Original article Cytokine and Ig-production by CG-containing sequences with phosphorodiester backbone and dumbbell-shape Atopic dermatitis (AD) belongs to the atopic diseases, such as allergic rhinitis or allergic asthma, and is a frequently occurring chronic skin disease primarily affect- ing younger age groups (1). The extrinsic AD is charac- terized by a deviated immune response with hyper reactivity to various, normally tolerated antigens of the environment. Clinically AD is characterized by subacute and chronic eczematous skin lesions. Especially severely affected patients exhibit elevations of total serum immu- noglobulin (Ig)E and the presence of several specific IgE levels towards common allergens. The affected skin sites are infiltrated mostly by CD4 + T cells and eosinophils, but also harbor an increased number of mast cells (2, 3). The pathophysiology of AD includes several immunolo- gical deviations: an unknown genetic predisposition leads to an imbalanced T H 2-type dominated immune response with consecutive interleukin (IL)-4, IL-5, IL-6 and IL-10 production resulting in eosinophil activation, but also IgE production. Subsequently, skin infiltrating macrophages and eosinophils produce IL-12, promoting interferon (IFN)-c production and initiating the chronic phase of AD (4, 5). Thus, molecules with T H 1-promoting properties may be useful for the prevention of allergic diseases, including AD, if the primary T H 2-type response is inhibited. One group of such immunostimulatory compounds are olig- odeoxynucleotide with nonmethylated cytosine-guanine motifs (CpG-ODN), which resemble microbial DNA and serve as Ôdanger signalsÕ. These CpG-ODNs not only increase the production of the pro-inflammatory cytokine Background: Immunostimulatory oligodeoxynucleotides (CpG-ODN) usually contain phosphorothioate (PS) backbones for nucleotide protection, which may result in some nonspecific side-effects like prolongation of coagulation time. Objective: The aim of the present study was to investigate the immunomodu- latory potential of DNA molecules without PS backbones. Thus, we designed phosphorodiester (PO) molecules with a dumbbell-like covalently-closed struc- ture (dSLIM-30L1). Methods: We analyzed their effects on peripheral blood mononuclear cells (PBMC) from spontaneous high and low immunoglobulin (Ig)E producer (allergic and nonallergic donors) in comparison with linear CpG-ODN (lin-30L1) with PS backbones, using enzyme-linked immunosorbent assay and flow cytometry. Results: We observed a decrease of spontaneous IgE levels in PBMC from high IgE producer of approximately 27% with both dSLIM-30L1 and lin-30L1. In addition, both molecules enhanced the production of IgA, IgM and IgG1/IgG2, but with a slightly different pattern. Both molecules stimulated the secretion of the T H 1-like cytokines interleukin (IL)-2, interferon-c and IL-12p40 and the pro- inflammatory cytokine IL-6. The immunomodulatory potential of dSLIM-30L1 and lin-30L1 was also effective in PBMC from nonallergic donors, as was con- firmed for IL-2, IL-12p40, IgG1/IgG2 and IgM. Conclusion: Our data show an inhibition of IgE production but also enhance- ment of the inflammatory cytokine response in PBMC from allergic and non- allergic donors by covalently-closed PO-based dSLIM-30L1 with a pattern similar to that of linear PS-based lin-30L1, while avoiding PS-modifications and thus PS-mediated side-effects. Whether such molecules are useful for the treat- ment of allergic diseases will need further clarification by appropriate in vivo studies. M. Schmidt 1 , K. Anton 2 , C. Nordhaus 2 , C. Junghans 1 , B. Wittig 1,3 , M. Worm 2 1 Mologen AG, Berlin; 2 Department of Dermatology and Allergy, CharitØ Universitätsmedizin Berlin, Campus Mitte, Berlin; 3 Institute for Molecular Biology and Bioinformatics, CharitØ Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany Key words: allergy; CpG; cytokines; double stem-loop immunomodulator; immunoglobulins. Prof. Dr Margitta Worm Department of Dermatology and Allergy CharitØ-Campus Mitte Schumannstr. 20-21 10117 Berlin Germany Accepted for publication 26 April 2005 Abbreviations: AD, atopic dermatitis; CG, cytosine-guanine dinu- cleotide; dSLIM, double stem-loop immunomodulator; IL, inter- leukin; IFN-c, interferon-c; Ig, immunoglobulin; ODN, oligodeoxynucleotide; PBMC, peripheral blood mononuclear cells; PO, phosphorodiester; PS, phosphorothioate; T H , T helper cell. Allergy 2006: 61: 56–63 Copyright Ó Blackwell Munksgaard 2005 ALLERGY DOI: 10.1111/j.1398-9995.2005.00908.x 56