Peter J. Caspers

Combined In Vivo Confocal Raman Spectroscopy and Confocal Microscopy of Human Skin

In vivo confocal Raman spectroscopy is a noninvasive optical method to obtain detailed information about the molecular composition of the skin with high spatial resolution. In vivo confocal scanning laser microscopy is an imaging modality that provides optical sections of the skin without physically dissecting the tissue. A combination of both techniques in a single instrument is described. This combination allows the skin morphology to be visualized and (subsurface) structures in the skin to be targeted for Raman measurements. Novel results are presented that show detailed in vivo concentration profiles of water and of natural moisturizing factor for the stratum corneum that are directly related to the skin architecture by in vivo cross-sectional images of the skin. Targeting of skin structures is demonstrated by recording in vivo Raman spectra of sweat ducts and sebaceous glands in situ. In vivo measurements on dermal capillaries yielded high-quality Raman spectra of blood in a completely noninvasive manner. From the results of this exploratory study we conclude that the technique presented has great potential for fundamental skin research, pharmacology (percutaneous transport), clinical dermatology, and cosmetic research, as well as for noninvasive analysis of blood analytes, including glucose.

In Vitro and In Vivo Raman Spectroscopy of Human Skin

Noninvasive techniques that provide detailed information about molecular composition, structure, and interactions are crucial to further our understanding of the relation between skin disease and biochemical changes in the skin, as well as for the development of penetration enhancers for transdermal drug administration. In this study we present in vitro and in vivo Raman spectra of human skin. Using a Raman microspectrometer, in vitro spectra were obtained of thin cross sections of human skin. They provided insight into the molecular composition of different skin layers. Evidence was found for the existence of a large variation in lipid content of the stratum corneum. A simple experimental setup for in vivo confocal Raman microspectroscopy of the skin was developed. In vivo Raman spectra of the stratum corneum were obtained at different positions of the arm and hand of three volunteers. They provided evidence for differences in the concentration of natural moisturizing factor at these positions.

Loss-of-Function Mutations in the Filaggrin Gene Lead to Reduced Level of Natural Moisturizing Factor in the Stratum Corneum

TO THE EDITOR Filaggrin is a key protein required for the formation of the stratum corneum (SC) barrier. Filaggrin is also essential for SC hydration, as it acts as a source of hygroscopic amino acids and their derivatives, known as natural moisturizing factor (NMF). The human gene encoding filaggrin (FLG) is highly polymorphic and to date, 15 null mutations have been detected of which four (R501X, 2282del4, R2447X, and S3247X) are prevalent at varying frequencies in the white European population (Sandilands et al., 2007). Homozygous or compound heterozygous FLG mutations underlie the common skin-keratinizing disorder ichthyosis vulgaris, and have been shown to be a major genetic predisposing factor for atopic dermatitis (AD) (Sandilands et al., 2006). Diminished filaggrin expression has been demonstrated in both ichthyosis vulgaris and AD skin (Seguchi et al., 1996; Sugiura et al., 2005; Smith et al., 2006). As filaggrin is the precursor protein for the amino-acid-derived components of the NMF, we hypothesized that carriers of FLG-null mutations have reduced level of NMF in the SC. To measure NMF in the SC of the palm (thenar eminence) and forearm skin, we used confocal Raman microspectroscopy (3510 Skin Composition Analyzer; River Diagnostics, Rotterdam, The Netherlands). The principles of this method and the procedure have extensively been described elsewhere (Caspers et al., 2001, 2003). The reference spectrum of NMF was constructed from a superposition of the spectra of pyrrolidone-5-carboxylic acid, ornithine, serine, proline, glycine, histidine, and alanine. In addition to NMF, skin barrier function as measured by transepidermal water loss was assessed on the volar forearm (Tewameter 210; Courage and Khazaka Electronic GmbH, Cologne, Germany). One hundred and forty-nine volunteers recruited by public advertisement, as well as 10 AD patients, were screened for four FLG mutations (R501X, 2282del4 R2447X, and S3247X). All subjects filled in a questionnaire on the history of skin diseases and allergies, and the Erlangen atopy questionnaire that also included a question on skin dryness. Signs of active disease (erythema, crusting, weeping, and lichenification) were assessed by a dermatologist. Having visible skin changes on the forearm was the exclusion criterion. Written informed consent was obtained from all subjects. The experimental protocol followed the Declaration of Helsinki Principles and was approved by the Ethical Committee of the Academic Medical Centre. Genomic DNA was extracted from buccal swab samples (Puregene DNA isolation kit; Gentra Systems, Minneapolis, MN). Polymorphisms were genotyped as reported previously (Sandilands et al., 2007). To compare data from two groups, we used twotailed Student’s t-test for unpaired samples. Sixteen carriers (12 female) of an FLG mutation and 23 individuals (15 female) wild type with respect to these mutations were included in the study. Of the 16 carriers, five were heterozygous for R501X, eight were heterozygous for 2282del4, and one was heterozygous for R2447X. One individual was homozygous for Abbreviations: AD, atopic dermatitis; FLG, human filaggrin-encoding gene; NMF, natural moisturizing factor; SC, stratum corneum

Automated depth‐scanning confocal Raman microspectrometer for rapid in vivo determination of water concentration profiles in human skin

An automated confocal Raman microspectrometer for rapid measurement of molecular concentration profiles in the skin is described. It permits the successive collection of Raman spectra at a range of depths below the skin surface. The axial resolution of the confocal Raman microspectrometer is 5.1±0.2 µm. The setup was applied to determine water concentration profiles of the stratum corneum and to determine changes therein as a result of hydration of the skin. Copyright

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

Filaggrin loss-of-function mutations are associated with enhanced expression of IL-1 cytokines in the stratum corneum of patients with atopic dermatitis and in a murine model of filaggrin deficiency

Background Filaggrin (FLG) mutations result in reduced stratum corneum (SC) natural moisturizing factor (NMF) components and consequent increased SC pH. Because higher pH activates SC protease activity, we hypothesized an enhanced release of proinflammatory IL-1 cytokines from corneocytes in patients with atopic dermatitis (AD) with FLG mutations (ADFLG) compared with that seen in patients with AD without these mutations (ADNON-FLG). Objectives We sought to investigate SC IL-1 cytokine profiles in the uninvolved skin of controls and patients with ADFLG versus patients with ADNON-FLG. We also sought to examine the same profiles in a murine model of filaggrin deficiency (Flgft/Flgft [FlgdelAPfal] mice). Methods One hundred thirty-seven patients were studied. NMF levels were ascertained using confocal Raman spectroscopy; transepidermal water loss and skin surface pH were measured. IL-1α, IL-1β, IL-18, IL-1 receptor antagonist (IL-1RA), and IL-8 levels were determined in SC tape strips from 93 patients. All subjects were screened for 9 FLG mutations. Flgft/Flgft (FlgdelAPfal) mice, separated from maFlgft/maFlgft (flaky tail) mice, were used for the preparation and culture of primary murine keratinocytes and as a source of murine skin. RT-PCR was performed using primers specific for murine IL-1α, IL-1β, and IL-1RA. Results SC IL-1 levels were increased in patients with ADFLG; these levels were inversely correlated with NMF levels. NMF values were also inversely correlated with skin surface pH. Skin and keratinocytes from Flgft/Flgft mice had upregulated expression of IL-1β and IL-1RA mRNA. Conclusions ADFLG is associated with an increased SC IL-1 cytokine profile; this profile is also seen in a murine homologue of filaggrin deficiency. These findings might have importance in understanding the influence of FLG mutations on the inflammasome in the pathogenesis of AD and help individualize therapeutic approaches.

Discriminating basal cell carcinoma from perilesional skin using high wave-number Raman spectroscopy

An expanding body of literature suggests Raman spectroscopy is a promising tool for skin cancer diagnosis and in-vivo tumor border demarcation. The development of an in-vivo diagnostic tool is, however, hampered by the fact that construction of fiber optic probes suitable for Raman spectroscopy in the so-called fingerprint region is complicated. In contrast, the use of the high wave-number region allows for fiber optic probes with a very simple design. We investigate whether high wave-number Raman spectroscopy (2800 to 3125 cm(-1)) is able to provide sufficient information for noninvasive discrimination between basal cell carcinoma (BCC) and noninvolved skin. Using a simple fiber optic probe, Raman spectra are obtained from 19 BCC biopsy specimens and 9 biopsy specimens of perilesional skin. A linear discriminant analysis (LDA)-based tissue classification model is developed, which discriminates between BCC and noninvolved skin with high accuracy. This is a crucial step in the development of clinical dermatological applications based on fiber optic Raman spectroscopy.

Caspase-14 Is Required for Filaggrin Degradation to Natural Moisturizing Factors in the Skin

Caspase-14 is a protease that is mainly expressed in suprabasal epidermal layers and activated during keratinocyte cornification. Caspase-14-deficient mice display reduced epidermal barrier function and increased sensitivity to UVB radiation. In these mice, profilaggrin, a protein with a pivotal role in skin barrier function, is processed correctly to its functional filaggrin (FLG) repeat unit, but proteolytic FLG fragments accumulate in the epidermis. In wild-type stratum corneum, FLG is degraded into free amino acids, some of which contribute to generation of the natural moisturizing factors (NMFs) that maintain epidermal hydration. We found that caspase-14 cleaves the FLG repeat unit and identified two caspase-14 cleavage sites. These results indicate that accumulation of FLG fragments in caspase-14(-/-) mice is due to a defect in the terminal FLG degradation pathway. Consequently, we show that the defective FLG degradation in caspase-14-deficient skin results in substantial reduction in the amount of NMFs, such as urocanic acid and pyrrolidone carboxylic acid. Taken together, we identified caspase-14 as a crucial protease in FLG catabolism.

Tissue characterization using high wave number Raman spectroscopy.

Raman spectroscopy is a powerful diagnostic tool, enabling tissue identification and classification. Mostly, the so-called fingerprint (approximately 400-1800 cm(-1)) spectral region is used. In vivo application often requires small flexible fiber-optic probes, and is hindered by the intense Raman signal that is generated in the fused silica core of the fiber. This necessitates filtering of laser light, which is guided to the tissue, and of the scattered light collected from the tissue, leading to complex and expensive designs. Fused silica has no Raman signal in the high wave number region (2400-3800 cm(-1)). This enables the use of a single unfiltered fiber to guide laser light to the tissue and to collect scattered light in this spectral region. We show, by means of a comparison of in vitro Raman microspectroscopic maps of thin tissue sections (brain tumors, bladder), measured both in the high wave number region and in the fingerprint region, that essentially the same diagnostic information is obtained in the two wave number regions. This suggests that for many clinical applications the technological hurdle of designing and constructing suitable fiber-optic probes may be eliminated by using the high wave number region and a simple piece of standard optical fiber.

Monitoring the Penetration Enhancer Dimethyl Sulfoxide in Human Stratum Corneum in Vivo by Confocal Raman Spectroscopy

The stratum corneum (SC) barrier typically consists of layers of corneocytes embedded in a lipid continuum that regulates barrier function. The lipid domain containing ceramides, cholesterol, and free fatty acids provides the major pathway for most drugs permeating across SC. Penetration enhancers diminish the SC barrier function. The classic enhancer is dimethyl sulfoxide (DMSO). Its mechanisms of action remain unclear, although DMSO disrupts lipid organisation and may displace protein-bound water. Here we use confocal Raman spectroscopy to probe molecular interactions between a finite (depleting) dose of DMSO and SC, as functions of depth and time, providing novel information about residence time and location of DMSO in human SC in vivo