Level of contact hypersensitivity response to diphencyprone and keratinocyte cancer.

BACKGROUND: Keratinocyte cancers (KC) are common and pose a significant financial burden globally. Ultraviolet radiation is a significant factor in their development, through mutagenesis promotion but also through local and systemic immunosuppression. Although systemic immunosuppression is well understood, cutaneous immunity has been more difficult to evaluate. OBJECTIVES: This study used a contact sensitizer, diphencyprone (DPCP), which elicits a contact hypersensitivity reaction in skin, to compare the degree of reactivity to DPCP in patients with a high KC burden versus those with a low KC burden. METHODS: A prospective study was performed in immunocompetent patients aged 70 ± 5 years of age, comparing patients with a high KC burden (>10 previous KC) with those with a low KC burden (<2 previous KC). All patients were sensitized with 2% DPCP and then patch tested two weeks later with eight different concentrations of DPCP with the threshold concentration and total degree of reaction recorded. RESULTS: Nine patients were recruited, 5 in the 'high cancer' group and 4 in the 'low cancer' group. All patients were Fitzpatrick skin type 1 or 2. All patients developed a reaction to DPCP. Patients in the low cancer group developed a reaction at a significantly lower threshold DPCP concentration than the high cancer group (P = 0.039). The cumulative intensity of reaction was higher in the low cancer group (P = 0.087). CONCLUSION: Patients with a high KC burden required a higher threshold concentration of DPCP to elicit a hypersensitivity reaction, supporting the concept of a lower skin immunity in these patients. DPCP reactivity threshold could be a useful tool in the evaluation of skin immunity and propensity to develop keratinocyte cancers.

Anti-inflammatory potency testing of topical corticosteroids and calcineurin inhibitors in human volunteers sensitized to diphenylcyclopropenone.

AIMS: To quantify the anti-inflammatory potency of topical corticosteroids and topical calcineurin inhibitors by measuring the contact allergic response to a diphenylcyclopropenone (DPCP) challenge in de novo sensitized human volunteers. METHODS: Two randomized, double-blind, vehicle-controlled studies were performed encompassing 76 volunteers: 29 in the first and 47 in the second study. Topical drugs were applied pre- and/or post-treatment in block designs. The compounds were tested simultaneously under occluded patch tests covering DPCP-induced dermatitis. Inhibitory responses were assessed by visual scoring and measurements of the oedema thickness with ultrasound. RESULTS: When applied both before and after the DPCP challenge, significant anti-inflammatory effects were seen in descending order for tacrolimus 0.1% ointment, clobetasol propionate ointment, betamethasone valerate ointment and hydrocortisone butyrate ointment, while pimecrolimus cream, hydrocortisone ointment and vehicles had no significant effect. Only tacrolimus ointment (P < 0.01) demonstrated a consistent significant pre-treatment inhibitory effect compared with an untreated DPCP control. CONCLUSIONS: This human testing method in which the inflammation of experimentally induced allergic patch test reactions is quantified by objective measurement allows an analysis of the anti-inflammatory potency of not only topical corticosteroids, but also of drugs that have no effect on vasoconstriction. The method allowed comparison of the potencies of four topical corticosteroids and two calcineurin inhibitors.

Carbohydrate supplementation does not blunt the prolonged exercise-induced reduction of in vivo immunity.

BACKGROUND: Carbohydrate (CHO) supplementation during prolonged exercise is widely acknowledged to blunt in vitro immunoendocrine responses, but no study has investigated in vivo immunity. PURPOSE: To determine the effect of CHO supplementation during prolonged exercise on in vivo immune induction using experimental contact hypersensitivity with the novel antigen diphenylcyclopropenone (DPCP). METHODS: In a double-blind design, 32 subjects were randomly assigned to 120 min of treadmill exercise at 60 % [Formula: see text] with CHO (Ex-CHO) or placebo (Ex-PLA) supplementation. Responses were also compared to 16 resting control (CON) subjects from a previous study (for additional comparison with a resting non-exercise condition). Standardised diets (24 h pre-trial) and breakfasts (3.5 h pre-trial) were provided. Subjects received a primary DPCP exposure (sensitisation) 20 min after trial completion, and exactly 28 days later the strength of immune reactivity was quantified by magnitude of the cutaneous response (skin-fold thickness and erythema) to a low dose-series DPCP challenge. Stress hormones and leucocyte trafficking were also monitored. RESULTS: CHO supplementation blunted the cortisol and leucocyte trafficking responses, but there was no difference (P > 0.05) between Ex-CHO and Ex-PLA in the in vivo immune responses (e.g. both ~46 % lower than CON for skin-fold response). CONCLUSIONS: CHO supplementation does not influence the decrease in in vivo immunity seen after prolonged exercise. The effects with more stressful (or fasted) exercise remain to be determined. However, there appears to be no benefit under the conditions of the present study, which have practical relevance to what many athletes do in training or competition.

Unique microRNAs appear at different times during the course of a delayed-type hypersensitivity reaction in human skin.

Diphencyprone (DPCP) is a hapten that induces delayed-type hypersensitivity (DTH) reactions. MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and have been implicated in various inflammatory skin diseases, but their role in DTH reactions is not well understood. We generated global miRNA expression profiles (using next-generation sequencing) of DPCP reactions in skin of seven healthy volunteers at 3, 14 and 120 days after challenge. Compared to placebo-treated sites, DPCP-challenged skin at 3 days (peak inflammation) had 127 miRNAs significantly deregulated. At 14 days (during resolution of inflammation), 43 miRNAs were deregulated and, at 120 days (when inflammation had completely resolved), six miRNAs were upregulated. While some miRNAs have been observed in psoriasis or atopic dermatitis, most of the deregulated miRNAs have not yet been studied in the context of skin biology or immunology. Across the three time points studied, many but not all miRNAs were uniquely expressed. As various miRNAs may influence T cell activation, this may indicate that the miRNAs exclusively expressed at different time points function to promote or resolve skin inflammation, and therefore, may inform on the paradoxical ability of DPCP to treat both autoimmune conditions (alopecia areata) and conditions of ineffective immunity (melanoma).

Exercise Intensity and Duration Effects on In Vivo Immunity.

PURPOSE: To examine the effects of intensity and duration of exercise stress on induction of in vivo immunity in humans using experimental contact hypersensitivity (CHS) with the novel antigen diphenylcyclopropenone (DPCP). METHODS: Sixty-four healthy males completed either 30 min running at 60% V˙O2peak (30MI), 30 min running at 80% V˙O2peak (30HI), 120 min running at 60% V˙O2peak (120MI), or seated rest (CON). Twenty min later, the subjects received a sensitizing dose of DPCP; and 4 wk later, the strength of immune reactivity was quantified by measuring the cutaneous responses to a low dose-series challenge with DPCP on the upper inner arm. Circulating epinephrine, norepinephrine and cortisol were measured before, after, and 1 h after exercise or CON. Next, to understand better whether the decrease in CHS response on 120MI was due to local inflammatory or T-cell-mediated processes, in a crossover design, 11 healthy males performed 120MI and CON, and cutaneous responses to a dose series of the irritant, croton oil (CO), were assessed on the upper inner arm. RESULTS: Immune induction by DPCP was impaired by 120MI (skinfold thickness -67% vs CON; P < 0.05). However, immune induction was unaffected by 30MI and 30HI despite elevated circulating catecholamines (30HI vs pre: P < 0.01) and greater circulating cortisol post 30HI (vs CON; P < 0.01). There was no effect of 120MI on skin irritant responses to CO. CONCLUSIONS: Prolonged moderate-intensity exercise, but not short-lasting high- or short-lasting moderate-intensity exercise, decreases the induction of in vivo immunity. No effect of prolonged moderate-intensity exercise on the skin's response to irritant challenge points toward a suppression of cell-mediated immunity in the observed decrease in CHS. Diphenylcyclopropenone provides an attractive tool to assess the effect of exercise on in vivo immunity.

Systemic immunogenicity of para-Phenylenediamine and Diphenylcyclopropenone: two potent contact allergy-inducing haptens.

p-Phenylenediamine (PPD) and Diphenylcyclopropenone (DPCP) are two potent haptens. Both haptens are known to cause delayed-type hypersensitivity, involving a cytokine response and local infiltration of T-cell subpopulations, resulting in contact dermatitis. We investigated the systemic immune effects of PPD and DPCP, two relatively unexplored skin allergens. The dorsal sides of the ears of BALB/c mice were exposed to PPD or DPCP (0.1% w/v or 0.01% w/v), or vehicle alone. Mice were treated once daily for 3 days (induction period) and subsequently twice per week for 8 weeks. Local and systemic immune responses in the auricular and pancreatic lymph nodes, spleen, liver, serum, and ears were analyzed with cytokine profiling MSD, flow cytometry, and qPCR. Ear swelling increased significantly in mice treated with 1% PPD, 0.01% DPCP or 0.1% DPCP, compared with vehicle treatment, indicating that the mice were sensitized and that there was a local inflammation. Auricular lymph nodes, pancreatic lymph nodes, spleen, and liver showed changes in regulatory T-cell, B-cell, and NKT-cell frequencies, and increased activation of CD8(+) T cells and B cells. Intracellular cytokine profiling revealed an increase in the IFN-γ- and IL-4-positive NKT cells present in the liver following treatment with both haptens. Moreover, we saw a tendency toward a systemic increase in IL-17A. We observed systemic immunological effects of PPD and DPCP. Furthermore, concentrations too low to increase ear thickness and cause clinical symptoms may still prime the immune system. These systemic immunological effects may potentially predispose individuals to certain diseases.

High altitude impairs in vivo immunity in humans.

The aim was to assess the effect of high altitude on the development of new immune memory (induction) using a contact sensitization model of in vivo immunity. We hypothesized that high-altitude exposure would impair induction of the in vivo immune response to a novel antigen, diphenylcyclopropenone (DPCP). DPCP was applied (sensitization) to the lower back of 27 rested controls at sea level and to ten rested mountaineers 28 hours after passive ascent to 3777 m. After sensitization, mountaineers avoided strenuous exercise for a further 24 hours, after which they completed alpine activities for 11-18 days. Exactly 4 weeks after sensitization, the strength of immune memory induction was quantified in rested mountaineers and controls at sea level, by measuring the response to a low, dose-series DPCP challenge, read at 48 hours as skin measures of edema (skinfold thickness) and redness (erythema). Compared with control responses, skinfold thickness and erythema were reduced in the mountaineers (skinfold thickness,-52%, p=0.01, d=0.86; erythema, -36%, p=0.02, d=0.77). These changes in skinfold thickness and erythema were related to arterial oxygen saturation (r=0.7, p=0.04), but not cortisol (r<0.1, p>0.79), at sensitization. In conclusion, this is the first study to show, using a contact sensitization model of in vivo immunity, that high altitude exposure impairs the development of new immunity in humans.

The contact sensitizer diphenylcyclopropenone has adjuvant properties in mice and potential application in epicutaneous immunotherapy.

BACKGROUND: Epicutaneous vaccination has gained increasing interest during the past decade as it offers a safe, needle-free, and patient-friendly alternative to invasive vaccine administrations. Recently, the safety and early efficacy of epicutaneous immunotherapy were also demonstrated in patients with hay fever, as an alternative to conventional subcutaneous allergen-specific immunotherapy (SCIT). One major challenge to epicutaneous vaccination is the barrier function of the stratum corneum, which must be overcome either by abrasive methods or by hydration. Such barrier function of the stratum corneum also hampers the use of common adjuvants used to enhance the efficacy of vaccination. METHODS: In a mouse model of allergy, we tested the adjuvant potential of diphenylcyclopropenone (DCP), a strong contact sensitizer, which is currently used for the treatment of a T cell-mediated hair loss disease (alopezia areata). RESULTS: Diphenylcyclopropenone enhanced antigen-specific IgG2a antibody responses as well as IL-10 cytokine production after epicutaneous immunization with ovalbumin (OVA). Epicutaneous allergen-specific immunotherapy (EPIT) with OVA and DCP also protected sensitized mice from anaphylaxis and asthma. The protective effect was more robust than that of conventional SCIT, which did not significantly alleviate the symptoms of allergy in the murine models of anaphylaxis and asthma. CONCLUSIONS: This preclinical study confirmed previous clinical data that have demonstrated the potential of the skin as a target for allergen immunotherapy. The study also suggests that epicutaneous immunization or immunotherapy can be improved when an appropriate adjuvant such as DCP is used.

Impaired hapten sensitization in patients with autoimmune disease.

An inverse relation between contact allergy and autoimmune diseases is suggested from epidemiological studies. The aim of this study was to investigate susceptibility and reactivity in patients with psoriasis, patients with diabetes and healthy controls in an experimental sensitization study. We sensitized 68 adult individuals (23 patients with psoriasis, 22 patients with diabetes and 23 healthy controls) with diphenylcyclopropenone (DPCP) and assessed challenge responses with visual scoring and ultrasound. Skin biopsies from challenged skin were investigated for differences in down-regulatory mechanisms with immunohistochemistry and gene-expression profiles using microarray technology. The sensitization ratios were 26%, 36% and 65% for the psoriatic, diabetic and healthy groups, respectively. Logistic regression analysis gave an odds ratio (OR) for a patient with psoriasis or diabetes type I of being sensitized to 0·18 [95% confidence interval (CI): 0·039-0·85], P = 0·031 and 0·74 (95% CI: 0·548-1·008), P = 0·056, respectively. A high degree of forkhead box P3-positive (FoxP3(+) ) cells were found in biopsies of positively challenged reactions, but only limited numbers in negatively challenged reactions, with no difference among the groups. No specific mRNA expression was found in the challenged skin of negative elicitation reactions, also indicating no sign of active down-regulation. The study contibutes strongly to the evidence of a decreased susceptibility to develop contact allergy in individuals with autoimmune diseases such as psoriasis.

Exercise-induced stress inhibits both the induction and elicitation phases of in vivo T-cell-mediated immune responses in humans.

Little is known about the influence of exercise on induction and elicitation phases of in vivo immunity in humans. We used experimental contact-hypersensitivity, a clinically relevant in vivo measure of T cell-mediated immunity, to investigate the effects of exercise on induction and elicitation phases of immune responses to a novel antigen. The effects of 2 h-moderate-intensity-exercise upon the induction (Study One) and elicitation of in vivo immune memory (Study Two) to diphenylcyclopropenone (DPCP) were examined. Study One: matched, healthy males were randomly-assigned to exercise (N=16) or control (N=16) and received a primary DPCP exposure (sensitization), 20 min after either 2 h running at 60% V O(2peak) (EX) or 2 h seated rest (CON). Four weeks later, participants received a low, dose-series DPCP challenge (elicitation) on their upper inner arm, which was read at 24 and 48 h as clinical score, oedema (skinfold thickness) and redness (erythema). Study Two: pilot; 13 healthy males were sensitized to DPCP. Elicitation challenges were repeated every 4 weeks until responses reached a reproducible plateau. Then, N=9 from the pilot study completed both EX and CON trials in a randomized order. Elicitation challenges were applied and evaluated as in Study One. Results demonstrate that exercise-induced stress significantly impairs both the induction (oedema -53% at 48 h; P<0.001) and elicitation (oedema -19% at 48 h; P<0.05) phases of the in vivo T-cell-mediated immune response. These findings demonstrate that prolonged moderate-intensity exercise impairs the induction and elicitation phases of in vivo T-cell-mediated immunity. Moreover, the induction component of new immune responses appears more sensitive to systemic-stress-induced modulation than the elicitation component.

Adjuvant effect of cationic liposomes and CpG depends on administration route.

In this study we explored the immunization route-dependent adjuvanticity of cationic liposomes loaded with an antigen (ovalbumin; OVA) and an immune potentiator (CpG). Mice were immunized intranodally, intradermally, transcutaneously (with microneedle pre-treatment) and nasally with liposomal OVA/CpG or OVA/CpG solution. In vitro, OVA/CpG liposomes showed enhanced uptake by DCs of both OVA and CpG compared to OVA+CpG solution. A similar enhanced uptake by DCs was observed in vivo when fluorescent OVA/CpG liposomes were administered intranodally. However, after transcutaneous and nasal application a lower uptake of OVA/CpG liposomes compared to an OVA+CpG solution was observed. Moreover, the IgG titers after nasal and transcutaneous administration of OVA/CpG liposomes were reduced compared to administration of an OVA+CpG solution. Although serum IgG titers may suggest limited added value of liposomes to the immunogenicity, for all routes, OVA/CpG liposomes resulted in elevated IgG2a levels, whereas administration of OVA+CpG solutions did not. These data show that encapsulation of antigen and adjuvant into a cationic liposome has a beneficial effect on the quality of the antibody response in mice after intranodal or intradermal immunization, but impairs proper delivery of antigen and adjuvant to the lymph nodes when the formulations are administered transcutaneously or nasally.

Diphencyprone and topical tacrolimus as two topical immunotherapeutic modalities. Are they effective in the treatment of alopecia areata among Egyptian patients? A study using CD4, CD8 and MHC II as markers.

OBJECTIVE: To evaluate the efficacy of two topically applied immunomodulative agents through the detection of lymphocyte subsets using monoclonal antibodies against CD4, CD8 and MHC II. METHODS: Fifty patients from the Departments of Medical Biochemistry, Dermatology and Pathology at Cairo University with different degrees of alopecia areata (AA) were included in this study. They were classified into two groups each of 25 patients. Each patient was treated with the immunomodulative agent on one side of the scalp and the other side was left as a control. Biopsies were taken from all patients at the beginning of treatment and at the end of the study. Tissue specimens were prepared for histologic and immunophenotypic analysis. The main outcome measures were the uses of diphencyprone (DPCP) and topical tacrolimus as two topical immunotherapeutic modalities in the treatment of AA. RESULTS: A clinical response of 68% was achieved in group A (treated with DPCP) while group B (treated with 0.1% tacrolimus) showed an insignificant clinical response. Decreased expression of CD4 and increased expression of CD8 and MHC II was detected in the post-treated areas compared with pretreated areas in cases treated with DCPC. In tacrolimus-treated cases, there was a decrease in CD4 and MHC II, with no change in CD8 between the pre- and post-treated areas. CONCLUSION: DCPC is one of the most accepted therapeutic modalities in the treatment of AA, with a favourable prognosis among patchy hair loss. MHC II expression was the one correlating with clinical response. Tacrolimus, though beneficial in other dermatoses, could not be considered effective in the treatment of AA.

Cytokines and other mediators in alopecia areata.

Alopecia areata, a disease of the hair follicles with multifactorial etiology and a strong component of autoimmune origin, has been extensively studied as far as the role of several cytokines is concerned. So far, IFN-gamma, interleukins, TNF-alpha, are cytokines that are well known to play a major role in the pathogenesis of the disease, while several studies have shown that many more pathways exist. Among them, MIG, IP-10, BAFF, HLA antigens, MIG, as well as stress hormones are implicated in disease onset and activity. Within the scope of this paper, the authors attempt to shed light upon the complexity of alopecia areata underlying mechanisms and indicate pathways that may suggest future treatments.

No adaptation to UV-induced immunosuppression and DNA damage following exposure of mice to chronic UV-exposure.

It is well known that ultraviolet (UV) radiation induces erythema, immunosuppression and carcinogenesis. We hypothesized that chronic exposure to solar UV radiation induces adaptation that eventually prevents the suppression of acquired immunity. We studied adaptation for UV-induced immunosuppression after chronic exposure of mice to a suberythemal dose of solar simulated radiation (SSR) with Cleo Natural lamps, and subsequent exposure to an immunosuppressive dose of solar or UVB radiation (TL12). After UV dosing, the mice were sensitized and challenged with either diphenylcyclopropenone (DPCP) or picryl chloride (PCl). To assess the adaptation induced by solar simulated radiation, we measured the proliferative response and cytokine production of skin-draining lymph node cells after immunization to DPCP, the contact hypersensitivity (CHS) response to PCl, and thymine-thymine (T-T) cyclobutane dimers in the skin of mice. After induction of immunosuppression by SSR or by TL12 lamps, the proliferative response of draining lymph node cells after challenge with DPCP, or the CHS after challenge with PCl, showed significant suppression of the immune response. Chronic irradiation from SSR preceding the immunosuppressive dose of UV failed to restore the suppressed immune response. Reduced lipopolysaccharide-triggered cytokine production (of IL-12p40, IFN-gamma, IL-6 and TNF-alpha) by draining lymph node cells of mice sensitized and challenged with DPCP indicated that no adaptation is induced. In addition, the mice were not protected from T-T dimer DNA damage after chronic solar irradiation. Our studies reveal no evidence that chronic exposure to low doses of SSR induces adaptation to UV-induced suppression of acquired immunity.

Topical sensitizers in alopecia areata.

Though many therapies exist for alopecia areata, one of the most unique is topical sensitization. By altering the pathogenic inflammatory response with few side effects, sensitizers offer an attractive treatment option for many patients with alopecia areata, including those who have previously failed more traditional treatments and those who have extensive disease.

Age dependence of diphenylcyclopropenone sensitization in patients with alopecia areata.

Contact sensitization has been an accepted topical immunotherapy in the treatment of alopecia areata (1, 2). Whether the age of patients is a significant factor in the treatment results is a matter of controversy. In our study, we investigated the correlation between age of the patients and diphenylcyclopropen-one contact sensitization in vivo and in vitro (3, 4).

The therapeutic use of topical contact sensitizers in benign dermatoses.

Topical therapy using contact sensitizers has been practised since the 1960s to treat conditions associated with an altered immunological state. Dinitrochlorobenzene, squaric acid dibutyl ester and diphencyprone are most commonly employed in the therapy of alopecia areata and viral warts. Few dermatology departments in the U.K. provide such treatment. This systematic review discusses the various contact sensitizers used for topical immunotherapy, the methodology of treatment, factors influencing efficacy and likely adverse effects.

Impaired responses of peripheral blood mononuclear cells to T-cell stimulants in alopecia areata patients with a poor response to topical immunotherapy.

BACKGROUND: Topical immunotherapy with a contact allergen is effective in alopecia areata (AA). However, the mechanism of the effect is still unknown, and pretreatment prediction of the outcome of therapy in each patient remains difficult. OBJECTIVES: To predict the clinical effect of this therapy in AA patients, we investigated the relationship between clinical responses to topical immunotherapy and in vitro proliferative responses of peripheral blood mononuclear cells (PBMC) to T-cell stimulants. METHODS: PBMC were taken from 67 AA patients before or during diphenylcyclopropenone immunotherapy and from 14 healthy controls, and proliferative responses to phytohaemagglutinin and staphylococcal enterotoxin B were evaluated by measuring [3H]-thymidine incorporation. RESULTS: PBMC from the AA patients with a good clinical response to immunotherapy showed a normal level of proliferation, whereas PBMC from the poor responders showed a markedly suppressed proliferative response and interleukin (IL)-2 production, but increased IL-4 production compared with the controls. CONCLUSIONS: The proliferative response of PBMC to T-cell stimulants may be one of the indicators of the clinical effect of topical immunotherapy for AA.