Ethanol treatment of nanoPGA/PCL composite scaffolds enhances human chondrocyte development in the cellular microenvironment of tissue-engineered auricle constructs.

A major obstacle for tissue engineering ear-shaped cartilage is poorly developed tissue comprising cell-scaffold constructs. To address this issue, bioresorbable scaffolds of poly-ε-caprolactone (PCL) and polyglycolic acid nanofibers (nanoPGA) were evaluated using an ethanol treatment step before auricular chondrocyte scaffold seeding, an approach considered to enhance scaffold hydrophilicity and cartilage regeneration. Auricular chondrocytes were isolated from canine ears and human surgical samples discarded during otoplasty, including microtia reconstruction. Canine chondrocytes were seeded onto PCL and nanoPGA sheets either with or without ethanol treatment to examine cellular adhesion in vitro. Human chondrocytes were seeded onto three-dimensional bioresorbable composite scaffolds (PCL with surface coverage of nanoPGA) either with or without ethanol treatment and then implanted into athymic mice for 10 and 20 weeks. On construct retrieval, scanning electron microscopy showed canine auricular chondrocytes seeded onto ethanol-treated scaffolds in vitro developed extended cell processes contacting scaffold surfaces, a result suggesting cell-scaffold adhesion and a favorable microenvironment compared to the same cells with limited processes over untreated scaffolds. Adhesion of canine chondrocytes was statistically significantly greater (p ≤ 0.05) for ethanol-treated compared to untreated scaffold sheets. After implantation for 10 weeks, constructs of human auricular chondrocytes seeded onto ethanol-treated scaffolds were covered with glossy cartilage while constructs consisting of the same cells seeded onto untreated scaffolds revealed sparse connective tissue and cartilage regeneration. Following 10 weeks of implantation, RT-qPCR analyses of chondrocytes grown on ethanol-treated scaffolds showed greater expression levels for several cartilage-related genes compared to cells developed on untreated scaffolds with statistically significantly increased SRY-box transcription factor 5 (SOX5) and decreased interleukin-1α (inflammation-related) expression levels (p ≤ 0.05). Ethanol treatment of scaffolds led to increased cartilage production for 20- compared to 10-week constructs. While hydrophilicity of scaffolds was not assessed directly in the present findings, a possible factor supporting the summary data is that hydrophilicity may be enhanced for ethanol-treated nanoPGA/PCL scaffolds, an effect leading to improvement of chondrocyte adhesion, the cellular microenvironment and cartilage regeneration in tissue-engineered auricle constructs.

Studies on the anti-psoriasis effects and its mechanism of a dual JAK2/FLT3 inhibitor flonoltinib maleate.

Psoriasis is a chronic, inflammatory autoimmune disease mediated by T cells, and characterized with abnormal proliferation and differentiation of keratinocytes, and inflammatory infiltration. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway has been identified to play essential roles in mediating various of biological processes, and is closely related to autoimmune diseases. Dendritic cells (DCs) are important antigen presenting cells and play an important regulatory role in T cells. The proliferation, differentiation and function of DCs are regulated by JAK and FMS-like tyrosine kinase 3 (FLT3) signal pathways. Flonoltinib maleate (FM), a high selectivity dual JAK2/FLT3 inhibitor with IC50 values of 0.8 nM and 15 nM for JAK2 and FLT3, respectively, was developed by our laboratory. Moreover, FM was a potent JAK2 inhibitor with 863-fold and 696-fold selectivity over JAK1 and JAK3, respectively. In this study, the anti-psoriasis activity of FM was evaluated both in vitro and in vivo. FM effectively inhibited the proliferation of HaCaT, the inflammatory keratinocyte induced by M5 and markedly suppressed the generation and differentiation of DCs from bone marrow (BM), and inhibited the expression of FLT3 in DCs in vitro. FM effectively inhibited the ear thickening and improved the pathological changes of the ear in interleukin (IL)-23-induced psoriasis-like acanthosis mouse model. Further in keratin 14-vascular endothelial growth factor (K14-VEGF) transgenic homozygous mice model, FM could obviously improve the psoriatic symptom and pathological changes, significantly inhibit the generations of Th1 and Th17 cells in the spleen, and the accumulations of DCs in the ears. FM could also significantly reduce the expression of various inflammatory factors both in C57BL/6 and K14-VEGF mice ears, and the serum of K14-VEGF mice. Mechanism revealed that FM effectively suppressed the phosphorylation of JAK2, STAT3 and STAT5 in inflammatory keratinocytes and the mice ears of C57BL/6 and K14-VEGF, as well as the phosphorylation of FLT3 in K14-VEGF mice ears. In conclusion, FM plays an excellent anti-psoriasis activity, including inhibiting keratinocyte proliferation and regulating inflammatory response through inhibiting JAK2 and FLT3 signaling pathway.

Fe2+: Fe3+ Molar Ratio Influences the Immunomodulatory Properties of Maghemite (γ-Fe2O3) Nanoparticles in an Atopic Dermatitis Model.

Here, we report the different antioxidant and physiological effects of maghemite nanoparticles (γ-Fe2O3 NPs) obtained using various Fe2+: Fe3+ molar ratios (FM1 = 1: 1, FM2 = 1: 2, and FM3 = 2: 3) via coprecipitation from ferrous/ferric salts. We investigated the physical, optical, and antioxidant properties of FM1, FM2, and FM3 nanoparticles by conducting UV, Raman, FTIR, and EDX spectroscopic analyses along with DPPH radical scavenging activity. Results showed the highest DPPH scavenging activity in the FM2 group (50.76%), while the activity in the FM1 and FM3 groups was 23.60% and 34.63%, respectively. In addition, topical application of nanoparticles induced significant but different anti-inflammatory and immunomodulatory effects in Dermatophagoides farinae extract/2,4-dinitrochlorobenzene (DFE/DNCB)-sensitized BALB/c mice. The FM2 treatment alleviates more effectively the DFE/DNCB-induced atopic dermatitis-like (AD-like) symptoms in mouse ears (edema, excoriation, scaling, and hemorrhage). In comparison with the DFE/DNCB-sensitized mice, FM2 treatment greatly reduced the size and weight of the spleen and the lymph nodes. It also suppressed mast cell infiltration (2-fold) and reduced dermal and epidermal thickness in mice. In addition, FM2 treatment exhibited better inhibition of the mRNA levels of Th1 (IFN-γ and TNF-α) and Th2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, and IL-31), as well as the levels of various inflammation-related proteins (COX-2, iNOS, and TNF-α). Moreover, we demonstrated that an increasing proportion of Fe3+ in Fe2+: Fe3+ enhances the antioxidant activity and increases the anti-inflammatory and immunomodulatory effects of γ-Fe2O3 NPs in an AD mouse model. Thus, γ-Fe2O3 NPs could be used in the formulation of nonsteroidal drugs for AD treatment.

The scabicide effect of moxidectin in vitro and in experimental animals: Parasitological, histopathological and immunological evaluation.

Scabies is considered one of the commonest dermatological diseases that has a global health burden. Current treatment with ivermectin (IVM) is insufficient and potential drug resistance was noticed. Moxidectin (MOX), with a better pharmacological profile may be a promising alternative. The efficacy of moxidectin against Sarcoptes scabiei was assessed both in vitro and in vivo in comparison with ivermectin. For the in vitro assay, both drugs were used in two concentrations (50 µg/ml and 100 µg/ml). For the in vivo assay, twenty rabbits infected with Sarcoptes scabiei were divided into three groups: untreated, moxidectin-treated and ivermectin-treated with the same dose of 0.3 mg/kg once. Another four rabbits were used as a normal control non-infected group. Treatment efficacy was evaluated by clinical assessment, parasitological evaluation and histopathological examination of skin samples using Hematoxylin and eosin and toluidine blue for mast cell staining. Immune response was also assessed by immunohistochemical staining of CD3 T cells in skin samples. Our results showed that moxidectin had a high efficacy (100%) in killing mites when used in both concentrations (50 µg/ml, 100 µg/ml) in the in vitro assay. Concerning the in vivo assay, on day 14 post-treatment, all MOX-treated rabbits were mite-free with full clinical cure by the end of the study (D21) showing (100%) reduction of mites count. Also, marked improvement in the epidermis with absence of mites in skin samples were shown. Poor clinical and parasitological improvements were noted in the ivermectin-treated rabbits, when given as a single dose with a percentage reduction (60.67%) in the 2nd week and progressive increase in lesions and mites count in the 3rd week post-treatment. Regarding the immune response, MOX-treated group showed mild infiltration with both mast cells and CD3 T cells in comparison to severe infiltration with both types of cells in the untreated and IVM-treated group. On conclusion, our results demonstrated that a single dose of MOX was more effective than IVM, supporting MOX as a valuable therapeutic approach for scabies therapy.

Ethyl Alcohol Versus Botulinum Toxin A: A Comparative Study of the Visual and Histopathological Outcomes in the Rabbit Anterior Auricular Muscle Model.

BACKGROUND: Botulinum toxin has long been known for its paralytic effects at the neuromuscular junction. Although it has been widely used for vascular and nervous tissues, there has been no study of the aesthetic effects of the application of ethanol to muscle tissues to date. OBJECTIVE: The authors aimed to demonstrate the effects of the application of ethanol to muscle tissues after an intramuscular injection and to compare the effects of botulinum toxin A (BTA) and ethanol. METHODS AND MATERIALS: A total of 28 rabbits were divided into 4 groups (n = 7 each). Botulinum toxin A (5 units) and different concentrations of ethanol (5 cc) were injected into the left and right anterior auricular muscles of all rabbits, respectively. Ear ptosis was assessed, and histopathological examination was performed after all rabbits were euthanized in the eighth week. RESULTS: Muscle function was affected earlier in ethanol-treated ears than in botulinum-treated ears; however, the ptotic effect lasted for a significantly shorter duration in ethanol-injected ears than in BTA-applied ears. CONCLUSION: Ethanol can block muscle function reversibly and can serve as an alternative to BTA, particularly when rapid results are desirable.

Discovery of a novel and selective fungicide that targets fungal cell wall to treat dermatomycoses: 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride.

BACKGROUND: Fungal infections are highly prevalent and are responsible for high rates of morbidity and mortality. In this context, the search for new treatment alternatives is very relevant. OBJECTIVES: Analyse chemical compounds for antifungal potential against dermatomycosis fungi. METHODS: The antifungal activity of 121 compounds, intermediates or derivatives of 1,3-bis(aryloxy)propane substituted at C-2 (111 compounds) and isothiouronium derivatives (10 compounds) was investigated through susceptibility tests, mechanism of action, toxicity and hydrogel incorporation. RESULTS: The compound 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride (2j) was the most active fungicide against dermatophytes and Candida spp., at very low concentrations (0.39-3.12 µg/mL), including action on resistant and multidrug-resistant clinical strains. Compound 2j has presented a promising toxicity profile, showing selectivity index >10, relative to human lymphocytes. The compound was classified as non-irritant by the HET-CAM test and did not cause histopathological alterations in pig ear skin, thus presenting an excellent perspective for topical application. 2j targets the fungal cell wall, which was confirmed by scanning electron microscopy, which also indicated the additional ability of 2j to inhibit the Candida albicans pseudohyphae formation and biofilm of Microsporum canis. Compound 2j was incorporated in a hydrogel with bioadhesive potential. The results of the human skin permeation showed that 2j remained significantly in the epidermis, ideally for the dermatomycosis treatment. CONCLUSIONS: Therefore, the compound 2j demonstrated the potential for antifungal drug development, with a action mechanism elucidated and already applied in a semisolid formulation as a new therapeutic option for fungal skin infections.

Anti-inflammatory effect of lanoconazole on 12-O-tetradecanoylphorbol-13-acetate- and 2,4,6-trinitrophenyl chloride-induced skin inflammation in mice.

BACKGROUND: Lanoconazole (LCZ) is a topical antifungal agent clinically used to treat fungal infections such as tinea pedis. LCZ has not only antifungal effects but also anti-inflammatory effects, which have the potential to provide additional clinical benefits. However, the characteristic features of the inhibitory effects of LCZ on skin inflammation remain unclear. OBJECTIVE: We evaluated the inhibitory effects of topical application of LCZ, and compared the effects of LCZ with those of other antifungal agents including liranaftate, terbinafine and amorolfine. METHODS: Each antifungal agent was topically applied on 12-O-tetradecanoylphorbol-13-acetate-induced irritant dermatitis and 2,4,6-trinitrophenyl chloride-induced contact dermatitis in mice (BALB/c). The ear thickness, myeloperoxidase activity and inflammatory mediator contents were evaluated. RESULTS: LCZ dose-dependently suppressed 12-O-tetradecanoylphorbol-13-acetate-induced irritant dermatitis, suppressed the production of neutrophil chemotactic factors such as keratinocyte-derived chemokine and macrophage inflammatory protein-2, and inhibited neutrophil infiltration to the inflammation site. Moreover, 1% LCZ reduced the ear swelling in mice with 2,4,6-trinitrophenyl chloride-induced contact dermatitis in accordance with the inhibition of interferon-γ production. The inhibitory potency of LCZ on these types of dermatitis in mice was stronger than that of other types of antifungal agents. CONCLUSION: The anti-inflammatory effects of LCZ were exerted through the inhibition of inflammatory mediator production. These effects may contribute to the relief of dermatitis symptoms in patients with tinea pedis.

Hypochlorous-Acid-Generating Electrochemical Scaffold for Treatment of Wound Biofilms.

Biofilm formation causes prolonged wound infections due to the dense biofilm structure, differential gene regulation to combat stress, and production of extracellular polymeric substances. Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa are three difficult-to-treat biofilm-forming bacteria frequently found in wound infections. This work describes a novel wound dressing in the form of an electrochemical scaffold (e-scaffold) that generates controlled, low concentrations of hypochlorous acid (HOCl) suitable for killing biofilm communities without substantially damaging host tissue. Production of HOCl near the e-scaffold surface was verified by measuring its concentration using needle-type microelectrodes. E-scaffolds producing 17, 10 and 7 mM HOCl completely eradicated S. aureus, A. baumannii, and P. aeruginosa biofilms after 3 hours, 2 hours, and 1 hour, respectively. Cytotoxicity and histopathological assessment showed no discernible harm to host tissues when e-scaffolds were applied to explant biofilms. The described strategy may provide a novel antibiotic-free strategy for treating persistent biofilm-associated infections, such as wound infections.

Topical Oxandrolone Reduces Ear Hypertrophic Scar Formation in Rabbits.

BACKGROUND: Wound healing is a complex process. Despite extensive studies, hypertrophic and keloid scars still occur, and can be functionally and cosmetically problematic. In an attempt to prevent hypertrophic scar formation, the effects of topical oxandrolone, using hyaluronic acid as a biomaterial, were studied on ear wounds in rabbits. METHODS: Deep second-degree burns were inflicted on each ear in 10 New Zealand rabbits. On the left ears, considered the control side, hyaluronic acid gel was applied, whereas on the right ears, the study side, a combination of oxandrolone and hyaluronic acid was applied. Dressings were changed every 2 days for 2 weeks. At week 10, biopsy specimens from the postburn scars were harvested for histologic and immunohistochemical examinations. RESULTS: Fourteen wounds were studied, half on the control side and half on the study side. Six hypertrophic scars were encountered on the control side and only one scar was encountered on the study side. In addition, an increased degree of inflammation, an increased amount of collagen and fibroblast cellularity, increased vascularization, and increased myofibroblast activity were observed on the control side. CONCLUSION: Topical administration of oxandrolone using hyaluronic acid as a biomaterial led to better healing and prevented hypertrophic scar formation.

Expression of cytokines and chemokines in mouse skin treated with sulfur mustard.

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7 days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7 days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.

PPARD is an Inhibitor of Cartilage Growth in External Ears.

Peroxisome proliferator-activated receptor beta/delta (PPARD) is an important determinant of multiple biological processes. Our previous studies identified a missense mutation in the PPARD gene that significantly reduces its transcription activity, and consequently causes enlarged external ears in pigs. However, the mechanisms underlying the causality has remained largely unknown. Here, we show that PPARD retards the development of auricular cartilage by accelerating the apoptosis of cartilage stem/progenitor cells (CSPCs), the terminal differentiation of cartilage cells and the degradation of cartilage extracellular matrix in the auricle. At the transcription level, PPARD upregulates a set of genes that are associated with CSPCs apoptosis and chondrogenic differentiation, chondroblast differentiation and extracellular matrix degradation. ChIP-seq identified direct target genes of PPARD, including a well-documented gene for cartilage development: PPARG. We further show that compared to wild-type PPARD, the G32E mutant up-regulates the expression of PPARG and subsequently leads to the downregulation of critical genes that inhibit cartilage growth. These findings allow us to conclude that PPARD is an inhibitor of auricular cartilage growth in pigs. The causative mutation (G32E) in the PPARD gene attenuates the PPARD-mediated retardation of cartilage growth in the auricle, contributing to enlarged ears in pigs. The findings advance our understanding of the mechanisms underlying auricular development in mammals, and shed insight into the studies of innate pinna disorders and cartilage regeneration medicine in humans.

Detection of local inflammation induced by repeated exposure to contact allergens by use of IVIS SpectrumCT analyses.

BACKGROUND: Contact allergy is characterized by local skin inflammation that, in some cases, can result in systemic immune activation. OBJECTIVES: To investigate whether IVIS SpectrumCT analyses can be used to detect the immune response induced by contact allergens. METHODS: Mice were repeatedly exposed to vehicle or allergens on the ears. The local and systemic responses were analysed at different times with the ProSense 750 FAST probe in IVIS SpectrumCT measurements. In addition, changes in ear thickness, cytokine profile in the skin and immunological phenotype in the draining lymph nodes and spleen were determined. RESULTS: Local inflammation was detected by ProSense 750 FAST and correlated with changes in ear thickness, cytokine profile and immunological phenotype following exposure to the strong contact allergen 2,4-dinitrofluorobenzene. Analysis of the systemic response with ProSense 750 FAST did not show any difference between allergen-exposed and control mice, although fluorescence-activated cell sorting analysis of the spleen showed increased numbers of γδ T cells and CD11b+ CD11c+ MHCII+ cells in allergen-treated mice. CONCLUSIONS: IVIS SpectrumCT analyses with ProSense 750 FAST as the probe can be used to detect local immune responses induced by contact allergens.

Cross-reactivity between methylisothiazolinone, octylisothiazolinone and benzisothiazolinone using a modified local lymph node assay.

BACKGROUND: In the light of the exceptionally high rates of contact allergy to the preservative methylisothiazolinone (MI), information about cross-reactivity between MI, octylisothiazolinone (OIT) and benzisothiazolinone (BIT) is needed. OBJECTIVES: To study cross-reactivity between MI and OIT, and between MI and BIT. METHODS: Immune responses to MI, OIT and BIT were studied in vehicle and MI-sensitized female CBA mice by a modified local lymph node assay. The inflammatory response was measured by ear thickness, cell proliferation of CD4+ and CD8+ T cells, and CD19+ B cells in the auricular draining lymph nodes. RESULTS: MI induced significant, strong, concentration-dependent immune responses in the draining lymph nodes following a sensitization phase of three consecutive days. Groups of MI-sensitized mice were challenged on day 23 with 0·4% MI, 0·7% OIT and 1·9% BIT - concentrations corresponding to their individual EC3 values. No statistically significant difference in proliferation of CD4+ and CD8+ T cells was observed between mice challenged with MI compared with mice challenged with BIT and OIT. CONCLUSIONS: The data indicate cross-reactivity between MI, OIT and BIT, when the potency of the chemical was taken into account in choice of challenge concentration. This means that MI-sensitized individuals may react to OIT and BIT if exposed to sufficient concentrations.

Mycophenolate mofetil embryopathy: A newly recognized teratogenic syndrome.

Mycophenolate mofetil (MMF) is probably the most common employed immunosuppressant drug in recipients of solid organ transplant and in many autoimmune diseases. In vitro studies, a significant number of single clinical observations and a recent study from a group of different European teratogen information services, have provided very consistent data supporting the existence of a specific MMF embryopathy. The typical malformative pattern of MMF embryopathy includes external ear anomalies ranging from hypoplastic pinna (microtia) to complete absence of pinna (anotia); cleft lip, with or without cleft palate, and ocular anomalies as iris or chorioretinal coloboma and anophthalmia/microphthalmia. Other less frequent features are congenital heart defects, distal limbs anomalies, esophageal atresia, vertebral malformations, diaphragmatic hernia, and kidney and central nervous system anomalies. Neurodevelopmental outcome seems favorable in the small number of patients where information about this issue is available, but neurological deficits have been documented. Physicians in charge of women under MMF therapy should be aware of the potential risk of this drug to cause a specific embryopathy and the need of interrupting the treatment at least six weeks before becoming pregnant.

Recombinant human endostatin reduces hypertrophic scar formation in rabbit ear model through down-regulation of VEGF and TIMP-1.

BACKGROUND: Recombinant human endostatin (Endostar) has been widely used to suppress angiogenesis in carcinoma patients. Hypertrophic scar (HS) tissue, much like a carcinoma, is often associated with angiogenesis. However, there have been few studies conducted on the effects of Endostar on HS or its mechanism. OBJECTIVE: This paper investigated the effects Endostar on the HS of rabbit ears and studied the effects of Endostar on VEGF and TIMP-1 expression. METHODS: Sixteen New Zealand white rabbits were used to establish HS models. Then, rabbit ears containing HS were randomly assigned to either the Endostar group or the control group. The changes of appearance and histology were evaluated using the naked eye, hematoxylin eosin staining, and a scar elevation index. The VEGF and TIMP-1 expressions were detected by immunohistochemical staining, RT-PCR, and western blot. RESULTS: The thickness of the connective tissue in the Endostar group were thinner, the numbers of micro vessels and fibroblasts were fewer, and the collagen fibers were smoother. Moreover, the mRNA and protein expressions of VEGF and TIMP-1 in the Endostar group were significantly lower than those in the control group. CONCLUSION: The results suggested that Endostar reduced the formation of HS by down-regulation of VEGF and TIMP-1 expressions.

Protective effects of ginsenoside F2 on 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation in mice.

Topical use of ginsenosides, the major bioactive substances in Panax ginseng, has been used for the treatment of irritated skin complaints. However, the protective mechanisms of ginsenosides remain unclear. In the present study, we investigated the anti-inflammatory role of ginsenoside F2 (GF2) on the skin inflammation. To induce irritant dermatitis, 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied on the surface of the mouse ears with or without treatments of GF2 and dexamethasone for 24 h. Protective effects of GF2 on edema and inflammation were assessed by measuring ear thickness, weights of skin punch, and inflammatory responses. In gross findings, treatments with GF2 significantly decreased skin thickness and weight compared to those of TPA-treated groups, which was comparable with the protective effects of dexamethasone. In addition, expression of inflammatory mediators was remarkably reduced in GF2-treated ears compared to that of vehicle-treated ears of mice. Interestingly, immunohistochemistry and flow cytometry analyses revealed that TPA treatment significantly increased infiltration of interleukin-17 (IL-17) producing dermal γδ T cells, while frequencies of γδ T cells was decreased by GF2 treatment, subsequently ameliorating inflammation in skin. Concomitantly, TPA-mediated skin inflammation was significantly ameliorated in IL-17A knock out mice. Furthermore, GF2 treatment inhibited infiltration and generation of reactive oxygen species (ROS) of neutrophils in damaged ears compared with vehicle-treated mice. These results clearly suggest that GF2 treatment ameliorates TPA-induced dermal inflammation by inhibiting production of IL-17 and ROS in γδ T cells and neutrophils, respectively. Therefore, as a natural compound, application of GF2 may be a novel therapeutic approach for treating skin inflammation.

Histamine Induces Vascular Hyperpermeability by Increasing Blood Flow and Endothelial Barrier Disruption In Vivo.

Histamine is a mediator of allergic inflammation released mainly from mast cells. Although histamine strongly increases vascular permeability, its precise mechanism under in vivo situation remains unknown. We here attempted to reveal how histamine induces vascular hyperpermeability focusing on the key regulators of vascular permeability, blood flow and endothelial barrier. Degranulation of mast cells by antigen-stimulation or histamine treatment induced vascular hyperpermeability and tissue swelling in mouse ears. These were abolished by histamine H1 receptor antagonism. Intravital imaging showed that histamine dilated vasculature, increased blood flow, while it induced hyperpermeability in venula. Whole-mount staining showed that histamine disrupted endothelial barrier formation of venula indicated by changes in vascular endothelial cadherin (VE-cadherin) localization at endothelial cell junction. Inhibition of nitric oxide synthesis (NOS) by L-NAME or vasoconstriction by phenylephrine strongly inhibited the histamine-induced blood flow increase and hyperpermeability without changing the VE-cadherin localization. In vitro, measurements of trans-endothelial electrical resistance of human dermal microvascular endothelial cells (HDMECs) showed that histamine disrupted endothelial barrier. Inhibition of protein kinase C (PKC) or Rho-associated protein kinase (ROCK), NOS attenuated the histamine-induced barrier disruption. These observations suggested that histamine increases vascular permeability mainly by nitric oxide (NO)-dependent vascular dilation and subsequent blood flow increase and maybe partially by PKC/ROCK/NO-dependent endothelial barrier disruption.

Trans-pinnal movement of methimazole: an in vitro study showing that methimazole can cross from the inner to outer pinna of cats.

OBJECTIVES: The aim of the study was to determine if methimazole applied in a transdermal formulation to the internal pinna will cross to the external pinna in an in vitro Franz cell model. METHODS: The ears from six cats were harvested soon after death. Whole ears were mounted onto Franz-type diffusion cells with the stratum corneum of the inner pinnae uppermost. A commercial transdermal preparation containing methimazole (0.1 ml/10 mg) was applied to the inner pinnae. At 1, 2, 4, 6, 8, 12, 18, 24 and 30 h, a 200 µl sample of reservoir solution was removed to determine the methimazole concentration by high-performance liquid chromatography. The ears were then dissected, separating the internal pinna from the cartilage and the external pinna, before the methimazole concentration was measured at each site. The thickness of the different regions of the ear was measured on paraffin histology sections. RESULTS: Mean ± SD methimazole concentrations at 30 h for the right and left ear, respectively, were: inner ear, 1.25 ± 0.53 mg/g, 0.39 ± 0.26 mg/g; cartilage, 1.36 ± 0.47 mg/g, 0.33 ± 0.20 mg/g; and outer ear, 1.0 ± 0.32 mg/g, 0.33 ± 0.14 mg/g. There was a difference between the left and right ears (P <0.001). Minimal methimazole concentrations were detected in the receptor fluid. The mean methimazole concentration absorbed by the skin after application of 10 mg was, for the right ear, 3.65 ± 1.27 mg/g and, for the left, 1.08 ± 0.27 mg/g. There was no correlation between methimazole concentrations and thickness of each region of the ear. CONCLUSIONS AND RELEVANCE: Methimazole in a lipophilic vehicle applied to the inner pinna will penetrate to the outer pinna of cats in an in vitro model, which may have safety implications for humans associated with cats treated with transdermal methimazole. Substantial inter-individual variation was found. Further research is required in the area of transdermal penetration of drugs in cats.

Topical atorvastatin ameliorates 12-O-tetradecanoylphorbol-13-acetate induced skin inflammation by reducing cutaneous cytokine levels and NF-κB activation.

Atorvastatin is a 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitor used in the treatment of atherosclerosis and dyslipidemia. Studies have evaluated the utility of statins in the treatment of skin inflammation but with varied results. In the present study, we investigated the effect of atorvastatin on TNF-α release and keratinocyte proliferation in vitro and in acute and chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) induced skin inflammation in vivo. Atorvastatin significantly inhibited lipopolysacharide induced TNF-α release in THP-1 cells and keratinocyte proliferation in HaCaT cells. In an acute study, topical atorvastatin showed dose dependent reduction in TPA induced skin inflammation with highest efficacy observed at 500 µg/ear dose. In chronic study, topical atorvastatin significantly reduced TPA induced ear thickness, ear weight, cutaneous cytokines, MPO activity and improved histopathological features comparable to that of dexamethasone. Atorvastatin also inhibited TPA stimulated NF-κB activation in mouse ear. In conclusion, our results suggest that atorvastatin ameliorates TPA induced skin inflammation in mice at least in part, due to inhibition of cytokine release and NF-κB activation and may be beneficial for the treatment skin inflammation like psoriasis.