P.E.J. van Erp

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases.

Memory effector (CD45RO+) and cytotoxic (CD8+) T cells appear early in the margin zone of spreading psoriatic lesions in contrast to cells expressing natural killer receptors, which appear late

Background  An influx of immunocytes, increased epidermal proliferation and abnormal keratinization are hallmarks of the psoriatic lesion. T‐lymphocyte subsets in particular activated effector memory T cells and natural killer (NK) T cells have been suggested to play an important role in the pathogenesis of psoriasis.

Pretreatment to Enhance Protoporphyrin IX Accumulation in Photodynamic Therapy

The response rates of photodynamic therapy (PDT) vary widely. Limited uptake of topically applied 5-aminolaevulinic acid (ALA), or its methyl ester (MAL), and suboptimal production of protoporphyrin IX (PpIX) may account for these differences. Recently, we demonstrated that hyperkeratosis is an important negative factor in ALA uptake. This review has its focus on pretreatment of the skin in order to improve the clinical outcome of ALA/MAL PDT. Pretreatment of hyperkeratosis can be achieved with keratolytics, curettage/debulking, tape stripping, microdermabrasion or laser ablation. Penetration enhancers may alter the composition or organization of the intercellular lipids of the stratum corneum. Several studies have been performed on the use of dimethyl sulfoxide, azone, glycolic acid, oleic acid and iontophoresis to increase the penetration of ALA. As PpIX production is also dominated by temperature-dependent processes, elevating skin temperature during ALA application may also improve treatment results. Another approach is the use of additives that interact with the heme biosynthetic pathway, e.g. by removing ferrous iron with iron-chelating substances such as: ethylenediaminetetraacetic acid; 3-hydroxypyridin-4-ones; 1,2-diethyl-3-hydroxypyridin-4-one-hydrochloride; and desferrioxamine. In conclusion, simple pretreatments or additions to the regular practice of PDT, aimed to optimize intralesional PpIX content, improve the clinical outcome.

Keratin 17: a useful marker in anti-psoriatic therapies.

The investigation of changes that take place during development or therapeutical regression of psoriatic lesions can give us a clearer insight into processes that are of importance in the pathogenesis and treatment of this disease. In previous studies, different parameters have been used to investigate increased epidermal cell proliferation and disturbed differentiation in psoriasis. The histological appearance [6, 10, 11], percentage of cells in SG2Mphase [1], incorporation of 3H-thymidine [2], or BrdUrd [13], Ki-67-staining [3, 13], and upor down-regulation of distinct keratins [7] have been used as parameters. The aim of the present study was to investigate further the effect of anti-psoriatic therapies on keratin expression in human epidermis. In psoriasis, keratins are expressed that are only found in small amounts or not at all in normal human skin. Also down-regulation of keratins that are related to differentiation takes place [14]. Assessing the amount of keratin numbers 2, 10, 16 and 18 in psoriatic skin has proved to be a useful approach in monitoring therapies. Keratin 16, especially, correlates well with progression or regression of the lesions [4, 7]. Keratin 17 is not present in normal human epidermis; it is restricted to the lower part of the outer root sheath of the hair follicle, and to myoepithelial cells of the sebaceous gland. It is expressed by basal cell carcinomas, which has led to the hypothesis that basal cell carcinomas have a follicular origin [8, 12]. A correlation of keratin 17 expression and hyperproliferation has also previously been suggested [8, 12]. Recently the expression of keratin 17 has been shown in lesional psoriatic epidermis of the scalp [16]. In this study the presence and localization of keratin 17 in psoriatic lesions was investigated further and the

Molecular diagnostics of psoriasis, atopic dermatitis, allergic contact dermatitis and irritant contact dermatitis.

Background  Microarray studies on the epidermal transcriptome in psoriasis and atopic dermatitis (AD) have revealed genes with disease‐specific expression in keratinocytes of lesional epidermis. These genes are possible candidates for disease‐specific pathogenetic changes, but could also provide a tool for molecular diagnostics of inflammatory skin conditions in general.

A sequential double immunoenzymic staining procedure to obtain cell kinetic information in normal and hyperproliferative epidermis.

SummaryA sequential double immunoenzymic staining procedure was developed using the monoclonal antibody anti-BrdUrd and Ki67 in order to determine whether hyperproliferative skin disorders, such as psoriasis, are characterized by an increased growth fraction rather than a much shorter cell cycle time of all germinative cells. Ki67 binds to a proliferation-associated nuclear antigen in a variety of human cell types, and anti-BrdUrd can be used to identify DNA-synthesizing cells. Although in hyperproliferative epidermis the absolute numbers of BrdUrd-positive cells as well as Ki67-positive cells were grossly increased, the ratio of these values was not changed compared to the ratio found in the epidermis of the clinically uninvolved skin of psoriatic patients and in normal epidermis. This suggests an increased growth fraction in hyperproliferative epidermis.Our data show that immunohistochemical double-staining techniques can be a valuable tool in the study of cell cycle kinetics in epithelial tissues.

The effect of the combination of calcipotriol and betamethasone dipropionate versus both monotherapies on epidermal proliferation, keratinization and T-cell subsets in chronic plaque psoriasis

Abstract: Several reports have indicated that the combination of calcipotriol ointment and potent or ultrapotent corticosteroids are more effective and better tolerated, as compared to the monotherapies.

A placebo-controlled randomized study on the clinical effectiveness, immunohistochemical changes and protoporphyrin IX accumulation in fractionated 5-aminolaevulinic acid-photodynamic therapy in patients with psoriasis

Background  Topical 5‐aminolaevulinic acid (ALA)‐photodynamic therapy (PDT) for the treatment of psoriasis has been evaluated in a few studies. In these studies different treatment parameters were used, there was a variable clinical response, and a nonhomogeneous fluorescence was seen after irradiation with Woods light.

Identification of lesional CD4+ CD25+ Foxp3+ regulatory T cells in psoriasis

Background: Depletion of CD4+ CD25+ Foxp3+ naturally occurring regulatory T cells (Treg) induces autoimmune phenomena. These cells have not yet been fully characterized in the skin of psoriatic patients. Objectives: To prove that the Zenon immunofluorescent labeling technique is suitable for the demonstration of co-localization of T-cell markers and in particular to show the distribution of Treg in psoriatic skin. Methods: In biopsies derived from normal and psoriatic skin, CD4+ CD25+, CD4+ CD45RO+, CD8+ CD25+, CD8+ CD45RO+ and CD4+ CD25+ Foxp3+ cells in the dermis and in the epidermis were immunophenotyped, using a quantitative immunofluorescent labeling technique (Zenon), analyzed and compared using image analysis. Results:The immunofluorescent labeling technique was shown to be an easy and reliable tool to demonstrate co-localization of T-cell markers. In psoriasis, all pathogenic T-cell subsets (CD4+ CD25+, CD4+ CD45RO+, CD8+ CD25+ and CD8+ CD45RO+ cells) were significantly increased in the dermis and in the epidermis, as compared to normal skin (all p < 0.05). Using this labeling technique we were able to reveal CD4+ CD25+ Foxp3+ Treg in psoriatic dermis, but not in the dermis of normal skin (p < 0.0001). Conclusions:The Zenon immunofluorescence technique in combination with image analysis is suitable for the demonstration of co-localization of T-cell markers in tissue. Increased numbers of pathogenic T cells (CD4+ CD25+, CD4+ CD45RO+, CD8+ CD25+ and CD8+ CD45RO+) were shown in the dermis and epidermis, whereas CD4+ CD25+ Foxp3+ Treg were identified in psoriatic skin with a predilection for the upper dermis.

Repeated tape stripping of normal skin: a histological assessment and comparison with events seen in psoriasis

The aim of the present study was to investigate the response of normal human skin to repeated courses of Sellotape stripping. The skin of healthy volunteers was stripped five times at 24-h intervals. Skin biopsies were taken before stripping (day 0) and on days 2, 4, 7 and 10. The responses were studied using H & E staining and an immunohistochemical analysis of several aspects of epidermal proliferation and keratinization. Although increased proliferation (nuclear binding to Ki-67 binding), acanthosis and parakeratosis were observed, the overall histological picture did not resemble psoriatic histology completely: no micropustules of Kogoj and no thinning of the suprapapillary plate were observed. Involucrin staining followed the recruitment of cycling epidermal cells showing a statistically significant elevation of positive cell layers from day 2 onwards. Filaggrin expression showed an increase from day 2 onwards, which was statistically significant on day 7 and day 10. Using the anti-keratin antibodies KS8.12 (K13 and K16) and RKSE60 (K10) we observed a fast induction of K13/K16 expression, while the staining of keratin 10 showed the same overall intensity at different time intervals. In conclusion, the response to repeated courses of tape stripping provides an adequate model for studies on epidermal proliferation, hypergranulosis and hyperkeratosis. This approach causes a more prolonged induction of these phenomena than a single course of stripping. In contrast to the situation following a single course of stripping, repeated tape stripping induced the expression of filaggrin. Therefore the repeated tape stripping model is less compatible with psoriasis than a single course of stripping.