Evi Houben

Validation of the VapoMeter, a closed unventilated chamber system to assess transepidermal water loss vs. the open chamber Tewameter

Background/aim: Transepidermal water loss (TEWL) is one of the most important biophysical parameters for evaluating the efficiency of the human skin water barrier. Different approaches exist to measure TEWL. The most commonly used methodology consists of the open chamber diffusion technique in which the water vapor pressure gradient is measured in g/h m2 according to Ficks law. A typical apparatus is the Tewameter®. Recently, a portable device – the VapoMeter – became available with a humidity sensor in a closed chamber.

A keratinocyte's course of life.

An adequate permeability barrier function of the mammalian epidermis is guaranteed by the characteristic architecture of the stratum corneum. This uppermost layer consists of a highly organized extracellular lipid compartment which is tightly joined to the corneocytes. The generation of the extracellular lipid compartment and the transformation of the keratinocytes into corneocytes are the main features of epidermal differentiation. However, equally important is the continuous renewal of the stratum corneum, which is insured by a careful balance between the replenishment of new keratinocytes from the proliferating basal layer, and the well-orchestrated loss of the most superficial cells after the so-called ‘epidermal programmed cell death’. In this overview, the complete life of keratinocytes is described, from the proliferative organization to the process of desquamation.

Differentiation-associated expression of ceramidase isoforms in cultured keratinocytes and epidermis

Ceramides (Cers) accumulate within the interstices of the outermost epidermal layers, or stratum corneum (SC), where they represent critical components of the epidermal permeability barrier. Although the SC contains substantial sphingol, indicative of ceramidase (CDase) activity, which CDase isoforms are expressed in epidermis remains unresolved. We hypothesized here that CDase isoforms are expressed within specific epidermal compartments in relation to functions that localize to these layers. Keratinocytes/epidermis express all five known CDase isoforms, of which acidic and alkaline CDase activities increase significantly with differentiation, persisting into the SC. Conversely, neutral and phytoalkaline CDase activities predominate in proliferating keratinocytes. These differentiation-associated changes in isoform activity/protein are attributed to corresponding, differentiation-associated changes in mRNA levels (by quantitative RT-PCR). Although four of the five known CDase isoforms are widely expressed in cutaneous and extracutaneous tissues, alkaline CDase-1 occurs almost exclusively in epidermis. These results demonstrate large, differentiation-associated, and tissue-specific variations in the expression and activities of all five CDase isoforms. Because alkaline CDase-1 and acidic CDase are selectively upregulated in the differentiated epidermal compartment, they could regulate functions that localize to the distal epidermis, such as permeability barrier homeostasis and antimicrobial defense.

Hydrolytic Pathway Protects against Ceramide-Induced Apoptosis in Keratinocytes Exposed to UVB

Although ceramides (Cers) are key constituents of the epidermal permeability barrier, they also function as apoptogenic signals for UVB irradiation-induced apoptosis in epidermal keratinocytes. As epidermis is continuously exposed to UV irradiation, we hypothesized that Cer hydrolysis protects keratinocytes from UVB-induced apoptosis by attenuating Cer levels. Both low-dose UVB (L-UVB) (< 35 mJ cm(-2)) and high-dose UVB (H-UVB) (> or = 45 mJ cm(-2)) irradiation inhibited DNA synthesis in cultured human keratinocytes, but apoptosis occurred only after H-UVB. Whereas Cer production increased after both L- and H-UVB, it normalized only in L-UVB-exposed keratinocytes, but remained elevated after H-UVB. Both acidic ceramidase (aCDase) and neutral ceramidase (nCDase) activities declined after L- and H-UVB, but returned to normal only in L-UVB cells, with decreased CDase activities or mRNA or protein levels being sustained in H-UVB cells. Inhibition of CDase using either a CDase inhibitor, N-oleoylethanolamine, or small interfering RNA (siRNA) (either to a- and/or n-CDase(s)) sensitized keratinocytes to L-UVB-induced apoptosis in parallel with further Cer accumulation. Blockade of sphingosine kinase 1 (SPHK1) (but not SPHK2) by siRNA also increased apoptosis in L-UVB keratinocytes, revealing that conversion of sphingosine to sphingosine-1-phosphate (S1P) further protects keratinocytes from UVB-induced cell death. Thus, Cer → sphingosine → S1Pmetabolic conversion protects against UVB-induced, Cer-mediated apoptosis in keratinocytes, but excessive UVB overwhelms this mechanism, thereby leading to keratinocyte apoptosis.

Skin condition associated with intensive use of alcoholic gels for hand disinfection: a combination of biophysical and sensorial data.

Although hand hygiene is an important and inexpensive measure to prevent nosocomial infections in clinical settings, the compliance of healthcare workers remains low. In Europe, alcoholic hand disinfection is first choice, but there exists a limited user acceptability due to estimated adverse effects on skin condition. This study was designed to investigate skin tolerance to alcohol‐based disinfecting gels and changes in skin condition depending on humectant concentration, alcohol grades, as well as type of alcohol used. A comparison of 6 alcohol‐based gels was made based on a randomized double‐blind study under in use conditions for 1 day. Skin condition was evaluated by measuring transepidermal water loss (TEWL), stratum corneum hydration, apparent skin pH, redness and degree of scaliness. With respect to user acceptability, all gels were sensorially evaluated using a questionnaire. We saw that none of the alcohol‐based gels, applied under in use conditions, altered TEWL or caused irritation. All gels hydrated the skin, proportionally to their glycerine content, and decreased skin pH. Elevated ethanol concentrations resulted in increased scaliness. Sensorial assessment revealed less appreciation for isopropanol. From this study, it was concluded that gels containing an elevated glycerine concentration and 70% (v/v) ethanol are preferred.

Analysis of Epidermal Lipids of the Healthy Human Skin: Factors Affecting the Design of a Control Population

The intervariability of studies on the lipids of human epidermis and stratum corneum is high because of the different origin of the skin samples and the variety of extraction methods used. In the present work, a high-performance thin-layer chromatographic technique has been used to study the parameters age, sex, and anatomical site for their effects on the lipid profiles recovered from healthy epidermal skin biopsy specimens. It was found that sex-related differences were seen at the level of the total ceramide concentration. Observed decreases in lipid concentration, due to ageing, depended on the anatomical site. Therefore, these variables should be controlled in a reproducible and standardized way in order to be able to study the direct relationship between skin condition and barrier lipid composition. Only when this relation is established, results of topical treatment can be scientifically evaluated.

Epidermal ceramidase activity regulates epidermal desquamation via stratum corneum acidification.

The acidic pH of the outer surface of the mammalian skin plays several important roles in the epidermal barrier function. The 2 endogenous pathways that are currently known to elicit this acidic pH are the generation of free fatty acids from phospholipids and the exchange of protons for sodium ions by non-energy-dependent sodium-proton exchangers. In this study, we propose a third endogenous pathway, i.e. epidermal ceramidase activity, generating free fatty acids from ceramides. By topical application of N-oleylethanolamine, a well-known ceramidase inhibitor, we could demonstrate a significant increase in the stratum corneum pH and a corresponding decrease in the epidermal free fatty acid content. Moreover, we could show that the resulting change in the apparent skin pH also provoked a delay in early barrier recovery and an increased epidermal desquamation, corresponding to earlier observations made for the already known endogenous mechanisms.

Kinetic characteristics of acidic and alkaline ceramidase in human epidermis.

It has recently become evident that at least five ceramidase (CDase) isoforms are present in human epidermis, and that specifically acidic CDase (aCDase) and alkaline CDase (alkCDase) activities increase during keratinocyte differentiation, and thus might play a pivotal role(s) in permeability barrier function. Prior to investigating their possible roles in the epidermal barrier function, it is necessary to characterize basic kinetic parameters for these enzymes, as well as to determine the effects of the established CDase inhibitors and their activities. In this study, assays for both aCDase and alkCDase activities in fully differentiated human epidermis were optimized using a radiolabeled substrate. These studies revealed that aCDase activity is substantially higher than alkCDase activity, and that both isoenzymes are inhibited by a CDase inhibitor N-oleylethanolamine. These findings were also confirmed using an in situ enzyme assay.

Seasonal Effects on the Nasolabial Skin Condition

In the present work, nasolabial skin condition and the influence of seasonal changes during autumn and winter were studied in 16 healthy female volunteers. Apart from visual scoring of erythema and skin scaliness, transepidermal water loss (TEWL), skin hydration, apparent skin pH, skin colour and skin desquamation were biophysically measured. The study results showed that nasolabial TEWL was significantly higher during wintertime than in autumn. Also skin colour measurements and squamometry scorings revealed higher values, indicating a more reddish and scaly nasolabial skin during winter compared to autumn. Results from tape stripping and skin surface lipid analysis by high-performance thin-layer chromatography demonstrated significant differences for triglycerides and cholesterol esters, indicating a functionally inferior hydrolipidic layer during the winter season.

Effects of lotioned disposable handkerchiefs on skin barrier recovery after tape stripping

Background/purpose: In the present work, it was studied whether repeated use of lotioned disposable handkerchiefs on tape‐stripped forearm skin was able to improve skin barrier recovery.