Value of component based diagnostics in IgE-mediated hymenoptera sting reactions.

BACKGROUND: Stings by insects can precipitate many signs and symptoms of dermatological and ocular diseases. Of particular importance is the anaphylaxis after Hymenoptera stings. Selection of the appropriate venom for immunotherapy requires a precise diagnosis, which is frequently difficult to confirm since the history presented by the patient is many times not conclusive and diagnostic tests are often positive for bee venom (BV) and vespula venom (VV). This double positivity is either caused by true double sensitization or by antibodies cross-reactive to homologous peptide sequences or to cross-reactive carbohydrate determinants (CCDs). In this study, we analyzed in 39 patients, tested positive for specific immunoglobulin E (sIgE) against BV and VV and CCDs whether the routine detection of sIgE against the recombinant species-specific major allergens (SSMAs) rApi m1 and rVes v5 enables the discrimination between genuine double sensitization and cross reactivity and therefore may be superior to other in vitro assays such as IgE-inhibition test or the basophil activation test. MATERIALS AND METHODS: Thirty-nine patients each with allergic reactions to vespula and/or honey bee stings and tested positive for sIgE antibodies against CCDs were analyzed for sIgE against BV, VV, CCDs (MUFX3) and SSMAs by UNICAP (CAP) and to BV, VV, bromelain, horseradish peroxidase and ascorbat oxidase by Immulite 2000 (IMMU). In 12 cases results from a basophil activation test, in nine cases results from IgE-inhibition assays and in 10 cases an unambiguous history of the patient were taken into consideration. RESULTS: A definite diagnosis could be assigned to each patient: sensitization to BV n = 7, sensitization to VV n = 29 and true double sensitization to both venoms n = 3. Detection of sIgE against BV and VV by CAP leads in three cases to the diagnosis BV allergy, in 35 cases to the diagnosis double sensitization and in one case to the diagnosis VV allergy. Detection of sIgE against BV and VV by IMMU leads in five cases to the diagnosis BV allergy, in 27 cases to the diagnosis double sensitization and in seven cases to the diagnosis VV allergy. Detection of sIgE against rApi m1 and rVes v5 by CAP leads in six cases to the diagnosis BV allergy, in eight cases to the diagnosis double sensitization, in 21 cases to the diagnosis VV allergy and in four cases to a false double-nagative result implicating no allergy. DISCUSSION AND CONCLUSION: Detection of sIgE to rApi m 1 and rVes v 5 by CAP is the most reliable diagnostic procedure to discriminate between true double sensitization and cross reactivity in patients with double-positive IgE results to venom extracts in the presence of sIgE against CCDs. In this study, however, we demonstrate that in nine of 39 patients tested positive for sIgE against CCDs, even the allergen component based diagnostic produces false double-positive and also false double-negative test results. Thus, we conclude that especially in hard to diagnose CCD positive patients beside the detection of sIgE, in vitro assays such as the IgE-inhibition test or the basophil activation test are still of importance. Detection of sIgE against only two SSMAs is not sufficient for a precise diagnosis. We propose inclusion of further SSMAs in diagnostic procedures.

Skin: major target organ of allergic reactions to small molecular weight compounds.

Skin is a major target organ for allergic reactions to small molecular weight compounds. Drug allergic reactions may be life-threatening such as in the case of anaphylactic reactions or bullous drug reactions and occur in about 5% of all hospitalized patients. Allergic contact dermatitis has an enormous influence on the social life of the patient because it is the most frequent reason for occupational skin diseases and the treatment and prevention of this disease cost approximately euro 3 billion per year in Germany. The different proposed pathophysiological pathways leading to a drug eruption are discussed in this paper. All major enzymes which are involved in the metabolism of xenobiotica were shown to be present in skin. Evidence supporting the role of metabolism in the development of drug allergy and allergic contact dermatitis is demonstrated in the example of sulphonamides and fragrances.

Enhanced expression levels of IL-31 correlate with IL-4 and IL-13 in atopic and allergic contact dermatitis.

BACKGROUND: IL-31 is produced by activated T lymphocytes, preferentially by TH2 cells. Transgenic mice overexpressing IL-31 have a phenotype resembling allergic dermatitis in human subjects. OBJECTIVE: We sought to evaluate the potential importance of IL-31 in the pathogenesis of human T cell-mediated skin diseases. METHODS: We analyzed total RNA taken from 149 skin biopsy specimens from patients with atopic dermatitis (AD), allergic contact dermatitis (ACD), or psoriasis in comparison with specimens taken from patients with healthy skin (n = 13) by using quantitative real-time PCR for the expression of TH1/TH2 cytokines. RESULTS: We found statistically increased mRNA levels of IL-31 in biopsy specimens taken from patients with AD, irrespective of the severity of the disease and serum IgE levels. Moreover, IL-31 mRNA levels were strongly increased in many biopsy specimens taken from patients with ACD. However, no increased transcription of IL-31 could be detected in biopsy specimens taken from psoriatic plaques. A comparison of mRNA levels of IL-31 with TH1 or TH2 cytokines demonstrates a correlation of the expression of IL-31 with IL-4 and IL-13 but not with IFN-gamma. No significant increase of IL-31 receptor mRNA could be detected in any disease, whereas the second receptor subunit of IL-31, the oncostatin M receptor, seems to be enhanced transcribed in patients with psoriasis. CONCLUSION: IL-31 expression is not only increased in patients with AD but also in those with ACD, 2 pruritic skin disorders. In both types of eczema, expression of IL-31 is associated with the expression of the TH2 cytokines IL-4 and IL-13. CLINICAL IMPLICATIONS: IL-31 might contribute not only to the development of AD but also to ACD-provoked skin inflammation.

Differentiation-specific factors modulate epidermal CYP1-4 gene expression in human skin in response to retinoic acid and classic aryl hydrocarbon receptor ligands.

Human epidermal keratinocytes express subsets of cytochromes P450 (P450) (CYP gene products) that are strongly up-regulated, not regulated, or down-regulated by differentiation-specific factors. We investigated how drug exposure affects epidermal expression of CYP1-4 genes, which encode many drug-metabolizing P450s. Real-time polymerase chain reaction (PCR) assays measured CYP1-4 mRNA levels in epidermal keratinocytes differentiated in vitro in the presence of drug or vehicle for 6 days. We confirmed the spinous phenotype at day 6 by changes in cellular morphology and upregulation of cytokeratin 10 and transglutaminase (TGM)1 mRNA in the differentiating keratinocytes. Effects of drug exposure depended on the influence of differentiation-specific factors in controlling epidermal CYP1-4 expression. CYP2C18, 2C19, 2C9, 2W1, 3A4, and 4B1 are up-regulated by cellular differentiation; mRNA levels for these CYP genes were inhibited in differentiating keratinocytes exposed to retinoic acid and aryl hydrocarbon receptor (AhR) ligands. These same drugs effected

Skin retinoid concentrations are modulated by CYP26AI expression restricted to basal keratinocytes in normal human skin and differentiated 3D skin models.

Cellular levels of all-trans retinoic acid (RA) are meticulously regulated utilizing an array of systems to balance uptake, biosynthesis, catabolism, and efflux transport. Metabolic transformation of all-trans RA to 4-hydroxylated RA appears to be primarily catalyzed by the cytochrome P450 (CYP) 26AI. Analysis of monolayer cultures of normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts by quantitative real-time PCR and reverse transcription-PCR revealed no basal levels of CYP26AI mRNA expression, whereas specific transcripts were detectable following addition of 10(-6) M all-trans RA. Immunofluorescence and Western blot analysis showed a weak expression of CYP26AI in NHEK, which was increased by stimulation with all-trans RA. Using a newly developed peptide antibody, we further examined the localization of CYP26AI expression in normal skin and three-dimensional (3D) skin models. In contrast to cell culture monolayers where CYP26AI was only weakly detectable, strong constitutive expression of CYP26AI in vivo and in organotypic culture was found to be restricted to basal epidermal keratinocytes, as well as eccrine sweat glands and sebaceous glands. These studies verify the capacity of human skin to metabolize RA, although substantial differences exist in CYP expression between normal skin and 3D skin models compared to monolayer cultures. Complex metabolic processes that maintain retinoid homeostasis may therefore be better studied in model systems more closely resembling in vivo skin. In light of our prior studies documenting the functional activity of RA metabolites, expression of CYP26 in the sebaceous gland epithelium supports the suggestion that altered RA metabolism may be involved in the pathogenesis of acne.

Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells.

Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.