In vivo evaluation of sunscreen application by multispectral imaging: A new tool for educating sunscreen users.

BACKGROUND: The sun protection factor (SPF) of sunscreens is evaluated using standardized protocols based on the application of 2 mg/cm2 of product. However, the amount of product applied by sunscreen users in real life is likely to be much lower. OBJECTIVES: To evaluate a new multispectral imaging approach for determining the actual quantity of sunscreen applied by users and to assess the benefits of an application guide for the use of an SPF 50+ sunscreen. MATERIALS AND METHODS: Analyses of the reflectance spectra obtained from multispectral images were used to determine the actual dose of sunscreen that 26 healthy volunteers applied to their face following three application modalities: a single application, reapplication after 30 min, and application according to an instruction guide. RESULTS: Without the application guide, volunteers applied an average of 1.04 mg/cm2 of sunscreen during the single application and 1.23 mg/cm2 during the repeated application. With the application guide, the amount of sunscreen applied was 1.45 mg/cm2 : around 40% higher than during the single application. Spreading of the sunscreen was also less uniform with the unguided single application than with the other application modalities. CONCLUSIONS: This study showed that the multispectral imaging approach can be used to measure the amount of sunscreen applied in vivo. Our findings confirmed that the standard dose used for SPF measurements and other sunscreen tests is far higher than that applied by users in practice. Providing users with precise guidelines could increase the amount of sunscreen applied, resulting in more adequate photoprotection.

DNA photoproducts released by repair in biological fluids as biomarkers of the genotoxicity of UV radiation.

UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.

High bacterial colonization and lipase activity in microcomedones.

BACKGROUND: Although acne vulgaris has a multifactorial aetiology, comedogenesis and bacteria colonization of the pilosebaceous unit are known to play a major role in the onset of inflammatory acne lesions. However, many aspects remain poorly understood such as where and when is the early stage of the Propionibacterium acnes colonization in follicular unit? Our research aimed at providing a precise analysis of microcomedone's structure to better understand the interplay between Propionibacterium acnes and follicular units, and therefore, the role of its interplay in the formation of acne lesions. METHODS: Microcomedones were sampled using cyanoacrylate skin surface stripping (CSSS). Their morphology was investigated with multiphoton imaging and their ultrastructure with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bacterial lipase activity in the microcomedones was quantified using a dedicated enzymatic test as well as a Fourier Transform Infra-Red (FTIR) analysis. The porphyrin produced by bacteria was analysed with HPTLC and fluorescence spectroscopy. RESULTS: The imaging analysis showed that microcomedones' structure resembles a pouch, whose interior is mostly composed of lipids with clusters of bacteria and whose outer shell is made up of corneocyte layers. The extensive bacteria colonization is clearly visible using TEM. Even after sampling, clear lipase activity was still seen in the microcomedone. A high correlation, r = .85, was observed between porphyrin content measured with HPTLC and with fluorescence spectroscopy. These observations show that microcomedones, which are generally barely visible clinically, already contain a bacterial colonization.

Correction: Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin.

Correction for 'Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin' by Sana Tfaili et al., Analyst, 2012, 137, 3673-3682, DOI: .

Raman characterization of human skin aging.

BACKGROUND: Skin aging is a complex biological process mixing intrinsic and extrinsic factors, such as sun exposure. At the molecular level, skin aging affects in particular the extracellular matrix proteins. MATERIALS AND METHODS: Using Raman imaging, which is a nondestructive approach appropriate for studying biological samples, we analyzed how aging modifies the matrix proteins of the papillary and reticular dermis. Biopsies from the buttock and dorsal forearm of volunteers younger than 30 and older than 60 were analyzed in order to identify chronological and photoaging processes. Analyses were performed on skin section, and Raman spectra were acquired separately on the different dermal layers. RESULTS: We observed differences in dermal matrix structure and hydration state with skin aging. Chronological aging alters in particular the collagen of the papillary dermis, while photoaging causes a decrease in collagen stability by altering proline and hydroxyproline residues in the reticular dermis. Moreover, chronological aging alters glycosaminoglycan content in both dermal compartments. CONCLUSION: Alterations of the papillary and reticular dermal matrix structures during photo- and chronological aging were clearly depicted by Raman spectroscopy.

Visualization of dendritic cells' responses in atopic dermatitis: Preventing effect of emollient.

Atopic dermatitis (AD) is a chronic and multifactorial inflammatory skin disease involving various dendritic cells such as epidermal Langerhans cells (LC) and inflammatory dendritic epidermal cells (IDECs). Most of the clinical studies was performed on isolated cells, and thus, it would be useful to characterize directly on the human epidermal tissue the first cellular events occurred during the AD. The suction blister method was used to obtain whole epidermis samples and interstitial cutaneous fluids. Employing multiphoton microscopy, we analyzed the early dynamic behavior of inflammatory cells using Dermatophagoides pteronyssinus atopy patch test (Derp-APT) and evaluated the effects of emollient pre-application. Derp-APT application provoked rapid and strong infiltration of IDECs, and proliferation and activation of LC in the AD subjects' epidermis. Moreover, emollient pre-application strengthened the defective skin barrier and had positive effects on inflammatory cells' behavior, characterized by the complete inhibition of IDEC influx and the presence of immature LC.

Sub-optimal Application of a High SPF Sunscreen Prevents Epidermal DNA Damage in Vivo.

The cyclobutane pyrimidine dimer (CPD) is a potentially mutagenic DNA photolesion that is the basis of most skin cancers. There are no data on DNA protection by sunscreens under typical conditions of use. The study aim was to determine such protection, in phototypes I/II, with representative sunscreen-user application. A very high SPF formulation was applied at 0.75, 1.3 and 2.0 mg/cm2. Unprotected control skin was exposed to 4 standard erythema doses (SED) of solar simulated UVR, and sunscreen-treated sites to 30 SED. Holiday behaviour was also simulated by UVR exposure for 5 consecutive days. Control skin received 1 SED daily, and sunscreen-treated sites received 15 (all 3 application thicknesses) or 30 (2.0 mg/cm2) SED daily. CPD were assessed by quantitative HPLC-tandem mass spectrometry (HPLC-MS/MS) and semi-quantitative immunostaining. In comparison with unprotected control sites, sunscreen significantly (p ≤ 0.001-0.05) reduced DNA damage at 1.3 and 2.0 mg/cm2 in all cases. However, reduction with typical sunscreen use (0.75 mg/cm2) was non-significant, with the exception of HPLC-MS/MS data for the 5-day study (p <0.001). Overall, these results support sunscreen use as a strategy to reduce skin cancer, and demonstrate that public health messages must stress better sunscreen application to get maximal benefit.

Vibrational spectroscopies for the analysis of cutaneous permeation: experimental limiting factors identified in the case of caffeine penetration.

Caffeine is utilised as a reference for permeation studies in dermatology and cosmetology. The present work aimed to monitor the permeation of a caffeine solution through the skin. For this purpose, Raman and infrared studies were performed. Raman microspectroscopy permitted a dynamic follow-up of the caffeine diffusion. In complementary, infrared microimaging provided information of the caffeine localization in the skin by applying multivariate statistical processing on skin tissue sections. Herein, we prove the possibility of tracking low concentrations of caffeine through the skin and we highlight some experimental limitations of vibrational spectroscopies.

Nuclear and Urinary Measurements Show the Efficacy of Sun-Protection Factor 50+ Sunscreen against DNA Photoproducts upon Real-Life Recreational Exposure.

Sunscreens have been shown to protect against UVR-induced DNA damage in human skin under laboratory conditions. We presently extended these observations to real-life conditions in volunteers after their ordinary exposure habits during summer holidays. Volunteers were randomly assigned to a control group and an educated group supplied with a SPF ≥50 sunscreen and receiving instructions for use. A questionnaire was used to determine the extent of exposure. No difference in average solar UVR exposure was found between the two groups. DNA photoprotection was first assessed by, to our knowledge, a previously unreported noninvasive assay on the basis of the quantification of pyrimidine dimers released by DNA repair in urine. Damage was also quantified in the nuclear DNA extracted from the roof of suction blisters collected after recreational exposure. The urinary concentration of photoproducts was significantly higher in the control than in the educated group. The same trend was observed for the level of photoproducts in the DNA from suction blisters. The unambiguous observation of an efficient photoprotection against DNA damage afforded by sunscreen under real-life conditions provides strong support for the efficiency of the sunscreens. In addition, the results validate the use of urinary DNA photoproducts as a noninvasive assay applicable to photoprotection.

Shedding light on the laser wavelength effect in Raman analysis of skin epidermises.

Confocal Raman microspectroscopy is a promising technique which enables measuring the molecular composition of the skin layers, non-destructively and without extrinsic markers. The Raman approach is increasingly used in skin research but with various experimental conditions. In addition to the different skin types, one of the varying parameters is the wavelength of laser excitation. This parameter contributes strongly in the skin Raman response. The present work aimed to evaluate this effect for 3 different wavelengths, 532, 633 and 785 nm, on pig ear skin models. The Raman signal was assessed in the spectral fingerprint region. According to the Raman response for stability, repeatability, variability and fluorescence contribution, the 785 nm excitation wavelength was shown to be the most suitable for epidermis depth profiling in the fingerprint region.

Confocal Raman microspectroscopy for skin characterization: a comparative study between human skin and pig skin.

The present paper provides a spectral comparison between abdominal human skin (Transkin) and pig ear skin using confocal Raman microspectroscopy at 660 nm. Pig ear skin is usually utilized as a substitute for human skin for active ingredients assessment in dermatological and cosmetics fields. Herein, the comparison is made at the level of the stratum corneum (SC), the SC/epidermis junction and the viable epidermis. The 660 nm excitation source appears to be the most appropriate wavelength for such skin characterization. From Raman signatures of both skin types, a tentative assignment of vibrations was performed in the fingerprint and the high wavenumber spectral regions. Significant differences were highlighted for lipid content in in-depth spectra and for hyaluronic acid (HA) and carotenoid in SC spectra. Marked tissular variability was also revealed by certain Raman vibrations. These intrinsic molecular data probed by confocal Raman microspectroscopy have to be considered for further applications such as cutaneous drug permeation.

EczemaPred: A computational framework for personalised prediction of eczema severity dynamics.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease leading to substantial quality of life impairment with heterogeneous treatment responses. People with AD would benefit from personalised treatment strategies, whose design requires predicting how AD severity evolves for each individual. OBJECTIVE: This study aims to develop a computational framework for personalised prediction of AD severity dynamics. METHODS: We introduced EczemaPred, a computational framework to predict patient-dependent dynamic evolution of AD severity using Bayesian state-space models that describe latent dynamics of AD severity items and how they are measured. We used EczemaPred to predict the dynamic evolution of validated patient-oriented scoring atopic dermatitis (PO-SCORAD) by combining predictions from the models for the nine severity items of PO-SCORAD (six intensity signs, extent of eczema, and two subjective symptoms). We validated this approach using longitudinal data from two independent studies: a published clinical study in which PO-SCORAD was measured twice weekly for 347 AD patients over 17 weeks, and another one in which PO-SCORAD was recorded daily by 16 AD patients for 12 weeks. RESULTS: EczemaPred achieved good performance for personalised predictions of PO-SCORAD and its severity items daily to weekly. EczemaPred outperformed standard time-series forecasting models such as a mixed effect autoregressive model. The uncertainty in predicting PO-SCORAD was mainly attributed to that in predicting intensity signs (75% of the overall uncertainty). CONCLUSIONS: EczemaPred serves as a computational framework to make a personalised prediction of AD severity dynamics relevant to clinical practice. EczemaPred is available as an R package.

Long-Term Use of Silybum marianum fruit extract Contributes to Homeostasis in Acne-Prone Skin-A 12-Month Follow-Up International "Real Life" Cohort Study.

BACKGROUND: Homeostasis in the differentiation programme of sebaceous stem cells has been identified as a key step in comedogenesis and should be a target for acne-prone skin care. OBJECTIVE: To report on a multicentre, year-long/real-life use study of a patented natural product containing S. marianum fruit extract proven to modulate molecular actors in the initial steps of comedogenesis. METHODS: An open-label multicentric international study, with a 12 month follow-up, included 54 teenage and young adult subjects with mild to moderate facial acne. The study was aimed at reproducing a real-life use context. RESULTS: Total lesion count mean was 88.3 at inclusion. There was a sustained, highly significant decrease over the months of clinical lesion counts (45.6% improvement after 6 months and 59.6% at 12 months) and on other efficacy markers, associated with a significant decrease in global microcomedone quantity on cyanoacrylate superficial skin surface biopsies. Importantly, the study protocol allowed the dermatologist to prescribe, if needed as in real life, any of the acne drugs registered in the acne guidelines. The exposure to these acne drugs during the whole year was calculated as a percentage of S. marianum fruit extract/352 days of use and happened to be very limited at less than 4%, which indicates a marginal contribution to the sustained clinical improvement. (Oral and local acne treatments: Lymecycline 1.46%; Doxycycline 0.24%; Adapalene 0.16% or gel association with Benzoyl peroxide 1.17%; Clindamycin 0.04%; Benzoyl peroxide 1.5%; Erythromycin 0.75%). The tolerance with daily S. marianum fruit extract long-term use was good. LIMITATIONS: The association with routine prescription acne drugs when needed, even if limited, does not allow a full evaluation of the intrinsic quantitative efficacy of S. marianum fruit extract in lesion reduction. CONCLUSION: This open, real-life, year-long multicentre study confirms a previous 48-week proof of concept study and qualifies the use of S. marianum fruit extract as a "field-dermo cosmetic" contributing to homeostasis of acne-prone skin in association with acne drugs.

Lipid Droplet Proteins in Acne Skin: A Sound Target for the Maintenance of Low Comedogenic Sebum and Acne-Prone Skin Health.

In adipocytes and sebocytes, lipid droplet proteins control the storage of lipids in organized droplets and their release on demand. The contribution of lipid droplet proteins to the pathogenesis of acne is plausible because they control the levels of comedogenic free fatty acids. The expression of two lipid droplet proteins, CIDEA and PLIN2, was analyzed in the skin of patients with acne by immunohistochemistry and western blotting. The design of clinical protocols allowed correlating the expression of CIDEA and PLIN2 with both comedogenesis and the release of free fatty acids. Both proteins were detected by immunohistochemistry in the sebaceous glands of patients with acne, with a disturbed expression pattern of PLIN2 compared with that in the controls. Higher levels of PLIN2 and CIDEA, as detected by western blotting in the infundibulum, significantly correlated with lower ongoing comedogenesis over 48 weeks of Silybum marianum fruit extract application. Accordingly, free fatty acid release from sebum triglycerides was significantly decreased, as shown with two distinct methods. The data are consistent with the expected role of PLIN2 and CIDEA in the prevention of comedogenic free fatty acid release. Modulation of PLIN2 and CIDEA expression appears as a sound target for the maintenance of low comedogenic sebum and acne-prone skin health.

Assessment of the clinical efficacy of a hyaluronic acid-based deep wrinkle filler using new instrumental methods.

BACKGROUND: The efficacy of numerous hyaluronic acid (HA)-based fillers has been demonstrated by semi-quantitative and qualitative methods, useful in clinical practice, but poorly reliable. OBJECTIVE: To objectively evaluate the efficacy of a HA gel in treating nasolabial folds (NLFs) over a 9-12-month follow-up period. METHODS: A total of 47 adult patients with moderate to severe NLFs received one or two injections of HA gel. Efficacy was assessed by measuring NLF depth at time intervals up to 12 months subjectively by blind and open clinical scoring using the Lemperle scale, and objectively using skin replicas and in vivo 3D imaging methods. Tissue characterization and dermal thickness were also assessed using radiofrequency ultrasonography and high-resolution ultrasound imaging, respectively. RESULTS: The filler injection highly significantly decreased the depth of NLFs (p < 0.0001) at all time points, with an improvement of at least 1 grade in the Lemperle score in 77% and 89% of the subjects at 9 and 12 months, respectively. NLF volume measured on replicas and 3D images significantly decreased after injection and this improvement was maintained over 12 months. CONCLUSION: This HA gel is well tolerated and provides a significant and long-lasting correction of moderate to severe NLFs, as objectively demonstrated by instrumental methods.

Efficacy of D-pigment dermocosmetic lightening product for solar lentigo lesions of the hand: A randomized controlled trial.

Solar lentigo, benign lesions which mostly appear on chronically, sun-exposed surfaces, are associated with ageing. Patients are increasingly requesting a more uniform skin texture, especially for hands. Treatment options include dermoabrasion, intense pulsed light, cryotherapy, peelings, and laser therapy. Topical compounds can be employed, in alternative or associated with dermatologic procedures. The current study was designed to evaluate solar lentigo hyperpigmentation, skin architecture and clinician and patient assessments comparing a dermocosmetic lightening product (active) with a moisturizing product (control) according to clinical, digital and subjective analyses in 72 lesions over 12-month follow up period. Statistically significant differences were observed between the lesions treated with the active compared to the control in terms of papillary brightness (p = 0.03) and contrast (p = 0.03), and in the limitation of dermal-epidermal junction destructuring (p = 0.03) according to dermal-epidermal junction destructuring score at Reflectance Confocal Microscopy. Luminance (p = 0.04) and redness (p = 0.03) were improved at color analysis, and physician and patient evaluations favored the active in efficacy and patient satisfaction investigations. The dermocosmetic lightening product utilized in the current study proved to be more effective, according to clinical, digital and subjective analyses in reducing lesion hyperpigmentation, stabilizing the lesion skin architecture and increasing patient satisfaction compared to the control in a cohort of 36 subjects, over a 12-month period. Beside demonstrating the efficacy of this topical lightening product, we propose a "destructuring score", which improves the robustness of solar lentigo's evaluation, and can be used in future studies to standardize the quantitative comparisons of different treatment options.

Dermal fiber structures and photoaging.

The use of multiphoton imaging has become a standard technique to visualize the dermis fibers as it requires no specific staining. The density and organization of collagen and elastin are common markers of skin intrinsic aging and photoaging; thus, there is a need of grading this skin aging with quantitative indicators able to provide a robust evaluation of the dermis fibers' state. We propose a systematic analysis of multiphoton images of skin biopsies taken on the buttock and the forearm of patients of different ages. The intensity histograms of images were analyzed through their moments, a wavelet decomposition was done, and the wavelet coefficients distribution was fitted by a generalized Gaussian distribution. Different parameters relative to the collagen or elastin densities, organizations, and structures were calculated and exhibit phenomena specific to intrinsic or extrinsic aging. Those indicators could become a standard method to analyze the degree of skin aging (intrinsic or extrinsic) through multiphoton imaging.

Joint segmentation and characterization of the dermis in 50 MHz ultrasound 2D and 3D images of the skin.

We propose a novel joint segmentation and characterization algorithm for the assessment of skin aging using 50 MHz high-frequency ultrasound images. The proposed segmentation method allows a fine determination of the envelope signal's statistics in the dermis as a function of depth. The sequence of statistical estimates obtained is then combined into a single aging score. The segmentation is based on tailored recursive non-linear filters. The epidermis and the dermis are jointly segmented with a non-parametric active contour combining a texture criterion, an epidermis indicator map and the geometric constraint of horizontal continuity. The algorithm is designed to apply to 2D and 3D images as well. We evaluated skin photo-aging on ultrasound images with an experimental study on a cohort of 76 women separated into 2 groups of different ages. Two aging scores are computed from the images: local dermal contrast and skin roughness. We show that these scores are much better at identifying the two groups (p-value ≈10-6) than the previously used MGVR indicator (p-value 0.046). Moreover, we find that a combined score more reliably evaluates skin photo-aging, with 84% success, than a scoring of the ultrasound images by 4 experts.

The use of suction blisters to measure sunscreen protection against UVR-induced DNA damage.

The formation of DNA photoproducts caused by solar UVR exposure needs to be investigated in-vivo and in particular in order to assess sunscreens' level of protection against solar genotoxicity. The study's purposes were: i) to evaluate if the roof of suction blisters is an appropriate sampling method for measuring photoproducts, and ii) to measure in-vivo sunscreen protection against cyclobutane pyrimidine dimers. Skin areas on the interior forearms of eight healthy volunteers were exposed in-vivo to 2 MED of simulated solar radiation (SSR) and to 15 MED on a sunscreen protected area. After irradiation, six suction blisters were induced and the blister roofs were collected. Analysis of SSR-induced CPDs was performed by two independent methods: a chromatography coupled to mass spectroscopy (HPLC-MS/MS) approach and a 3D-imaging of CPD immunostaining by multiphoton microscopy on floating epidermal sheets. HPLC-MS/MS analyses showed that SSR-unexposed skin presented no CPD dimers, whereas 2 MED SSR-exposed skin showed a significant number of TT-CPD. The sunscreen covered skin exposed to 15 MED appeared highly protected from DNA damage, as the amount of CPD-dimers remained below the detection limit. The multiphoton-immunostaining analysis consistently showed that no CPD staining was observed on the non-SSR-exposed skin. A significant increase of CPD staining intensity and number of CPD-positive cells were observed on the 2 MED SSR-exposed skin. Sunscreen protected skin presented a very low staining intensity and the number of CPD-positive cells remained very close to non-SSR-exposed skin. This study showed that suction blister samples are very appropriate for measuring CPD dimers in-vivo, and that sunscreens provide high protection against UVR-induced DNA damage.