Can shelter dog observers score behavioural expressions consistently over time?

A substantial number of dogs live in animal shelters worldwide. Stressors within the shelter environment can compromise their welfare, and scientific evaluations of feasible welfare assessment methods are therefore needed. Qualitative Behaviour Assessment (QBA) is a "whole-animal" approach used to assess welfare by observing animals' expressive behaviour. To investigate whether observers can score dogs' behavioural expressions consistently over time, this study replicated and extended previous research, by evaluating intra- and inter-observer reliability of QBA based on video recordings of shelter dogs. In Part I, nine veterinary nurse students received theoretical and practical training, and then scored 12 2 min video recordings of shelter dogs using a fixed list of behavioural descriptors. Three of the students undertook further practice and calibration using direct observations of dog behaviour in a local shelter. In Part II, the videos from Part I were scored by these three observers a second time, 15 months later. QBA data were analysed using principal component analysis (PCA), and reliability was assessed using Kendall's coefficient of concordance (W). In Part I, the inter-observer reliability was high for both components (0.78 for PC1 and 0.85 for PC2). In Part II, the inter-observer reliability was very high and moderate for PC1 and PC2, respectively (0.90 for PC1 and 0.65 for PC2). The intra-observer reliability was high for both components (W ≥ 0.86). Our results indicate that the fixed list of behavioural descriptors for shelter dogs can be used reliably when assessing videos, and that observers can score dogs' behavioural expressions consistently after a break of 15 months following the initial assessment. Nevertheless, the reduction in inter-observer-reliability of PC2 in Part II can indicate that some retraining and calibration may be required to avoid observer drift.

Investigation of the interaction between modified ISCOMs and stratum corneum lipid model systems.

The modified ISCOMs, so-called Posintro nanoparticles, provide an opportunity for altering the surface charge of the particles, which influences their affinity for the negatively charged antigen sites, cell membranes and lipids in the skin. Hypothetically, this increases the passage of the ISCOMs (or their components) and their load through the stratum corneum. The subsequent increase in the uptake by the antigen-presenting cells results in enhanced transcutaneous immunization. To understand the nature of penetration of Posintro nanoparticles into the intercorneocyte space of the stratum corneum, the interaction between the nanoparticles and lipid model systems in form of liposomes and/or supported lipid bilayer was studied. As a lipid model we used Stratum Corneum Lipid (SCL), a mixture similar in composition to the lipids of the intercorneocyte space. By Förster Resonance Energy Transfer (FRET), Atomic Force Microscopy (AFM), Electrochemical Impedance Spectroscopy (EIS) and cryo-Transmission Electron Microscopy (cryo-TEM) it was shown that application of nanoparticles to the SCL bilayers results in lipid disturbance. Investigation of this interaction by means of Isothermal Titration Calorimetry (ITC) confirmed existence of an enthalpically unfavorable reaction. All these methods demonstrated that the strength of electrostatic repulsion between the negatively charged SCL and the nanoparticles affected their interaction, as decreasing the negative charge of the Posintro nanoparticles leads to enhanced disruption of lipid organization.

In vitro cutaneous application of ISCOMs on human skin enhances delivery of hydrophobic model compounds through the stratum corneum.

This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of -33 to -26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.

Status and future prospects of lipid-based particulate delivery systems as vaccine adjuvants and their combination with immunostimulators.

Vaccines seek to adopt pathogen-like characteristics but not true pathogen characteristics to activate the immune system without causing life-threatening disease. Vaccine formulations are therefore often particulate in nature, with dimensions comparable to pathogens, and often contain highly conserved pathogen-associated molecular patterns as adjuvants stimulating the immune system. Only a few adjuvants have been approved for human use. There is therefore an unmet medical need for the development of effective and safe adjuvants that can stimulate cellular, humoral or mucosal immunity, or combinations thereof, depending on the requirements, to prevent the specific disease. Lipid-based particulate systems are in this respect promising and versatile adjuvants that can be customized rationally towards specific vaccine targets by varying their composition. In this review, current progress in the development of lipid-based vaccine delivery systems is discussed, with a special focus on emulsions, liposomes and immune-stimulating complexes, and their combination with immunostimulatory compounds. Formulations, adjuvant mechanisms and alternative administration routes are highlighted.