Topical treatment of mild to moderate plaque psoriasis with 0.3% tacrolimus gel and 0.5% tacrolimus cream: the effect on SUM score, epidermal proliferation, keratinization, T-cell subsets and HLA-DR expression.

BACKGROUND: Tacrolimus gel 0.3% and tacrolimus cream 0.5% were studied and compared with calcipotriol ointment 0.005%, as topical treatment for mild to moderate plaque psoriasis. Tacrolimus is able to inhibit several cellular processes thought to be important in the pathogenesis of psoriasis, e.g. the transcription of proinflammatory cytokines, keratinocyte hyperproliferation and the expression of HLA-DR in lesional psoriatic skin. METHOD: In the present study we investigated the effects of preparations of tacrolimus and calcipotriol ointment on SUM score, hyperproliferation (Ki67-positive keratinocytes), keratinization (percentage keratin 10 (K10)-positive epidermal surface), T-cell subsets (CD4, CD8, CD45RO, CD45RA, CD2, CD25), cells expressing natural killer receptors and HLA-DR expression. The following three topical treatments were studied in chronic plaque psoriasis over a 12-week treatment period: calcipotriol ointment 0.005% twice daily, tacrolimus gel 0.3% twice daily and tacrolimus cream 0.5% twice daily. RESULTS: The mean reductions in SUM score between day 0 and week 12 for calcipotriol ointment, tacrolimus gel and cream were significant. Calcipotriol ointment, and tacrolimus gel and cream had a comparable effect on epidermal proliferation (Ki67-positive cells), but calcipotriol is significantly more effective in normalizing differentiation (K10-positive epidermal surface). Calcipotriol and tacrolimus gel both reduced several lesional T-cell subsets significantly, whereas the effect induced by tacrolimus cream was modest. CONCLUSIONS: Calcipotriol and tacrolimus gel are comparable in reducing the SUM score, the number of Ki67-positive cells and T-cell subsets and HLA-DR expression, although calcipotriol induces a more substantial improvement of keratinization.

Dermal connective tissue development in mice: an essential role for tenascin-X.

Deficiency of the extracellular matrix protein tenascin-X (TNX) causes a recessive form of Ehlers-Danlos syndrome (EDS) characterized by hyperextensible skin and hypermobile joints. It is not known whether the observed alterations of dermal collagen fibrils and elastic fibers in these patients are caused by disturbed assembly and deposition or by altered stability and turnover. We used biophysical measurements and immunofluorescence to study connective tissue properties in TNX knockout and wild-type mice. We found that TNX knockout mice, even at a young age, have greatly disturbed biomechanical properties of the skin. No joint abnormalities were noted at any age. The spatio-temporal expression of TNX during normal mouse skin development, during embryonic days 13-19 (E13-E19), was distinct from tropoelastin and the dermal fibrillar collagens type I, III, and V. Our data show that TNX is not involved in the earliest phase (E10-E14) of the deposition of collagen fibrils and elastic fibers during fetal development. From E15 to E19, TNX starts partially to colocalize with the dermal collagens and elastin, and in adult mice, TNX is present in the entire dermis. In adult TNX knockout mice, we observed an apparent increase of elastin. We conclude that TNX knockout mice only partially recapitulate the phenotype of TNX-deficient EDS patients, and that TNX could potentially be involved in maturation and/or maintenance of the dermal collagen and elastin network.

A recessive form of the Ehlers-Danlos syndrome caused by tenascin-X deficiency.

BACKGROUND: The Ehlers-Danlos syndrome is a heritable connective-tissue disorder caused by defects in fibrillar-collagen metabolism. Mutations in the type V collagen genes account for up to 50 percent of cases of classic Ehlers-Danlos syndrome, but many other cases are unexplained. We investigated whether the deficiency of the tenascins, extracellular-matrix proteins that are highly expressed in connective tissues, was associated with the Ehlers-Danlos syndrome. METHODS: We screened serum samples from 151 patients with the classic, hypermobility, or vascular types of the Ehlers-Danlos syndrome; 75 patients with psoriasis; 93 patients with rheumatoid arthritis; and 21 healthy persons for the presence of tenascin-X and tenascin-C by enzyme-linked immunosorbent assay. We examined the expression of tenascins and type V collagen in skin by immunohistochemical methods and sequenced the tenascin-X gene. RESULTS: Tenascin-X was present in serum from all normal subjects, all patients with psoriasis, all patients with rheumatoid arthritis, and 146 of 151 patients with the Ehlers-Danlos syndrome. Tenascin-X was absent from the serum of the 5 remaining patients with Ehlers-Danlos syndrome, who were unrelated. Tenascin-X deficiency was confirmed in these patients by analysis of skin fibroblasts and by immunostaining of skin. The expression of tenascin-C and type V collagen was normal in these patients. All five of these patients had hypermobile joints, hyperelastic skin, and easy bruising, without atrophic scarring. Tenascin-X mutations were identified in all tenascin-X-deficient patients; one patient had a homozygous tenascin-X gene deletion, one was heterozygous for the deletion, and three others had homozygous truncating point mutations, confirming a causative role for tenascin-X and a recessive pattern of inheritance. CONCLUSIONS: Tenascin-X deficiency causes a clinically distinct, recessive form of the Ehlers-Danlos syndrome. This finding indicates that factors other than the collagens or collagen-processing enzymes can cause the syndrome and suggests a central role for tenascin-X in maintaining the integrity of collagenous matrix.

Investigation on a novel and specific leukotriene B4 receptor antagonist in the treatment of stable plaque psoriasis.

The aim of the present study was to investigate the efficacy and clinical tolerability of the specific leukotriene B4 receptor antagonist VML295 in the treatment of stable plaque psoriasis. Immunohistochemical and flow cytometrical methods were used to assess the effects on inflammation and epidermal proliferation. VML295 in the treatment of chronic plaque psoriasis was shown to be safe and well tolerated. After treatment, there was a statistically significant difference between patients treated with VML295 and patients treated with placebo with respect to the leukotriene B4-induced CD11b up-regulation on the cell surface of polymorphonuclear leukocytes derived from peripheral blood. Ex vivo CD11b up-regulation in the VML295-treated group was completely inhibited after 7 days of treatment (P = 0.001). This effect persisted until the end of the treatment period (P = 0.004 on day 15 and P < 0.0001 after 4 weeks), whereas CD11b up-regulation in the placebo group remained unaffected. There was no statistically significant difference in the median psoriasis area and severity index between the treatment groups at the end of the treatment period. During treatment, no significant histological changes were observed in the markers for cutaneous inflammation and epidermal proliferation. Although not statistically significant, a tendency for the increased expression of some markers of cutaneous inflammation and epidermal proliferation was observed after 1 week of treatment with VML295, and a decreased expression of these markers was seen after 4 weeks of treatment with VML295. This observation could indicate anti-inflammatory effects of VML295 appearing between 2 and 4 weeks after the start of treatment.

Expression of endoglin in psoriatic involved and uninvolved skin.

Endoglin is a glycoprotein with TGF-beta binding capacity and is predominantly expressed on endothelial cells. In psoriasis, TGF-beta has appeared to play a role in the extravasation of peripheral blood mononuclear cells via the endothelium. In order to find out more about the role of endoglin in psoriasis, immunohistochemical staining with PN-E2, a novel anti-endoglin, and of PAL-E, recognizing vascular endothelium, was carried out in psoriatic involved, psoriatic uninvolved and normal skin. The expression of the antigens was assessed semi-quantitatively using a five-point scale. In psoriatic involved skin, a high endoglin expression was found. In psoriatic uninvolved skin, however, we found that endoglin expression was significantly decreased compared with normal skin. The relevance of these findings to the pathogenesis of psoriasis is discussed.

Expression of tenascin, biglycan and decorin in disorders of keratinization.

The distribution of three (recently discovered) extracellular matrix components (tenascin, biglycan and decorin) was studied in normal adult human skin and in a number of monogenic disorders of keratinization, using immunohistology. The expression of tenascin, which is sparsely distributed in normal human dermis, was found to be grossly increased in epidermolytic hyperkeratoses and in Darier's disease. Tenascin expression in three types of ichthyosis (X-linked recessive ichthyosis, autosomal dominant ichthyosis vulgaris, non-erythrodermic lamellar ichthyosis) was similar to that of normal skin. The presence of biglycan and decorin did not show a marked variation between the different disorders studied, suggesting that their expression is subject to regulatory mechanisms distinct from those of tenascin. The increased expression of tenascin in two disorders of keratinization with a hyperproliferative phenotype, lends further support to the hypothesis that dermal tenascin expression is increased as a result of epidermal hyperproliferation.

Cytokeratin expression in alopecia areata hair follicles.

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.

Immunohistochemical localization of SKALP/elafin in psoriatic epidermis.

Recently we have reported the purification and biochemical characterization of a new, inducible elastase inhibitor [skin-derived antileukoproteinase (SKALP)], which could be extracted in high amounts from psoriatic skin but not from normal human skin. Here we demonstrate the immunohistochemical localization of SKALP in psoriatic epidermis. SKALP was found exclusively in the upper layers of the suprabasal compartment and stratum corneum of lesional psoriatic epidermis. Basal keratinocytes were always negative. No immunoreactive SKALP was found in normal epidermis and non-lesional psoriatic epidermis, in accordance with findings in functional assays. Western blots of skin extracts from psoriatic and normal skin confirmed the immunohistochemical findings and revealed two major bands with apparent molecular weights of 10.5 and 11.5 kDa. We would hypothesize that SKALP could act as a modulator of epidermal inflammation by interfering with polymorphonuclear leukocyte trafficking, and that it could protect structural proteins against elastase-mediated damage.

The inter-relation between inflammation and epidermal proliferation in normal skin following epicutaneous application of leukotriene-B4--an immunohistochemical study.

Topical application of leukotriene-B4 (LTB4) on normal skin has been used as an in-vivo model to investigate cutaneous inflammation and epidermal proliferation, which are important phenomena in the pathogenesis of psoriasis. The aim of the present investigation is to further elucidate the interrelation between inflammation and epidermal proliferation, using specific monoclonal antibodies as markers for the different cell types involved. Aliquots of LTB4 were applied on the upperarms of eight healthy volunteers. After LTB4-application, biopsies were taken at consecutive time intervals. On frozen sections, epidermal proliferation was assessed by Ks8.12-(keratin 16) and Ki-67-binding (cycling cells), inflammation was characterized using anti-elastase (PMN), T11 (T-lymphocytes), pan-B (B-lymphocytes), WT 14 (CD14-positive cells) and OKT 6 (Langerhans cells). New observations were that the density of CD14-positive cells was increased even at 8 h and decreased slightly at 72 h. A striking rearrangement of Langerhans cells was seen in close vicinity to intra-epidermal accumulations of PMN. Remarkably an increased density of these cells in the dermis at 72 h was seen and a decrease in the epidermis. In line with previous studies, the accumulation of PMN reached a maximum 24 h after LTB4-challenge. The identity of the mononuclear infiltrate cells which have been reported 48-72 h after LTB4 proved to be T-lymphocytes. No B-lymphocytes were observed. Ki-67-positive nuclei were maximally increased 72 h after LTB4-application, which implies that recruitment of cycling cells is of relevance for the LTB4-induced proliferation in vivo. The hyperproliferation-related keratin 16 was expressed inconsistently in the suprabasal compartment.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal proliferation and keratinization following standardized elicitation with diphenylcyclopropenone.

Diphenylcyclopropenone (DCP) was applied to the upper arms of five alopecia areata patients using 10% of the concentration that had been applied previously to the scalp during topical immunotherapy. DCP applied in this concentration evoked a mild eczematous reaction. Biopsies were taken before DCP application and after 24, 48 and 96 h. A large increase in T-lymphocytes and CD14-positive cells in the dermis was seen after 24 h. Migration of these cells into the epidermis was mainly observed during the first 48 h. This was followed by epidermal proliferation as assessed by the number of Ki-67-positive nuclei and the degree of Ks8.12-binding. Both showed their main increase after 48 h; but after 24 h the increase of Ki-67-positive nuclei was significant (P < 0.04). Involucrin and filaggrin showed a gradual increase which became substantial after 96 h (both P < 0.04). As the invasion of inflammatory cells into the epidermis preceded the main increase in epidermal proliferation, cytokines are suggested as possible mediators for the initial phase of the proliferative response after DCP application.

Abnormal expression of Ki-67 antigen in hair follicle of alopecia areata.

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.

Clinical and immunohistochemical responses of plantar warts to topical immunotherapy with diphenylcyclopropenone.

A 30-year-old man with bilateral plantar warts of the mosaic type which had been resistant to standard treatment modalities was treated with diphenylcyclopropenone. After 10 weeks, the treated warts had disappeared; the untreated warts, although showing some involution, still persisted. The untreated warts, serving as a control to prove the effectiveness of topical immunotherapy, responded likewise to subsequent treatment with diphenylcyclopropenone. Wart regression was reflected histopathologically by decreases in acanthosis, papillomatosis, granular vacuolation, and hyperkeratosis. Immunohistochemically, Ki-67 expression was markedly reduced, and a reversal of the CD4/CD8 ratio was seen. These findings suggest a major role of a cell-mediated immune response in the spontaneous resolution of warts.

Clinical and biochemical effects of an oral leukotriene biosynthesis inhibitor (MK886) in psoriasis.

Antipsoriatic agents have been shown to decrease skin levels of arachidonic acid and its metabolites including 12-monohydroxy-eicosatetranoic acid (12-HETE), and leukotriene B4 (LTB4). In addition, specific systemic and topical lipoxygenase inhibitors have been reported to be effective in the treatment of psoriasis. The objective of this study was to investigate the effect of a potent oral leukotriene biosynthesis inhibitor (MK886) in patients with chronic plaque psoriasis. Clinical response together with the changes of LTB4 levels in lesional skin biopsy specimens, and urinary leukotriene E4 (LTE4) excretion were evaluated. In addition, markers of inflammation, proliferation and keratinization were studied immunohistochemically. No change in clinical scores or lesional LTB4 levels were observed with a 10 1/3-day course of MK886. A statistically significant reduction in urinary LTE4 excretion was observed: mean LTE4 (ng/h) were 5.14 before treatment and 1.51 on day 11 with MK886; and 7.55 before treatment and 6.57 on day 11 with placebo treatment. Epidermal accumulation of polymorphonuclear leukocytes (PMN) tended to diminish in the MK886 treatment group. These results indicate that although a reduction (greater than 70%) in urinary LTE4 excretion was found, and a slight decrease of epidermal PMN accumulation was observed, no correlative changes in clinical scores or LTB4 levels in skin lesion were found with a short course of MK886.

Tenascin expression in hyperproliferative skin diseases.

The expression of tenascin, a recently discovered extracellular matrix glycoprotein, was studied by immunohistochemistry in normal human skin and in a number of skin diseases with epidermal hyperproliferation such as psoriasis, basal cell carcinoma, Bowen's disease and solar keratosis. Tenascin expression in the upper dermis of normal skin was found to vary from almost absent to patchy along the basal membrane. Staining was continuous and intense around blood vessels, hair follicles and eccrine sweat ducts. In basal cell carcinoma a marked expression of tenascin was found in the tumour stroma, especially adjacent to the basal membrane surrounding the tumour cell nests. In Bowen's disease and solar keratosis, tenascin expression was found in the dermis next to the keratinocytes. In psoriasis the dermal papillae of clinically involved skin were intensely stained and a continuous band of tenascin was present in the upper dermis along the basal membrane. The distribution of tenascin differed from other known extracellular matrix components.