In vitro characterization of IMI-Gel.

<p>To characterize IMI-Gel and Aldara, imiquimod containing formulations were analyzed in terms of <b>A)</b> presence of imiquimod crystals in IMI-Gel using electron microscopy, <b>B)</b> sizes (mean+SD) of imiquimod-particles in IMI-Gel immediately or 9 months after manufacturing (under room conditions) and <b>C)</b> flow curves defining rheological characteristics.</p

Delayed tumor growth and tumor protection after IMI-Gel application.

<p>E.G7 thymoma cells (4×10⁵ s.c.), expressing SIINFEKL, were injected into the flank of C57BL/6 mice. After the tumor was palpable mice were immunized as indicated on two consecutive days in weekly intervals over a period of three weeks or left untreated. <b>A)</b> The tumor size and <b>B)</b> the survival were monitored. Depicted are the cumulative results of three independent experiments with n = 23 for emulsion gel and Aldara and n = 17 for untreated control. *Significant difference with p<0.05 by Mantel Cox test compared to the untreated control group.</p

IMI-Gel and Aldara are equally potent in inducing primary CTL-responses.

<p>C57BL/6 mice were shaved on their backs and afterwards immunized on two consecutive days with either Aldara (50 mg) together with SIINFEKL (100 µg) or IMI-Gel (50 mg) and officinal cremor basalis together with SIINFEKL or left untreated (untreated control). <b>A)</b> The frequency of peptide-specific CD8⁺ T cells in the blood (mean and SD) and <b>B)</b><i>in vivo</i> cytolytic activity 24 hours (mean and SD) after transfer of peptide-loaded target cells was assessed. Depicted are the cumulative results of two independent experiments with n = 6 for the tetramer staining and three independent experiments with n = 9 for the cytotoxicity assay. *Significant difference with p<0.05 by one-way ANOVA with Bonferroni’s posttest.</p

The route of IMI-Gel application influences vaccination efficacy.

<p>C57BL/6 mice were shaved on their backs and received the following treatments: untreated (untreated control), IMI-Gel with SIINFEKL (100 µg) (IMI-Gel TCI) on two consecutive days, IMI-Gel alone (IMI-Gel without SIIN) on two consecutive days, oral IMI-Gel (50 mg) with SIINFEKL (100 µg) on two consecutive days (IMI-Gel p. o.) or s. c. once with IMI-Gel (100 mg) diluted with SIINFEKL (200 µg) and distilled water into the neck. <b>A)</b> The frequency of peptide-specific CD8⁺ T cells in the blood (mean and SD) and <b>B)</b><i>in vivo</i> cytolytic activity 24 hours (mean and SD) after transfer of peptide-loaded target cells was assessed. Depicted are the cumulative results of two independent experiments with n = 7 for IMI-Gel treated groups and n = 4 for controls. *Significant difference with p<0.05 by Students <i>t</i> test.</p

LPS stimulation can be enhanced by facilitating binding to CD14.

<p>Human PMN (2×10⁵ cells per well) were incubated with LPS (100 ng/ml) with or without the addition of aPL (10 µg/ml) or LPS-binding protein (LBP, 10 ng/ml). ROS formation was measured in a fluorescence reading device. Detection of specific fluorescence index (SFI) over time was calculated by subtraction of the background fluorescence of labelled cells incubated in medium. Data from one representative experiment with two replicates is shown.</p

A Role for Toll-Like Receptor Mediated Signals in Neutrophils in the Pathogenesis of the Anti-Phospholipid Syndrome

<div><p>The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.</p> </div

Imiquimod passes mouse skin <i>in vitro</i> more rapidly when formulated in Aldara than in IMI-Gel.

<p><b>A)</b> Release of imiquimod as the active component was detected with a modified Franz-diffusion-cell model. <b>B)</b> Shaved skin of C57BL/6 mice (n = 6) was ablated and afterwards treated with Aldara or IMI-Gel (each 50 mg). The imiquimod concentration in the acceptor medium was determined after various time points as indicated using HPLC (UV absorption 245 nm). *Significant difference with p<0.05 by Wilcoxon signed rank test. <b>C)</b> C57BL/6 mice (n = 3) were with Aldara or IMI-Gel (each 50 mg/6 cm²) on. After 3 hours mice were sacrificed and remaining formulation was removed with gauze. 1 cm² of treated skin was prepared, fat removed and subsequently hackled with an ultra turrax. The amount of imiquimod recovered from the skin surface (left panel) and within the skin (right panel) was determined using HPLC.</p

Synergistic activation of PMN with aPL and TLR2 ligation.

<p>Human PMN (2×10⁵ cells per well) were incubated with the TLR2 ligand Pam₃Cys (100 ng/ml, 1 µg/ml, 10 µg/ml). A) Production of radical oxygen species was determined after 170 minutes. B) Phagocytosis rate was analysed defining percentage of PMN taken up PE-labelled microspheres. C) Shedding of L-Selectin was determined by surface staining of expressed molecules. D) Supernatants of stimulated PMN culture were harvested and an IL-8 specific ELISA was performed after 2 hours (left panel) or 6 hours (right panel) of incubation. Data shown are from one representative experiment out of 3 independent with 2 replicates per group. *Indicates significant difference of Pam₃Cys-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p

Synergistic induction of L-Selectin shedding mediated via aPL and LPS.

<p>PMN (2×10⁵ cells per well) were incubated with medium, aPL or hIgG in the presence or absence of titrated LPS. Shedding of L-Selectin was determined by surface staining of expressed molecules and analyzed by flow cytometry. Data shown are from one representative experiment out of 3 independent with 2 replicates per group. *Indicates significant difference of LPS-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p

PMN release IL-8 upon stimulation with aPL and LPS.

<p>PMN (2×10⁵ cells per well) were incubated with medium, aPL or hIgG (10 µg/ml) in the presence or absence of LPS. Supernatants were harvested and an IL-8 specific ELISA was performed after A) 2 hours or B) 6 hours of incubation (37°C). Data shown are from one representative experiment out of 3 independent with 2 replicates per group. * Indicates significant difference of LPS-stimulated groups compared to corresponding hIgG controls (p<0.05 in a Tukey’s multiple comparison test).</p