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Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (ACS) inhibitor to enter preclinical development. Our studies demonstrated attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound showed exceptional in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocked P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identified ACS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. MMV693183 was well adsorbed after oral administration in mice, rats and dogs. Pharmacokinetic – pharmacodynamic modelling predicted that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. In conclusion, the ACS-targeting compound MMV693183 represents a promising addition to the portfolio of antimalarials in (pre)clinical development with a novel mode of action for the treatment of malaria and blocking transmission.

Contains fulltext : 248277.pdf (Publisher’s version ) (Open Access) Psoriasis and atopic dermatitis are chronic inflammatory skin diseases characterized by keratinocyte (KC) hyperproliferation and epidermal acanthosis (hyperplasia). The milieu of disease-associated cytokines and soluble factors is considered a mitogenic factor; however, pinpointing the exact mitogens in this complex microenvironment is challenging. We employed organotypic human epidermal equivalents, faithfully mimicking native epidermal proliferation and stratification, to evaluate the proliferative effects of a broad panel of (literature-based) potential mitogens. The KC GF molecule, the T-helper 2 cytokines IL-4 and IL-13, and the psoriasis-associated cytokine IL-17A caused acanthosis by hyperplasia through a doubling in the number of proliferating KCs. In contrast, IFN-γ lowered proliferation, whereas IL-6, IL-20, IL-22, and oncostatin M induced acanthosis not by hyperproliferation but by hypertrophy. The T-helper 2‒cytokine‒mediated hyperproliferation was Jak/signal transducer and activator of transcription 3 dependent, whereas IL-17A and KC GF induced MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase‒dependent proliferation. This discovery that key regulators in atopic dermatitis and psoriasis are direct KC mitogens not only adds evidence to their crucial role in the pathophysiological processes but also highlights an additional therapeutic pillar for the mode of action of targeting biologicals (e.g., dupilumab) or small-molecule drugs (e.g., tofacitinib) by the normalization of KC turnover within the epidermal compartment.

IntroductionPatients with sepsis who present to an emergency department (ED) have highly variable underlying disease severity, and can be categorized from low to high risk. Development of a risk stratification tool for these patients is important for appropriate triage and early treatment. The aim of this study was to develop machine learning models predicting 31-day mortality in patients presenting to the ED with sepsis and to compare these to internal medicine physicians and clinical risk scores.MethodsA single-center, retrospective cohort study was conducted amongst 1,344 emergency department patients fulfilling sepsis criteria. Laboratory and clinical data that was available in the first two hours of presentation from these patients were randomly partitioned into a development (n = 1,244) and validation dataset (n = 100). Machine learning models were trained and evaluated on the development dataset and compared to internal medicine physicians and risk scores in the independent validation dataset. The primary outcome was 31-day mortality.ResultsA number of 1,344 patients were included of whom 174 (13.0%) died. Machine learning models trained with laboratory or a combination of laboratory + clinical data achieved an area-under-the ROC curve of 0.82 (95% CI: 0.80–0.84) and 0.84 (95% CI: 0.81–0.87) for predicting 31-day mortality, respectively. In the validation set, models outperformed internal medicine physicians and clinical risk scores in sensitivity (92% vs. 72% vs. 78%;p0.02). The model had higher diagnostic accuracy with an area-under-the-ROC curve of 0.85 (95%CI: 0.78–0.92) compared to abbMEDS (0.63,0.54–0.73), mREMS (0.63,0.54–0.72) and internal medicine physicians (0.74,0.65–0.82).ConclusionMachine learning models outperformed internal medicine physicians and clinical risk scores in predicting 31-day mortality. These models are a promising tool to aid in risk stratification of patients presenting to the ED with sepsis.

BACKGROUND: Psychological stressors might contribute to the severity of chronic inflammatory diseases such as psoriasis by dysregulating hypothalamic-pituitary-adrenal (HPA) axis activity. OBJECTIVES: To evaluate the role of cortisol, a key component of the HPA axis, in reaction to psychological stress in patients with psoriasis. METHODS: Serum cortisol, clinical indicators of disease severity (Psoriasis Area and Severity Index) and self-report measures of daily stressors were measured monthly for 6 months in 62 patients with psoriasis. RESULTS: In addition to the previous findings in this sample showing that peak levels of daily stressors predicted an increase in disease severity a month later, the peak levels of daily stressors were also significantly associated with a lower cortisol level. Moreover, patients who persistently experienced higher levels of daily stressors had lower mean cortisol levels than patients who experienced lower levels of daily stressors. CONCLUSIONS: Results suggest that daily stressors influence disease outcome in patients with psoriasis by affecting cortisol levels at moments of high stress. Furthermore, patients with persistently high levels of stressors seem to have a specific psychophysiological profile of lowered cortisol levels and may be particularly vulnerable to the influence of stressors on their psoriasis.

Psoriasis is a multifactorial skin disease characterised by epidermal abnormalities and infiltration by lymphocytes and polymorphonuclear leukocytes (PMN). Skin-derived antileukoproteinase (SKALP), also known as elafin, is a potent inhibitor of human leukocyte elastase and proteinase 3, two PMN-derived proteinases implicated in tissue destruction and leukocyte migration. We have shown that, at least at the protein level, SKALP is significantly decreased in lesional skin of patients with pustular psoriasis compared with plaque-type psoriasis. This finding raised the possibility that SKALP could be one of the candidate genes for pustular forms of psoriasis. We therefore performed single strand conformation polymorphism (SSCP) analysis on the SKALP gene to screen for mutations/polymorphisms in the exons of 30 patients with plaque-type psoriasis, 15 patients with pustular psoriasis and 48 healthy controls. In exon 1 a polymorphism was detected at position +43 relative to the translation start site, resulting in a substitution of threonine for alanine in the signal peptide. In the promoter region a dinucleotide repeat polymorphism was identified. Both polymorphisms were not associated with pustular psoriasis, or psoriasis in general. Our data indicate that the decrease in SKALP activity in pustular psoriasis is not caused by mutations in the coding region of the gene, and that there is no allelic association between pustular psoriasis and SKALP gene polymorphisms.

Contains fulltext : 153351.pdf (Publisher’s version ) (Closed access) BACKGROUND: Schnitzler's syndrome (SchS) is an autoinflammatory disease characterized by a chronic urticarial rash, a monoclonal component and signs of systemic inflammation. Interleukin (IL)-1beta is pivotal in the pathophysiology. OBJECTIVES: Here we investigated the cellular source of proinflammatory mediators in the skin of patients with SchS. METHODS: Skin biopsies of lesional and nonlesional skin from eight patients with SchS and healthy controls, and patients with cryopyrin-associated periodic syndrome (CAPS), delayed-pressure urticaria (DPU) and cold-contact urticaria (CCU) were studied. We studied in vivoIL-1beta, IL-17 and antimicrobial protein (AMP) expression in resident skin cells and infiltrating cells. In addition we investigated the in vitro effect of IL-1beta, IL-17 and polyinosinic-polycytidylic acid (poly:IC) stimulation on cultured epidermal keratinocytes. RESULTS: Remarkably, we found IL-1beta-positive dermal mast cells in both lesional and nonlesional skin of patients with SchS, but not in healthy control skin and CCU, and fewer in CAPS. IL-17-positive neutrophils were observed only in lesional SchS and DPU skin. In lesional SchS epidermis, mRNA and protein expression levels of AMPs were strongly increased compared with nonlesional skin and that of healthy controls. When exposed to IL-1beta, poly:IC or IL-17, patient and control primary human keratinocytes produced AMPs in similar amounts. CONCLUSIONS: Dermal mast cells of patients with SchS produce IL-1beta. This presumably leads to activation of keratinocytes and neutrophil influx, and further amplification of inflammation by IL-17 (from neutrophils and mast cells) and epidermal AMP production leading to chronic histamine-independent neutrophilic urticarial dermatosis.

In the present study we compared the effect of a ceramide 3-containing emollient (Locobase(R) Repair) with a control emollient (vaselinum album/cremor lanette ana) and untreated damaged skin using clinical, bioengineering and immunohistochemical methods in two different models of experimentally induced skin barrier dysfunction. In model A (n = 13) skin barrier dysfunction was inflicted at three investigation sites by tape stripping. In model B (n = 13) the volunteers were patch tested at three investigation sites with sodium dodecyl sulphate (0.2%) for 4 h a day for 4 consecutive days. The investigation sites were treated once a day with the above-mentioned agents. Irritant reaction was assessed daily by erythema scoring and measurements of transepidermal water loss (TEWL). After 5D, punch biopsies were taken from all sites. Immunohistochemical assessment was carried out with respect to epidermal proliferation, epidermal differentiation and Langerhans cells. Tape stripping resulted in an erythematous reaction and an increase of TEWL associated with up-regulation of cycling cells, involucrin and expression of cytokeratin 16. At day 4, ceramide 3-containing emollient significantly decreased (p < 0.03) the erythema score, TEWL and cycling cells in comparison with the untreated site. Repetitive exposure to SDS induced a variable degree of erythema, gradual increase of TEWL, an increase of cycling cells, and up-regulation of involucrin, E-FABP and SKALP. The treatment with the control emollient significantly prevented erythema, increase of TEWL and cycling cells at day 4 compared to the untreated site. In summary, the present study demonstrated that both tested emollients improve skin barrier in different conditions compared to the untreated skin. There is some indication that formulations containing skin-related lipids might be of benefit in barrier disruption following tape stripping. Different models and clinical trials are needed to establish the usefulness in specific conditions of emollients containing skin-related lipids.

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.

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.

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a 'bright' population of highly fluorescent small cells and a 'dim' population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocytes was dependent on oligonucleotide concentration, incubation time and temperature. This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be considered when specific oligonucleotides are used against skin disease-specific genes.

The role of epidermal keratinocytes in the early phases of normal unimpaired wound healing has been studied extensively. However, little is known about the cell biological processes in the epidermis and the basal membrane zone during the later phases of dermal matrix formation and remodelling of the scar tissue, This study investigated epidermal growth and differentiation and maturation of the basal membrane zone. Biopsies mere taken from (clinically) hypertrophic and non-hypertrophic scars at 3 and 12 months after a breast-reduction operation, Tissues were analysed using immunohistochemical techniques. The data showed that epidermal abnormalities with respect to differentiation persist up to 3 months, as witnessed by the expression of cytokeratin 16, Remarkably, hypertrophic scars that remained hypertrophic throughout the period of analysis (up to 12 months) showed significantly more cytokeratin 16 expression at 3 months, when compared tither with normal scars or with hypertrophic scars that became normal after 12 months, Staining for Ki-67 antigen, a marker for cell proliferation, revealed an increase in basal keratinocyte proliferation rate in 3-month-old hypertrophic scars compared with non-hypertrophic scars. After 12 months, this difference had disappeared completely and the number of cycling basal cells had returned to normal values, Three-month-old hypertrophic scars showed more acanthosis than non-hypertrophic scars of the same age, irrespective of whether they remained hypertrophic or became normal scars. After 12 months, this difference was no longer present, Staining for various heparan sulphate proteoglycan epitopes revealed that restoration of the basal membrane was incomplete at 3 months, but was complete at 12 months with respect to this component, No differences in the expression of several components of the basal membrane zone (heparan sulphate proteoglycan, laminin, tenascin) were noted between hypertrophic and non-hypertrophic scars, These data show that in the early phase of hypertrophic scarring, epidermal abnormalities are found compared with normal wound healing, In addition, early (3 months) epidermal abnormalities are associated with the clinical outcome at 12 months, These findings raise the possibility that the epidermal compartment is involved in the pathogenic process, (C) 1998 John Wiley & Sons, Ltd.

Summary Impetigo herpetiformis (IH) is a rare pustular dermatosis with unknown aetiology, typically occurring during pregnancy. Based upon a similar clinical and histological presentation, i.e. spongiform accumulation of polymorphonuclear leucocytes in the stratum corneum, several authors consider IH as a variant of generalized pustular psoriasis (GPP), while others state that IH is a separate entity. Skin-derived antileucoproteinase (SKALP) is a strong and specific inhibitor of human leucocyte elastase (HLE) and proteinase 3, two neutral proteinases that have been implicated in leucocyte migration and tissue destruction. Previously, we reported decreased SKALP activity in pustular forms of psoriasis compared with plaque psoriasis. In this study we present a case study of a patient with IH, where SKALP activity was measured using biochemical and immunochemical techniques. Epidermal scales and sera were collected during the course of the disease. Comparison was made with three patients with GPP and six patients with plaque psoriasis. Initially, anti-HLE activity in epidermal scales of the patient with IH was comparable with values in patients with GPP, i.e. decreased compared with plaque psoriasis. During the course of the disease, anti-elastase activity dropped to undetectable levels, concomitant with the appearance of free elastase activity. This finding suggests a total saturation of epidermal anti-HLE activity. Low levels of SKALP, presumably complexed with HLE, could be measured immunochemically in scale extracts. Serum levels of total SKALP correlated with the disease activity. We suggest that a reduced amount of epidermal SKALP contributes to an imbalance between elastase and its inhibitor, resulting in the formation of epidermal pustules. This mechanism of pustule formation could apply both to GPP and IH, suggesting a final common pathway in the pathogenic mechanisms of IH and GPP.

Background. After the injection of rabbit anti-mouse glomerular basement membrane (GBM ) antibody into normal C57BL/6J mice severe albuminuria develops. which reaches a peak at 24 h. This early albuminuria is dependent on polymorphonuclear granulocytes (PMN) and is completely absent in the congenic beige mutant strain (C57BL/6J, bg/bg), which is genetically deficient in leukocytic neutral proteinase activity. We now studied the development of anti-GBM nephritis in beige mice during the later heterologous phase. Methods. In untreated beige mice we assessed the albuminuria and glomerular lesions on days 1-5 after i.v. injection of anti-GBM antibody. Secondly, effector mechanisms involved in the later days of the heterologous phase were studied by substitution of whole anti-GBM antibodies by F(ab') 2 fragments, by leukocyte depletion (total body irradiation), scavenging of reactive oxygen metabolites (dimethylsulfoxide treatment), and complement depletion (cobra venom factor treatment). Results. In the later part of the heterologous phase (days 2-5), when there is still no sign of autologous antibody formation, i.v. injection of anti-GBM antibodies in beige mice induces nephritis with gradually increasing albuminuria, that reaches levels similar to those in non-deficient, congenic controls by day 3. This late albuminuria did not occur after injection of F(ab') 2 fragments of the antibody, could be prevented by leukocyte depletion, and was greatly reduced by treatment with dimethylsulfoxide, a scavenger of hydroxyl radicals. The late albuminuria was not influenced by complement depletion with cobra venom factor. Histologic and immunohistologic studies gave no indication for a role of glomerular macrophages or lymphocytes. Conclusions. The heterologous phase in murine anti-GBM nephritis is a biphasic process, with sequential involvement of different and independent mediating systems : both phases are PMN-dependent, but only the early albuminuria depends on leukocytic neutral proteinase activity, whereas the albuminuria and the glomerular damage at later days are effected by reactive oxygen metabolites, most probably originating from PMN accumulating in the glomerulus.

Skin-derived antileukoproteinase, also described as elafin or as elastase specific inhibitor, is a serine proteinase inhibitor which is thought to play a regulatory role in inflammation. Research in the last few years has increased our knowledge on the structural, biochemical, and cell biological aspects of this molecule. Here we will review the most relevant literature presently available on this proteinase inhibitor.

Recent genome-wide association studies have identified several genetic risk factors for psoriasis, but data on their association with age at onset are lacking.To compare the association between known risk alleles and psoriasis in well-defined cohorts with paediatric- and adult-onset psoriasis.Based on previous studies we selected seven genes and loci associated with psoriasis. Patients with paediatric-onset (18 years) and adult-onset psoriasis (≥ 18 years) and controls were genotyped. Genotype frequencies were compared between controls (n = 450) and all cases (n = 217), and between controls and cases stratified for confirmed age at onset (paediatric onset n = 80, adult onset n = 85).Paediatric-onset psoriasis showed a significant association with single nucleotide polymorphisms in the ERAP1 (P = 0.042) and IL23R loci (P = 0.042), LCE3C_LCE3B-del (P = 0.003) and HLA-C*06 (P = 1.72 × 10(-19)) when compared with the control group. A significant association of these four genes was also demonstrated when all psoriasis cases were compared with controls. In adult-onset psoriasis a significant association was found for HLA-C*06 (P = 5.11 × 10(-6)) and for LCE3C_LCE3B-del (P = 0.042). No associations were found for the IFIH1, IL12B and TRAF3IP2 loci.Notwithstanding the small cohort sizes, we demonstrated an association with established and recently discovered genetic risk factors in paediatric-onset psoriasis including genes involved in epidermal barrier function and adaptive immunity. Our data suggest that heritable factors may play a more important role in paediatric-onset psoriasis than in adult-onset psoriasis.

Recent studies have emphasized the importance of heritable and acquired skin barrier abnormalities in common inflammatory diseases such as psoriasis and atopic dermatitis (AD). To date, no comprehensive studies on the effect of experimental barrier disruption on cornified envelope protein expression have been performed.To analyse the effect of experimental skin barrier disruption on the expression of cornified envelope structural proteins and keratinocyte differentiation-regulating proteins.We examined mRNA (day 1, 3 and 7) and protein (day 1, 2, 4 and 9) expression levels of structural proteins and regulatory molecules after sodium dodecyl sulphate (SDS) application on normal skin, and tape stripping of uninvolved epidermis of patients with psoriasis and AD and healthy controls.Upon tape stripping, several structural molecules were significantly downregulated (at the mRNA level as well as the protein level), including LCE5A, LCE2B, FLG, FLG2 and LOR, whereas others were upregulated: IVL, SPRR1, SPRR2, HRNR and most notably LCE3A. The epidermal crosslinking enzymes TGM1, TGM3 and TGM5 were all upregulated, whereas proteases involved in the desquamation process (CTSV, KLK5 and KLK7) were downregulated or unaffected. Most results were similar in SDS-instigated irritant contact dermatitis. There was no significant difference in response between normal epidermis and nonlesional skin of patients with psoriasis and AD.Skin barrier disruption induces a temporary barrier repair response composed of increased expression of several cornification-related proteins, and decreased expression of some structural and desquamation-related proteins.

In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-beta 1. It was found that human keratinocytes grown in KGM plus TGF-beta 1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and paralleled the decrease of cells in S-phase. Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-beta 1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.

Summary 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.

In this study we performed a cell kinetic characterization of the growth and differentiation of human keratinocytes. Using a combination of immunohistochemical and flow cytometric techniques it was possible to obtain a detailed description of these processes. The proliferative activity of the cell cultures was analysed with flow cytometric techniques, measuring relative DNA content, iododeoxyuridine incorporation and the expression of the antigen recognized by Ki-67. In addition to a standard monolayer culture technique, cells were maintained in suspension. Under these conditions these cells were not capable of dividing, started to lose their nuclei, and the expression of differentiation-related proteins such as involucrin and filaggrin was induced, suggesting that the cells changed towards a differentiated phenotype. Binding of the antibody Ks8.12, recognizing keratins 13 and 16, occurred under all culture conditions, independent of cell density, and also in suspension, suggesting that it is a marker for abnormal differentiation rather than for hyperproliferation.

Psychological stressors might contribute to the severity of chronic inflammatory diseases such as psoriasis by dysregulating hypothalamic-pituitary-adrenal (HPA) axis activity.To evaluate the role of cortisol, a key component of the HPA axis, in reaction to psychological stress in patients with psoriasis.Serum cortisol, clinical indicators of disease severity (Psoriasis Area and Severity Index) and self-report measures of daily stressors were measured monthly for 6 months in 62 patients with psoriasis.In addition to the previous findings in this sample showing that peak levels of daily stressors predicted an increase in disease severity a month later, the peak levels of daily stressors were also significantly associated with a lower cortisol level. Moreover, patients who persistently experienced higher levels of daily stressors had lower mean cortisol levels than patients who experienced lower levels of daily stressors.Results suggest that daily stressors influence disease outcome in patients with psoriasis by affecting cortisol levels at moments of high stress. Furthermore, patients with persistently high levels of stressors seem to have a specific psychophysiological profile of lowered cortisol levels and may be particularly vulnerable to the influence of stressors on their psoriasis.

Tenascin-X is a large extracellular matrix glycoprotein that is widely distributed within connective tissues and is associated with an autosomal recessive type of Ehlers-Danlos syndrome (EDS). Tenascin-X represents the first EDS susceptibility gene that does not code for a fibrillar collagen or collagen-processing enzyme. We describe a paediatric case of tenascin-X deficiency and review the literature.

A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using non-denaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.

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.To analyse if gene expression signatures as found in purified epidermal cells from AD are also present in other eczematous conditions such as allergic and irritant contact dermatitis.We used real-time quantitative polymerase chain reaction, immunohistochemistry and bioinformatics to investigate gene expression in different forms of eczema. Normal epidermis and psoriatic epidermis were analysed for comparison.Carbonic anhydrase II was highly induced in epidermis from all forms of eczema but not in psoriasis. Remarkably, the presumed neuron-specific Nel-like protein 2 showed a strong induction only in AD epidermis. Interleukin-1F9, elafin, beta-defensin-2 and vanin-3 were strongly induced in psoriasis, but not in any type of eczema. High levels of the chemokines CCL17 and CXCL10 were predominantly found in epidermis of allergic contact dermatitis. The chemokine CXCL8 was highly expressed in psoriasis, AD and allergic contact dermatitis, but not in irritant contact dermatitis. Cluster analysis or multinomial logistic regression indicated that expression levels of a set of seven genes are a strong predictor of the type of inflammatory response.These observations contribute to molecular diagnostic criteria for inflammatory skin conditions.

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.

We analysed the diversity of tenascin-X (TNX) species in serum and studied parameters that could affect determination of TNX levels in serum. Using western blot analysis we identified at least seven distinct TNX species, ranging from 75 kDa to the presumably full-length 450 kDa form. Purification of serum TNX followed by sequence analysis positively identified two major TNX species of 75 and 140 kDa. We found that serum TNX binds to tropoelastin but not to fibrillar collagens. We conclude that serum TNX is composed of distinct molecular species that retain functional activity.

Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders with characteristic skin and joint involvement. The concept that EDS is a disease of fibrillar collagen was challenged by the identification of a clinically distinct, recessive type of EDS caused by deficiency of the extracellular matrix protein tenascin-X (TNX). Interestingly, haploinsufficiency of TNX is associated with the dominantly inherited hypermobility type of EDS. In this study, we examined whether missense mutations in the TNX gene can account for some of the cases of hypermobility type EDS. Furthermore, we studied whether missense mutations or heterozygosity for truncating mutations in the TNX gene lead to alterations in the dermal connective tissue. Sequence analysis revealed three missense mutations in TNX in hypermobility type EDS patients, which were not present in 192 control alleles. Morphometric analysis of skin biopsies of these patients showed altered elastic fibers in one of them, suggesting that this missense mutation is disease causing. Light microscopic and ultrastructural changes of the elastic fibers were observed in TNX-haploinsufficient hypermobility type EDS patients, which were not found in hypermobility type EDS patients in whom TNX mutations were excluded. Our results indicate that the observed alterations in elastic fibers are specific for hypermobility type EDS patients with mutations of TNX.

We recently described a new autosomal recessive type of Ehlers-Danlos syndrome (EDS) based on a deficiency of the extracellular matrix protein tenascin-X (TNX). TNX-deficient patients have hypermobile joints, hyperextensible skin and show easy bruising. Because of the reported cardiovascular abnormalities in other EDS types and the excessive haematoma formation after mild trauma in TNX-deficient individuals, we investigated whether cardiovascular or coagulation abnormalities occur in these patients.We examined seven TNX-deficient patients. One of them had a mitral valve prolapse and died postoperatively after valve replacement, before the study was completed.Bleeding time and coagulation factors (INR, APTT, PT and fibrinogen) were all within the normal range. Ultrasonographic examination of the carotid and femoral arteries showed normal vessel wall compliance and distensibility. Echocardiography showed a slight billowing of the mitral valve in two patients from one family. All patients had normal diameters of aortic root and ascending aorta.Although the patient group is small, there are no indications of generalised cardiovascular abnormalities in this type of EDS. We would recommend echocardiography for all these patients at the first evaluation and when a cardiac murmur appears.

The exact role of epidermal fatty acid binding protein (E-FABP) in skin is unknown. A restoration of the barrier function may be associated with an upregulation of E-FABP. Moreover, E-FABP is upregulated in a variety of cells in response to oxidative stress. A recent observation that dithranol induced irritation is not associated with skin barrier impairment prompted us to investigate the expression of E-FABP in this skin condition to elucidate the unknown function of this protein in skin. This study shows lack of E-FABP upregulation after a single application of dithranol on uninvolved skin of patients with psoriasis. The expression of E-FABP in dithranol irritation correlates with the unimpaired skin barrier function as assessed by measurements of TEWL. Furthermore, we did not find evidence for the recently introduced hypothesis that E-FABP functions as an antioxidant protein in the skin irritation induced by dithranol as oxidative stressor.

In the present study we compared the effect of a ceramide 3-containing emollient (Locobase(R) Repair) with a control emollient (vaselinum album/cremor lanette ana) and untreated damaged skin using clinical, bioengineering and immunohistochemical methods in two different models of experimentally induced skin barrier dysfunction. In model A (n = 13) skin barrier dysfunction was inflicted at three investigation sites by tape stripping. In model B (n = 13) the volunteers were patch tested at three investigation sites with sodium dodecyl sulphate (0.2%) for 4 h a day for 4 consecutive days. The investigation sites were treated once a day with the above-mentioned agents. Irritant reaction was assessed daily by erythema scoring and measurements of transepidermal water loss (TEWL). After 5D, punch biopsies were taken from all sites. Immunohistochemical assessment was carried out with respect to epidermal proliferation, epidermal differentiation and Langerhans cells. Tape stripping resulted in an erythematous reaction and an increase of TEWL associated with up-regulation of cycling cells, involucrin and expression of cytokeratin 16. At day 4, ceramide 3-containing emollient significantly decreased (p0.03) the erythema score, TEWL and cycling cells in comparison with the untreated site. Repetitive exposure to SDS induced a variable degree of erythema, gradual increase of TEWL, an increase of cycling cells, and up-regulation of involucrin, E-FABP and SKALP. The treatment with the control emollient significantly prevented erythema, increase of TEWL and cycling cells at day 4 compared to the untreated site. In summary, the present study demonstrated that both tested emollients improve skin barrier in different conditions compared to the untreated skin. There is some indication that formulations containing skin-related lipids might be of benefit in barrier disruption following tape stripping. Different models and clinical trials are needed to establish the usefulness in specific conditions of emollients containing skin-related lipids.

Item does not contain fulltext Oral reconstructions for cleft palate repair are often complicated by a shortage of mucosal tissue. This shortage causes scar tissue formation leading to impaired growth of the dento-maxillary complex. The overall aim of our research is to develop a substitute. which limits the iatrogenic effects of cleft palate surgery. This study describes the culture and characterization of mucosal substitutes containing keratinocytes. Epidermal and oral keratinocytes from a beagle dog were cultured on several skin-derived and collagen-based substrates. Oral keratinocytes cultured on the skin-derived substrates closely resembled normal oral epithelium of the dog. A multi-layered epithelium was formed showing parakeratosis, expression of cytokeratin 16 and the formation of a basement membrane. Epidermal keratinocytes cultured on the skin-derived substrates formed an epithelium which was similar to dog epidermis. In contrast, keratinocytes cultured on the collagen-based substrates invaded the substrate without the formation of a multi-layered epithelium. In conclusion, this study shows that oral canine keratinocytes cultured on skin-derived substrates exhibit a tissue organization that resembles normal oral mucosa. This type of mucosal substitute will therefore be used in further studies for implantation on the palate of beagle dogs. These studies might eventually lead to an improvement of cleft palate surgery in humans.

Item does not contain fulltext Using serial analysis of gene expression on cultured human keratinocytes we found high expression levels of genes putatively involved in host protection and defense, such as proteinase inhibitors and antimicrobial proteins. One of these expressed genes was the recently discovered cysteine proteinase inhibitor cystatin M/E that has not been characterized so far at the protein level with respect to tissue distribution and additional biologic properties. Here we report that cystatin M/E has a tissue-specific expression pattern in which high expression levels are restricted to the stratum granulosum of normal human skin, the stratum granulosum/spinosum of psoriatic skin, and the secretory coils of eccrine sweat glands. Low expression levels were found in the nasal cavity. The presence of cystatin M/E in skin and the lack of expression in a variety of other tissues was verified both at the protein level by immunohistochemistry or western blotting, and at the mRNA level by reverse transcriptase polymerase chain reaction or northern blotting. Using biotinylated hexapeptide probes we found that cystatin M/E is an efficient substrate for tissue type transglutaminase and for transglutaminases extracted from stratum corneum, and that it acts as an acyl acceptor but not as an acyl donor. Western blot analysis showed that recombinant cystatin M/E could be cross-linked to a variety of proteins extracted from stratum corneum. In vitro, we found that cystatin M/E expression in cultured keratinocytes is upregulated at the mRNA and protein level, upon induction of differentiation. We demonstrate that cystatin M/E, which has a putative signal peptide, is indeed a secreted protein and is found in vitro in culture supernatant and in vivo in human sweat by enzyme-linked immunosorbent assay or western blotting. Cystatin M/E showed moderate inhibition of cathepsin B but was not active against cathepsin C. We speculate that cystatin M/E is unlikely to be a physiologically relevant inhibitor of intracellular lysosomal cysteine proteinases but rather functions as an inhibitor of self and nonself cysteine proteinases that remain to be identified.

To protect against the extracellular activity of serine proteinases, the lung is equipped with serine proteinase inhibitors including secretory leukocyte proteinase inhibitor (SLPI) and elafin. Both SLPI and elafin are locally produced by airway epithelial cells, but the mechanisms that regulate the expression of these proteinase inhibitors are relatively unknown. Previous studies using airway epithelial cell lines indicated that neutrophil elastase (NE) increases SLPI mRNA transcripts while decreasing SLPI protein release. Similar results were observed for elafin. The aim of the present study was to investigate the effect of NE on SLPI and elafin synthesis in cultures of human primary bronchial epithelial cells (PBEC).Subcultures of human PBEC were incubated with NE, followed by preparation of cell-free supernatants and cellular lysates and determination of SLPI and elafin protein levels by enzyme-linked immunoadsorbent assay. The effect of NE on SLPI mRNA transcripts was determined by Northern blot analysis.The results showed that NE increased SLPI mRNA expression while decreasing SLPI protein release. This NE-induced decrease was associated with an increase in cell-associated SLPI, providing an explanation for the apparent paradox of increased SLPI mRNA transcripts and decreased SLPI protein levels present in supernatants. In addition, NE had a stimulatory effect on the release of elafin by airway epithelial cells, whereas no increase in cell-associated elafin was observed.The results from the present study indicate that NE may play a role in the regulation of the antiproteinase screen in the lung and the formation of a protective surface at the epithelial site.

Two new elastase inhibitors (SKALP, skin-derived antileucoproteases) were recently described in the lesional skin in psoriasis. The present study investigated the distribution of SKALP activity in the marginal zone of spreading psoriatic plaques. In a 4-mm zone immediately adjacent to the erythemato-squamous plaques, SKALP activity was slightly increased compared to distant uninvolved skin. Within the lesion the anti-elastase activity was pronounced, but was significantly higher in the central zone of the plaque compared to the periphery. The appearance of SKALP in the psoriatic lesion appears to be a late event compared to endothelial involvement, intraepidermal accumulation of PMNs, epidermal proliferation and abnormal keratinization. This observation lends further support for the hypothesis that the induction of anti-elastase activity is associated with the off-switch of cutaneous inflammation.

Targeted and selective inhibition of keratinocyte gene expression in human epidermis could be an efficient and safe pharmacologic approach in many skin diseases. In this study we investigated whether topical application of antisense oligodeoxynucleotides (ODN) on intact human skin can be used to inhibit expression of a gene in the differentiated compartment of the epidermis. We applied a variety of 20-mer antisense and control ODN designed to hybridize to different regions on the mRNA of the inducible epidermal proteinase inhibitor skin-derived antileukoproteinase (SKALP)/elafin that was used as a model target gene. When nuclease-resistant fully phosphorothioate ODN were applied to explant cultures of human skin, they were found to be either ineffective at low doses or severely toxic at higher doses which could be attributed to the extremely high degree of protein binding found with this type of ODN. When chimeric ODN with a phosphodiester core and phosphorothioate 5' and 3' ends were applied to intact skin, no toxicity was noted. One of the tested chimeric ODN, that exhibit only minor protein binding, was found to inhibit SKALP expression at the protein level in a dose-dependent manner. The observed inhibition on SKALP expression levels was specific as evaluated by application of strict criteria. Sequence specificity was assessed by the addition of sense and scrambled ODN which were ineffective. Furthermore the expression levels of three other differentiation-related genes (involucrin, cytokeratin 16, and secretory leukocyte proteinase inhibitor) were not affected, indicating that the inhibition was gene specific. Confocal laser scanning analysis of fluorescently labeled ODN confirmed that these molecules can easily penetrate the epidermis and localize in the cytoplasm of differentiated keratinocytes. We conclude that topical application of antisense ODN can be used to modulate epidermal gene expression, and could potentially be useful to inhibit expression of genes that are relevant in skin diseases.