Patient characteristics and mRNA expression of FcεRI α, β, and γ receptor chains^a.

a<p>mRNA expression was evaluated by RT-PCR after immunomagnetic purification of eosinophils.</p>b<p>reported as IU/ml, normal range ≤100.</p>c<p>expressed as Index Units, positive test ≥19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p>e<p>Not done.</p><p>Patient characteristics and mRNA expression of FcεRI α, β, and γ receptor chains^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107725#nt110" target="_blank">a</a>}.</p

Disease activity and autoantibody profiles in patients with BP.

<p>Subjects (n = 48) were enrolled before receiving any immunosuppressive treatment for BP. Since patients were enrolled over several years, disease activity was scored with a BP Index (A) or the more comprehensive Bullous Disease Area Index (BPDAI) criteria (B). A strong correlation (Spearman’s r = 0.7193, p<0.0001) was observed between these two scoring systems (C). Sera were collected from untreated BP patients (BP sera; BPS), patients evaluated for other autoimmune skin diseases (other autoimmune sera; OAS), or age- and gender-matched controls (normal human sera; NHS) and evaluated for BP180 IgG, BP230 IgG, total IgE and BP180 IgE by ELISA. Each point represents the average of duplicate samples from an individual patient with the N per group indicated. Mann-Whitney U-test, *p≤0.05, **p≤0.01, ***p≤0.001.</p

Eosinophils from active BP patients degranulate in response to BP180 protein.

<p>For degranulation, peripheral blood was incubated with 10 µg/ml NC16A or GST control protein in duplicate and EDN release was measured by ELISA. Mean GST values (background) were subtracted from NC16A and results are expressed as percent maximal (100 nM ionomycin) release. Each point represents the mean of replicate samples from the same patients. The number of patients per group is indicated. A Kruskal-Wallis test was performed, * = p≤0.05.</p

Surface expression of FcεRI on BP eosinophils.

<p>Interaction of FcεRIα with IgE or FcεRIβ was evaluated using the proximity ligation assay on non-permeabilized preparations of circulating granulocytes or skin cryosections from BP patients or controls. Eosinophils were identified by their unique nuclear morphology (bi-lobed nucleus stained with DAPI) using high resolution confocal microscopy. Interaction of FcεRIα/IgE on peripheral blood and tissue eosinophils from BP patient (A, B) or controls (C, D). Insets are enlarged to show nuclear morphology. Scale bar = 25 uM. Interaction of FcεRIα/FcεRIβ on eosinophils in lesional biopsies (E–H). Panel H is an image of the same BP sample depicted in panel G, captured at higher magnification for resolution of nuclear morphology. Scale bar = 50 uM.</p

Evaluation of IgE receptor expression on circulating eosinophils from BP patients.

<p>Peripheral granulocytes were stained with fluorescently tagged antibodies specific for human CD203c, CD49d, CD16, and FcεRI-α, FcεRIβ, CD23 or IgE for flow cytometric analysis. Eosinophils were identified by gating on the CD16⁻/CD49d⁺/CD203c⁻ population. Eosinophils from healthy controls (A), active BP patients (B), or basophils (CD16⁻/CD49d⁺/CD203c⁺) from active BP patients (C) were evaluated. The degree of specific staining is indicated by the open histogram compared to appropriate isotype control shown by the shaded histogram. Staining is representative of 10 BP patients and 11 age- and gender-matched controls.</p

Correlation^a between antibody levels, eosinophil counts, and disease severity in untreated BP patients.

a<p>determined using Spearman’s rank correlation coefficient (r) and p = *<0.05, **<0.01, ***0.001.</p>b<p>reported as IU/ml, normal range ≤100.</p>c<p>expressed as Index Units, positive test ≥19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p><p>Correlation^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107725#nt101" target="_blank">a</a>} between antibody levels, eosinophil counts, and disease severity in untreated BP patients.</p

Samples evaluated for FcεRI expression using the proximity ligation assay.

a<p>eosinophils were enriched from peripheral blood using density centrifugation and adhered to glass slides.</p>b<p>expressed as IU of IgE, normal range ≤100 IU.</p>c<p>ELISA Index Value, normal range ≤19.</p>d<p>reported as BP index/BPDAI as described in the Methods.</p>e<p>not done.</p><p>Samples evaluated for FcεRI expression using the proximity ligation assay.</p