Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

Lipidomic Analysis Reveals Specific Differences between Fibroblast and Keratinocyte Ceramide Profile of Patients with Psoriasis Vulgaris.

Ceramides are important lipid metabolites for primal skin functions. There is increasing evidence that alteration of the profile and metabolism of ceramides is associated with skin diseases, such as psoriasis vulgaris. Most studies have reported alteration in ceramide content in the stratum corneum, but these have been scarcely reported for other skin layers. In the present work, we aimed to explore changes in the ceramide profile of fibroblasts and keratinocytes in patients with psoriasis vulgaris and healthy subjects. Using the reversed-phase liquid chromatography-quadrupole-time-of-flight-tandem-mass spectrometry (RPLC-QTOF-MS/MS) platform, we identified ceramide containing non-hydroxy fatty acid ([N]), α-hydroxy fatty acid ([A]), and esterified ω-hydroxy fatty acid ([EO]) and 3 sphingoid bases, dihydrosphingosine ([DS]), sphingosine ([S]), and phytosphingosine ([P]). We found that in the keratinocytes of patients with psoriasis, CER[NS], CER[NP], CER[AS], CER[ADS], CER[AP] and CER[EOS] tended to be expressed at higher relative levels, whereas CER[NDS] tended to be expressed with lower levels than in healthy subjects. In the case of fibroblasts, significant differences were observed, mainly in the three ceramide classes (CER[AS], CER[ADS] and CER[EOS]), which were expressed at significantly higher levels in patients with psoriasis. The most significant alteration in the fibroblasts involved elevated levels of CER[EOS] that contained ester-linked fatty acids. Our findings provide insights into the ceramide profile in the dermis and epidermis of patients with psoriasis and contribute for the research in this field, focusing on the role of keratinocyte-fibroblast crosstalk in the development of psoriasis vulgaris.

Poly(lactic acid)/Carbon Nanotube Composite Microneedle Arrays for Dermal Biosensing.

Minimally invasive, reliable and low-cost in vivo biosensors that enable real-time detection and monitoring of clinically relevant molecules and biomarkers can significantly improve patient health care. Microneedle array (MNA)-based electrochemical sensors offer exciting prospects in this respect, as they can sample directly from the skin. However, their acceptability is dependent on developing a highly scalable and cost-effective fabrication strategy. In this work, we evaluated the potential for poly(lactic acid)/carboxyl-multiwalled carbon nanotube (PLA/ f-MWCNT) composites to be developed into MNAs and their effectiveness for dermal biosensing. Our results show that MNAs are easily made from solvent-cast nanocomposite films by micromolding. A maximum carbon nanotube (CNT) loading of 6 wt % was attained with the current fabrication method. The MNAs were mechanically robust, being able to withstand axial forces up to 4 times higher than necessary for skin insertion. Electrochemical characterization of these MNAs by differential pulse voltammetry (DPV) produced a linear current response toward ascorbic acid, with a limit of detection of 180 µM. In situ electrochemical performance was assessed by DPV measurements in ex vivo porcine skin. This showed active changes characterized by two oxidative peaks at 0.23 and 0.69 V, as a result of the diffusion of phosphate-buffered saline. The diagnostic potential of this waveform was further evaluated through a burn wound model. This showed an attenuated oxidative response at 0.69 V. Importantly, the impact of the burn could be measured at progressive distances from the burn site. Overall, alongside the scalable fabrication strategy, the DPV results promise efficient electrochemical biosensors based on CNT nanocomposite MNAs.

Lipid profiling of suction blister fluid: comparison of lipids in interstitial fluid and plasma.

BACKGROUND: Recent technical advances in the extraction of dermal interstitial fluid (ISF) have stimulated interest in using this rather unexploited biofluid as an alternative to blood for detection and prediction of disease. However, knowledge about the presence of useful biomarkers for health monitoring in ISF is still limited. In this study, we characterized the lipidome of human suction blister fluid (SBF) as a surrogate for pure ISF and compared it to that of plasma. METHODS: Plasma and SBF samples were obtained from 18 healthy human volunteers after an overnight fast. Total lipids were extracted and analyzed by liquid chromatography-tandem mass spectrometry. One hundred ninety-three lipid species covering 10 complex lipid classes were detected and quantified in both plasma and SBF using multiple reaction monitoring. A fraction of the lipid extract was subjected to alkaline transesterification and fatty acid methyl esters were analyzed by gas chromatography-mass spectrometry. RESULTS: The total concentration of lipids in SBF was 17% of the plasma lipid concentration. The molar fraction of lipid species within lipid classes, as well as total fatty acids, showed a generally high correlation between plasma and SBF. However, SBF had larger fractions of lysophospholipids and diglycerides relative to plasma, and consequently less diacylphospholipids and triglycerides. Principal component analysis revealed that the interindividual variation in SBF lipid profiles was considerably larger than the within-subject variation between plasma and SBF. CONCLUSIONS: Plasma and SBF lipid profiles show high correlation and SBF could be used interchangeably with blood for the analysis of major lipids used in health monitoring.

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.

Galectin-1 and Galectin-3 and Their Potential Binding Partners in the Dermal Thickening of Keloid Tissues.

Keloids are defined histopathologically as an inflammatory disorder characterized by exhibiting numerous fibroblasts, abnormal vascularization, increased number of proinflammatory immune cells as well as uncontrolled cell proliferation, and exacerbated and disorganized deposition of extracellular matrix (ECM) molecules. Importantly, many of these ECM molecules display N- and O-linked glycan residues and are considered as potential targets for galectin-1 (Gal-1) and galectin-3 (Gal-3). Nevertheless, the presence and localization of Gal-1 and Gal-3 as well as the interactions with some of their binding partners in keloid tissues have not been considered. Here, we show that in the dermal thickening of keloids, versican, syndecan-1, fibronectin, thrombospondin-1, tenascin C, CD44, integrin ß1, and N-cadherin were immunolocalized in the elongated fibroblasts that were close to the immune cell infiltrate, attached to collagen bundles, and around the microvasculature and in some immune cells. We also show that Gal-1 and Gal-3 were present in the cytoplasm and along the cell membrane of some fibroblasts and immune and endothelial cells of the dermal thickening. We suggest that Gal-1 and Gal-3, in concert with some of the ECM molecules produced by fibroblasts and by immune cells, counteract the inflammatory response in keloids. We also proposed that Gal-1 and Gal-3 through their binding partners may form a supramolecular structure at the cell surface of fibroblasts, immune cells, endothelial cells, and in the extracellular space that might influence the fibroblast morphology, adhesion, proliferation, migration, and survival as well as the inflammatory responses.

In situ type I oligomeric collagen macroencapsulation promotes islet longevity and function in vitro and in vivo.

Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5 -3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D.

Gender differences in the fluorescence of human skin in young healthy adults.

BACKGROUND: Human skin naturally contains many endogenous fluorophores; therefore, fluorescence techniques can be used for monitoring of the human skin even in in vivo mode. The aim of this work was to study skin autofluorescence in vivo regarding the possible effect of gender. MATERIALS AND METHODS: Fluorescence emission spectra of young healthy Caucasian adults in 3 anatomical regions (forehead, hand, and inner upper arm) were taken with excitation at 280, 325, or 400 nm. RESULTS: Three emission bands were found in the spectra for both men and women: (1) an intensive band peaked at 340/280 nm (peak emission/excitation wavelength), corresponding to aromatic amino acids of proteins in epidermis; (2) a broad band with emission between 360 nm and 480 nm (excitation 325 nm) with a base peak around 390 nm and 2 side peaks at 420 and 450 nm, mainly due to collagen cross-links in dermis with a possible weak contribution of elastin and mitochondrial NADPH; (3) a weak but distinct peak at 600/400 nm corresponding presumably to skin unmetalled porphyrins. CONCLUSION: The intensity of skin autofluorescence showed differences between genders and among anatomical regions. The 340 nm intensity was 1.4 times higher in the male group in all 3 anatomical regions studied. The highest intensity of skin autofluorescence for the peaks at 340/280 nm and 600/400 nm was found on the forehead, whereas the 390/325 nm band was most intensive on the inner upper arm in both genders.

Quantification of acyclovir in dermal interstitial fluid and human serum by ultra-high-performance liquid-high-resolution tandem mass spectrometry for topical bioequivalence evaluation.

Time-concentration curves for the topical anti-viral drug acyclovir can provide valuable information for drug development. Open flow microperfusion is used for continuous sampling of dermal interstitial fluid but it requires validated methods for subsequent sample analysis. Therefore, we developed a sensitive, selective and high-throughput ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry method to determine acyclovir in human dermal interstitial fluid and serum. We validated the method over a concentration range of 0.1-25 ng/mL for a sample volume of just 20 µL and employed cation-exchange solid-phase extraction in a fully automated sample treatment procedure. Short- and long-term sample stability data and the analysis of 5000 samples from a clinical trial demonstrate the successful application of our method.

Migration ability and Toll-like receptor expression of human mesenchymal stem cells improves significantly after three-dimensional culture.

While the conventional two-dimensional (2D) culture protocol is well accepted for the culture of mesenchymal stem cells (MSCs), this method fails to recapitulate the in vivo native three-dimensional (3D) cellular microenvironment, and may result in phenotypic changes, and homing and migration capacity impairments. MSC preparation in 3D culture systems has been considered an attractive preparatory and delivery method recently. We seeded human umbilical cord-derived MSCs (hUCMSCs) in a 3D culture system with porcine acellular dermal matrix (PADM), and investigated the phenotypic changes, the expression changes of some important receptors, including Toll-like receptors (TLRs) and C-X-C chemokine receptor type 4 (CXCR4) when hUCMSCs were transferred from 2D to 3D systems, as well as the alterations in in vivo homing and migration potential. It was found that the percentage of CD105-positive cells decreased significantly, whereas that of CD34- and CD271-positive cells increased significantly in 3D culture, compared to that in 2D culture. The mRNA and protein expression levels of TLR2, TLR3, TLR4, TLR6, and CXCR4 in hUCMSCs were increased significantly upon culturing with PADM for 3 days, compared to the levels in 2D culture. The numbers of migratory 3D hUCMSCs in the heart, liver, spleen, and bone marrow were significantly greater than the numbers of 2D hUCMSCs, and the worst migration occurred in 3D + AMD3100 (CXCR4 antagonist) hUCMSCs. These results suggested that 3D culture of hUCMSCs with PADM could alter the phenotypic characteristics of hUCMSCs, increase their TLR and CXCR4 expression levels, and promote their migratory and homing capacity in which CXCR4 plays an important role.

Development of a Two-Dimensional Model for Predicting Transdermal Permeation with the Follicular Pathway: Demonstration with a Caffeine Study.

PURPOSE: The development of a new two-dimensional (2D) model to predict follicular permeation, with integration into a recently reported multi-scale model of transdermal permeation is presented. METHODS: The follicular pathway is modelled by diffusion in sebum. The mass transfer and partition properties of solutes in lipid, corneocytes, viable dermis, dermis and systemic circulation are calculated as reported previously [Pharm Res 33 (2016) 1602]. The mass transfer and partition properties in sebum are collected from existing literature. None of the model input parameters was fit to the clinical data with which the model prediction is compared. RESULTS: The integrated model has been applied to predict the published clinical data of transdermal permeation of caffeine. The relative importance of the follicular pathway is analysed. Good agreement of the model prediction with the clinical data has been obtained. The simulation confirms that for caffeine the follicular route is important; the maximum bioavailable concentration of caffeine in systemic circulation with open hair follicles is predicted to be 20% higher than that when hair follicles are blocked. CONCLUSIONS: The follicular pathway contributes to not only short time fast penetration, but also the overall systemic bioavailability. With such in silico model, useful information can be obtained for caffeine disposition and localised delivery in lipid, corneocytes, viable dermis, dermis and the hair follicle. Such detailed information is difficult to obtain experimentally.

Cold-Associated Perniosis of the Thighs ("Equestrian-Type" Chilblain): A Reappraisal Based on a Clinicopathologic and Immunohistochemical Study of 6 Cases.

Cold-associated perniosis of the thighs ("equestrian cold panniculitis") is an unusual and still enigmatic entity. The authors retrieved 6 cases for a re-evaluation of their clinicopathologic features and for an immunohistochemical assessment with antibodies anti-CD3, anti-CD20, and anti-CD123. All patients were women, aged 17-45 years. One of them had elevated antinuclear antibody titers. Available anamnestic data confirmed the triggering role of prolonged/intermittent exposure to cold (not necessarily for equestrian activities). The lesions affected the thighs, with a preferential, although not exclusive involvement of the upper lateral surface. The histopathological pattern was perivascular, superficial, and deep, extending toward the superficial fat lobules, with lymphocytic vasculitis and mucin deposition; clumps of CD123 cells were found in 4 of 6 cases. Cold-associated perniosis of the thighs cannot be considered as a panniculitis. The histopathological features considerably overlap with perniosis at other sites of the body and with chilblain lupus erythematosus.

Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes.

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10µl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, 'high-throughput' profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.

Laser capture microdissection followed by next-generation sequencing identifies disease-related microRNAs in psoriatic skin that reflect systemic microRNA changes in psoriasis.

Psoriasis is a systemic disease with cutaneous manifestations. MicroRNAs (miRNAs) are small non-coding RNA molecules that are differentially expressed in psoriatic skin; however, only few cell- and region-specific miRNAs have been identified in psoriatic lesions. We used laser capture microdissection (LCM) and next-generation sequencing (NGS) to study the specific miRNA expression profiles in the epidermis (Epi) and dermal inflammatory infiltrates (RD) of psoriatic skin (N = 6). We identified 24 deregulated miRNAs in the Epi and 37 deregulated miRNAs in the RD of psoriatic plaque compared with normal psoriatic skin (FCH > 2, FDR < 0.05). Interestingly, 9 of the 37 miRNAs in RD, including miR-193b and miR-223, were recently described as deregulated in circulating peripheral blood mononuclear cells (PBMCs) from patients with psoriasis. Using flow cytometry and qRT-PCR, we found that miR-193b and miR-223 were expressed in Th17 cells. In conclusion, we demonstrate that LCM combined with NGS provides a robust approach to explore the global miRNA expression in the epidermal and dermal compartments of psoriatic skin. Furthermore, our results indicate that the altered local miRNA changes seen in the RD are reflected in the circulating immune cells, suggesting that miRNAs may contribute to the pathogenesis of psoriasis.

Dermal dendrocytes FXIIIa+ are essential antigen-presenting cells in indeterminate leprosy.

Indeterminate leprosy (IL) is the early phase of Hansen disease and reword (APCs). Langerhans cells and dermal dendrocytes FXIIIa positive (DDFXIIIa) are the major APCs in the skin and can be identified by the expression of CD1a and FXIIIa, respectively, by immunohistochemical techniques. Plasmacytoid dendritic cells (PDCs) are another type of dermal dendrocytes with a questionable antigen-presenting function and can be highlighted by anti-CD123 expression. To our knowledge, there are no studies evaluating DDFXIIIa and PDC in IL. The purpose was to investigate the involvement of these cells in the pathogenesis of IL. The authors performed a retrospective study on 18 cases of IL (10 confirmed and 8 suspected) to investigate expression of FXIIIa, CD1a, and CD123. The results were compared with normal skin (for CD1a and FXIIIa only). A higher amount of FXIIIa-positive cells (P , 0.05) in confirmed and suspected IL cases was noted when comparing with normal skin. However, CD1a showed no quantitative differences in the epidermis of IL lesions when comparing with normal skin and CD123 expression was negligible. Based on these findings, the authors postulate that Langerhans cells and PDCs do not have a major role in IL and that DDFXIIIa may be the main APCs in IL. Further study is required to establish this.

Histopathological analysis of the progression pattern of subungual melanoma: late tendency of dermal invasion in the nail matrix area.

Subungual melanoma is a rare subtype of melanoma that usually originates and spreads from the nail matrix. Because of its poor prognosis and short matrix-to-bone distance, amputation has been traditionally performed. Recently, conservative surgery has been attempted for early subungual melanoma, but the evidence supporting this practice is sparse. As little is known about the progression pattern of subungual melanoma, further advances on the subject may provide better guidance on the optimal surgical approach. Histopathology slides, clinical records, and photographs of 23 cases of subungual melanoma were reviewed. For all cases, each area of the nail unit-proximal nail fold, nail matrix, nail bed, and/or hyponychium-in longitudinal sections was available for histological examination. Growth pattern, dermal invasion, and thickness were assessed in each area of the nail unit. There were five cases of melanoma in situ. Eighteen cases showed dermal invasion in at least one area of the nail unit. There were no cases showing dermal invasion in the nail matrix area only. In four cases, dermal invasion involved areas of the nail unit other than the nail matrix. In 14 cases, dermal invasion involved the nail matrix area as well as other areas of the nail unit. Except for one case, the nail matrix area showed thinner dermal invasion compared with dermal invasion in other areas of the nail unit. In conclusion, dermal invasion of subungual melanoma in the nail matrix area tends to occur later than other areas of the nail unit. Longitudinal incisional biopsy is necessary to accurately evaluate melanoma invasion. The findings of this study suggest that conservative surgical treatment for early subungual melanoma may be justified as the nail matrix area, an area of thin dermis and close proximity to the underlying bone, appears to be more resistant to invasion.

Similar appearance, different mechanisms: xerosis in HIV, atopic dermatitis and ageing.

Xerosis is one of the most common dermatologic disorders occurring in the elderly and in patients with atopic dermatitis (AD) and human immunodeficiency virus (HIV) infection. Xerosis has been linked to an impaired skin barrier function of the stratum corneum. Using Raman microspectroscopy, we concentrated on deeper skin layers, viable epidermis and dermis of 47 volunteers and associated molecular alterations to the evolution of xerosis and the skin barrier, for example, lipid, water and antioxidant content. A decrease in lipids within the viable epidermis is found for elderly and HIV-patients. Lipid and water values of AD patients and their healthy reference group are similar. Decreases in lipids and simultaneous increases in water are found in the dermis for HIV and AD patients in comparison to their healthy reference groups. Excessive levels of epidermal carotenoids, mainly lycopene, in HIV-patients were found potentially leading to adverse effects such as premature skin ageing.

Acral persistent papular mucinosis: is it an under-diagnosed disease?

Acral persistent papular mucinosis is a subtype of localized lichen myxedematosus. It presents as acrally located papules with a benign, but persistent course. It is a scarcely reported disease. We present a female with both the clinical and histopathological described criteria.

Amyloidosis cutis dyschromia: a rare form of primary cutaneous amyloidosis.

Amyloidosis cutis dyschromia is a rare form of primary cutaneous amyloidosis. Amyloid deposition in the skin occurs without systemic manifestations and produces hypopigmented and hyperpigmented macules. A 19-year-old woman is presented with progression of this condition over 16 years.

A novel method to assess collagen architecture in skin.

BACKGROUND: Texture within biological specimens may reveal critical insights, while being very difficult to quantify. This is a particular problem in histological analysis. For example, cross-polar images of picrosirius stained skin reveal exquisite structure, allowing changes in the basketweave conformation of healthy collagen to be assessed. Existing techniques measure gross pathological changes, such as fibrosis, but are not sufficiently sensitive to detect more subtle and progressive pathological changes in the dermis, such as those seen in ageing. Moreover, screening methods for cutaneous therapeutics require accurate, unsupervised and high-throughput image analysis techniques. RESULTS: By analyzing spectra of images post Gabor filtering and Fast Fourier Transform, we were able to measure subtle changes in collagen fibre orientation intractable to existing techniques. We detected the progressive loss of collagen basketweave structure in a series of chronologically aged skin samples, as well as in skin derived from a model of type 2 diabetes mellitus. CONCLUSIONS: We describe a novel bioimaging approach with implications for the evaluation of pathology in a broader range of biological situations.