Dawn Cooper, Jonathan Hales, Richard Camp

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IgE-Dependent Activation of T cells by Allergen in Atopic Dermatitis: Pathophysiologic Relevance Dawn Cooper, Jonathan Hales, Richard Camp Journal of Investigative Dermatology Volume 123, Issue 6, Pages 1086-1091 (December 2004) DOI: 10.1111/j.0022-202X.2004.23484.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Proliferative responses of peripheral blood mononuclear cells (PBMC) from atopic dermatitis (AD) patients to Dermatophagoides pteronyssinus (Der p) allergen in the presence of unmodified or heated autologous serum. Responses in the presence of medium containing 10% fresh, unmodified autologous serum (shaded symbols) or 10% fresh, autologous serum that had been heated at 56°C for 60 min (open symbols) are shown. The results of independent experiments with PBMC and autologous sera from six different patients are given. Results are expressed as counts per minute (cpm), each point showing the mean of triplicate assays, with SD values. Where SD values are not shown, they are less than the diameter of the symbol. Journal of Investigative Dermatology 2004 123, 1086-1091DOI: (10.1111/j.0022-202X.2004.23484.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effects of non-atopic serum on responses of peripheral blood mononuclear cells (PBMC) from an atopic dermatitis (AD) patient to Dermatophagoides pteronyssinus (Der p) allergen. The serum used was either autologous or from a healthy, non-atopic donor, and was either unmodified or heated at 56°C for 60 min. Symbols representing the different sera used are indicated in the figure. Results are expressed as counts per minute (cpm) and are the mean of triplicate assays. SD values were less than 30% of the means. Similar results were obtained in a second, independent experiment. Journal of Investigative Dermatology 2004 123, 1086-1091DOI: (10.1111/j.0022-202X.2004.23484.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IgE depletion and complement activation in atopic serum, and dependence of peripheral blood mononuclear cell (PBMC) responses on autologous serum concentration. (A) Immunoreactive IgE levels in atopic serum before (unshaded column) and after depletion by the Dynabead immunoprecipitation method. IgE levels following each of three rounds of treatment with Dynabeads coupled to control antibody (shaded columns) or anti-IgE antibody (hatched columns), are shown. IgE levels were determined by ELISA after dilution of serum 2–100-fold with the sample diluent provided, and the mean results of duplicate assays are shown. The limit of detection of the ELISA was about 2 IU per mL. Results are representative of three independent experiments. (B) Immunoreactive iC3b levels in untreated atopic sera (unshaded columns) or sera treated with Dynabeads (Db) coupled to isotype control monoclonal antibody (shaded columns) or anti-IgE monoclonal antibody (hatched columns). Serum samples were freshly prepared on ice and antibody-coupled Dynabeads added at time zero. The samples were then kept on ice for 15 and 60 min prior to ELISA, which was carried out as described under Materials and Methods. Data represent the means of duplicate ELISA measurements, and are from four independent experiments. (C) Responses of PBMC from an AD patient to Der p allergen in medium supplemented with four different concentrations of fresh, unmodified autologous serum (1%, 2.5%, 5%, and 10%). The responses of PBMC to Der p allergen in the presence of 10% autologous serum heated at 56°C for 60 min are shown for comparison. Each point shows the mean of triplicate assays. SD values are less than 30% of the means. Results are representative of four independent experiments. Journal of Investigative Dermatology 2004 123, 1086-1091DOI: (10.1111/j.0022-202X.2004.23484.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Proliferative responses of peripheral blood mononuclear cells (PBMC) from atopic dermatitis (AD) patients to Dermatophagoides pteronyssinus (Der p) allergen in the presence of IgE-depleted serum. Medium was supplemented either with 5% control autologous serum (shaded symbols) or 5% autologous serum depleted of IgE by immunoprecipitation (open symbols). Results are expressed as counts per minute (cpm) and are from independent experiments with PBMC and sera from six different patients. Each point shows the mean of triplicate assays with SD values. Journal of Investigative Dermatology 2004 123, 1086-1091DOI: (10.1111/j.0022-202X.2004.23484.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions