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.

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.

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.

Cyclosporine in the treatment of palmoplantar pustulosis. A randomized, double-blind, placebo-controlled study.

BACKGROUND AND DESIGN: Palmoplantar pustulosis (PPP) is an inflammatory skin disease characterized by pustule formation, erythema, induration, and scaling of the affected skin of the palms and soles. Palmoplantar pustulosis is usually resistant to treatment. In a double-blind study (phase 1) of 4 weeks, 40 patients with PPP were randomized to receive oral cyclosporine, 2.5 mg/kg per day, or placebo. An open-label dose-finding phase 2 with cyclosporine doses of 1.25, 2.5, and 3.75 mg/kg per day was performed in the following 3 months. The patients were then followed for at least 2 months after termination of cyclosporine treatment. Response to treatment was judged by the number of fresh pustules. Patients displaying a reduction of 50% or greater in the number of pustules, compared with baseline, were defined as responders. RESULTS: Of the patients who completed phase 1, 17 of 19 patients in the cyclosporine group and four of 15 in the placebo group were classified as responders (P < .001). Cyclosporine, but not placebo, significantly reduced formation of new pustules (P = .001). In the subsequent open phase, a daily cyclosporine dose of 1.25 mg/kg appeared to be an effective treatment of PPP in approximately half of the treated patients. Many patients relapsed after initial success with cyclosporine. However, only one patient studied totally failed to respond to cyclosporine treatment. At the end of phase 3, most of the studied parameters had returned to pretreatment levels. The most common side effect was headache in the 2.5 mg/kg per day dosage group; no significant side effects were observed in the 1.25 mg/kg per day dosage group. CONCLUSIONS: Low-dose cyclosporine treatment (1.25 to 2.5 mg/kg per day) is effective in PPP.

Cellular kinetics of delayed hypersensitivity test reactions to topical glucocorticosteroids.

The phenotypes of the infiltrating cells in 13 patients with delayed hypersensitivity to topical glucocorticosteroids (GCS) were studied from sequential biopsies of positive epicutaneous test reactions by using the avidin-biotin-complex (ABC) technique. Monoclonal antibodies were used to identify the cells with the following phenotypes: T3, T4/T4a, T6, T8, T9, T11, M1, Ia1 (HLA-DR), interleukin-2 receptor/T26a, and dendritic reticular cell. The cellular kinetics of GCS hypersensitivity reactions were compared with delayed hypersensitivity reactions caused by allergens not related to GCS. In both GCS and non-GCS reactions the epidermal dendritic T6+ cells were more numerous than dendritic Ia1+ cells. There was a decrease in the number of both cell types during these reactions; in GCS reactions the decrease in the number of T6+ cells was seen later than in non-GCS reactions. Ia1+ keratinocytes were seen at sites near dermal infiltrates. Compared with the non-GCS delayed hypersensitivity reaction, there were fewer pan T (T11+/T3+) in the GCS reaction. The relative numbers of M1+ monocytes and the T4/T8 ratio were substantially lower in the latter; these findings can be explained as a GCS effect which modulates the delayed type hypersensitivity reaction.

Topical FK506: suppression of allergic and irritant contact dermatitis in the guinea pig.

Topical FK506 has recently been shown to have an anti-inflammatory effect in vivo in humans. In this study its effects in contact dermatitis were studied in the guinea pig model. Topical FK506 suppressed both irritant and allergic patch-test reactions. The most prominent suppressive effect was seen when skin sites were pretreated with FK506. Topical FK506 did not impair the induction of contact allergy as assessed by challenges, although it suppressed local lymph node cell accumulation during contact allergy induction. Topical FK506 may hold promise as a treatment for skin disorders that respond to oral FK506 or cyclosporin A.

A local lymph-node assay validation study of a structure-activity relationship model for contact allergens.

A structure-activity relationship model for prediction of contact allergenic potential of chemicals had previously been developed. The model had been shown to be able to classify known allergens and nonallergens using data on physicochemical and reactivity parameters of functional groups by discriminant two-value multiple regression analysis. To investigate the model, six selected chemicals which had not been previously investigated for allergenicity were studied with both the model and a murine local lymph-node assay. The same compounds were predicted to be allergens (3-bromo-2-coumaranone, 1-nitrocyclohexene and alpha-acryloyloxy-beta, beta-dimethyl-beta-butyrolactone) and nonallergens (1-carbethoxy-4-piperidone, 6,7-dimethoxy-2-tetralone and 9-acetylanthracene) by both the model and the local lymph-node assay.

Patch test reactions to inhalant allergens in atopic dermatitis.

To study whether inhalant allergens could induce eczematous reactions on normal skin of atopic patients we applied birch pollen and house dust mite antigens at 500 times the concentration used for prick testing as epicutaneous tests. Six out of 17 patients with atopic dermatitis in remission had positive delayed type reactions to birch pollen and three to house dust mite. Only one out of 13 atopic patients without history of atopic dermatitis but with seasonal allergic rhinitis had a positive patch test reaction to birch pollen and no patient had positive test reactions to house dust mite. No positive patch test reactions to birch pollen or house dust mite were seen in the ten healthy control subjects. In patients with positive test reactions biopsies from the test sites revealed epidermal spongiosis and vesiculation. Immunostaining of the epidermis revealed keratinocytes displaying both CD1 and HLA-DR. The present study suggests that inhalant allergens can exacerbate atopic dermatitis.

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.

Screening for corticosteroid contact sensitivity. Comparison of tixocortol pivalate, hydrocortisone-17-butyrate and hydrocortisone.

3 corticosteroids have so far been tried as markers for corticosteroid contact sensitivity: hydrocortisone, tixocortol pivalate and hydrocortisone-17-butyrate. The present study compared these steroids for screening in addition to a standard patch test series. Of 727 patients, 28 (3.9%) reacted to tixocortol pivalate and 10 (1.4%) to hydrocortisone-17-butyrate; hydrocortisone gave an allergic reaction in 2 of 521 (0.4%) patients. Serial dilutions suggested that tixocortol pivalate, not marketed in Finland, caused allergic reactions which could possibly be cross-reactions to hydrocortisone. In contrast to previously published data, frequent cross-reactions occurred with hydrocortisone-17-butyrate and tixocortol pivalate. All allergic reactions to other corticosteroids found by testing with tixocortol pivalate concurred with reactions to hydrocortisone-17-butyrate. The study suggests that the most effective choice for routine testing for corticosteroid contact sensitivity would be both tixocortol pivalate and hydrocortisone-17-butyrate.

Contact allergy to corticosteroids.

Recent evidence shows that contact allergy to topical corticosteroids is more frequent than earlier believed. This review summarizes the current knowledge of this condition, including methods for clinical diagnosis.

Topical FK506--clinical potential or laboratory curiosity?

Cyclosporine A (CsA) is an immunosuppressant that is efficacious in several inflammatory skin disorders. Its use is, however, limited due to systemic side-effects. Topical forms of CsA have been tried but they do not seem to be effective presumably due to insufficient penetration to skin. Tacrolimus (FK506) is another immunosuppressant that has almost similar effects on the cellular level as CsA. FK506 is 10-100 times more potent than CsA and it has lower molecular weight. Recent studies suggest that topical FK506 penetrates human skin in amounts exceeding those of topical CsA. Topical FK506 also seems to suppress skin inflammation in man. These findings prompt for further clinical studies of efficacy and safety of topical FK506.

Detection of contact hypersensitivity to topical corticosteroids with hydrocortisone-17-butyrate.

We showed earlier that most patients with contact dermatitis due to corticosteroids show cross-reactions when patch tested with hydrocortisone-17-butyrate (H-17-B). To test whether H-17-B could be used for detecting topical corticosteroid allergy, we screened patients undergoing routine patch testing with H-17-B. Patients with clearly allergic or doubtful/mildly irritant patch test reactions to H-17-B, and with a history suggesting topical corticosteroid allergy, were further tested with a large panel of steroid preparations. 20 out of 4039 patients (0.5%) showed definite allergic test reactions to corticosteroids. A further 165 patients with clinically suspected corticosteroid allergy were directly tested with a panel of steroid preparations; 14 patients showed positive patch test reactions. Altogether, 33 out of 34 patients with corticosteroid allergy had positive test reactions to H-17-B. Inclusion of 1.0% H-17-B in ethanol in the standard patch test series improves the diagnosis of topical corticosteroid hypersensitivity.

Contact allergies in patients with familial benign chronic pemphigus (Hailey-Hailey disease)

To determine whether patients with familial benign chronic pemphigus have an increased risk for the development of contact allergies to topical therapy, we performed epicutaneous tests in 15 patients with this disease. Nine had positive patch test reactions of which seven were allergic to compounds used in local treatments. Allergic reactions in more than one patient were seen to neomycin sulfate (three patients), bacitracin (two), and hydrocortisone-17-butyrate and betamethasone-17-valerate (two). Although no comparable control group was similarly studied, the frequency of sensitization appears to be increased in patients with familial benign chronic pemphigus.