Dutch translation, adaptation and validation of the OT-10 scale for orthostatic tremor.

Introduction: There are currently no effective treatments for primary orthostatic tremor (POT). An adequate disease-specific POT severity scale is a prerequisite to conduct clinical trials and monitor disease severity in clinical practice. Recently, the English OT-10 scale has been developed for this purpose. Here we aimed to obtain a scale to measure the severity of POT in Dutch speaking individuals. Methods: An established translation, adaptation and validation approach was employed to obtain a Dutch version of the OT-10 scale. Validation was performed in a Dutch POT cohort (n = 46). Results: A Dutch OT-10 scale was obtained which showed good internal consistency (Cronbach's alpha > 0.80), total score test-retest reliability (intraclass correlation coefficient > 0.80), and concurrent validity (Pearson correlation > 0.80). Item-to-total correlation was good (weighted kappa > 0.40) for all items, and item test-retest reliability was good (weighted kappa > 0.40) for eight out of ten items. Overall, the Dutch OT-10 scale demonstrated acceptable validity. Conclusions: We obtained and validated a Dutch version of the OT-10 scale, capturing POT severity. Next to its use in clinical practice, translation and validation of the OT-10 scale in more languages will help to find evidence-based treatments for POT.

Development and application of monoclonal antibodies against SKALP/elafin and other trappin family members.

SKALP/elafin is an epithelial proteinase inhibitor with antimicrobial properties that is not normally expressed in human epidermis, but is induced under inflammatory conditions and in some types of skin cancer. SKALP is a member of the recently described trappin gene family, which encodes a new class of proteins, characterized by a four-disulphide core and a transglutaminase substrate domain. Polyclonal antisera against SKALP have been shown to be useful for monitoring disease activity in psoriasis and tumour differentiation in squamous cell carcinoma. We developed ten different mouse monoclonal antibodies (mAbs) against synthetic peptides corresponding to a hexapeptide epitope in the transglutaminase substrate domain and three mAbs recognizing an epitope in the proteinase-inhibiting domain. The antibodies could be used with high specificity by immunohistochemistry on formalin-fixed tissue, by affinity chromatography, by Western blotting, and by enzyme-linked immunoadsorbent assay (ELISA) for the detection of SKALP/elafin. These antibodies have several advantages over existing polyclonal antisera, such as a defined epitope, the detection of full-length SKALP/elafin and unlimited supply. An antibody against the hexapeptide epitope, which is common to all known human, simian, bovine and swine trappin family members, was used to immunolocalize bovine trappins expressed in trachea, that have recently been discovered. These mAbs will serve as important new tools to measure SKALP/elafin and trappin family members in research and diagnostics.

In situ demonstration of phosphorylated c-jun and p38 MAP kinase in epidermal keratinocytes following ultraviolet B irradiation of human skin.

Ultraviolet B (UVB) irradiation is known to induce activation of cellular stress response pathways in cultured cells or intact human skin, as demonstrated by phosphorylation of MAP kinase family members and up- or down-stream targets, using biochemical assays. This study demonstrates by immunohistochemistry that low-dose UVB irradiation of normal human skin induces rapid and reversible phosphorylation of c-jun (a target of c-jun N-terminal kinase) and p38 mitogen activated protein kinase (p38 MAP kinase). Phosphorylation was maximal at 4-8 h and returned to normal levels at 48 h after irradiation. Nuclear localization of these phosphorylated substrates was found using antisera against the epitope containing the phosphorylated serine-73 of c-jun, and the dually phosphorylated epitope (threonine-180 and tyrosine-182) of p38 MAP kinase. Nearly all epidermal cells were positive for c-jun phosphorylation, whereas p38 phosphorylation was seen predominantly in the differentiated layers. In contrast to the massive activation of c-jun and p38, only a small population of the suprabasal cells showed nuclear translocation of nuclear factor kappa B (NFkappaB), and a few scattered cells became apoptotic, as determined by TUNEL (TdT mediated dUTP nick end labelling) staining. The expression of involucrin and skin-derived anti-leukoproteinase (SKALP)/elafin, two genes putatively under control of the c-jun and p38 pathways, was found to be increased. These findings establish the first cellular localization of UVB-induced protein phosphorylation of stress response proteins in human epidermis, thereby providing a link between cellular activation and gene expression in defined cell populations.

Expression of tenascin-C splice variants by human skin cells.

Tenascin-C is an extracellular matrix glycoprotein that is expressed in a spatially and temporally restricted pattern. Various functionally different tenascin-C isoforms can be expressed as a result of alternative splicing of the pre-mRNA. Previously we identified human epidermal keratinocytes as a source of tenascin-C in healing wounds. In this study, we investigated whether different tenascin-C transcripts are expressed by epidermal keratinocytes and dermal fibroblasts. In addition, we compared expression of tenascin-C splice variants at the mRNA and protein levels in tissue samples of normal and diseased skin. Northern blot analysis revealed two major tenascin-C mRNA transcripts of approximately 7500 and 5800 nucleotides in cultured epidermal keratinocytes and fibroblasts, and in biopsies. Although both dermal fibroblasts and epidermal keratinocytes predominantly expressed the larger tenascin-C mRNA, epidermal keratinocytes expressed smaller transcripts at higher levels than dermal fibroblasts. In keratinocytes the levels of the two mRNAs were differentially affected by inflammatory cytokines that increased tenascin-C expression in these cells. The addition of IFN gamma slightly increased the proportion of large transcripts. In contrast, TNF alpha favoured expression of smaller tenascin-C transcripts, and IL-4 equally affected the expression of large and small tenascin-C mRNAs. To enable detection of tenascin-C transcripts that are expressed at very low levels, we amplified by polymerase chain reaction the fibronectin type III repeats whose expression is regulated by alternative splicing. In cDNA of cultured keratinocytes and fibroblasts, and in skin biopsies, several tenascin-C transcripts could be detected that corresponded to tenascin-C variants including different numbers of fibronectin type III repeats. Distribution of tenascin-C isoforms at the protein level was studied immunohistochemically in healthy skin, wounds, psoriatic lesions and epidermal tumours and hyperplasia. No differences were observed in reactivity between an antibody that binds all tenascin-C isoforms and antibodies that bind fibronectin type III repeats that can be spliced out from smaller tenascin-C isoforms. We conclude that the tenascin-C isoforms that are translated from transcripts that we identified at the mRNA level seem to be distributed similarly in the conditions investigated.

TNF-alpha and serum induce SKALP/elafin gene expression in human keratinocytes by a p38 MAP kinase-dependent pathway.

Keratinocytes of inflamed epidermis (psoriasis, wound healing) are hyperproliferative and display an abnormal differentiation programme. This regenerative differentiation pathway is characterized by the induction of genes that are not expressed by keratinocytes in normal skin, such as the cytokeratins CK6, CK16, CK17, and the proteinase inhibitor SKALP/elafin. In the study reported here we investigated the induction and regulation of SKALP expression as a marker for regenerative differentiation in epidermal keratinocytes. Various cytokines and growth factors known to be present in psoriatic epidermis were examined for their ability to induce SKALP gene expression in cultured human keratinocytes. Tumour necrosis factor-alpha (TNF-alpha) and serum were found to be potent inducers of SKALP expression at both the mRNA and the protein levels. SB202190 or SB203580, two specific p38 MAP kinase inhibitors almost completely blocked the induction of SKALP expression by TNF-alpha and serum. These results suggest that in keratinocytes, p38 activity is crucial for the induction of SKALP gene expression. These findings could be relevant for the elucidation of the mechanisms involved in normal and disturbed epidermal differentiation.

Tenascin-C degradation in chronic wounds is dependent on serine proteinase activity.

Degradation of extracellular matrix (ECM) components by proteinases is part of the physiological remodelling process during normal wound healing. Excessive degradation of the ECM, however, is likely to create an environment that can no longer support keratinocyte migration and is thought to play a role in the impaired healing of chronic ulcers. Tenascin-C is an ECM component that is markedly upregulated in acute and chronic wounds. Here we report on our investigations into the degradation of tenascin-C in chronic venous leg ulcers. We found proteolytic fragments of tenascin-C in leg ulcer exudate. We also detected fragments of fibronectin in the wound fluid and in addition observed breakdown of fibronectin by wound fluid in vitro, as has previously been reported by others. Wound fluid of four out of six chronic leg ulcers degraded purified human tenascin-C in vitro, and degradation of tenascin-C correlated with high levels of functionally active leucocyte elastase and metalloproteinases in the wound fluid. To identify which proteinases were involved in tenascin-C degradation, we tested the effect of specific proteinase inhibitors. The addition of EDTA or E64 did not protect tenascin-C from degradation, suggesting that neither metalloproteinases nor cysteine proteinases are responsible for cleavage. Tenascin-C breakdown was inhibited by PMSF and SKALP/elafin, and we therefore conclude that leucocyte elastase and possibly other serine proteinases are the tenascin-C-degrading enzymes in ulcer exudate. Taking into account the possible effects of tenascin-C and tenascin-C fragments on cell behaviour, we hypothesize that degradation of tenascin-C could affect the healing process in chronic venous ulcers.

Tenascin-C is not a useful marker for disease activity in psoriasis.

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.

The effects of oral liarozole on epidermal proliferation and differentiation in severe plaque psoriasis are comparable with those of acitretin.

The imidazole derivative liarozole is a potent inhibitor of cytochrome P450-dependent 4-hydroxylation of endogenous all-trans retinoic acid, thereby increasing the levels of all-trans retinoic acid in both plasma and skin. As part of a large, double-blind, randomized clinical study, we investigated the cell biological alterations in uninvolved and lesional skin of 20 patients with severe plaque psoriasis, who were treated with either liarozole or acitretin. The extent and severity of the skin lesions, as recorded by the Psoriasis Area and Severity Index score, was significantly reduced (P

Transcription factor C/EBPalpha: novel sites of expression and cloning of the human gene.

We describe the characterization of recombinant clones for the human transcription factor CCAAT/enhancer binding protein alpha (hC/EBP alpha). The intronless hC/EBP alpha gene is almost 90% homologous to its rat and mouse counterparts. The gene copies of more distant species are less conserved, but the alignment reveals a striking homology in five regions, of which four may be involved in transactivation functions while the fifth concerns the carboxy-terminal bZip sequences (basic region and leucine zipper) mediating sequence specific DNA-binding. In addition to the usual expression sites, significant transcript levels were detected in the epidermal compartment of human skin and in rat aorta by northern analysis. The presence of hC/EBP alpha is further documented by immunohistochemical analysis of human skin biopsies and cultured keratinocytes showing the nuclear presence of the protein, notably in the suprabasal layers of the epidermis and in human keratinocytes induced to differentiate.

Human epidermal keratinocytes are a source of tenascin-C during wound healing.

Tenascin-C is a large hexameric extracellular matrix glycoprotein that is expressed in a temporally and spatially restricted pattern associated with stromal-epithelial interactions. In adult human skin, the expression level of tenascin-C is low, but tenascin-C is abundantly present in the dermal compartment during embryogenesis and wound healing and in skin tumors. Herein we have investigated the cellular source of tenascin-C production in human skin, both in vivo and in vitro, by using immunohistochemistry, mRNA in situ hybridization, western blotting, and an enzyme-linked immunosorbent assay. In addition we studied the cell-matrix interaction between epidermal keratinocytes and purified tenascin-C. By using in vitro culture models, we found that keratinocytes not only synthesize and secrete tenascin-C but can also deposit tenascin-C in de-epidermized dermis in a pattern that is very similar to that in vivo. In vivo, during wound healing of normal human skin, we found tenascin-C extracellularly in the wound bed and also in a granular pattern within the neo-epidermis. By mRNA in situ hybridization, we could identify the basal migrated keratinocytes as the main source of tenascin-C in the early phase of wound healing. In the granulation phase, tenascin-C expression by the keratinocytes is downregulated. Cultured keratinocytes were found to adhere poorly to tenascin-C, and those that did adhere retained a rounded morphology. We conclude that human keratinocytes are a major source of tenascin-C during the early phase of wound healing, and we hypothesize that tenascin-C is unlikely to be an adhesive substrate for migrating keratinocytes.

Skin-derived antileukoproteinase (SKALP) and epidermal fatty acid-binding protein (E-FABP): two novel markers of the psoriatic phenotype that respond differentially to topical steroid.

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.

Basal membrane heparan sulphate proteoglycan expression during wound healing in human skin.

Heparan sulphate proteoglycans (HSPGs) are integral components of the basement membrane (BM) in various tissues. HSPGs are important in the assembly and structure of the BM, and their putative functions include regulation of basement membrane permeability, binding of growth factors, and a role in cellular adhesion. In this study the expression of HSPG was examined during wound healing in human skin, using monoclonal antibodies (MAbs) that recognize the HSPG core protein and two different heparan sulphate (HS) epitopes, and the dynamics of HSPG expression were investigated in relation to epidermal cellular proliferation and permeability of the BM. Healing of excisional wounds in healthy volunteers was studied from day 0 up to 1 year. Intact human skin showed strong continuous staining of the dermo-epidermal BM and the vascular BM with all MAbs. Up to day 4 after wounding, staining for HSPG was absent under the ingrowing epidermis, with any of the MAbs, indicating that no complete BM was present. From day 7 onwards, the BM of the neo-epidermis showed positive staining for the HSPG core protein and a low sulphated HS epitope, and after day 14, the staining intensity was similar to normal skin. The staining patterns of these HSPG epitopes were similar to that of laminin. The staining pattern with a MAb against an epitope in the highly sulphated part of HS was found to be distinct from the other BM markers studied. This epitope was absent under the neo-epidermis up to 2 months after wounding. One year after wounding, the epitope was found to be present again. We observed that only in the time period between 2 months and 1 year had the epidermis normalized with respect to the number of cycling cells and the absence of high molecular weight plasma proteins. These findings suggest a correlation between normalization of epidermal proliferation, BM permeability, and regeneration of BM HS. It is proposed that complete BM maturation following skin wounding is a slow process and may account for the epidermal abnormalities that persist for a considerable period of time after wound healing.

Tenascin expression during wound healing in human skin.

In adult human skin, the expression of the extracellular matrix glycoprotein tenascin is limited. Under hyperproliferative conditions such as psoriasis and epidermal tumours, dermal tenascin expression is strongly upregulated. The aim of this study was to investigate the pattern and kinetics of tenascin expression in human skin during wound healing and to address the question of whether keratinocytes can directly interact with tenascin during re-epithelialization. Tenascin expression was investigated in excisional wounds in normal human skin, in explants of normal human skin, and in chronic venous ulcers, using immunohistochemistry. No tenascin staining was found directly underneath the leading edge of the sheet of migrating keratinocytes in the excisional wounds and explants. In the excisional wounds and the ulcers, dermal tenascin was strongly upregulated in areas adjacent to hyperproliferative epidermis. These hyperproliferative areas are located approximately 10-50 cells behind the leading edge, as assessed by staining for the Ki-67 antigen and the proliferating cell nuclear antigen (PCNA). At the later stages of normal wound healing and in the chronic ulcers, tenascin was also detected in the wound bed. In these areas, the dermal-epidermal junction stained positive for laminin but was negative for heparan sulphate. The absence of the latter basement membrane component suggests that the formation of a new basement membrane is not completed in these wounds. These findings suggest that tenascin is not a substrate for migrating keratinocytes; that the rapid induction of tenascin expression in the papillary dermis during wound healing results from interaction with the hyperproliferative epidermis; and that in the later stages of wound healing, keratinocytes can potentially interact with tenascin in the wound bed, because the basement membrane of the neo-epidermis is incomplete.

Comparison between the hypoosmotic swelling test and morphology evaluation using strict criteria in predicting in vitro fertilization (IVF).

BACKGROUND: The role of male factor with respect to sperm morphology, progressive motility, and density is studied under in vitro conditions. METHODS: The semen samples of 67 males participating in an in vitro fertilization program were evaluated by the conventional WHO criteria of spermatogram, by morphology evaluation using strict criteria (MEUSC), and by the hypoosmotic swelling test (HOST). All sperm tests were performed in the original semen sample as delivered on the day of IVF, before further sperm treatment. The correlations between these parameters and the fertilization outcome were evaluated and their predictive values were calculated. RESULTS: When the patients were divided into two groups, namely, fertile (fertilization rate per oocyte greater than 0%) and infertile (fertilization rate per oocyte = 0%), only mean sperm density and morphology were significantly different between the groups (P less than 0.05). The correlation with fertilization rate in vitro was in favor of MEUSC. CONCLUSIONS: Our results show that the HOST is inferior to MEUSC and conventional WHO sperm analysis in predicting fertilization in vitro.

Epidermal transglutaminase in the ichthyoses.

Membrane-bound transglutaminase (TGm) is responsible for the cross-linking of proteins to form the cornified envelope. Since abnormalities have been reported in the envelope in certain ichthyoses, we have carried out a survey of TGm concentrations in scales from these disorders. Surprisingly, a striking and specific increase in enzyme activity was found in patients with non-erythrodermic autosomal recessive lamellar ichthyosis. It is not clear how this increase is related to the underlying recessive mutation.

Enzymatic distinction between two subgroups of autosomal recessive lamellar ichthyosis.

It has been proposed that the autosomal recessive lamellar ichthyoses may be divided into two subgroups, the erythrodermic (EARLI) and non-erythrodermic (NEARLI) forms. We report measurements of the enzymes beta-glucosidase, a recently described phosholipase, a short-chain carboxylesterase ("butyrase"), and a long-chain carboxylesterase ("palmitase") in aqueous extracts of scales from patients diagnosed according to clinical and micromorphologic criteria, and show that beta-glucosidase and phospholipase tend to be lower in the EARLI group, whereas butyrase is relatively low in the NEARLI group. The internal ratio of either butyrase/glucosidase or butyrase/phospholipase yields a clear separation of the two subgroups, supporting the concept of heterogeneity in this group of diseases.

Membrane-bound phospholipase C activity in normal and psoriatic epidermis.

We report the quantification of a membrane-bound phospholipase C in human epidermis which is active against the physiologically relevant substrate, phosphatidylinositol 4,5-bisphosphate. The level of this enzyme is significantly increased in the psoriatic lesion, both on a weight and protein basis. Etiological implications of this observation are discussed.