Jens M. Baron

Clinical usefulness of microarray-based IgE detection in children with suspected food allergy

Background:  Component‐resolved diagnostics using microarray technology has recently been introduced into clinical allergology, but its applicability in children with food allergy has hardly been investigated so far. The aim of this study was to evaluate the utility of microarray‐based IgE detection in the diagnostic workup of food allergy and to compare this new diagnostic tool with established methods of allergen‐specific IgE detection.

Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.

IL-31 regulates differentiation and filaggrin expression in human organotypic skin models

BACKGROUND Atopic dermatitis (AD) is an inflammatory skin disease affecting 10% to 20% of children and 1% to 3% of adults in industrialized countries. Enhanced expression of IL-31 is detected in skin samples of patients with AD, but its physiological relevance is not known. OBJECTIVE We sought to determine the role of IL-31 in skin differentiation. METHODS We used human 3-dimensional organotypic skin models with either primary keratinocytes or HaCaT keratinocytes with inducible IL-31 receptor α to evaluate the effect of IL-31. The consequences were studied by using histology, the expression of markers analyzed by immunofluoresence and quantitative RT-PCR, and gene expression arrays. RESULTS We observed that IL-31 interferes with keratinocyte differentiation. Gene expression analysis revealed a limited set of genes deregulated in response to IL-31, including IL20 and IL24. In HaCaT keratinocytes with inducible IL-31 receptor α, IL-31 inhibited proliferation upon induction of IL-31 receptor α by inducing cell cycle arrest. As in primary cells, IL-31-treated HaCaT cells elicited a differentiation defect in organotypic skin models, associated with reduced epidermal thickness, disturbed epidermal constitution, altered alignment of the stratum basale, and poor development of the stratum granulosum. The differentiation defect was associated with a profound repression of terminal differentiation markers, including filaggrin, an essential factor for skin barrier formation, and a reduced lipid envelope. The highly induced proinflammatory cytokines IL-20 and IL-24 were responsible for part of the effect on FLG expression and thus for terminal differentiation. CONCLUSION Our study suggests that IL-31 is an important regulator of keratinocyte differentiation and demonstrates a link between the presence of IL-31 in skin, as found in patients with AD, and filaggrin expression.

Frontiers in sebaceous gland biology and pathology.

Abstract:  The development of experimental models for the in vitro study of human sebaceous gland turned down the theory of a phylogenetic relict and led to the identification of several, unknown or disregarded functions of this organ. Such functions are the production of foetal vernix caseosa, the influence of three‐dimensional organization of the skin surface lipids and the integrity of skin barrier and the influence on follicular differentiation. In addition, the sebaceous gland contributes to the transport of fat‐soluble antioxidants from and to the skin surface, the natural photoprotection, the pro‐ and antiinflammatory skin properties and to the innate antimicrobial activity of the skin. It is mainly responsible for skin’s independent endocrine function, the hormonally induced skin ageing process, the steroidogenic function of the skin as well as its thermoregulatory and repelling properties and for selective control of the hormonal and xenobiotical actions of the skin. Interestingly, sebocytes, at least in vitro, preserve characteristics of stem‐like cells despite their programming for terminal differentiation. This review reports on various sebaceous gland functions, which are currently under investigation, including its role on the hypothalamus–pituitary–adrenal‐like axis of the skin, the impact of acetylcholine on sebocyte biology, the activity of ectopeptidases as new targets to regulate sebocyte function, the effects of vitamin D on human sebocytes, the expression of retinoid metabolizing cytochrome P450 enzymes and the possible role of sebum as vehicle of fragrances. These multiple homeostatic functions award the sebaceous gland the role ‘brain of the skin’ and the most important cutaneous endocrine gland.

Signaling by IL-31 and functional consequences

Cytokines are key to control cellular communication. Interleukin-31 (IL-31) was recently discovered as a new member of the IL-6 family of cytokines. IL-31 signals through a heterodimeric receptor composed of OSMR and IL-31RA, a complex that stimulates the JAK-STAT, the RAS/ERK and the PI3K/AKT signal transduction pathways. The available data suggests that IL-31 is important for both innate and adaptive immunity in tissues that are in close contact with the environment, i.e. the skin, the airways and the lung, and the lining of the intestine. Enhanced expression of IL-31 is associated with a number of diseases, including pruritic diseases such as atopic dermatitis, but also in allergy and inflammatory bowel disease. In these tissues IL-31 coordinates the interaction of different immune cells, including T-cells, mast cells, and eosinophils, with epithelial cells. In this review we have summarized the available data on IL-31 and its receptor, their expression pattern and how they are regulated. We describe the current state of knowledge of the involvement of IL-31 in diseases, both in humans and in mouse models. From these studies it is becoming clear that IL-31 plays an important role in the proper functioning of the skin and of airway and intestinal epithelia. The findings available suggest that IL-31 might be an interesting target for directed drug therapy.

Y‐box protein‐1 is actively secreted through a non‐classical pathway and acts as an extracellular mitogen

Y‐box protein (YB)‐1 of the cold‐shock protein family functions in gene transcription and RNA processing. Extracellular functions have not been reported, but the YB‐1 staining pattern in inflammatory glomerular diseases, without adherence to cell boundaries, suggests an extracellular occurrence. Here, we show the secretion of YB‐1 by mesangial and monocytic cells after inflammatory challenges. It should be noted that YB‐1 was secreted through a non‐classical mode resembling that of the macrophage migration inhibitory factor. YB‐1 release requires ATP‐binding cassette transporters, and microvesicles protect YB‐1 from protease degradation. Two lysine residues in the YB‐1 carboxy‐terminal domain are crucial for its release, probably because of post‐translational modifications. The addition of purified recombinant YB‐1 protein to different cell types results in increased DNA synthesis, cell proliferation and migration. Thus, the non‐classically secreted YB‐1 has extracellular functions and exerts mitogenic as well as promigratory effects in inflammation.

Routine detection of herpes simplex virus and varicella zoster virus by polymerase chain reaction reveals that initial herpes zoster is frequently misdiagnosed as herpes simplex

The differential diagnosis of herpes simplex and zoster may require virological confirmation, yet virus typing is not regarded as necessary in routine dermatological assessment. In an attempt to evaluate the clinical benefits of the routine detection of herpes simplex virus (HSV) and varicella zoster virus (VZV), we analysed skin swabs from 110 patients who were diagnosed at the first clinical visit as having herpes simplex (n = 45) or zoster (n = 65). Viruses were typed using the polymerase chain reaction (PCR) with the general primer pair GPHV‐RU. PCR analysis showed that at the initial clinical presentation, herpes simplex in these patients was not mistaken for zoster but that zoster was incorrectly diagnosed as herpes simplex in nine cases. Thus these results suggest that initial zoster often mimics herpes simplex, hence routine PCR diagnosis of HSV and VZV or alternative rapid diagnostic approaches may be beneficial in these cases.

Cytokines and the Skin Barrier

The skin is the largest organ of the human body and builds a barrier to protect us from the harmful environment and also from unregulated loss of water. Keratinocytes form the skin barrier by undergoing a highly complex differentiation process that involves changing their morphology and structural integrity, a process referred to as cornification. Alterations in the epidermal cornification process affect the formation of the skin barrier. Typically, this results in a disturbed barrier, which allows the entry of substances into the skin that are immunologically reactive. This contributes to and promotes inflammatory processes in the skin but also affects other organs. In many common skin diseases, including atopic dermatitis and psoriasis, a defect in the formation of the skin barrier is observed. In these diseases the cytokine composition within the skin is different compared to normal human skin. This is the result of resident skin cells that produce cytokines, but also because additional immune cells are recruited. Many of the cytokines found in defective skin are able to influence various processes of differentiation and cornification. Here we summarize the current knowledge on cytokines and their functions in healthy skin and their contributions to inflammatory skin diseases.

Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens

Contact sensitization is caused by low molecular weight compounds which penetrate the skin and bind to protein. In many cases, these compounds are activated to reactive species, either by autoxidation on exposure to air or by metabolic activation in the skin. Geraniol, a widely used fragrance chemical, is considered to be a weak allergen, although its chemical structure does not indicate it to be a contact sensitizer. We have shown that geraniol autoxidizes and forms allergenic oxidation products. In the literature, it is suggested but not shown that geraniol could be metabolically activated to geranial. Previously, a skin-like CYP cocktail consisting of cutaneous CYP isoenzymes, was developed as a model system to study cutaneous metabolism. In the present study, we used this system to investigate CYP-mediated activation of geraniol. In incubations with the skin-like CYP cocktail, geranial, neral, 2,3-epoxygeraniol, 6,7-epoxygeraniol and 6,7-epoxygeranial were identified. Geranial was the main metabolite formed followed by 6,7-epoxygeraniol. The allergenic activities of the identified metabolites were determined in the murine local lymph node assay (LLNA). Geranial, neral and 6,7-epoxygeraniol were shown to be moderate sensitizers, and 6,7-epoxygeranial a strong sensitizer. Of the isoenzymes studied, CYP2B6, CYP1A1 and CYP3A5 showed high activities. It is likely that CYP1A1 and CYP3A5 are mainly responsible for the metabolic activation of geraniol in the skin, as they are expressed constitutively at significantly higher levels than CYP2B6. Thus, geraniol is activated through both autoxidation and metabolism. The allergens geranial and neral are formed via both oxidation mechanisms, thereby playing a large role in the sensitization to geraniol.

N-Cyano Sulfoximines: COX Inhibition, Anticancer Activity, Cellular Toxicity, and Mutagenicity

From insects to cancer: N-Cyano sulfoximines were evaluated for COX inhibition and antiproliferative activity against a panel of cancer cell lines. The most active compound exhibited potent COX-2 inhibition, some selectivity for COX-2 over COX-1, only slight cytotoxicity towards healthy cells (HaCaT skin cells), and no mutagenic potential (as determined by an Ames assay).