Comparative in situ topoproteome analysis reveals differences in patch test-induced eczema: cytotoxicity-dominated nickel versus pleiotrope pollen reaction.

A subgroup of patients with atopic eczema develops acute eczematous reactions to type I allergy-inducing agents such as pollen that clinically resemble type IV allergies induced by haptens like metal ions. To clarify the underlying immunologic mechanisms, this study was designed to map the inflammatory in situ topoproteome of eczematous responses to grass/birch pollen and nickel by using atopy patch test (APT) and nickel patch test (NPT) as an appropriate clinical model, respectively. Biopsies from NPT (n = 6) and APT (n = 6) with positive reactions at 72 h were analysed by multiple epitope ligand cartography (MELC), which enabled to investigate coexpression of 49 different epitopes immunohistochemically in a single given tissue section. Colocalisation of IgE and FcepsilonRI was investigated by confocal microscopy. Compared with APT responses, NPT reactions were dominated by cytotoxic TIA-1 + and CD8 + T cells. In contrast, the immune response in APT reactions appeared more pleiotrope - as detected by colocalisation analysis. Multiple combinatorial molecular phenotype (CMP) motifs containing naive, early maturation and memory T cell (CD45RA, CD7, CD44, CD45R0), and general activation markers (CLA, HLA-DR, CD13, CD29, CD58, CD71, CD138) were significantly higher expressed in APT when compared with NPT reactions. APT response was confirmed to be accompanied by IgE bound to FcepsilonRI. In summary, our results demonstrate that the NPT reaction is clearly dominated by cytotoxic events, while the APT reaction to pollen grains is more heterogeneous and elicits a combined humoral and cellular immune reaction.

In-situ-topoproteome analysis of cutaneous lymphomas: perspectives of assistance for dermatohistologic diagnostics by Multi Epitope Ligand Cartography (MELC).

BACKGROUND: Immunophenotyping is essential for diagnostics of cutaneous lymphomas. In this regard we present a skin tissue-adapted application platform of MELC technology. PATIENTS AND METHODS: This topoproteome analysis allows the subcellular colocalization of at least n = 100 epitopes in situ. For this purpose the specimen is processed by a Toponome Imaging Cycler for a n-fold repetition of the following cycle: 1) staining with a fluorophore-labeld antibody, 2) fluorescence-imaging, and 3) photobleaching. Overlay and binarization of fluorescence images lead to combinatorial molecular phenotypes (CMP), which relate to a pixel or microtopographic unit (450 x 450 nm2, 20x objective). Skin biopsies were derived from patients with mycosis fungoides (patch/plaque lesions), psoriasis, atopic eczema and from healthy skin donors. RESULTS: In orientation to the WHO-EORTC-classification of cutaneous lymphomas a MELC-library of 23 markers was established. According to an inaugurative detailed procedure the CMP frequency was determined in a normalization to 100 microm horizontal skin width. By a TopoMiner strategy mycosis fungoides could be separated from the other states with a maximum of significance (p < or = 0.03) by at least 10-fold overexpression of the following tumor cell-representative CMP-motif: CD3+/CD4+/CD1a-/CD7-/CD8-/CD45R0+/CD45RA-/CD11a+. CONCLUSIONS: The skin tissue-adapted MELC-application-platform extends substantially conventional lymphoma diagnostics by an unprecedented dimension of in-situ-analysis of marker combinatorics including its exact quantification and visualization.