The number of BAL infiltrating leukocytes in pre-vaccinated and control animals were analyzed after sensitization. dorsal mouse skin. The grass pollen allergen Phl p 5 was administered by patch with or without the T helper 1-promoting CpG oligodeoxynucleotide 1826 as adjuvant, or was subcutaneously injected. Protection from allergic immune responses was tested by sensitization via injection of allergen adjuvanted with alum, followed by intranasal instillation. In a therapeutic setting, pre-sensitized mice were treated NSC 405020 either by the standard method using subcutaneous injection or via laser-generated micropores. Sera were analyzed for IgG antibody subclass distribution by ELISA and for IgE antibodies by a basophil mediator release assay. Cytokine profiles from supernatants of re-stimulated lymphocytes and from bronchoalveolar lavage fluids were assessed by flow cytometry using a bead-based assay. The cellular composition of lavage fluids was determined by flow cytometry. Results Application of antigen via micropores induced T helper NSC 405020 2-biased immune responses. Addition of CpG balanced the response and prevented from allergic sensitization, i.e. IgE induction, airway inflammation, and expression of T helper 2 cytokines. Therapeutic efficacy of transcutaneous immunotherapy was equal compared to subcutaneous injection, but was superior with respect to suppression of already established IgE responses. Conclusions Transcutaneous immunotherapy via laser-generated micropores provides an efficient novel platform for treatment of type I allergic diseases. Furthermore, immunomodulation with T helper 1-promoting adjuvants can prevent the risk for new sensitization. 1.?Introduction Effective allergen-specific immunotherapy (SIT), regardless if performed by subcutaneous injections (SCIT) or sublingual application using droplets or tablets (SLIT), is perceived as an intervention to redirect inappropriate and exaggerated TH2 responses against allergens. Crucial events for this immune deviation are the preferential production of TH1 cytokines such as IFN-, and the induction of IL-10/TGF- secretion by T regulatory cells in blood and inflamed airways [1]. Furthermore, suppression of allergen-specific IgE and induction of IgG4, and suppression of mast cells, basophils, and eosinophils contribute to the control of allergen-specific immune responses associated with SIT [2]. Despite its verifiable clinical success [3,4], only a small percentage of patients prefer this therapy to symptomatic treatment [5,6], and drop-out rates are considerable [7,8], mainly due to therapy duration of 3-5 years and local (SLIT) and/or systemic (SCIT) side effects [9,10]. The ideal SIT approach should therefore (i) target a tissue rich in immunocompetent cells to increase efficacy, NSC 405020 thereby reducing the number of required doses, (ii) employ a needle-free administration method, and (iii) avoid contact with the circulation to minimize NSC 405020 the risk of systemic side effects. SUV39H2 Cutaneous delivery perfectly fulfills these prerequisites as the skin is easily accessible, harbors high numbers of antigen presenting cells, provides non-vascularized superficial layers, and delivery techniques avoiding needle and syringe have become available. In our current study, we used one of these platforms, the P.L.E.A.S.E? (Precise Laser Epidermal System) device for fractional ablation of superficial skin layers and the creation of micropores. This novel technology employs a diode-pumped Er:YAG laser, which emits light at 2.94?m, corresponding to a major absorption peak of water molecules. Their excitation and evaporation leads to formation of aqueous micropores with a diameter of approx. 150?m. Due to extremely short energy pulses, NSC 405020 heat transfer to neighboring tissue is negligible. Using a scanning laser technology, an array of several hundred identical micropores of pre-defined number, density and depth can be sequentially created within a few seconds. In contrast to other transcutaneous vaccination methods, the P.L.E.A.S.E? laserporation system is easily adaptable to target appropriate skin layers in different species/individuals by adjusting the number of pulses per pore. Originally intended for increased delivery of small molecular weight compounds [11C13], in vivo transport of functionally intact proteins, such as antibodies, via P.L.E.A.S.E? -generated micropores has also been demonstrated [14]. Epicutaneous immunotherapy performed by application of allergen extracts to an area at the volar forearm, which had been pre-treated by needle scarification, was already described more than 50 years ago [15]. Recently, studies in mice and humans revisited.