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The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14+ DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system. Conventional co-culture systems often lack physiological complexity of the tumor microenvironment. Here, the authors report an organotypic skin melanoma culture and use this model to investigate the tumor induced suppression on dendritic cells. [ABSTRACT FROM AUTHOR]

Diseases of the skin are amenable to RNAi-based therapies and targeting key components in the pathophysiology of psoriasis using RNAi may represent a successful new therapeutic strategy. We aimed to develop a straightforward and highly reproducible in vitro psoriasis model useful to study the effects of gene knockdown by RNAi and to identify new targets for topical RNAi therapeutics. We evaluated the use of keratinocytes derived from psoriatic plaques and normal human keratinocytes (NHKs). To induce a psoriatic phenotype in NHKs, combinations of pro-inflammatory cytokines (IL-1α, IL-17A, IL-6 and TNF-α) were tested. The model based on NHK met our needs of a reliable and predictive preclinical model, and this model was further selected for gene expression analyses, comprising a panel of 55 psoriasis-associated genes and five micro-RNAs (miRNAs). Gene silencing studies were conducted by using small interfering RNAs (siRNAs) and miRNA inhibitors directed against potential target genes such as CAMP and DEFB4 and miRNAs such as miR-203. We describe a robust and highly reproducible in vitro psoriasis model that recapitulates expression of a large panel of genes and miRNAs relevant to the pathogenesis of psoriasis. Furthermore, we show that our model is a powerful first step model system for testing and screening RNAi-based therapeutics. [ABSTRACT FROM AUTHOR]

There is evidence to suggest that genetic factors play an important role in the development of basal cell carcinomas (BCCs), and that skin neoplasms might be a sign for a genetic predisposition to cancer. We investigated whether the incidence of visceral and skin malignancies among first-degree relatives of BCC-patients was increased. Postal questionnaires were sent to 249 BCC-patients, who were divided into two groups (young = BCC under the age of 51 years and older = BCC over the age of 50 years), and asked them about cancer in their first-degree relatives. The reported numbers of cancer among the relatives were compared with the expected numbers based on sex and age-specific population-based incidence rates. The accuracy of the reported diagnoses was verified. A total of 157 BCC-patients reported 277 malignancies in 1,272 relatives. The incidence of the following cancers was higher than expected in relatives from young BCC-patients: bone and soft tissue (O/E = 3.91; 95% CI: 1.43–8.66), skin (O/E = 2.13; 95% CI: 1.30–3.29) and digestive tract (O/E = 1.59; 95% CI: 1.10–2.23). In relatives of older BCC-patients, only the incidence of digestive tract cancer was higher than expected (O/E = 1.44; 95% CI: 1.08–1.89). Diagnoses that were verified turned out to be accurate in 87% of the cases. This study suggests that the risk of certain cancers, particularly that of the digestive tract, in first-degree relatives of BCC-patients is increased. These findings may indicate a genetic predisposition to both skin and visceral malignancies in this patient group. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

Background. New therapeutic approaches have to be considered in the treatment of irritant contact dermatitis (ICD). Recently, phosphodiesterase 4 (PDE-4) inhibitors have been introduced as nonsteroidal, antiinflammatory agents. These agents inhibit the secretion of the cytokines thought to be involved in the pathogenesis of ICD. We investigated the effect of a new selective PDE-4 inhibitor (cipamfylline) in human models using single and repeated exposures to an irritant in a blind, randomized pilot study with healthy volunteers. We compared the effect of cipamfylline ointment with a strong corticosteroid (betamethasone-17-valerate) and with a placebo ointment. Methods. Ten volunteers were patch tested at four investigation sites with sodium dodecyl sulphate (1%) for 24 h. In a model that simulates chronic damage, 11 volunteers were patch tested with sodium dodecyl sulphate (0.2%) for 4 h daily for four consecutive days. The investigation sites were treated once a day with the above-mentioned agents. One site was left untreated. We used erythema scoring, measurements of transepidermal water loss (TEWL) and several immunohistochemical markers for epidermal proliferation and differentiation. Results. Repeated application revealed that betamethasone-17-valerate caused a statistically significant reduction in erythema and TEWL compared to cipamfylline and placebo. We also observed a significant suppression of proliferating cells and cytokeratin 16 expression at sites treated with betamethasone compared to the other sites. In the model for acute ICD, no significant differences were seen between the investigated sites. Conclusions. Our results show that betamethasone-17-valerate may modulate the response in ICD. In this human model of ICD we could not confirm the efficacy of cipamfylline. Clinical studies are needed before the effect of PDE-4 inhibitors in ICD can be refuted with certainty. [ABSTRACT FROM AUTHOR]

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-α) 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-α 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. [ABSTRACT FROM AUTHOR]

We investigated the mechanism of cartilage degradation by pancreatic elastase as a model system for the action of cationic proteases such as leukocyte elastase and cathepsin-G. It is shown that the cationic properties of elastase contribute to its proteolytic potential with respect to cartilage degradation. Elastase preparations with neutral or negative charge, obtained by chemical modification were far less effective in cartilage degradation than the cationic, native enzyme. Modification of elastase did not affect the active site of the enzyme as shown by kinetic studies, nor did it alter the complexing with alpha-1-proteinase inhibitor. Quantitative and autoradiographic studies indicate that the positive charge of the enzyme favors the penetration in cartilage and interaction with the polyanionic proteoglycan substrate. It is concluded that the potent cartilage-degrading activity of elastase is dependent on charge-mediated interactions with its substrate. [ABSTRACT FROM AUTHOR]

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 K8.12, 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. [ABSTRACT FROM AUTHOR]

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 chromatrography 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 nondenaturing 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. [ABSTRACT FROM AUTHOR]

Tenascin, a recently discovered extracellular matrix protein, was demonstrated in perifollicular connective tissue of normal human scalp using immunohistochemistry. Its localization was different from other well-known extracellular matrix components, like fibronectin, laminin and heparan sulphate proteoglycan. A comparison between alopecia areata and normal scalps did not reveal major qualitative differences, except for an increased expression near heavily infiltrated follicles. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Recurrent Neuropathy Associated With Ehlers–Danlos Syndrome," that was published in the previous issues of the "Journal of Neurology."