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Allergic contact dermatitis (ACD) is the most prevalent occupational disease and the most common form of immunotoxicity in humans. Preventing exposure to the triggering allergens is the mainstay of treatment. However, avoidance is not always possible in an occupational setting. From a pathophysiological point of view, a variety of events are involved in the development of ACD, including the formation of immunogenic complexes following the stable association of the allergen with skin proteins, which is thought to be the molecular initiating event responsible for the development of ACD. Previously, the team identified molecules that exhibited higher antiallergic potential due to their capacity to block the interaction between allergens and skin proteins. These assumptions were the starting point for the design of this work aiming to develop and characterize a new hydrogel containing the active ingredients lysine and N-acetyl cysteine under the premises of quality- and safety- by design. Two factorial plannings were established envisioning the optimization of the hydrogel in terms of mechanical and rheological properties. In vitro release and permeation studies supported its skin surface barrier effect. In addition, the selected hydrogel proved to be safe without causing human skin irritation or skin sensitization.

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Atopic dermatitis (AD) is a chronic inflammatory skin disease associated with excessive inflammation and defective skin barrier function. Activated protein C (APC) is a natural anticoagulant with anti-inflammatory and barrier protective functions. However, the effect of APC on AD and its engagement with protease activated receptor (PAR)1 and PAR2 are unknown. Methods: Contact hypersensitivity (CHS), a model for human AD, was induced in PAR1 knockout (KO), PAR2KO and matched wild type (WT) mice using 2,4-dinitrofluorobenzene (DNFB). Recombinant human APC was administered into these mice as preventative or therapeutic treatment. The effect of APC and PAR1KO or PARKO on CHS was assessed via measurement of ear thickness, skin histologic changes, inflammatory cytokine levels, Th cell phenotypes and keratinocyte function. Results: Compared to WT, PAR2KO but not PAR1KO mice displayed less severe CHS when assessed by ear thickness; PAR1KO CHS skin had less mast cells, lower levels of IFN-γ, IL-4, IL-17 and IL-22, and higher levels of IL-1ß, IL-6 and TGF-ß1, whereas PAR2KO CHS skin only contained lower levels of IL-22 and IgE. Both PAR1KO and PAR2KO spleen cells had less Th1/Th17/Th22/Treg cells. In normal skin, PAR1 was present at the stratum granulosum and spinosum, whereas PAR2 at the upper layers of the epidermis. In CHS, however, the expression of PAR1 and PAR2 were increased and spread to the whole epidermis. In vitro, compared to WT cells, PAR1KO keratinocytes grew much slower, had a lower survival rate and higher para permeability, while PAR2KO cells grew faster, were resistant to apoptosis and para permeability. APC inhibited CHS as a therapeutic but not as a preventative treatment only in WT and PAR1KO mice. APC therapy reduced skin inflammation, suppressed epidermal PAR2 expression, promoted keratinocyte growth, survival, and barrier function in both WT and PAR1KO cells, but not in PAR2KO cells. Conclusions: APC therapy can mitigate CHS. Although APC acts through both PAR1 and PAR2 to regulate Th and mast cells, suppression of clinical disease in mice is achieved mainly via inhibition of PAR2 alone. Thus, APC may confer broad therapeutic benefits as a disease-modifying treatment for AD.

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INTRODUCTION: Because minipig skin is similar to human skin in anatomy and physiology, establishing an atopic dermatitis (AD) minipig model seems meaningful. MATERIAL AND METHODS: We applied 1-fluoro-2,4-dinitrobenzene (DNFB) or ovalbumin onto the back skin of five Yucatan minipigs aged 8-10 months and 19 kg in median weight. Two minipigs with the same parameters served as controls. RESULTS: Both DNFB and ovalbumin mediated epithelial hyperplasia, spongiosis, and immune cell infiltration in the dermis, which is a typical histopathological feature of AD. Moreover, AD upregulated the Th1- and Th2-related cytokine expressions in DNFB- or in ovalbumin-treated skin. Notably, AD-induced minipigs exhibited greater cytokine serum concentrations. CONCLUSION: Histopathological finding and cytokine analysis revealed that DNFB or ovalbumin mediates AD. However, ovalbumin-treated minipig is a more reliable and precise AD model owing to the DNFB-induced severe skin damage. In summary, ovalbumin-treated skin shows similar AD as human in histopathological and molecular analysis.

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Atopic dermatitis (AD) is a chronic and relapsing inflammatory disease that affects 1%-20% of people worldwide. Despite affecting many people, AD current treatments, such as corticosteroids and calcineurin inhibitors, have not only harmful secondary effects but are also often ineffective. Therefore, natural nontoxic compounds are on high demand for developing new effective AD treatments. Panax ginseng Meyer has been used traditionally for its promising healing and restorative properties to treat many diseases including skin disorders, reason why in this review we want to explore the research performed with AD and P. ginseng as well as determining its potential for new drug development. Previous researches have shown that P. ginseng has positive effects in AD patients such as lower eczema area and severity index, transepidermal water loss, and immunoglobulin E levels and better quality of sleep. In vivo animal models, as well, have shown positive results to P. ginseng and derived ginsenosides, such as the decrease of transepidermal water loss, immunoglobulin E levels in serum, allergy-related cytokines, and downregulation of NF-κB, MAPK, and Ikaros pathways. All of these previous data suggest that P. ginseng and its derived ginsenosides are undoubtedly a nontoxic effective option to treat AD.

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BACKGROUND: Long-term use of most immunosuppressants to treat allergic contact dermatitis (ACD) generates unavoidable severe side effects, warranting discovery or development of new immunosuppressants with good efficacy and low toxicity is urgently needed to treat this condition. Hispidulin, a flavonoid compound that can be delivered topically due to its favorable skin penetrability properties, has recently been reported to possess anti-inflammatory and immunosuppressive properties. However, no studies have investigated the effect of hispidulin on Th1 cell activities in an ACD setting. METHODS: A contact hypersensitivity (CHS) mouse model was designed to simulate human ACD. The immunosuppressive effect of hispidulin was investigated via ear thickness, histologic changes (i.e., edema and spongiosis), and interferon-gamma (IFN-γ) gene expression in 1-fluoro-2,4-dinitrobenzene (DNFB)-sensitized mice. Cytotoxicity, total number of CD4+ T cells, and percentage of IFN-γ-producing CD4+ T cells were also investigated in vitro using isolated CD4+ T cells from murine spleens. RESULTS: Topically applied hispidulin effectively inhibited ear swelling (as measured by reduction in ear thickness), and reduced spongiosis, IFN-γ gene expression, and the number of infiltrated immune cells. The inhibitory effect of hispidulin was observed within 6 h after the challenge, and the observed effects were similar to those effectuated after dexamethasone administration. Hispidulin at a concentration up to 50 µM also suppressed IFN-γ-producing CD4+ T cells in a dose-dependent manner without inducing cell death, and without a change in total frequencies of CD4+ T cells among different concentration groups. CONCLUSION: The results of this study, therefore, suggest hispidulin as a novel compound for the treatment of ACD via the suppression of IFN-γ production in Th1 cells.

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Levels of IL36α are known to be increased in specimens from patients with atopic dermatitis and psoriasis. In addition, it has been reported that IL-36α is crucial for development of imiquimod-induced psoriatic dermatitis in mice. On the other hand, the role of IL-36α in induction of allergic contact dermatitis/contact hypersensitivity (ACD/CHS) is poorly understood. We found that IL-36α was produced in keratinocytes of mice during imiquimod-induced psoriatic dermatitis, but it was hardly detectable in the skin of mice during either fluorescein isothiocyanate (FITC)- or 1-fluoro-2, 4-dinitrobenzene (DNFB)-induced CHS. Although IL-36α can enhance activation of dendritic cells (DCs) and T cells, in CHS, IL-36α was not essential for DC migration from the skin to draining LNs, but it was required for induction or activation of hapten-specific T cells such as Th/Tc1 or Th17 cells. However, local inflammation, assessed by measurement of ear skin thickness, was comparable between wild-type and IL-36α-deficient mice during both FITC- and DNFB-induced CHS. These observations indicate that IL-36α is involved in induction and/or activation of hapten-specific T-cell subsets in the sensitization phase of CHS, but not essential for induction of local inflammation in the elicitation phase.

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Accumulating evidence suggests that activated mast cells are involved in contact hypersensitivity, although the precise mechanisms of their activation are still not completely understood. We investigated the potential of common experimental allergens to induce mast cell activation using murine bone marrow-derived cultured mast cells and rat peritoneal mast cells. Among these allergens, 1-chloro-2,4-dinitrobenzene and 1-fluoro-2,4-dinirobenzene (DNFB) were found to induce degranulation of rat peritoneal mast cells. DNFB-induced degranulation is accompanied by cytosolic Ca2+ mobilization and is significantly inhibited by pertussis toxin, U73122 (a phospholipase C inhibitor), and BAPTA (a Ca2+ chelator), raising the possibility that DNFB acts on the G protein-coupled receptors and activates Gi , which induces activation of phospholipase C, as well as known mast cell secretagogues, such as compound 48/80. DNFB could induce mast cell degranulation in the absence of serum proteins and IgE. Structure-activity relationship analyses revealed an inverse correlation between the degree of degranulation and the electron density of the C1 carbon of the DNFB derivatives. These findings raise a possibility that DNFB functions as a potent contact allergen through induction of cutaneous mast cell degranulation.

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The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A2 via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca2+]i) in MHS macrophage cells. Ionomycin increased [Ca2+]i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca2+→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca2+ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca2+→ ERK pathway, which plays a key role in suppression of inflammatory reactions.

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Recent changes in regulatory requirements and social views on animal testing have accelerated the development of reliable alternative tests for predicting skin sensitizing potential of chemicals. In this study, we aimed to develop a new in vitro skin sensitization assay using reconstructed human epidermis, RhE model, which is expected to have broader applicability domain rather than existing in vitro assays. Microarray analysis revealed that the expression of five genes (ATF3, DNAJB4, GCLM, HSPA6 and HSPH1) related to cellular stress response were significantly up-regulated in RhE model after 6h treatment with representative skin sensitizers, 1-fluoro-2,4-dinitrobenzene and oxazolone, but not a non-sensitizer, benzalkonium chloride. The predictive performance of five genes was examined with eight skin sensitizers (e.g., cinnamic aldehyde), four non-sensitizers (e.g., sodium lauryl sulfate) and four pre-/pro-haptens (e.g., p-phenylenediamine, isoeugenol). When the positive criteria were set to obtain the highest accuracy with the animal testing (LLNA), ATF3, DNAJB4 and GCLM exhibited a high predictive accuracy (100%, 93.8% and 87.5%, respectively). All tested pre-/pro-haptens were correctly predicted by both ATF3 and DNAJB4. These results suggested that the RhE-based assay, termed epidermal sensitization assay (EpiSensA), could be an useful skin sensitization assay with a broad applicability domain including pre-/pro-haptens.

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