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The diversity and dynamics of the skin microbiome in health and disease have been studied recently, but adequate model systems to study skin microbiotas in vitro are largely lacking. We developed an in vitro system that mimics human stratum corneum, using human callus as substrate and nutrient source for bacterial growth. The growth of several commensal and pathogenic bacterial strains was measured for up to one week by counting colony-forming units or by quantitative PCR with strain-specific primers. Human skin pathogens were found to survive amidst a minimal microbiome consisting of 2 major skin commensals: Staphylococcus epidermidis and Propionibacterium acnes. In addition, complete microbiomes, taken from the backs of healthy volunteers, were inoculated and maintained using this system. This model may enable the modulation of skin microbiomes in vitro and allow testing of pathogens, biological agents and antibiotics in a medium-throughput format.

Epidermal keratinocytes are heterogeneous and can be divided into stem cells (strong beta1-integrin expression) with unlimited clonogenic potential, transient amplifying cells (weaker beta1-integrin expression) with restricted proliferative capacity and terminally differentiated cells (no beta1-integrin expression) that have lost the capacity to divide. We tested the hypothesis that cell kinetic characteristics of the epidermal subpopulations differ. Single cell suspensions from small human skin punch biopsies were sorted flow cytometrically into a beta1-integrin weakly positive (dim) and strongly positive (bright) subpopulation and the clonogenic potential was compared in cell culture experiments. Image analysis was used to determine growth characteristics of the colonies. We found that cell size in the beta1-integrin bright subpopulation increased when colonies aged, whereas this was constant in the dim subpopulation. The total number of colonies formed and the growth rate of the colonies were higher in the beta1-integrin dim cells than in the bright subpopulation. Experimental data from this study confirm the hypothesis that cell kinetic characteristics of beta1-integrin dim and bright cells are different. Combining flow cytometric sorting, cell culture and image analysis provides powerful means for phenotypical and functional characterization of epidermal subpopulations.

Tenascin-C is an extracellular matrix glycoprotein that is markedly upregulated in the dermis of psoriatic skin. In this study, we have addressed the question whether the presence of tenascin-C in the lesion or in serum is a marker for disease activity. Immunohistochemical staining of tenascin-C before and after treatment with different topical and systemic medication showed that tenascin-C remained abundant after clinical remission of lesions, indicating that downregulation of tenascin-C to normal values is a slow process. By using a sensitive enzyme-linked immunosorbent assay to measure levels of serum tenascin-C in psoriatic patients and unaffected individuals, we found that tenascin-C levels in most patients were within the normal range. Moreover, tenascin-C values did not correlate with disease activity. We conclude that tenascin-C is not useful as a marker for disease activity in psoriasis.

Heparan sulfate proteoglycans (HSPGs) are components of the basement membrane (BM) of various tissues. They consist of a core protein and negatively charged glycosaminoglycan side chains: the heparan sulfate (HS) moieties. In psoriasis, uninvolved skin of psoriasis patients and in normal skin, the distribution of HSPGs was studied immunohistochemically by means of three different monoclonal antibodies: JM-72, directed against the HSPG-core protein, JM-13 against the sulfated domains of HS and JM-403 against the unmodified/low sulfated parts of HS. In psoriasis JM-13 staining was consistently absent in the tips of the dermal papillae, whereas JM-13 showed a continuous staining in the BM of uninvolved and normal skin. JM-403 staining was present in BM of all specimens. In addition, a honeycomb-like staining was found in epidermis of normal skin and to a lesser extent in uninvolved skin, due to binding with plasma membrane-associated HS. In psoriasis this JM-403 staining of the epidermis was invariably absent. JM-72 showed a continuous staining of BM in all biopsies. In conclusion, normal human skin and involved psoriatic skin show clear differences in expression of HS. These data may provide insight into the role of HSPGs in psoriasis, which remains further to be elucidated.

Recently we have described two novel markers for disturbed epidermal differentiation, which are strongly upregulated in psoriatic epidermis: skin-derived antileukoproteinase (SKALP) and epidermal fatty acid-binding protein (E-FABP). No data are available on the kinetics of SKALP and E-FABP expression in vivo and the relation with epidermal growth and differentiation. We used treatment of lesional psoriatic skin with topical steroid as a model to correlate the expression pattern of SKALP and E-FABP with known cell biological events during regression of the psoriatic lesion. Expression of these markers was studied using immunohistochemistry and Northern blot analysis. After 4 weeks of treatment a substantial clinical improvement was induced by the topical steroid, whereas no significant improvement had occurred at the placebo-treated sides. The expression of SKALP following treatment with steroid was nearly undetectable both at the protein and mRNA level. Mitotic activity, as measured by Ki-67 staining, and cytokeratin 16 expression were downregulated to normal levels in the steroid-treated epidermis. In contrast, although there was a marked decrease of E-FABP mRNA, the staining pattern for E-FABP at the protein level was not affected. After 4 weeks of treatment with steroid the complete suprabasal compartment remained positive, even after considerable clinical improvement of the lesion. We conclude that SKALP and cytokeratin 16 are markers that are downregulated even before complete macroscopic clearance of the lesion. The kinetics of E-FABP expression is distinct from the other molecules and lags behind the clinical signs of psoriasis.

In this study we have investigated epidermal growth and differentiation during wound healing in human skin. The studies were performed in excisional wounds in normal skin and in chronic venous ulcers. Tissues were analyzed by immunohistochemical staining for proliferation-associated nuclear antigens (PCNA and Ki-67 antigen) and cytokeratin 16. Healing of excisional wounds was studied from day 2 to 14. Recruitment of resting (G0) epidermal cells started within 2 days after wounding; the number of cycling cells was maximal at day 4 and continued to be increased (compared to baseline levels in normal skin) after wound closure (7-14 days). Cytokeratin 16, a proliferation-associated keratin, was induced within 48 h and was expressed in the suprabasal keratinocytes of the wound edge. Cytokeratin 16 expression was maximal at day 4 and was still present in the neo-epidermis after restoration of epidermal continuity (7-14 days). Surprisingly, in chronic venous ulcers, cycling cells were present in the wound edges of all stages of the leg ulcers studied. Both the number and localization of cycling cells were similar to those in normal wound healing. Cytokeratin 16 was strongly expressed in all these ulcers. Our in vivo data demonstrate that recruitment of G0-cells into the cell cycle is not impaired in venous ulcers, which suggests that epidermal proliferation is not a limiting factor in the healing process of chronic venous ulcers.

We have assessed the distribution pattern of a new epidermal elastase inhibitor, skin-derived antileukoproteinase (SKALP) and polymorphonuclear leukocytes (PMN) in a patient with annular pustular psoriasis, using immunohistochemical methods. In clinically uninvolved skin SKALP was not expressed and only occasionally a few PMN could be identified. In the erythematous distal margin of the lesion, expression of SKALP was shown in suprabasal keratinocytes. Although PMN were present in the dermis, no transepidermal PMN migration had occurred at this stage. In the pustular region a dense SKALP-positive zone was demonstrated in the suprabasal compartment. In the centrally healed area neither SKALP expression nor PMN accumulation was shown. The present case report suggests that the induction of SKALP expression does not result from the passage of PMN through the epidermis. The offswitch of SKALP expression coincides with the disappearance of the PMN infiltration and not with the resolution of the mononuclear infiltrate.

Alopecia areata is a human hair disease of unknown etiology. Immunological mechanisms, alterations in the extracellular matrix and follicular growth abnormalities have been suggested as a possible cause. Here we compare the expression of cytokeratins in normal hair follicles to that of alopecia areata using immunohistology with monoclonal antibodies. A number of cytokeratins were specifically expressed in defined anatomical parts of the follicle; however, no gross qualitative or quantitative differences were found between normal and diseased scalp. Interestingly, the expression of cytokeratin 16, which is modulated by conditions that affect the rate of keratinocyte proliferation, was found to be unchanged in the outer root sheet of alopecia areata follicles. This is in contrast with earlier observations of a decrease in the expression of the proliferation-associated, Ki-67 nuclear antigen.

The monoclonal antibody Ki-67 was used to determine the numbers of cycling cells in hair follicles both in alopecia areata and in normal scalp skin. Pronounced nuclear staining was limited to the area below the critical line of Auber and the exterior part of the outer root sheath. In alopecia areata there is reduced nuclear Ki-67 binding in the bulb of anagen hair follicles. These findings indicate that inhibition of keratinocyte proliferation might be a pathogenetic mechanism in alopecia areata.

Horizontal sectioning of scalp biopsies is especially useful in hair diseases characterized by a reduction of follicles in size or number. Horizontal sectioning using standard cryo-microtomes is hampered by the differences in cutting properties between hair follicles and fatty tissue, resulting in loss of topography. We report a simple, inexpensive method to temporarily cool the specimen to an appropriate temperature by means of a conduction system using liquid nitrogen. Immunohistological methods such as immunofluorescence and immunoperoxidase techniques can be applied without restrictions.

Elastase inhibiting activity (EIA) has been observed in normal skin as a response to surface trauma, immediately following the intra-epidermal accumulation of polymorphonuclear leukocytes (PMN). In order to elucidate the relation between EIA and inflammation, the inhibiting activity was assessed in skin samples of scaling dermatoses (a) without significant inflammation: erythrodermic autosomal recessive lamellar ichthyosis (EARLI), non-erythrodermic autosomal recessive lamellar ichthyosis (NEARLI), X-linked recessive ichthyosis (XLRI) and X-linked dominant chondrodysplasia punctata (XLD-CDP); (b) with predominantly mononuclear cell infiltration: atopic dermatitis; (c) with mixed infiltration of PMN and mononuclear cells: psoriasis and Netherton syndrome. All skin disorders investigated showed an increased EIA as compared with normal skin. Scales from psoriatic lesions, EARLI and Netherton syndrome showed a statistically significant increase in EIA above that observed in other monogenic disorders of keratinization NEARLI, XLRI XLD-CDP and above atopic dermatitis. EIA proved to be an indicator for abnormal keratinization with a marked expression when a mixed infiltrate is present in the skin.