The imbalance between Treg and Th17 cells caused by FTY720 treatment in skin allograft rejection

OBJECTIVES: FTY720 modulates CD4(+)T cells by the augmentation of regulatory T cell activity, secretion of suppressive cytokines and suppression of IL-17 secretion by Th17 cells. To further understand the process of graft rejection/acceptance, we evaluated skin allograft survival and associated events after FTY720 treatment. METHODS: F1 mice (C57BL/6xBALB/c) and C57BL/6 mice were used as donors for and recipients of skin transplantation, respectively. The recipients were transplanted and either not treated or treated with FTY720 by gavage for 21 days to evaluate the allograft survival. In another set of experiments, the immunological evaluation was performed five days post-transplantation. The spleens, axillary lymph nodes and skin allografts of the recipient mice were harvested for phenotyping (flow cytometry), gene expression (real-time PCR) and cytokine (Bio-Plex) analysis. RESULTS: The FTY720 treatment significantly increased skin allograft survival, reduced the number of cells in the lymph nodes and decreased the percentage of Tregs at this site in the C57BL/6 recipients. Moreover, the treatment reduced the number of graft-infiltrating cells and the percentage of CD4(+) graft-infiltrating cells. The cytokine analysis (splenocytes) showed decreased levels of IL-10, IL-6 and IL-17 in the FTY720-treated mice. We also observed a decrease in the IL-10, IL-6 and IL-23 mRNA levels, as well as an increase in the IL-27 mRNA levels, in the splenocytes of the treated group. The FTY720-treated mice exhibited increased mRNA levels of IL-10, IL-27 and IL-23 in the skin graft. CONCLUSIONS: Our results demonstrated prolonged but not indefinite skin allograft survival by FTY720 treatment. This finding indicates that the drug did not prevent the imbalance between Tr1 and Th17 cells in the graft that led to rejection.

Liquid Crystal Nanodispersions Enable the Cutaneous Delivery of Photosensitizer for Topical PDT: Fluorescence Microscopy Study of Skin Penetration

Topical photodynamic therapy (PDT) has been applied to almost all types of nonmelanoma skin cancer and numerous superficial benign skin disorders. Strategies to improve the accumulation of photosensitizer in the skin have been studied in recent years. Although the hydrophilic phthalocyanine zinc compound, zinc phthalocyanine tetrasulfonate (ZnPcSO4) has shown high photodynamic efficiency and reduced phototoxic side effects in the treatment of brain tumors and eye conditions, its use in topical skin treatment is currently limited by its poor skin penetration. In this study, nanodispersions of monoolein (MO)-based liquid crystalline phases were studied for their ability to increase ZnPcSO4 uptake by the skin. Lamellar, hexagonal and cubic crystalline phases were prepared and identified by polarizing light microscopy, and the nanodispersions were analyzed by dynamic light scattering. In vitro skin penetration studies were performed using a Franz's cell apparatus, and the skin uptake was evaluated in vivo in hairless mice. Aqueous dispersions of cubic and hexagonal phases showed particles of nanometer size, approximately 224 +/- 10 nm and 188 +/- 10 nm, respectively. In vitro skin retention experiments revealed higher fluorescence from the ZnPcSO4 in deeper skin layers when this photosensitizer was loaded in the hexagonal nanodispersion system when compared to both the cubic phase nanoparticles and the bulk crystalline phases (lamellar, cubic and hexagonal). The hexagonal nanodispersion showed a similar penetration behavior in animal tests. These results are important findings, suggesting the development of MO liquid crystal nanodispersions as potential delivery systems to enhance the efficacy of topical PDT.

A VALIDATED HPLC ANALYTICAL METHOD FOR THE ANALYSIS OF SOLASONINE AND SOLAMARGINE IN IN VITRO SKIN PENETRATION STUDIES

To assess topical delivery studies of glycoalkaloids, an analytical method by HPLC-UV was developed and validated for the determination of solasonine (SN) and solamargine (SM) in different skin layers, as well as in a topical formulation. The method was linear within the ranges 0.86 to 990.00 mu g/mL for SN and 1.74 to 1000.00 mu g/mL for SM (r = 0.9996). Moreover, the recoveries for both glycoalkaloids were higher than 88.94 and 93.23% from skin samples and topical formulation, respectively. The method developed is reliable and suitable for topical delivery skin studies and for determining the content of SN and SM in topical formulations.

Antimicrobial activity of two techniques for arm skin disinfection of blood donors in Brazil

Objective: Evaluation of the antimicrobial effect of skin disinfection techniques is essential to avoid the transmission of infectious agents during blood transfusion. The aim of this study was to examine the effectiveness of two methods of arm skin disinfection used in blood donors at a Hemotherapy Center in Brazil that represents an important centre for distributing haemocomponents to many cities in the country. Methods: Two skin disinfection techniques in 50 blood donors were evaluated. For the first arm, 10% povidone-iodine/two-stage technique was used. On the opposite arm, 0.5% chlorhexidine digluconate alcohol solution/one-stage technique was used. The swabs were seeded on three culture media: blood agar, mannitol salt agar and Mac Conkey agar. Automated bacterial classification based on biochemical tests/specific substrates was performed. Donor characteristics were collected using the computerised system of the Hemotherapy Center. Results: We found that microbial reduction was significantly higher for 10% povidone-iodine technique (98.57-98.87%) when compared with 0.5% chlorhexidine technique (94.38-95.06%). The species Leuconostoc mesenteroides and Staphylococcus hominis showed resistance to both disinfection techniques. We did not find statistically significant relationships between donor characteristics and microbial reduction. Conclusions: Arm skin disinfection with 10% povidone-iodine produced better antimicrobial activity. We must acknowledge that 10% povidone-iodine technique has the limitation of being a two-stage method. However, prevention of adverse events due to bacterial contamination and transfusion reactions should be prioritised. Production of hypoallergenic and stronger antiseptics that allowed a safe one-stage disinfection technique should be encouraged in health systems, not only in Brazil but also around the world.

The Use of Nanotechnology in Cosmetic Formulations: The Influence of Vehicle in the Vitamin A Skin Penetration

Despite the efficacy of topical retinoic acid, skin reactions have limited its acceptance by patients. Other retinoids, like Retinyl Palmitate (RP), are considerably less irritating, but they are also less effective. In order to enhance the performance of retinoids, in this work RP has been added to cosmetic formulations such as nanoemulsions, which can provide better penetration of this active substance. Because the vehicle can directly influence the skin penetration and the effectiveness of RP, two skin care products containing 5000 UI RP have been developed and investigated, namely a nanoemulsifying system and a classic gel cream. In vitro penetration tests were conducted by using Franz diffusion cells and placing porcine ear skin and iso-propanol in the receptor compartment. The RP concentration in the skin layers was analyzed by high performance liquid chromatography, and a Zeta-Sizer system was employed for measurement of the the particle size distribution. The penetration tests revealed a large difference between the vehicles in terms of the RP concentrations in each skin layer. The classic gel cream furnished better RP penetration in both the stratum corneum and the epidermis without stratum corneum + dermis, as compared to the self-nanoemulsifying system. The two vehicles displayed the same particle size (between 100 and 200 nm). Better understanding of RP skin delivery using different vehicles has been acquired, and the importance of evaluating the efficacy of nanocosmetics. Results from the present study should also contribute to the assessment of commercial self-nanoemulsifying systems with potential application in the facile production of nanoemulsions.

Skin Penetration Time-Profiles for Continuous 810nm and Superpulsed 904nm Lasers in a Rat Model

Objective: The purpose of this study was to investigate the rat skin penetration abilities of two commercially available low-level laser therapy (LLLT) devices during 150 sec of irradiation. Background data: Effective LLLT irradiation typically lasts from 20 sec up to a few minutes, but the LLLT time-profiles for skin penetration of light energy have not yet been investigated. Materials and methods: Sixty-two skin flaps overlaying rat's gastrocnemius muscles were harvested and immediately irradiated with LLLT devices. Irradiation was performed either with a 810 nm, 200mW continuous wave laser, or with a 904 nm, 60mW superpulsed laser, and the amount of penetrating light energy was measured by an optical power meter and registered at seven time points (range, 1-150 sec). Results: With the continuous wave 810nm laser probe in skin contact, the amount of penetrating light energy was stable at similar to 20% (SEM +/- 0.6) of the initial optical output during 150 sec irradiation. However, irradiation with the superpulsed 904 nm, 60mW laser showed a linear increase in penetrating energy from 38% (SEM +/- 1.4) to 58% (SEM +/- 3.5) during 150 sec of exposure. The skin penetration abilities were significantly different (p < 0.01) between the two lasers at all measured time points. Conclusions: LLLT irradiation through rat skin leaves sufficient subdermal light energy to influence pathological processes and tissue repair. The finding that superpulsed 904nm LLLT light energy penetrates 2-3 easier through the rat skin barrier than 810nm continuous wave LLLT, corresponds well with results of LLLT dose analyses in systematic reviews of LLLT in musculoskeletal disorders. This may explain why the differentiation between these laser types has been needed in the clinical dosage recommendations of World Association for Laser Therapy.

Skin picking and trichotillomania in adults with obsessive-compulsive disorder

The objective of this study was to compare patients with obsessive-compulsive disorder (OCD) associated with pathologic skin picking (PSP) and/or trichotillomania, and patients with OCD without such comorbidities, for demographic and clinical characteristics. We assessed 901 individuals with a primary diagnosis of OCD, using the Structured Clinical Interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) Axis I disorders. Diagnoses of PSP and trichotillomania were made in 16.3% and 4.9% of the sample, respectively. After the logistic regression analysis, the following factors retained an association with OCD-PSP/trichotillomania: younger (odds ratio [OR] = 0.979; P = .047), younger at the onset of compulsive symptoms (OR = 0.941; P = .007), woman (OR = 2.538; P < .001), with a higher level of education (OR = 1.055; P = .025), and with comorbid body dysmorphic disorder (OR = 2.363; P = .004). These findings support the idea that OCD accompanied by PSP/trichotillomania characterizes a specific subgroup. (C) 2012 Elsevier Inc. All rights reserved.

Immediate and long-term effects of polysaccharides-based formulations on human skin

A new trend in cosmetic formulations is the use of biotechnological raw materials as the polysaccharides from Klebsiella pneumoniae, which are supposed to enhance cell renewal, improve skin hydration and micro-relief. Botanical extracts of Myrtus communis leaves contain different sugars, which may provide the same benefits. Thus, the objective of this study was to evaluate through objective and subjective analysis the immediate and long-term effects of cosmetic formulations containing polysaccharides biotechnologically-originated and / or the ones contained in Myrtus communis extracts. Three polysaccharide-based and placebo formulations were applied on the forearm skin of 40 volunteers. Skin hydration, transepidermal water loss (TEWL), viscoelasticity and skin micro-relief measurements were made before and 2 hours after a single application and after 15 and 30 day-periods of daily applications. Answers to a questionnaire about perceptions of formulation cosmetic features constituted the subjective analysis. All polysaccharide-based formulations enhanced skin hydration. Formulations with isolated or combined active substances improved skin barrier function as compared to placebo, in the short and long term studies. Formulations containing Myrtus communis extracts had the highest acceptance. Results suggest that daily use of formulations containing these substances is important for protection of the skin barrier function.

Effects of 830 and 670 nm Laser on Viability of Random Skin Flap in Rats

Objective: This study aimed to investigate the effect of 830 and 670 nm diode laser on the viability of random skin flaps in rats. Background data: Low-level laser therapy (LLLT) has been reported to be successful in stimulating the formation of new blood vessels and reducing the inflammatory process after injury. However, the efficiency of such treatment remains uncertain, and there is also some controversy regarding the efficacy of different wavelengths currently on the market. Materials and methods: Thirty Wistar rats were used and divided into three groups, with 10 rats in each. A random skin flap was raised on the dorsum of each animal. Group 1 was the control group, group 2 received 830 nm laser radiations, and group 3 was submitted to 670 nm laser radiation (power density = 0.5 mW/cm(2)). The animals underwent laser therapy with 36 J/cm(2) energy density (total energy = 2.52 J and 72 sec per session) immediately after surgery and on the 4 subsequent days. The application site of laser radiation was one point at 2.5 cm from the flap's cranial base. The percentage of skin flap necrosis area was calculated on the 7th postoperative day using the paper template method. A skin sample was collected immediately after to determine the vascular endothelial growth factor (VEGF) expression and the epidermal cell proliferation index (KiD67). Results: Statistically significant differences were found among the percentages of necrosis, with higher values observed in group 1 compared with groups 2 and 3. No statistically significant differences were found among these groups using the paper template method. Group 3 presented the highest mean number of blood vessels expressing VEGF and of cells in the proliferative phase when compared with groups 1 and 2. Conclusions: LLLT was effective in increasing random skin flap viability in rats. The 670 nm laser presented more satisfactory results than the 830 nm laser.

Discrimination of Basal Cell Carcinoma and Melanoma from Normal Skin Biopsies in Vitro Through Raman Spectroscopy and Principal Component Analysis

Objective: Raman spectroscopy has been employed to discriminate between malignant (basal cell carcinoma [BCC] and melanoma [MEL]) and normal (N) skin tissues in vitro, aimed at developing a method for cancer diagnosis. Background data: Raman spectroscopy is an analytical tool that could be used to diagnose skin cancer rapidly and noninvasively. Methods: Skin biopsy fragments of similar to 2 mm(2) from excisional surgeries were scanned through a Raman spectrometer (830 nm excitation wavelength, 50 to 200 mW of power, and 20 sec exposure time) coupled to a fiber optic Raman probe. Principal component analysis (PCA) and Euclidean distance were employed to develop a discrimination model to classify samples according to histopathology. In this model, we used a set of 145 spectra from N (30 spectra), BCC (96 spectra), and MEL (19 spectra) skin tissues. Results: We demonstrated that principal components (PCs) 1 to 4 accounted for 95.4% of all spectral variation. These PCs have been spectrally correlated to the biochemicals present in tissues, such as proteins, lipids, and melanin. The scores of PC2 and PC3 revealed statistically significant differences among N, BCC, and MEL (ANOVA, p < 0.05) and were used in the discrimination model. A total of 28 out of 30 spectra were correctly diagnosed as N, 93 out of 96 as BCC, and 13 out of 19 as MEL, with an overall accuracy of 92.4%. Conclusions: This discrimination model based on PCA and Euclidean distance could differentiate N from malignant (BCC and MEL) with high sensitivity and specificity.

Decreased RNA expression of interleukin 17A in skin of leprosy

Interleukin-17A (IL-17A) is a proinflamatory cytokine that plays an important role in fighting pathogens at mucosal interfaces, by summoning neutrophils and upregulating cytoplasmatic antimicrobial peptides. So far, the presence of IL-17A in leprosy has not been demonstrated. The expression of IL-17A and related cytokines (IL-6 and IL-23p19) was addressed through RNA extraction and cDNA quantitative amplification in macerated biopsies of active lesions of 48 leprosy patients and 20 fragments of normal skin of individuals. Blood levels of IL-17A, IL-23p19 and IL-6 were determined by ELISA. We found an abrogated mRNA IL-17A response in all biopsies of leprosy patients, as compared with controls. Circulating IL-17A and IL-23p19 were undetectable in both patients and controls, but IL-6 was higher in lepromatous patients. Although at low levels, IL-17A mRNA in lepromatous patients had an inverse linear correlation with bacillary burden. Low expression of IL-17A in patients may be a constitutive genetic feature of leprosy patients or a circumstantial event induced by the local presence of the pathogen, as an escape mechanism.

Clinical, histopathological and immunohistochemical assessment of human skin field cancerization before and after photodynamic therapy

Background The field cancerization concept in photodamaged patients suggests that the entire sun-exposed surface of the skin has an increased risk for the development of (pre)-malignant lesions, mainly epithelial tumours. Topical photodynamic therapy (PDT) is a noninvasive therapeutic method for multiple actinic keratosis (AK) with excellent outcome. Objectives To evaluate the clinical, histological and immunohistochemical changes in human skin with field cancerization after multiple sessions of PDT with methyl-aminolaevulinate (MAL). Methods Twenty-six patients with photodamaged skin and multiple AK on the face received three consecutive sessions of MAL-PDT with red light (37 J cm(-2)), 1 month apart. Biopsies before and 3 months after the last treatment session were taken from normal-appearing skin on the field-cancerized area. Immunohistochemical stainings were performed for TP-53, procollagen-I, metalloproteinase-1 (MMP-1) and tenascin-C (Tn-C). Results All 26 patients completed the study. The global score for photodamage improved considerably in all patients (P < 0.001). The AK clearance rate was 89.5% at the end of the study. Two treatment sessions were as effective as three MAL-PDT sessions. A significant decrease in atypia grade and extent of keratinocyte atypia was observed histologically (P < 0.001). Also, a significant increase in collagen deposition (P = 0.001) and improvement of solar elastosis (P = 0.002) were noticed after PDT. However, immunohistochemistry showed only a trend for decreased TP-53 expression (not significant), increased procollagen-I and MMP-1 expressions (not significant) and an increased expression of Tn-C (P = 0.024). Conclusions Clinical and histological improvement in field cancerization after multiple sessions of MAL-PDT is proven. The decrease in severity and extent of keratinocyte atypia associated with a decreased expression of TP-53 suggest a reduced carcinogenic potential of the sun-damaged area. The significant increase of new collagen deposition and the reduction of solar elastosis explain the clinical improvement of photodamaged skin.

Stem cells in embryonic skin development

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.

Effect of ultraviolet filters on skin superoxide dismutase activity in hairless mice after a single dose of ultraviolet radiation

Organic sunscreens may decrease their protective capability and also behave as photo-oxidants upon ultraviolet radiation (UVR) exposure. The present study investigated the effect of a cream gel formulation containing the UV filters benzophenone-3, octyl methoxycinnamate, and octyl salicylate on skin superoxide dismutase (SOD) after a single dose of UVR (2.87 J/cm(2)). The retention of these UV filters was first evaluated in vivo using hairless mice to guarantee the presence of the filters in the skin layers at the moment of irradiation. The in vivo effect of the UV filters on skin SOD was then assayed spectrophotometrically via the reduction of cytochrome c. The cream gel formulation promoted the penetration of the three UV filters into the epidermis and the dermis at one hour post-application. A significant decrease in SOD activity was observed in irradiated animals treated with sunscreen formulation. However, no effect on SOD activity in skin was observed by the isolated presence of the sunscreens, the formulation components, or the exposure to UVR. The sunscreens may have formed degradation products under UVR that may have either inhibited the enzyme or generated reactive species in the skin. (C) 2011 Elsevier B.V. All rights reserved.

Effect of topical clay application on the synthesis of collagen in skin: an experimental study

Background. Clay is often used in cosmetic treatments, although little is known about its action. Aim. To evaluate the effect of topical clay application on the histoarchitecture of collagen fibres in rat skin. Methods. Animals received a daily application of clay and retinoic acid (RA) 0.025% to the dorsal skin over 7 and 14 days, under vaporization at 37 degrees C for 40 min. Control skin was not vaporized. Samples from each region were excised, and stained with picrosirius red for collagen evaluation. Results. Seven days after clay treatment, an increase in the number of collagen fibres was observed in treated skin compared with control skin (51.74 +/- 1.28 vs. 43.39 +/- 1.79%, respectively, P < 0.01), whereas RA did not alter the collagen level (45.66 +/- 1.10%). Clay application over 14 days did not induce a further increase in skin collagen, whereas treatment with RA did (58.07 +/- 1.59%; P = 0.001 vs. control). Conclusion. Clay application promotes an increase in the number of collagen fibres, which may account for its beneficial effects.