Contact sensitizer 2,4-dinitrochlorobenzene is a highly potent human TRPA1 agonist.

2,4-Dinitrochlorobenzene (DNCB) is widely used in human clinical studies and in experimental animal studies to evoke allergic contact dermatitis. 2,4-Dinitrochlorobenzene is a potent immunogen capable of inducing contact sensitization in all humans exposed. However, the mechanism by which DNCB evokes such symptoms is presently unknown. TRPA1 is a nonselective cation channel that is expressed in peptidergic sensory neurons and fibroblasts. TRPA1 activation was recently implicated in the pathophysiology of atopic dermatitis especially in transducing cutaneous itch signals. Here, we test the hypothesis that DNCB acts as a TRPA1 agonist and thereby evokes allergic symptoms. We found that DNCB activates human TRPA1 dose dependently in FLIPR experiments with an EC50 of 167 nM, an effect that was fully blocked by selective TRPA1 antagonists Chembridge-5861528 and A-967079. Similarly, DNCB activated nonselective TRPA1 current in patch clamp studies. Neutralization of 3 critical cysteines in TRPA1 resulted in a loss of DNCB agonism.

Repeated epicutaneous exposures to ovalbumin progressively induce atopic dermatitis-like skin lesions in mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic skin disease in which environmental factors play a great role. A widely used murine model for AD has provided a useful tool to study the disease. OBJECTIVE: The purpose of this study is to investigate kinetically the induction of this AD model and the processes involved in the development of AD due to extrinsic allergen exposures. METHODS: BALB/c mice were epicutaneously exposed to ovalbumin (OVA) for 3 weeks; each week was separated by a 2-week resting period. Mice were killed after each exposure week. Skin biopsies and blood were obtained for histological study, RNA isolation and antibody analysis. RESULTS: There was a progressive and significant thickening of the epidermis and dermis in OVA-exposed mice. Significantly increased dermal cell infiltration of eosinophils, mast cells and total inflammatory cells, including CD3 and CD4 cells, was found after each OVA exposure week. Total IgE, IgG2a and OVA-specific IgE were significantly increased after the second and third exposure week, while OVA-specific IgG2a was significantly induced after the third exposure week. Gradual and/or significant increases in mRNA expression of IL-1beta, TNF-alpha, IL-4, IL-10, IL-13, IFN-gamma and IL-12p35 were found after each exposure week. Chemokines and their receptors involved in both T-helper type 1 (Th1)- and Th2-type cell recruitment (CCL1, CCL8, CCL11, CCL24, CXCL9, CXCL10, CCR1, CCR3, CCR5, CCR8 and CXCR3) were up-regulated significantly at different time-points. CONCLUSION: This study provides an insight into the dynamic nature and time-dependent transition of skin inflammation and systemic immune responses in a murine AD model induced by repeated epicutaneous exposures to OVA.

Patch test reactions to cosmetic allergens in 1995-1997 and 2000-2002 in Finland--a multicentre study.

Contact sensitivity to cosmetics is common, but the sensitizing chemicals vary between countries and study periods. The present survey aimed at revealing the recent trends in patch test sensitivity with cosmetic chemicals in Finland. We report a retrospective multicentre survey of patch test reactions focusing on cosmetic-related substances and comparing the test results in 1995-97 with those in 2000-02. The most striking increases in the frequency of the patch test sensitivity were found with balsam of Peru and propolis from 4.0% to 6.2% (P < 0.001) and from 0.5% to 1.4% (P < 0.001), respectively, whereas the most prominent decreases were found with methylchloro/methylisothiazolinone and chlorhexidine diglugonate from 2.4% to 1.3% (P < 0.001) and from 1.2% to 0.5% (P < 0.001), respectively. The level of patch test sensitivity to methyldibromo glutaronitrile increased, although not significantly, from 1.0% to 1.5%. An increasing tendency was also found with hair dye chemicals 4-aminophenol and toluene-2,5-diamine or toluene-2,5-diamine sulfate from 1.3% to 3.8% and from 1.4% to 5.2%, respectively, while such a tendency was not found among permanent wave chemicals. The sensitivity level of fragrance mix remained the same (6% - 7%). We conclude that surveys revealing the state of sensitivity to cosmetic chemicals should be performed periodically in different countries.

Phthalic anhydride allergy: development and characterization of optimized hapten-carrier conjugates for improved diagnosis.

BACKGROUND: At present the diagnosis of IgE-mediated hypersensitivity to phthalic anhydride (PA) is based on conjugates that are not characterized or standardized. The aim of this study was to develop optimized and molecularly characterized PA conjugates that can be used to improve the diagnosis of PA-allergy. METHODS: The PA conjugates were synthesized and the number of haptens bound on a carrier protein was estimated by matrix-assisted laser desorption/ionization time of light (MALDI-TOF) mass spectrometry. The ability of conjugates to bind IgE and IgG antibodies was measured by enzyme-linked immunosorbent assay (ELISA). Reactivity of the conjugates in vivo was evaluated by skin prick testing. RESULTS: The most active IgE-binding conjugates had a PA : HSA molar ratio of 80 : 1. In the optimal conjugates the average numbers of PA haptens per carrier molecule of human serum albumin (HSA) were 14-16. In ELISA, all 13 patients and none of the 20 controls had IgE antibodies to optimized PA conjugate. The sensitivity and specificity of the ELISA was comparable to commercial CAP RAST. PA conjugates elicited positive test results in skin prick testing showing that conjugates are immunologically active also in vivo. CONCLUSIONS: These results indicate that optimized and molecularly characterized PA-HSA conjugates can be used both in vitro and in vivo assays to improve the diagnosis of PA allergy.

Cryptococcosis during systemic glucocorticosteroid treatment.

Cryptococcosis is an opportunistic infection caused by a fungus, Cryptococcus neoformans. It is usually seen in immunocompromised patients with AIDS, leukaemia, lymphoma, sarcoidosis or immunosuppressive treatments. We describe a patient who was treated with systemic glucocorticosteroids for 4 years because of lung sarcoidosis. During the last year of treatment, a papular eruption developed which later became ulcerative. In a histopathological examination of a skin biopsy, there was granulomatous inflammation, and the disease was treated as sarcoidosis without success. After 1 year's unsuccessful treatment, another skin biopsy and skin fungal culture revealed C. neoformans. Cryptococcal antigen was found in blood and cerebrospinal fluid, too. The patient was successfully treated first with an amphotericin-B-flucytosine combination and later with fluconazole.

Tacrolimus ointment improves psoriasis in a microplaque assay.

Tacrolimus (FK506) is an effective and well tolerated immunosuppressant used to prevent allograft rejection. We describe the evaluation of two tacrolimus ointment formulations for treatment of chronic plaque-type psoriasis. This was a microplaque assay with randomized, double-blind design. Sixteen patients (15 men, one woman, all white and 28-69 years old) with chronic plaque-type psoriasis participated. Six different ointments were applied to discrete microplaques, 17 mm in diameter, on a descaled psoriasis lesion: these were tacrolimus ointment with diisopropyl adipate as penetration enhancer, tacrolimus ointment without diisopropyl adipate, 0.1% betamethasone 17alpha-valerate ointment, 0.005% calcipotriol ointment and, as controls, the ointment bases for tacrolimus and betamethasone. Ointments were reapplied and the area was sealed every 2-3 days during the 14-day treatment period. After 7 and 14 days, erythema and infiltration were graded on a scale of 0-4, and superficial blood flow was measured with a laser Doppler flowmeter. Epidermal thickness was measured histologically at the end of treatment. Compared with the vehicle controls, sites treated with tacrolimus ointment (with or without penetration enhancer) showed a significant reduction in erythema and infiltration (P < 0. 001), a significant reduction in superficial blood flow (P < 0.01) and a significant decrease in epidermal thickness (P < or = 0.001). Results for betamethasone and calcipotriol, when compared with the vehicle controls, were similar. These results suggest that, under conditions of descaling and occlusion, tacrolimus ointment is effective in the treatment of psoriasis.

Tacrolimus ointment does not affect collagen synthesis: results of a single-center randomized trial.

We conducted a randomized, double-blind, placebo-controlled trial to assess the atrophogenicity of tacrolimus ointment. In a combined group of atopic dermatitis patients (n = 14) and healthy volunteers (n = 12), 0.3% tacrolimus, 0.1% tacrolimus, betamethasone-valerate, and a vehicle control were applied in a randomized order to nonsymptomatic, 4 cm x 4 cm regions of abdominal skin. After 7 d of treatment under occlusion, the carboxy- and amino-terminal propeptides of procollagen I (PICP, PINP) and the amino-terminal propeptide of procollagen III (PIIINP) were measured from suction blister fluid with specific radioimmunoassays. In addition, ultrasound measurements of skin thickness were taken. Betamethasone-treated areas showed median PICP, PINP, and PIIINP concentrations of 17.0%, 17.6%, and 39.5% of the vehicle control at the end of the treatment period, respectively, whereas the 0.1% and 0.3% tacrolimus-treated areas showed median concentrations of approximately 100% of the vehicle control (p < 0.001). Betamethasone was also the only treatment to reduce skin thickness; the median decrease in skin thickness was 7.4% relative to 0.1% tacrolimus, 7.1% relative to 0.3% tacrolimus, and 8.8% relative to the vehicle control (p < 0.01). Results for atopic dermatitis patients and healthy volunteers were similar. These findings suggest that tacrolimus does not cause skin atrophy.

Topical FK506 suppresses cytokine and costimulatory molecule expression in epidermal and local draining lymph node cells during primary skin immune responses.

Recently, it has been shown that the immunosuppressive macrolide lactone, FK506, exerts good therapeutic efficacy in inflammatory skin diseases. The aim of this study was to analyze the influence of topical FK506 on molecular (IL-1alpha, IL-1beta, IL-2, IL-4, IL-12 p35, IL-12 p40, macrophage inflammatory protein-2 (MIP-2), granulocyte-macrophage CSF (GM-CSF), TNF-alpha, and IFN-gamma) and cellular (I-A+/CD80+, I-A+/CD54+, I-A+/CD69+, I-A+/B220+, and CD4+/CD25+) events in epidermal (EC) and local draining lymph node (LNC) cells during primary contact hypersensitivity responses. Cytokine mRNA levels for IL-1alpha, IL-1beta, GM-CSF, TNF-alpha, MIP-2, and IFN-gamma in EC and for IL-2, IL-4, IL-12 p35, IL-12 p40, and IFN-gamma in LNC were increased and resulted in significant LNC proliferation during oxazolone-induced contact hypersensitivity. Topical FK506 treatment dose-dependently suppressed oxazolone-induced LNC proliferation. This effect was correlated with decreased IL-1alpha, IL-1beta, GM-CSF, TNF-alpha, MIP-2, and IFN-gamma mRNA expression within the epidermis and decreased IL-12 p35 and p40 mRNA expression in LNC. Further analysis of the LNC cytokine pattern revealed that the production of both Thl (IFN-gamma and IL-2) and Th2 (IL-4) cytokines was dramatically impaired after topical FK506 treatment. Flow cytometric analysis showed that topical FK506 decreased the population of epidermis-infiltrating CD4+ T cells and suppressed the expression of CD54 and CD80 on I-A+ EC and LNC during hapten-induced contact hypersensitivity. Furthermore, topical FK506 profoundly impaired oxazolone-induced up-regulation of CD25 expression on CD4+ LNC and dramatically decreased hapten-induced expansion of I-A+/B220+ and I-A+/CD69+ LNC subsets. In conclusion, these results give new insights into the mechanisms of action of topical FK506 treatment.

Use of the newer immunosuppressive agents in dermatology.

Immune mechanisms play a central role in various diseases such as eczema and psoriasis, and in the past treatment tended to involve corticosteroids and cytostatic drugs. Organ transplantation has stimulated the development of newer immunosuppressants, some of which have also been found to be efficacious in the inflammatory dermatoses. The best studied such immunosuppressant is cyclosporin, which has shown efficacy especially in psoriasis and atopic dermatitis. The major limiting factor in the use of cyclosporin is its adverse effects, especially nephrotoxicity and hypertension. Therefore the risk : benefit ratio should always be considered before initiation of cyclosporin therapy, and the patient should be carefully followed for such adverse effects. Tacrolimus seems to share the efficacy and most of the adverse effects of cyclosporin when used systemically, presumably because of its similar intracellular mechanism of action. Unlike cyclosporin, tacrolimus is efficacious topically, which may allow lower systemic adverse effects to be combined with higher local efficacy. Other newer immunosuppressants include sirolimus (rapamycin) and monoclonal antibodies. Their use in dermatology is still in the research phase, and no conclusions about their clinical potential can yet be made.

A local lymph node assay to analyse immunosuppressive effects of topically applied drugs.

Topical glucocorticosteroids represent the mainstay of antiinflammatory therapy in the treatment of inflammatory skin diseases. Their clinical use, however, is limited by local and systemic side-effects. Thus, in dermatopharmacology there is a large demand for alternative non-steroidal antiinflammatories. Other than transplantation models, most of the frequently used in vivo test systems for assessment of drug-induced immunosuppression measure changes in inflammatory skin responses by means of skin erythema and edema after challenge of sensitized animals. The aim of this study was to develop an alternative mouse model to detect and analyse immunosuppressive effects of topically applied drugs. On the basis of a modified local lymph node assay, we analysed effects of topical hydrocortisone, dexamethasone, mometasone furoate and FK506 (tacrolimus) during the induction phase of contact hypersensitivity. On 4 consecutive days, NMRI mice were treated on the dorsal surfaces of both ears with increasing concentrations of test compound. During the last 3 days, the mice received in addition the contact sensitizer, oxazolone (1%). On day 5, draining auricular lymph nodes were removed in order to assess lymph node cell counts and perform flow cytometric analysis of lymph node cell subpopulations (CD4+/CD25+, Ia+/CD69+, Ia+/B220+). All test compounds proved to exert significant immunosuppressive effects after topical application, but showed differences in their immunomodulatory potential. In conclusion, the local lymph node assay serves as an appropriate model to characterize immunosuppressive effects of topically applied drugs by measuring immunologically relevant end-points.

Absorption of topical tacrolimus (FK506) in vitro through human skin: comparison with cyclosporin A.

Cyclosporin A (CsA) is efficacious in many dermatoses as an oral but not as a topical form, while tacrolimus (FK506) has been shown to be effective in both forms. As inadequate skin absorption has been proposed as the reason for inefficacy of topical CsA, factors contributing to percutaneous absorption of FK506 and CsA were studied. Partitioning of FK506 and CsA between octanol and water, stratum corneum and water, and stratum corneum and isopropyl myristate was determined. Absorption of FK506 and CsA through dermatomed human cadaver skin was determined with in vitro flow-through cells. In partitioning experiments, CsA was more lipophilic than FK506. Both drugs were seen in comparable amounts in skin layers, but FK506 permeated the skin to a greater extent than CsA.

Trimellitic anhydride-sensitive mouse as an animal model for contact urticaria.

The respiratory allergen trimellitic anhydride (TMA) has been shown to induce IgE production and immediate ear swelling in mice sensitized to it. We studied whether TMA sensitivity could be used as an animal model for immunological contact urticaria. BALB/C mice were sensitized to TMA by topical applications. Groups of animals were pretreated on the ears with the glucocorticosteroid (GCS) betamethasone-17,21-dipropionate, the antihistamine (AH) diphenhydramine hydrochloride, the non-steroidal anti-inflammatory drug (NSAID) indomethacin or vehicle (VEH). Ears were challenged with TMA and ear thickness was measured at baseline and 1, 2, 4, 8 and 24 h after challenge. Trimellitic anhydride caused a significant biphasic ear swelling response with an early peak at 1-2 h, a plateau at 4 h and a late peak at 24 h. However, there was also an early swelling by TMA in non-sensitized mice, suggesting that non-immunological as well as immunological mechanisms contribute to early swelling by TMA. Glucocorticosteroid suppressed significantly the early and to some extent the late TMA responses, while AH suppressed only early and NSAID only late TMA responses. Ear swelling in TMA-sensitive BALB/C mice may represent a combination of immunological and non-immunological contact urticaria and allergic contact dermatitis. Mice sensitive to TMA may be helpful in defining pharmacological agents affecting contact urticaria and the model is perhaps suitable for identification of some immunologically mediated contact urticants.

Delayed-type hypersensitivity in palmoplantar pustulosis: effect of cyclosporin A treatment on skin testing with recall antigens.

Delayed-type hypersensitivity reactions to skin antigens are an indirect measure of cellular immune response. We studied in a double-blind manner whether clinically effective doses of cyclosporin A in palmoplantar pustulosis would diminish delayed-type hypersensitivity reactions in vivo. For testing delayed-type hypersensitivity, we applied intradermally a standardized panel of seven recall antigens and a vehicle control in 30 patients with palmoplantar pustulosis, and 28 were tested both at baseline and after 4 weeks. For 4 weeks 14 patients were treated with 2.5 mg/kg/day cyclosporin A and 14 patients with placebo. Cyclosporin A but not placebo caused a significant decrease in clinical disease parameters. In contrast, no significant differences in delayed-type hypersensitivity reactions between treatment groups were observed. The results do not support the view that the efficacy of low-dose cyclosporin A in dermatological disorders can be entirely explained by cyclosporin A's inhibitory actions on effector T-cells.

Effect of topical cis-urocanic acid on local lymph node activation during contact sensitization in mouse, rat and guinea-pig.

Cis-urocanic acid (cUCA) has been suggested as a mediator of impairment of contact hypersensitivity induction by ultraviolet B (UVB) irradiation. We ascertained whether topical cUCA influences local lymph node activation during induction of contact hypersensitivity. Topical cUCA or vehicle was applied during the local lymph node assay to oxazolone. Local lymph node weight and cell number were assessed in all animals. Additionally, cell proliferation rate was studied in Hartley guinea-pigs and CBA/Ca mice, whereas activation of antigen-presenting cells was quantified in NMRI mice and Wistar rats. Topical cUCA suppressed all parameters of local lymph node activation due to oxazolone application in guinea-pigs. No effect, with the exception of a suppression of antigen-presenting cell activity, was seen in mice. No effect was seen in rats. The study shows that topical cUCA may suppress local lymph node activation during contact sensitization and suggests that differences between the effect of cUCA in different animal species may exist.