells. Autophagy inhibitor treatment abrogated cytoprotective effects of Aquatide™. In a clinical study, statistically significant increase in skin elasticity was observed after 4 and 8 weeks. Quantitative analysis of carbonylated proteins in SC also showed significant reduction in Aquatide™-treated group, which is consistent with the in vitro data. CONCLUSION: These results suggest that autophagy plays important roles in antioxidant system and aging process in skin, and topical autophagy activators can be potential cosmeceutical ingredients for skin antiaging.

Cellular and Metabolic Basis for the Ichthyotic Phenotype in NIPAL4 (Ichthyin)-Deficient Canines.

Mutations in several lipid synthetic enzymes that block fatty acid and ceramide production produce autosomal recessive congenital ichthyoses (ARCIs) and associated abnormalities in permeability barrier homeostasis. However, the basis for the phenotype in patients with NIPAL4 (ichthyin) mutations (among the most prevalent ARCIs) remains unknown. Barrier function was abnormal in an index patient and in canines with homozygous NIPAL4 mutations, attributable to extensive membrane stripping, likely from detergent effects of nonesterified free fatty acid. Cytotoxicity compromised not only lamellar body secretion but also formation of the corneocyte lipid envelope (CLE) and attenuation of the cornified envelope (CE), consistent with a previously unrecognized, scaffold function of the CLE. Together, these abnormalities result in failure to form normal lamellar bilayers, accounting for the permeability barrier abnormality and clinical phenotype in NIPA-like domain-containing 4 (NIPAL4) deficiency. Thus, NIPAL4 deficiency represents another lipid synthetic ARCI that converges on the CLE (and CE), compromising their putative scaffold function. However, the clinical phenotype only partially improved after normalization of CLE and CE structure with topical ω-O-acylceramide because of ongoing accumulation of toxic metabolites, further evidence that proximal, cytotoxic metabolites contribute to disease pathogenesis.

Promotion of hair growth by newly synthesized ceramide mimetic compound.

Based on the crucial roles of ceramides in skin barrier function, use of ceramides or their structural mimetic compounds, pseudoceramides, as cosmetic ingredients are getting more popular. While currently used pseudoceramides are intended to substitute the structural roles of ceramides in stratum corneum, development of bioactive pseudoceramides has been repeatedly reported. In this study, based on the potential involvement of sphingolipids in hair cycle regulation, we investigated the effects of newly synthesized pseudoceramide, bis-oleamido isopropyl alcohol (BOI), on hair growth using cultured human hair follicles and animal models. BOI treatment promoted hair growth in cultured human hair follicles ex vivo and induced earlier conversion of telogen into anagen. Although we did not find a significant enhancement of growth factor expression and follicular cell proliferation, BOI treatment resulted in an increased sphinganine and sphingosine contents as well as increased ceramides contents in cultured dermal papilla (DP) cells. Taken together, our data strongly suggest that biologically active pseudoceramide promotes hair growth by stimulating do novo synthesis of sphingolipids in DP cells.

Comparison of the Efficacy of Atopalm(®) Multi-Lamellar Emulsion Cream and Physiogel(®) Intensive Cream in Improving Epidermal Permeability Barrier in Sensitive Skin.

INTRODUCTION: The management of sensitive skin, which affects over 60% of the general population, has been a long-standing challenge for both patients and clinicians. Because defective epidermal permeability barrier is one of the clinical features of sensitive skin, barrier-enhancing products could be an optimal regimen for sensitive skin. In the present study, we evaluated the efficacy and safety of two barrier-enhancing products, i.e., Atopalm (®) Multi-Lamellar Emulsion (MLE) Cream and Physiogel (®) Intensive Cream for sensitive skin. METHODS: 60 patients with sensitive skin, aged 22-40 years old, were randomly assigned to one group treated with Atopalm MLE Cream, and another group treated with Physiogel Intensive Cream twice daily for 4 weeks. Lactic acid stinging test scores (LASTS), stratum hydration (SC) and transepidermal water loss (TEWL) were assessed before, 2 and 4 weeks after the treatment. RESULTS: Atopalm MLE Cream significantly lowered TEWL after 2 and 4 weeks of treatment (p < 0.01). In contrast, Physiogel Intensive Cream significantly increased TEWL after 2 weeks of treatment (p < 0.05) while TEWL significantly decreased after 4-week treatments. Moreover, both Atopalm MLE Cream and Physiogel Intensive Cream significantly increased SC hydration, and improved LASTS after 4 weeks of treatment. CONCLUSION: Both barrier-enhancing products are effective and safe for improving epidermal functions, including permeability barrier, SC hydration and LASTS, in sensitive skin. These products could be a valuable alternative for management of sensitive skin. FUNDING: Veterans Affairs Medical Center, San Francisco, California, USA, and NeoPharm Co., Ltd., Daejeon, Korea.

Altered epidermal lipid layers induced by long-term exposure to suberythemal-dose ultraviolet.

BACKGROUND: Although several studies have reported on the biological effects of ultraviolet (UV) radiation, there have been only a few reports on the changes in epidermal lipids following long-term UV irradiation at suberythemal dose (SED), to which people are usually exposed during their lifetime. OBJECTIVES: To investigate the changes of epidermal lipid properties after long-term UV radiation with SED. MATERIALS AND METHODS: Hairless mice were irradiated three times weekly for 15 weeks at an SED of UV (UVB: 20 mJ/cm(2) ; UVA: 14 J/cm(2) ). Every three weeks, transepidermal water loss (TEWL) was measured by a Tewameter. The morphological alterations of stratum corneum (SC) lipid lamellae were examined by electron microscopy (EM). Activities of three key enzymes for mRNA of serine palmitoyl transferase, fatty acid synthase, and HMG CoA reductase were analyzed with real time reverse transcriptase-polymerase chain reaction. We also measured the amount of ceramide, cholesterol sulfate, and free fatty acid in the SC by high-performance thin-layer chromatography with exposed times. RESULTS: The SED UV-irradiated group showed increased TEWL after 12 weeks. Following the irradiation period, EM revealed incomplete and separated lamellae at SC intercellular space. mRNA of three key enzymes was increased until six weeks of UV irradiation and decreased thereafter. However, three major lipid amounts gradually decreased throughout the exposed period, with a notable decrease in ceramide. CONCLUSIONS: Long-term UV irradiation even with SED influences skin barrier function and structure with prominent ceramide decrease in SC intercellular lipid.

Anti-ageing effects of a new synthetic sphingolipid (K6EAA-L12) on aged murine skin.

Recently, we reported on the anti-ageing effects of K6PC-5. This compound induced keratinocyte differentiation and fibroblast proliferation by increasing sphingosine-1 phosphate synthesis. We performed this study to confirm the anti-ageing effects of new synthetic products (the K6EAA series) derived from K6PC-5 through an amino group induction. Cellular responses such as differentiation, proliferation and calcium mobilization were investigated using cultured human keratinocytes and fibroblasts. Also, we measured the expressions of collagen mRNA and protein using real time RT-PCR and ELISA, respectively. The K6EAA-L12 product, selected by in vitro screening, was evaluated for anti-ageing effects on intrinsically and extrinsically (photo) aged models of hairless mice. In the intrinsically aged murine skin, K6EAA-L12 showed anti-ageing effects by activating collagen synthesis, eventually causing dermal thickening. Also, in the photo-aged skin, the dermal collagen density and dermal thickness were increased. In photo-aged murine skin, K6EAA-L12 increased stratum corneum integrity by increasing corneodesmosome density and improved the barrier recovery rate. However, there were no changes in the expressions of epidermal differentiation maker proteins. In conclusion, topical K6EAA-L12, a new synthetic K6PC-5 derivative, improves intrinsically and extrinsically (photo) aged skin by increasing the collagen density and improving the skin barrier function.