Nanoscale, Voltage-Driven Application of Bioactive Substances onto Cells with Organized Topography.

With scanning ion conductance microscopy (SICM), a noncontact scanning probe technique, it is possible both to obtain information about the surface topography of live cells and to apply molecules onto specific nanoscale structures. The technique is therefore widely used to apply chemical compounds and to study the properties of molecules on the surfaces of various cell types. The heart muscle cells, i.e., the cardiomyocytes, possess a highly elaborate, unique surface topography including transverse-tubule (T-tubule) openings leading into a cell internal system that exclusively harbors many proteins necessary for the cell's physiological function. Here, we applied isoproterenol into these surface openings by changing the applied voltage over the SICM nanopipette. To determine the grade of precision of our application we used finite-element simulations to investigate how the concentration profile varies over the cell surface. We first obtained topography scans of the cardiomyocytes using SICM and then determined the electrophoretic mobility of isoproterenol in a high ion solution to be -7 × 10(-9) m(2)/V s. The simulations showed that the delivery to the T-tubule opening is highly confined to the underlying Z-groove, and especially to the first T-tubule opening, where the concentration is ∼6.5 times higher compared to on a flat surface under the same delivery settings. Delivery to the crest, instead of the T-tubule opening, resulted in a much lower concentration, emphasizing the importance of topography in agonist delivery. In conclusion, SICM, unlike other techniques, can reliably deliver precise quantities of compounds to the T-tubules of cardiomyocytes

Allergen-Specific Immunotherapy with Monomeric Allergoid in a Mouse Model of Atopic Dermatitis.

Atopic dermatitis (AD) is a widespread and difficult to treat allergic skin disease and is a tough challenge for healthcare. In this study, we investigated whether allergen-specific immunotherapy (ASIT) with a monomeric allergoid obtained by succinylation of ovalbumin (sOVA) is effective in a mouse model of atopic dermatitis. An experimental model of AD was reproduced by epicutaneous sensitization with ovalbumin (OVA). ASIT was performed with subcutaneous (SC) administration of increasing doses of OVA or sOVA. The levels of anti-OVA antibodies, as well as cytokines, were detected by ELISA. Skin samples from patch areas were taken for histologic examination. ASIT with either OVA or sOVA resulted in a reduction of both the anti-OVA IgE level and the IgG1/IgG2a ratio. Moreover, ASIT with sOVA increased the IFN-γ level in supernatants after splenocyte stimulation with OVA. Histologic analysis of skin samples from the sites of allergen application showed that ASIT improved the histologic picture by decreasing allergic inflammation in comparison with untreated mice. These data suggest that ASIT with a succinylated allergen represents promising approach for the treatment of AD

Analysis of OVA-specific IgG.

<p>The levels of antibodies in sera obtained before, during, and after ASIT were detected and quantified by ELISA. The results are presented as mean antibodies concentrations (mean±SE, n = 8 for each). AD without ASIT: OVA-sensitized untreated mice; ASIT with OVA: OVA-sensitized mice treated by ASIT with OVA; ASIT with sOVA: OVA-sensitized mice treated by ASIT with sOVA; placebo: PBS-sensitized mice. (a) anti-OVA IgG1 response in mice with ASIT, (b) anti-OVA IgG2a response in mice with ASIT, (c) anti-OVA IgG response in mice with ASIT, (d) ratio of OVA-specific IgG1/IgG2a antibody in mice with ASIT, (*, p<0.05)</p

Level of Foxp3 expression.

<p>The specific mRNAs in OVA-stimulated mice splenocytes (incubated with OVA for 72hrs) were quantified by qRT-PCR. The results are presented as mean mRNA expression (mean±SE, n = 8 for each). The relative levels of Foxp3 expression were calculated by referring to the HPRT (hypoxanthine guanine phosphoribosyltransferase) in each sample. AD without ASIT: OVA-sensitized untreated mice; ASIT with OVA: OVA-sensitized mice treated by ASIT with OVA; ASIT with sOVA: OVA-sensitized mice treated by ASIT with sOVA; placebo: PBS-sensitized mice. *, p<0.05.</p

Levels of cytokines.

<p>The levels of cytokines in supernatants of OVA-stimulated mice splenocytes (incubated with OVA for 72hrs) were quantified by ELISA. The results are presented as mean IL concentration (mean±SE, n = 8 for each). AD without ASIT: OVA-sensitized untreated mice; ASIT with OVA: OVA-sensitized mice treated by ASIT with OVA; ASIT with sOVA: OVA-sensitized mice treated by ASIT with sOVA; placebo: PBS-sensitized mice. Level of (a) the IL-4, (b) IL-5, (c) IL-12, (d) IFN-γ, and (e) IL-17; (f) IL-4/IFN-γ ratio in mice with ASIT. *, p<0.05 versus “AD without ASIT”; **, p<0.05 versus “placebo”; #, p<0.05 versus “ASIT with OVA”.</p

Protocol of sensitization and ASIT with OVA or sOVA.

<p>Mice were sensitized with OVA (100 μg) or saline applied at 100 μl to a sterile patch. The patch was applied for 1-wk and then removed. ASIT was performed between the 1^st and 2^nd OVA applications by SC injection of increasing doses of OVA (a), sOVA (b), or PBS (as control). After ASIT, the mice had two 1-wk exposures to a patch separated by 2-wk intervals. The control group received PBS at the same time.</p

Histologic features of OVA- and sOVA-treated skin sites in BALB/c mice.

<p>(a) Mice sensitized with OVA and treated with PBS (“AD without ASIT”), (b) mice sensitized with OVA and treated with OVA (“ASIT with OVA”), (c) mice sensitized with OVA and treated with sOVA (“ASIT with sOVA”), and (d) mice sensitized with PBS (“placebo”). Skin sections were stained with H&E and examined at 100x. Scale bars 100 μm. There is marked hyperplasia of the epidermis, a dermal infiltrate (a-c). The cellular infiltrate consists of neutrophils, eosinophils, and lymphocytes. (e) Summary index of the main assessment criteria for histologic skin lesions.</p