Mieke Mommaas

Characterization of reconstructed skin models

The aim of the present study was to evaluate tissue architecture and lipid composition of commercially available reconstructed human skin models; EpiDerm™, SkinEthic™ and Episkin™ in comparison to in-house reconstructed epidermis on a de-epidermized dermis (RE-DED) model and native tissue. For this purpose, the tissue architecture was examined using light microscopy, electron microscopy and immunohistochemistry; epidermal lipid composition was analyzed by HPTLC. Histological examination showed a completely stratified epithelium in all skin models closely resembling normal human epidermis. Low intra-batch variation in tissue architecture was observed in all skin models, but moderate to considerable inter-batch variation was noticed. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis and RE-DED in EpiDerm, SkinEthic and Episkin models, the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. Evaluation of the expression and localization of a number of differentiation-specific protein markers revealed that all skin models showed an aberrant expression of keratin 6, skin-derived antileukoproteinase, small-proline-rich proteins, involucrin and transglutaminase. Although variation within batches was low, in particular keratin 6, involucrin and skin-derived antileukoproteinase expression demonstrated some inter-batch variation. In conclusion, all skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be diminished. All skin models tested reproduced many of the characteristics of normal human epidermis and therefore provide a morphologically relevant in vitro means to assess skin irritation and perform other skin-related studies.

Lipid and ultrastructural characterization of reconstructed skin models

The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm, SkinEthic and Episkin in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO(4) post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.

Barrier Function in Reconstructed Epidermis and Its Resemblance to Native Human Skin

One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.

Drosophila CPEB Orb2A Mediates Memory Independent of Its RNA-Binding Domain

Summary Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist.

Keratinocytes resident in normal human skin constitutively express, at low levels, the intercellular adhesion molecule-1. An in situ immunoelectronmicroscopy study

Summary The expression of intercellular adhesion molecule‐1 (ICAM‐1) on keratinocytes (KC) was previously demonstrated in biopsies from various inflammatory skin lesions. KC were, however, found virtually ICAM‐1 negative, in normal skin, when the same immunocytochemical techniques were employed. By contrast, epithelial cells resident in different organs constitutively express ICAM‐1, albeit weakly. The aim of the present study was to use an immunostaining system more sensitive than the conventional immunocytochemistry, namely the in situ immunogold labelling of ultracryosections, to investigate the constitutive ICAM‐1 expression by resting KC in normal skin, in vivo. The semiquantitative analysis performed on 500 resident KC, visualized within tissue ultracryosections of normal human skin, revealed that gold granules were present along the cell membrane in a small percentage (14·6%) of resident KC. The density of gold particles (10 nm sized) observed on the cell surface per KC section was as scarce as 13·72 ± 4·6 (mean ± standard deviation), although highly significant when compared with controls (P < 0·005). This indicates the presumably low expression of ICAM‐1 moieties on the plasma membrane of this KC subset. This ICAM‐1 expression could be important in modulating the trafficking to and from normal epidermis of migrating Langerhans cells and occasional leucocytes. The fact that the ICAM‐1 expression on KC in normal skin is limited can be considered favourable, because it can account for the prevention of inappropriate KC/leucocyte interactions in the resting cutaneous environment.

Differences in low density lipoprotein receptor expression in the suprabasal layer of normal and psoriatic epidermis

Previous morphological experiments on the distribution of binding sites for low density lipoprotein (LDL) on normal and psoriatic epidermis in situ, done with the LDL-gold technique [Mommaas-Kienhuis AM, et al. J Invest Dermatol 89: 513-517, 1987.] showed an unequivocal correlation between the ability to bind LDL-gold complexes and the state of keratinocyte differentiation. To determine the involvement of the LDL receptor in this phenomenon, we applied immunoelectronmicroscopical methods in conjunction with a monoclonal anti-LDL receptor antibody. Biopsy specimens of normal and psoriasis skin were fixed before being embedded in Lowicryl K4M. Ultrathin sections were incubated first with the anti-LDL receptor antibody, and then with a second antibody conjugated to colloidal gold. On basal cells of both normal and psoriatic epidermis the LDL receptor was distributed evenly between the cell surface and the cytoplasm. No obvious differences in the density of LDL receptors were observed. However, cells from the suprabasal layer showed two striking differences in the localization of the LDL receptor: 1) normal epidermis showed fewer LDL receptor molecules, whereas in psoriasis epidermis the number increased relative to those on basal cells; and 2) in normal suprabasal cells most of the LDL receptors were located inside the cell, but in psoriasis the majority was found on the cell surface. Both phenomena are discussed and we postulate that the higher expression of LDL receptors in psoriasis suprabasal cells and the high expression of the receptor on the cell surface is connected with the hyperproliferative state of the disorder.