Positive Patch Test Sites Research Articles

Contact sensitization to hair colours in acquired dermal macular hyperpigmentation: results from a patch and photo-patch test study of 108 patients.

Acquired dermal macular hyperpigmentation (ADMH) is an umbrella term including lichen planus pigmentosus, erythema dyschromicum perstans and pigmented contact/cosmetic dermatitis. To establish contact sensitization to hair colours as an aetiological factor for ADMH. Detailed clinical examination, skin biopsies, and patch and photo-patch testing with Indian standard series and patient's own cosmetic products were performed. Thirty-nine (36.1%) patients were found to demonstrate a positive patch/photo-patch test with 35/39 reacting to their own products (all were hair colours) and 16/39 reacting to antigens from commercial series (commonly paraphenylenediamine). Fourteen patients developed delayed hyperpigmentation on positive patch-test sites at 1 month. Higher mean age, symptomatic pigmentation (pruritus, burning and photosensitivity), hair margins involvement (outer surface, helix and lobule of ear; temples and preauricular area), ill-defined lesions, epidermal atrophy and epidermal melanization extending >3 layers were significantly common in patch-test-positive patients. Well-defined lesions, perioral involvement and associated lichen planus were clinical pointers towards patch-test negativity. Index study exemplifies that patch-test results have distinct clinical and histopathological correlates in ADMH. Hair dye contact sensitization appears to be an important aetiological factor in about one-third patients presenting with ADMH.

IL-9 Regulates Allergen-Specific Th1 Responses in Allergic Contact Dermatitis

The cytokine IL-9, derived primarily from T-helper (Th)-9 lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also plays a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch test sites from non-atopic patients were assayed using qPCR and immunohistochemistry. Along with Th2 associated cytokines, IFN-γ, IL-4, and IL-17A, expression of IL-9, and PU.1, a Th9-associated transcription factor, were elevated when compared to paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+CD3+, and PU.1+CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. PBMC from nickel-allergic patients, but not non-allergic controls, show significant IL-9 production in response to nickel. Blocking studies with monoclonal antibodies to HLA-DR (but not HLA-A, B, C) or chloroquine significantly reduced this nickel-specific IL-9 production. Additionally, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9−/− mice, shows enhanced Th1 lymphocyte immune responses, when compared to WT mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.

Flow Cytometric Analysis of Regulatory T Cells During Hyposensitization of Acquired Allergic Contact Dermatitis

We previously demonstrated that repeated intradermal steroid injections administered at weekly intervals into positive patch-test sites induce hyposensitization and desensitization. To examine changes in CD4CD25CD127lo/ regulatory T cells during the attenuation of the patch-test response. Ten patients with known allergic contact dermatitis were patch tested weekly for 10 weeks. The patch-test site was injected intradermally with 2 mg triamcinolone. At weeks 1 and 7, a biopsy was performed on the patch-test site in 6 patients, and flow cytometry was performed assessing CD4CD25CD127lo/ regulatory T cells. Secondary outcomes were clinical score, reaction size, erythema, and temperature. Statistical analysis included regression, correlation, and repeated-measures analysis of variance. The percentage of CD4CD25CD127lo/ regulatory T cells, measured by flow cytometry, increased from week 1 to week 7 by an average of 19.2%. The average grade of patch-test reaction decreased from +++ (vesicular reaction) to ++ (palpable erythema). The mean drop in temperature following treatment was 0.28°C per week. The mean area decreased 8.6 mm/wk over 10 weeks. Intradermal steroid injections of weekly patch-test reactions resulted in hyposensitization of the allergic contact dermatitis reaction. CD4CD25CD127lo/ regulatory T cells showed a tendency to increase; however, further studies are needed to determine if this is significant.

Hyposensitization and Desensitization in Allergic Contact Dermatitis

Acquired allergic contact dermatitis (ACD) causes significant morbidity. Certain occupations predispose workers to ACD, especially those that may damage the skin barrier. Allergic contact dermatitis has significant socioeconomic effects. Allergic contact dermatitis is difficult to treat, particularly if exposure is unavoidable. The objective of this study was to establish the efficacy of intradermal steroid injections in inducing specific antigen hyposensitization when administered weekly into shifted patch-test sites. Prospective, open-label trial included 10 patients with ACD. Each week for up to 8 weeks, patients were patch-tested to their known allergen, and the patch-test site was injected with triamcinolone. Primary outcomes were clinical score, reaction size, erythema, and thermal characteristics. Statistical analysis included regression, correlation, repeated measures, and analysis of variance with contrast variables. Overall results suggest significant decreases in patch-testing reactions as evidenced by improved clinical scores, reaction sizes, and reduced heat production. In 3 patients, the patch test became negative. This desensitized state persisted for 2 months in 1 patient and for 4 months in the other 2 patients. Repetitive treatments of weekly, shifted, positive patch test sites with intradermal steroid injections show a tendency toward allergen-specific hyposensitivity and in some cases to nonreactivity at repeat patch testing. This finding may influence future management of ACD.

Patients with Allergic and Irritant Contact Dermatitis are Characterized by Striking Change of Iron and Oxidized Glutathione Status in Nonlesional Area of the Skin

To assess the consequences of oxidative stress in allergic and irritant contact dermatitis, we compared the iron level, unsaturated iron-binding capacity, total iron binding capacity, the percentage saturation of iron-binding capacity, the amount of diene conjugates as well as the amounts of total glutathione, reduced glutathione, oxidized glutathione, and the oxidized glutathione/reduced glutathione ratio in skin homogenate from lesional and nonlesional skin. Lesional skin samples were obtained from positive patch test sites to 5% NiSO4 in five subjects, and from chronic contact dermatitis lesions on the hands, which had exacerbated over 3--9 wk in six subjects. Contact dermatitis caused at least a 4-fold increase in the iron level in the lesional skin area compared with the nonlesional skin area (p < 0.02). The increase in the iron level depended on the duration of contact dermatitis and was accompanied by high unsaturated iron-binding capacity and total iron-binding capacity values in the positive patch test sites (p < 0.05), and by a high percentage saturation value in the chronic contact dermatitis lesions (p < 0.05). We found high indices for iron, total iron-binding capacity and diene conjugates in the apparently healthy skin of the patients with persistent contact dermatitis that significantly (p < 0.05) exceeded the corresponding values in the patients with only patch test reactions. In summary, we have succeeded in providing evidence that generalized oxidative damage of the skin occurs as a consequence of contact dermatitis in a restricted area.

Expression of metallothioneins-I and -II isoforms at positive patch-test sites.

The expression and the distribution of metallothioneins (MT)-I and II isoforms were evaluated in 5 healthy volunteers and in 16 subjects with positive patch test reactions to various compounds. Skin specimens taken both from the healthy skin of the back and at positive patch test sites (at 48 h), were treated using a 3-step indirect immunoperoxidase procedure with a mouse monoclonal IgG1 antibody reactive against I and II isoforms of human, rat and horse MT. MT were expressed in the basal layer of the healthy skin of both controls and sensitive subjects, without any significant difference. At positive patch test sites, there was an overexpression of MT in basal and suprabasal layers of the epidermis. Overexpression of MT, related to the degree of the inflammatory reactions elicited by the penetrating compounds was observed in the dermis. The cells expressing MT in the dermis were mostly T lymphocytes and cells with dendritic morphology which positively stained in part for CD34 and in part for XIIIa markers and negatively for KP1, S100 and HLA-DR. Taken together, these results seem to indicate that MT represent a constitutive mechanism of defence expressed by different types of cells in the skin, which is triggered by contact with both metallic and non-metallic compounds. The biological significance of MT in the skin remains to be elucidated. Our preliminary findings do not permit evaluation of whether these nearly ubiquitous proteins exert their cytoprotective effects in the skin acting simultaneously as antioxidant, metal binding or zinc suppliers, or if they display these activities mainly depending on the nature of the penetrating substances.

Functional CD86 (B7-2/B70) is predominantly expressed on Langerhans cells in atopic dermatitis

Recently, we reported the functional expression of CD86 on cultured human Langerhans cells derived from normal epidermis. In the present study, we investigated the expression and function of co-stimulatory molecules in the pathogenesis of atopic dermatitis. In immunohistochemical analysis, CD80 and/or CD86 were detected on dendritic-shaped cells not only in the epidermis but also in the dermis in the inflammatory lesions of atopic dermatitis (n = 12). CD80 was expressed in only five cases (42%), while CD86 was expressed in all cases (100%). These molecules were not detected in normal control subjects (n = 8). In non-lesional skin of atopic dermatitis (n = 4), CD86 but not CD80 was detected in one case. CD86 was preferentially induced on dendritic-shaped cells in positive patch test sites to Dermatophagoides pteronyssinus or house dust allergen in atopic dermatitis (n = 4). The CD80- or CD86-positive cells were confirmed as Langerhans cells by double immunostaining using anti-CD1a monoclonal antibody. Neither CD86 nor CD80 was detected on keratinocytes. Similar results of the stronger expression of CD86 over that of CD80 were obtained from psoriasis vulgaris (n = 11) and from contact dermatitis (n = 7), although CD86 was expressed only in 57% of the contact dermatitis cases. The percentage of Langerhans cells positive for CD86 was higher than for CD80, i.e. 48% compared with 9%, respectively, in the epidermis of lesional skin of atopic dermatitis (n = 8). The expression rate of these molecules on Langerhans cells increased in the dermis. To investigate the function of co-stimulatory molecules on Langerhans cells in atopic dermatitis, we conducted an inhibition test with antibodies. Anti-CD86 monoclonal antibody almost completely inhibited T-cell proliferation stimulated with crude extract of D. pteronyssinus in the presence of epidermal cells as antigen-presenting cells, whereas anti-CD80 monoclonal antibody produced less of an inhibitory effect. These data indicate that CD86 expressed on Langerhans cells may play an important part in the pathogenesis of atopic dermatitis.

Constitutive endothelial and inducible nitric oxide synthase in inflammatory dermatoses

We have used immunohistochemistry to localize the expression of the constitutive endothelial and inducible forms of the enzyme nitric oxide synthase (NOS) in skin from involved and uninvolved sites in patients with atopic dermatitis (AD) and allergic contact dermatitis (CD). Endothelial NOS (eNOS) immunoreactivity was localized to vascular endothelium in the dermis of both involved and uninvolved skin from all patients. Inducible NOS (iNOS) immunoreactivity was found to be closely associated with the upper dermal microvasculature in all the involved AD biopsies, but only in two of 10 uninvolved AD biopsies. CD biopsies were taken from 10 positive skin patch test sites and iNOS immunoreactivity was detected in all of these. iNOS immunoreactivity was detected in only one of the negative patch test biopsies. Both the extent and intensity of iNOS immunoreactivity was lower in CD than in AD skin lesions. The presence of eNOS in the skin is necessary for constitutive NO-mediated dilatation of the dermal vasculature. Induction of iNOS in the dermal endothelium and in perivascular inflammatory cells may be significant with respect to the roles of NO in both the vasodilatory component of the inflammatory response and in the modulation of immune responses in the skin.

Immunohistological analysis of ‘negative’ patch test sites in atopic dermatitis

Variable results have been obtained when patients with atopic dermatitis (AD),) have been patch tested with allergens known to produce a positive prick test. The significance of patch test results in our understanding of the pathogenesis of AD therefore remains questionable. This study was designed to determine the relevance of either positive or negative patch test results in relation to the expression of cell mediated immunity to allergens in patients with AD. Thirty-five patients with AD exhibiting patch test positivity to one or more aeroallergens on 'tape stripped' areas of the back were retested without prior tape stripping. Nine patients again showed positivity to one or more allergens while 26 failed to show positive reactions. In six of the positive patients both positive and negative patch tests were observed. Skin biopsies were taken from these matched positive and negative patch test sites as well as from an area of uninvolved skin. Samples were frozen and cryostat sections were analysed with immunohistological techniques using monoclonal antibodies to investigate the distribution of immunocompetent cells. All positive patch tests exhibited characteristics of a cell mediated immune response. The negative patch test sites were also found to contain evidence of mononuclear cell infiltration. Both negative and positive patch test sites showed significantly greater proportions of T cells compared to uninvolved skin. No increase in numbers of RFD1 positive and RFD7 positive macrophages were observed in either positive or negative patch test sites. Expression of CD23 by CD1 positive Langerhans cells was raised in both negative and positive patch tests compared to uninvolved areas. A significant increase in the population (RFD7+, CD23+) was seen in positive patch test sites compared to uninvolved skin. An increase in the proportion of RFD1 positive cells expressing CD23 was also seen in both negative and positive patch tests compared to uninvolved skin. This paper demonstrates that immunological reactions are promoted at 'non-tape stripped' patch test sites where no clinical evidence of reactivity is seen. Together the data demonstrate that the presence of systemic cell mediated immunity to specific allergens identified in patients by positive patch test, may also be present when no clinical signs are seen at the patch test site.

Assessment of topical corticosteroid activity on experimentally induced contact dermatitis: echographic evaluation with binary transformation and image analysis.

A new echographic evaluation method employing a B scanner and a dedicated software (Dermavision 2D, Cortex Technology, Hadsund, Denmark) was used in assessing the potency of three different corticosteroids. Experimental lesions were induced by patch tests with nickel sulfate 5% in petrolatum in 10 sensitized subjects and treated with two medications of different steroids (clobetasol propionate, fluocinolone acetonide or clobetasone butyrate) performed 16 and 40 h after the application of the nickel patch tests. Clinical and echographic evaluations were carried out at the beginning of the experiment and 64 h after the induction of the reactions. After obtaining echographic images, these were processed by software, enabling the selection of amplitudes of interest, the highlighting of parts of images and their assessment by a value corresponding to the number of pixels (picture elements). For evaluations a low reflecting band was chosen, marking edema and inflammatory infiltration. At positive patch test sites we observed a progressive increase in the number of low reflecting pixels, in accordance with the intensity of the reaction. Therapeutic response was assessed as the difference between values of treated and untreated test sites. The rank order of the efficacy of test substances as determined echographically was identical to the rank order generally accepted for these steroids. This evaluation method of topical corticosteroid activity could be usefully employed besides traditional evaluation methods.

The duration of Grenz ray-induced suppression of allergic contact dermatitis and its correlation with the density of Langerhans cells in human epidermis.

Recent investigations have shown that Grenz rays can suppress the allergic contact dermatitis reaction completely and that Langerhans cells, identified by OKT6 antibodies and electron microscopy, disappear from the epidermis at the same time. It is not known for how long this suppression lasts. This has been investigated in 28 nickel-sensitive patients who were given Grenz rays (3 Gy) on the back, once a week for 3 weeks. The patients were then divided into four groups and tested with patch tests for nickel at 1, 7, 14 and 21 days after the last Grenz ray treatment. Biopsies were taken from positive patch test sites, and from the corresponding opposite control. They were labelled with OKT6 antibodies to detect Langerhans cells. The patch test reactions were suppressed and the Langerhans cell density was decreased initially. These changes were restored after 3 and 6 weeks, respectively. The results show that the effect of Grenz rays on eczematous reactions extends to a maximum of 3 weeks and imply that Langerhans cells are necessary for the elicitation of the efferent phase of allergic contact dermatitis.