Characterization of Ceramides with Phytosphingosine Backbone by Hydrogen-deuterium Exchange Mass Spectrometry

Ceramides are a lipid subclass of the sphingolipids that show large structural diversity. Structural characterization of the ceramides (CERs) can lead to better understanding of their role and function in the biological system. Here we investigated representatives of NP (CER III, CER IIIB) and AP ceramide classes (CER VI) that contain phytosphingosine (P) backbone. Ceramides were characterized in positive ionization mode by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Fragmentation in positive ionization mode of the CER III and CER VI resulted in abundant ions assigned to phytosphingosine moiety at m/z 282, 300 and 318. HDX-MS of fragments showed increase in m/z of corresponding ions confirming the exchange of deuterium. In negative ionisation spectra multiple fragment ions were assigned to fatty acyl (RCOO–) moiety. Presence of RCOO– allowed unambiguous identification of CER III and CER IIIB which were distinguished by the presence of double bond on fatty acyl chain. This work is licensed under a Creative Commons Attribution 4.0 International License

Changes in nano-mechanical properties of human epidermal cornified cells in children with atopic dermatitis

Background: Impaired skin barrier is an important etiological factor in atopic dermatitis (AD). The structural protein filaggrin (FLG) plays a major role in maintenance of the competent skin barrier and its deficiency is associated with enhanced susceptibility to mechanical injury. Here we examined biomechanical characteristics of the corneocytes in children with AD and healthy controls. Methods: We recruited 20 children with AD and 7 healthy children. They were genotyped for filaggrin gene ( FLG) loss-of-function mutations. Stratum corneum was collected from clinically unaffected skin by adhesive tapes. Cell stiffness (apparent elastic modulus, Ea) was determined by atomic force microscopy and filaggrin degradation products (NMF) by liquid chromatography. Skin barrier function was assessed through trans-epidermal water loss (TEWL) and disease severity by the SCORing Atopic Dermatitis (SCORAD) tool. Results: Corneocytes collected from AD patients showed a decreased elastic modulus which was strongly correlated with NMF and TEWL, but not with SCORAD. As compared with healthy controls, AD patients had reduced TEWL and NMF levels regardless of FLG mutations. NMF was strongly correlated with TEWL. Conclusion: Our findings demonstrate that AD patients have decreased corneocyte stiffness which correlates with reduced levels of filaggrin degradation products, NMF and skin barrier function. Altered mechanical properties of the corneocytes likely contribute to the loss of mechanical integrity of the SC and to reduced skin barrier function in AD

Systemic and stratum corneum biomarkers of severity in infant atopic dermatitis include markers of innate and T helper cell-related immunity and angiogenesis

BACKGROUND: Biomarkers of atopic dermatitis (AD) are largely lacking, especially in infant AD. Those that have been examined to date have focused mostly on serum cytokines with few on non-invasive biomarkers in the skin. OBJECTIVES: We aimed to explore biomarkers obtainable from non-invasive sampling of infant skin. We compared these to plasma biomarkers and structural and functional measures of the skin barrier. METHODS: We recruited 100 infants at first presentation with AD, who were treatment naïve to topical or systemic anti-inflammatory therapies and 20 healthy children. We sampled clinically unaffected skin by tape stripping the stratum corneum (SC). Multiple cytokines and chemokines and natural moisturizing factors (NMF) were measured in the SC and plasma. We recorded disease severity and skin barrier function. RESULTS: 19 SC and 12 plasma biomarkers showed significant difference between healthy and AD skin. Some biomarkers were common to both the SC and plasma, and others were compartment-specific. Identified biomarkers of AD severity included Th2 skewed markers (IL-13, CCL17, CCL22, IL-5), markers of innate activation (IL-18, Il-1α, IL1β, CXCL8), angiogenesis (Flt-1, VEGF) and others (sICAM-1, vCAM-1, IL-16, IL-17A). CONCLUSIONS: We identified clinically relevant biomarkers of AD, including novel markers, easily sampled and typed in infants. These markers may provide objective assessment of disease severity and suggest new therapeutic targets, or response measurement targets for AD. Future studies will be required to determine if these biomarkers, seen in very early AD, can predict disease outcomes or comorbidities

Characterization of silver particles in the stratum corneum of healthy subjects and atopic dermatitis patients dermally exposed to a silver-containing garment

Silver is increasingly being used in garments to exploit its antibacterial properties. Information on the presence of silver nanoparticles (AgNPs) in garments and their in vivo penetration across healthy and impaired skin from use is limited. We investigated the presence of AgNPs in a silver containing garment and in the stratum corneum (SC) of healthy subjects (CTRLs) and individuals with atopic dermatitis (AD). Seven CTRLs and seven AD patients wore a silver sleeve (13% Ag w/w) 8 h/day for five days on a forearm and a placebo sleeve on the other forearm. After five days, the layers of the SC were collected by adhesive tapes. The silver particles in the garment and SC were characterized by scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDX) and atomic force microscopy (AFM). AFM and SEM revealed the presence of sub-micrometre particles having a broad range of sizes (30\u2013500 nm) on the surface of the garment that were identified as silver. On the SC tapes collected from different depths, aggregates with a wide range of sizes (150 nm\u20132 \u3bcm) and morphologies were found. Most aggregates contained primarily silver, although some also contained chlorine and sulfur. There was no clear difference in the number or size of the aggregates observed in SC between healthy and AD subjects. After use, AgNPs and their aggregates were present in the SC at different depths of both healthy subjects and AD patients. Their micrometre size suggests that aggregation likely occurred in the SC

Effect of allergens and irritants on levels of natural moisturizing factor and corneocyte morphology

BACKGROUND: The irritant sodium lauryl sulfate (SLS) is known to cause a decrease in the stratum corneum level of natural moisturizing factor (NMF), which in itself is associated with changes in corneocyte surface topography. ----- OBJECTIVE: To explore this phenomenon in allergic contact dermatitis. ----- METHODS: Patch testing was performed on patients with previously positive patch test reactions to potassium dichromate (Cr), nickel sulfate (Ni), methylchloroisothiazolinone (MCI)/methylisothiazolinone (MI), or p-phenylenediamine. Moreover, a control (pet.) patch and an irritant (SLS) patch were applied. After 3 days, the stratum corneum from tested sites was collected, and NMF levels and corneocyte morphology, expressed as the amount of circular nanosize objects, quantified according to the Dermal Texture Index (DTI), were determined. ----- RESULTS: Among allergens, only MCI/MI reduced NMF levels significantly, as did SLS. Furthermore, only MCI/MI caused remarkable changes at the microscopic level; the corneocytes were hexagonal-shaped with pronounced cell borders and a smoother surface. The DTI was increased after SLS exposure but not after allergen exposure. ----- CONCLUSIONS: MCI/MI significantly decreased NMF levels, similarly to SLS. The altered corneocyte morphology suggests that skin barrier damage plays a role in the pathogenesis of MCI/MI contact allergy. The DTI seems to differentiate reactions to SLS from those to the allergens tested, as SLS was the only agent that caused a DTI increase

Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

Research Techniques Made Simple: Stratum Corneum Tape Stripping

Stratum corneum (SC)-derived biomarkers can provide relevant information on the skin's antimicrobial, physical, and immunological barriers. The SC is easily accessible, and collection by adhesive tapes (tape stripping [TS]) is robust and minimally invasive. Given its minimal invasiveness and simplicity, TS is particularly useful for studies in the pediatric population and when repetitive sampling over time is desirable, for example, in clinical trials. The palette of SC biomarkers is expanding in a wide variety of research areas, benefiting from advances in multiplex immunoassays and omics approaches, including proteomics, lipidomics, and transcriptomics. Although there is increasing interest in collecting SC samples, the lack of TS standardization hampers its broader implementation in research and clinical practice. In this article, we address the TS procedure as well as methodological challenges that should be considered in the development of an optimal sampling strategy

The role of skin barrier in occupational contact dermatitis

Skin diseases represent one of the most common work-related diseases and may have a detrimental effect on social, personal and occupational aspects of life. Contact dermatitis (CD), which comprises predominately irritant contact dermatitis (ICD) and allergic contact dermatitis (ACD), accounts for vast majority of occupational skin diseases, especially in occupations associated with frequent skin contact with irritants and contact allergens. Although ICD and ACD have similar clinical manifestation, their pathophysiology and the role of the skin barrier are different. In ICD, perturbation of the skin barrier is the primary event which sets into motion diverse metabolic processes and triggers activation of innate immunity without the involvement of adaptive immune system. In ACD, a type IV hypersensitivity reaction induced by contact allergens, the skin barrier impairment may evoke innate signalling pathways during the sensitization phase required for the activation of T-cell adaptive response. Thus, skin barrier impairment may increase the risk of ICD or ACD not only because of enhanced permeability and ingress of irritants and allergens but also by the generation of innate immune signal needed for the induction of allergic response. Hence, an efficient way to prevent CD is to avoid skin barrier damage in the workplace. This review focuses on the skin barrier, how it is affected by skin irritants and how its impairment contributes to the development of ICD and ACD

Stratum corneum levels of inflammatory mediators and natural moisturizing factor in patch test reactions to thiurams and fragrances and their possible role in discrimination between irritant and allergic reactions to hapten mixtures

Background: Patch test (PT) reactions to thiuram mix (TM) and fragrance mix (FM) I or II without concomitant reactions to their single constituents are potentially caused by the irritant properties of the mixes. Objective: Comparing inflammatory profiles of PT reactions to TM, FM I, FM II, and their constituents and assessing their potential in discrimination of irritant and allergic reactions. Patients and Methods: Levels of 14 cytokines and natural moisturizing factor (NMF) were determined in stratum corneum samples collected from PT reactions to TM, FM I or II, their constituents, and petrolatum (pet.) control sites in 36 individuals. Results: Levels of interleukin (IL)-16, chemokine (CXC motif) ligand (CXCL) 8, CXCL10, chemokine (CC motif) ligand (CCL) 17, and CCL22 were significantly increased in reactions (+, ++) to thiurams and fragrances compared to their pet. controls, except for PT reactions to FM I/II with negative breakdown testing in which, however, decreased levels of NMF were observed. In doubtful reactions to FM I/II with negative breakdown testing, NMF was significantly lower than in petrolatum controls. Conclusions: PT reactions to thiurams and fragrances indicate a Th2-skewed inflammation. The inflammatory profiles suggest that weak or doubtful FM I/II reactions without accompanying reaction to a constituent were irritant. IL-16 might be suitable to distinguish irritant from allergic reaction