A paper-based scaffold for enhanced osteogenic differentiation of equine adipose-derived stem cells.

OBJECTIVES: We investigated the applicability of single layer paper-based scaffolds for the three-dimensional (3D) growth and osteogenic differentiation of equine adipose-derived stem cells (EADSC), with comparison against conventional two-dimensional (2D) culture on polystyrene tissue culture vessels. RESULTS: Viable culture of EADSC was achieved using paper-based scaffolds, with EADSC grown and differentiated in 3D culture retaining high cell viability (>94 %), similarly to EADSC in 2D culture. Osteogenic differentiation of EADSC was significantly enhanced in 3D culture, with Alizarin Red S staining and quantification demonstrating increased mineralisation (p < 0.0001), and an associated increase in expression of the osteogenic-specific markers alkaline phosphatase (p < 0.0001), osteopontin (p < 0.0001), and runx2 (p < 0.01). Furthermore, scanning electron microscopy revealed a spherical morphology of EADSC in 3D culture, compared to a flat morphology of EADSC in 2D culture. CONCLUSIONS: Single layer paper-based scaffolds provide an enhanced environment for the in vitro 3D growth and osteogenic differentiation of EADSC, with high cell viability, and a spherical morphology.

Increased tumor localization and reduced immune response to adenoviral vector formulated with the liposome DDAB/DOPE.

UNLABELLED: We aimed to increase the efficiency of adenoviral vectors by limiting adenoviral spread from the target site and reducing unwanted host immune responses to the vector. We complexed adenoviral vectors with DDAB-DOPE liposomes to form adenovirus-liposomal (AL) complexes. AL complexes were delivered by intratumoral injection in an immunocompetent subcutaneous rat tumor model and the immunogenicity of the AL complexes and the expression efficiency in the tumor and other organs was examined. Animals treated with the AL complexes had significantly lower levels of beta-galactosidase expression in systemic tissues compared to animals treated with the naked adenovirus (NA) (P<0.05). The tumor to non-tumor ratio of beta-galactosidase marker expression was significantly higher for the AL complex treated animals. NA induced significantly higher titers of adenoviral-specific antibodies compared to the AL complexes (P<0.05). The AL complexes provided protection (immunoshielding) to the adenovirus from neutralizing antibody. Forty-seven percent more beta-galactosidase expression was detected following intratumoral injection with AL complexes compared to the NA in animals pre-immunized with adenovirus. CONCLUSIONS: Complexing of adenovirus with liposomes provides a simple method to enhance tumor localization of the vector, decrease the immunogenicity of adenovirus, and provide protection of the virus from pre-existing neutralizing antibodies.

Modification of liposomal concentration in liposome/adenoviral complexes allows significant protection of adenoviral vectors from neutralising antibody, in vitro.

Adenoviral vectors have been commonly used in gene therapy protocols, however the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced which limits further administration. This study examines the efficacy of complexing liposomes to adenovirus for the protection of the adenovirus from neutralising antibodies in an in vitro setting. Dimethyldioctadecylammonium bromide (DDAB)-dioleoyl-l-phosphatidylethanolamine (DOPE) liposomes were bound at varying concentrations to adenovirus to form AL complexes and tested these complexes' ability to prevent adenoviral neutralisation. It is shown that by increasing the concentration of liposomes in the adenoviral-liposome (AL) complexes we can increase the level of immuno-shielding afforded the adenovirus. It is also shown that the increase in liposomal concentration may lead to drawbacks such as increased cytotoxicity and reductions in expression levels.

Direct Conversion of Equine Adipose-Derived Stem Cells into Induced Neuronal Cells Is Enhanced in Three-Dimensional Culture.

The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with ßIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

In-vitro evaluation of ion-exchange microspheres for the sustained release of liposomal-adenoviral conjugates.

This study looks at the development of a novel combination vector consisting of adenovirus conjugated to liposomes (AL complexes) bound to cation-exchanging microspheres (MAL complexes). With adenovirus having a net negative charge and the liposomes a net positive charge it was possible to modify the net charge of the AL complexes by varying the concentrations of adenovirus to liposomes. The modification of the net charge resulted in altered binding and release characteristics. Of the complexes tested, the 5:1 and 2:1 ratio AL complexes were able to be efficiently bound by the microspheres and exhibited sustained release over 24 h. The 1:1 and 1:2 AL complexes, however, bound poorly to the microspheres and were rapidly released. In addition the MAL complexes also were able to reduce the toxicity of the AL complexes, which was seen with the 10:1 ratio. The AL complexes showed considerably more toxicity alone than in combination with microspheres, highlighting a potential benefit of this vector.

The detection of Chlamydia pneumoniae in atherosclerotic plaques of Australian subjects.

AIM: To determine whether the common respiratory pathogen, Chlamydia pneumoniae, was associated with atherosclerotic plaques in Australian subjects. METHODS: A total of 29 coronary atherosclerotic lesions and 18 normal coronary arterial samples were tested for the presence of C. pneumoniae by PCR and immunofluorescence methods. RESULTS: Chlamydia pneumoniae was detected in 15 of the atheromatous lesions as well as in three of the normal tissues; the immunofluorescence assay was more sensitive (P=0.028) than PCR (P=0.26). CONCLUSIONS: These findings contradict previous Australian studies which did not detect C. pneumoniae in atherosclerotic plaques, thereby discounting the speculation that its absence was likely due to geographical variation. The detection of the bacterium in some of the normal tissues suggests that C. pneumoniae infection might be an initial trigger of atherosclerotic development.

Microsphere-liposome complexes protect adenoviral vectors from neutralising antibody without losses in transfection efficiency, in-vitro.

Adenoviral vectors have been commonly used in gene therapy protocols but the success of their use is often limited by the induction of host immunity to the vector. Following exposure to the adenoviral vector, adenoviral-specific neutralising antibodies are produced, which limits further administration. This study examines the effectiveness of a novel combination of microspheres and liposomes for the shielding of adenovirus from neutralising antibodies in an in-vitro setting. We show that liposomes are effective in the protection of adenovirus from neutralising antibody and that the conjugation of these complexes to microspheres augments the level of protection. This study further reveals that previously neutralised adenovirus may still be transported into the cell via liposome-cell interactions and is still capable of expressing its genes, making this vector an effective tool for circumvention of the humoral immune response. We also looked at possible side effects of using the complexes, namely increases in cytotoxicity and reductions in transfection efficiency. Our results showed that varying the liposome:adenovirus ratio can reduce the cytotoxicity of the vector as well as increase the transfection efficiency. In addition, in cell lines that are adenoviral competent, transfection efficiencies on par with uncomplexed adenoviral vectors were achievable with the combination vector.

Pre-treatment with a non-therapeutic dose of cisplatin increases solid tumour response to liposomal-p53 gene therapy- An in vivo study.

Successful liposomal-mediated gene therapy is often limited by poor transfection efficiencies. One method previously shown to increase the efficiency of liposomal gene delivery is through the administration of a non-therapeutic dose of the chemotherapeutic drug cisplatin prior to lipofection. The currents study aims to utilise this method to deliver lipoplexes containing the p53 tumour suppressor gene with the aim of increasing therapeutic effect of the p53 gene on a solid tumour in vivo. Rats, implanted with solid salivary adenocarcinomas, were pre-treated with a low dose of cisplatin seven days prior to liposomal mediated p53 treatment. Following treatment with p53, tumour growth, p53 expression and levels of apoptosis were examined and compared to animals treated with p53 without cisplatin pre-treatment and a saline control. Tumours that had been pre-treated with cisplatin prior to p53-lipofection were significantly smaller than both the saline control and the non-cisplatin treated tumours. Saline treated tumours increased in size by an average of 164% over a 96-hour period compared to 64% and 101% for the cisplatin and non-cisplatin p53-liposome treated tumours. The cisplatin pre-treated tumours resulted in significantly higher levels of apoptosis surrounding the treatment site and exhibited prolonged p53 expression when compared to the non-cisplatin pre-treated tumours. The results suggest that the use of cisplatin to pre-sensitise tumours to lipofection has significant benefits when used in conjunction with p53.