Repeated monthly epicutaneous challenges with diphenylcyclopropenone result in a clinically reproducible level of contact allergy in de novo sensitized individuals

Dear Editor, Diphenylcyclopropenone (DPCP) has been used as an experimental contact allergen in humans and as topical immunotherapy for patients with alopecia areata. However, the efficacy is mostly based on case series. Randomized controlled clinical trials are lacking,1 as are detailed descriptions of how repeated topical exposures to DPCP affect the level of hypersensitivity. We studied the time course of the developing cutaneous immune reactions to DPCP in 10 de novo sensitized volunteers using visual grading and caliper readings of skinfold thickness. Ten healthy volunteers (one woman and nine men) between 20 and 41 years old (mean age 24·5 years) were recruited. Exclusion criteria were atopic dermatitis and treatment with topical corticosteroids or other immunosuppressive medication. Written informed consent and ethical committee approval were obtained (study‐ID: S‐20120085). Contact sensitivity to DPCP was induced by a single dose of DPCP (CAS no. 886‐38‐4; ThermoFisher Scientific, Karlsruhe, Germany) of 30 μg cm−2 (22·8 μL of 0·125% DPCP in acetone) applied to a filter paper in a 12‐mm Finn Chamber (SmartPractice, Phoenix, AZ, U.S.A.), and placed on the upper buttock for 48 h.2 Sensitization to DPCP was confirmed after 4 weeks by an elicitation challenge on the inner upper arm consisting of a dose series of five DPCP‐loaded patches. The DPCP doses were applied to filter‐paper‐lined, 8‐mm Finn Chambers as 10 μL of the appropriate concentration of DPCP in acetone to give the required dose range, increasing by 60% increments. The doses were 1·24, 1·98, 3·17, 5·1 and 8·12 μg cm−2. In addition, a vehicle control patch loaded with 10 μL of acetone was applied. Elicitation patches were removed exactly 6 h after application.2 The responses (numerical values in parentheses) were assessed visually at 24 h and 48 h after application and graded as negative (−) (0), doubtful (?+) (1), 1+ (2), 2+ (3), 3+ (4) or 4+ (5), using a modified version (based on our previous experience with DPCP) of the International Contact Dermatitis Research Group clinical scoring system. Reactions were also quantified as increase in skinfold thickness measured with a modified spring‐loaded skinfold caliper (Baty International, Camberley, U.K.). Each individual received a series of four or five repeated challenges at 4‐weekly intervals. Each rechallenge consisted of three challenge doses, which were applied with filter‐paper‐lined 8‐mm Finn Chambers and removed after 6 h. Visual assessment and skinfold thickness measurements were carried out on the three challenge doses at 48 h. Mean skinfold thickness was determined from duplicate measurements at each vehicle control and challenge site. Vehicle control values were subtracted from challenge site values and the results were summed (Σ) to give an approximation of the area under the dose–response curve, representative of the overall reactivity to DPCP from each participant.3 4 The data were analysed using the Friedman test (with a significance level of P <0·05). In order to avoid increased rechallenge reactivity at previously DPCP‐challenged sites,5 patches were applied on previously nonchallenged sites by using placement templates. Repeat challenges were also carried out alternately on the inner aspect of the left and right upper arms. Nine of the 10 volunteers sensitized with DPCP gave positive responses to the initial elicitation challenge at 48 h [the nonrespondent volunteer reacted at the subsequent challenge (i.e. rechallenge 1)]. The visual scores at the five challenged sites ranged from no reaction to 3+ and the summed scores for the five sites were mean ± SEM, 8·0 ± 1·45 (n =10). Due to the overall weak responses to the initial elicitation challenge and because of space limitations on the inner upper arm, the top three challenge doses were subsequently used in repeat challenges for nine of the participants, but for the 10th, who gave strong responses, three intermediate doses were used. The summed visual scores for the reactions at the top three challenge doses and the three intermediate doses for the 10th volunteer were mean ± SEM, 6·2 ± 0·99 (n =10) and the summed increase in skinfold thickness was 0·96 ± 0·2 mm (n =10) (Fig. 1a, b). Four weeks later, responses to the first repeat challenge with three doses of DPCP showed a significant increase in the strength and grade of the reactions, with some individuals developing vesicles or bullae (mean ± SEM, 10·5 ± 1·44; n =10). The increased clinical reactions were reflected in the increased skinfold thickness measurements (mean ± SEM, 3·55 ± 0·7 mm; n =10). At subsequent repeated challenges at 4‐weekly intervals, there was no further increase in the visual grades of the responses, the magnitude of the response having reached a plateau. The skinfold thickness reached a plateau at rechallenge 2 (Fig. 1b). The almost identical time course generated by caliper responses and visual scores was ascertained by calculating the correlation coefficient in a scatter plot. A strong positive correlation (R2 = 0·93) was identified between skinfold thickness measurements and visual scores (Fig. 2). Initial amplification of the immune response to diphenylcyclopropenone (DPCP) followed by a constant level of immune reactivity. Sensitized volunteers received an initial elicitation challenge (IEC) with DPCP followed by four or five repeated monthly challenges. (a) Visual assessment (numerical values) and (b) skinfold thickness measurements of responses at 48 h relative to a vehicle control site, were summed (Σ) for the three challenge sites (mean ± SEM, n = 10; vRc5, n = 5). The SEM was calculated using the mean values for area under the dose–response curve for the included volunteers. Significant changes from IEC are designated by: **P < 0·01; ***P < 0·001. VC, vehicle control; Rc, rechallenge. The strength of the linear association between skinfold thickness measurements and visual scores. A strong positive correlation (R2 = 0·9337) was identified between the caliper responses and the clinical scores. IEC, initial elicitation challenge; Rc, rechallenge; IEC–Rc4, n = 10; vRc5, n = 5. “v” in front of Rc5 is the abbreviation for 5, the number of volunteers included in the final rechallenge (Rc5). We have shown that immune responses to repeated monthly challenges with DPCP in de novo sensitized individuals reach a plateau after two challenges (by visual grading) and three challenges (by skinfold measurements), respectively. These findings indicate the presence of regulatory control mechanisms that prevent progressive augmentation of immune responses by repeated antigen exposure. However, the putative control mechanism(s) by which the repeated challenge responses are regulated remains to be determined. Few studies have attempted to quantify the time course of contact allergy. Of the few published studies, the majority have investigated the time course of repeated nickel challenges by assessing the visual grading of responses. It has been demonstrated that individuals with established contact allergy to nickel exhibit a reproducible and relatively constant level of reactivity after 6 months.6 This observation was concordant with another study in which 30 nickel‐allergic patients were tested with a nickel dilution series on four separate test occasions over 7 months. No upgrading or downgrading of the patch test reactivity was seen by visual scoring; however, a large intraindividual variation in threshold responses was observed.7 The experimental model presented here uses a continuous variable (skinfold thickness) that can more accurately detect differences in the level of immune response to repeated challenges with a contact allergen as opposed to relying solely on subjective (e.g. visual grading) forms of assessment. This more accurate detection is supported by the fact that visual scores reached a maximum after 8 weeks and caliper readings after a further 4 weeks (12 weeks after sensitization), indicating that skinfold measurements were more sensitive compared with visual grading. In addition, we can conclude that repeated applications with an appropriate dose series of DPCP at a fixed time interval ultimately result in cutaneous immune responses with constant levels of reactivity over time. We thank all healthy volunteers for participating in this study. The authors wish to acknowledge Kirsten H. Andersen (Dermatological Investigations Scandinavia, University of Southern Denmark, Denmark), Lars P. Christensen and Dorthe L. Christensen (Institute of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Denmark) for their excellent technical assistance. We are thankful to Neil P. Walsh and Bethany C. Diment (College of Health and Behavioural Sciences, Bangor University, U.K.) for input regarding study design. Funding sources: this work was supported by grants from LEO Pharma, the Region of Southern Denmark and the Faculty of Health Sciences, University of Southern Denmark. Conflicts of interest: M.A.R. and T.L. are employees of LEO Pharma, which partially financed this study. The remaining authors state no conflicts of interest.