069 mol/l, Acros GDC-0199 research buy Organics, Geel, Belgium) and loaded onto a 0.8% agarose gel supplemented with ethidium bromide. The gel was run at 100 V for 30 min in 0.5 × TAE (Tris–acetate–EDTA). Smeared DNA bands indicate DNA degradation. Complex stability before and after nebulisation was examined by measuring particle size and zeta potential as described in Section 2.2, and by agarose gel electrophoresis as described above. The absence of visible pDNA bands indicates pDNA condensation and therefore complex stability. Results in BGM cells allowed the selection of a candidate formulation of the DNA vaccine (brPEI polyplexes with an N/P ratio of 8) for subsequent in vivo studies. However, before starting in vivo

studies, we decided to check the cytotoxicity and transfection efficiency of brPEI polyplexes once again on an avian cell line, namely chicken embryo fibroblasts (DF-1 cells). Materials and methods are the same as described in Sections 2.3 and 2.4. The effect of parenteral (intramuscular injection; IM, m quadriceps) and mucosal (aerosol, AE) DNA vaccination

of turkeys was compared. For aerosol delivery LDN-193189 we used the Cirrus™ Nebulizer (Intersurgical), designed to give tracheo-bronchial deposition of particles (up to 5 μm) in humans. Twenty-one SPF turkeys (AFSSA, Ploufragan, France) were divided into four groups and reared in negative pressure isolation units (IM1500, Montair, Sevenum, The Netherlands). Three groups received a primary DNA inoculation at 1 day of age and one booster inoculation 3 weeks later. Groups 1 and 2 were twice immunised intramuscularly tuclazepam with respectively naked plasmid DNA or brPEI-pcDNA/MOMPopt, while group 3 was vaccinated at both time points through nebulisation of brPEI-pcDNA/MOMPopt. The control group (4) was left unvaccinated. Turkeys were challenged by aerosol infection at the age of 5.5 weeks using the Cirrus™ nebulizer. The challenge infection consisted of 108 TCID50 of Cp. psittaci strain 92/1293 (avian genotype D strain). All turkeys were euthanized at 25 days post-challenge (PC). The vaccination scheme and the experimental set-up

are presented in Table 1 and Table 2. The experimental design of the animal experiment was evaluated and approved by the Ethical Committee for Animal Experiments of Ghent University (Reference number: EC 2006/049). All turkeys were monitored daily for clinical signs. Pharyngeal and cloacal swabs were taken at day 1 of the experiment and subsequently every other day starting at 5 days PC until euthanasia. Swabs were stored at −80 °C in Cp. psittaci transport medium prior to isolation. Blood samples (v. ulnaris) for the detection of MOMP-specific serum antibody titres were taken immediately prior to each DNA vaccination, 1.5 weeks following booster vaccination, immediately prior to the challenge and at 2 and 3.5 weeks post-challenge.

As CARS produces anti-Stokes shifted signal (blueshifted with respect to excitation pulses), it is free from single photon CP-868596 order fluorescence, which hampers spontaneous Raman measurements. Unlike spontaneous Raman where the anti-Stokes scattering is much weaker than the Stokes scattering, the CARS process actively

drives molecules into a specific vibrational mode and therefore generates significantly more signal with reduced temperature sensitivity. CARS microscopy has been used to image a few pharmaceutical systems during drug release. Kang et al. [21], [22] and [23] published work where they imaged in situ release of paclitaxel from polymeric films in a static medium (phosphate buffer) using CARS microscopy. In the first work focusing on orally administered drugs and dosage forms, Windbergs et al. [24] and Jurna et al. [25] used CARS microscopy to image the distribution

of TP in lipid dosage forms and monitored the release of TP during dissolution in a flow through cell setup. They were able to image both drug release and conversion from TPa to TPm in real time. We have developed a new analytical technique to record the dissolved drug concentration and simultaneously monitor solid-state changes on the surface of the oral solid dosage form undergoing dissolution. Furthermore, we have applied hyperspectral CARS microscopy for improved solid-state form characterization. We illustrate the MK-2206 mouse use of these techniques using the model drug theophylline (TP) in different Thymidine kinase dissolution media. USP grade theophylline (TP, 1,3-dimethyl-7H-purine-2,6-dione) anhydrate and monohydrate were gifted from BASF (Ludwigshafen, Germany). Methyl cellulose (MC) (Methocel A4C premium) was gifted from Colorcon GmbH (Idstein, Germany). Weighed amounts of TPa and TPm (0.49 g) were directly compressed using a force feed tablet

press (IMA Kilian Pressima, Italy). The upper punch had a pre-compression height of 9.22 mm and a final compression height of 3.02 mm using a compaction force of about 13 kN, resulting in compacts which had a diameter of 12.02 mm and a thickness of about 3 mm. The compression did not result in changes in the solid-state form, which we confirmed using hyperspectral CARS microscopy. The CARS microscopy system is illustrated in Fig. 1 and is described in more detail elsewhere [26]. A Nd:YVO4 picosecond pulsed laser (Coherent Inc., USA) operating at a fundamental wavelength of 1064 nm was frequency doubled to pump an optical parametric oscillator (OPO) (APE Berlin GmbH, Germany), which produced two dependently tunable laser beams. The fundamental laser beam was combined with the signal beam from the OPO and directed into an inverted laser-scanning microscope (Olympus IX71/FV300, Japan) where they were focused onto the sample using a 20×/0.5 NA objective.