Brain-derived neurotrophic factor as an indicator of chemical neurotoxicity:an animal-free CNS cell culture model

Recent changes to the legislation on chemicals and cosmetics testing call for a change in the paradigm regarding the current 'whole animal' approach for identifying chemical hazards, including the assessment of potential neurotoxins. Accordingly, since 2004, we have worked on the development of the integrated co-culture of post-mitotic, human-derived neurons and astrocytes (NT2.N/A), for use as an in vitro functional central nervous system (CNS) model. We have used it successfully to investigate indicators of neurotoxicity. For this purpose, we used NT2.N/A cells to examine the effects of acute exposure to a range of test chemicals on the cellular release of brain-derived neurotrophic factor (BDNF). It was demonstrated that the release of this protective neurotrophin into the culture medium (above that of control levels) occurred consistently in response to sub-cytotoxic levels of known neurotoxic, but not non-neurotoxic, chemicals. These increases in BDNF release were quantifiable, statistically significant, and occurred at concentrations below those at which cell death was measureable, which potentially indicates specific neurotoxicity, as opposed to general cytotoxicity. The fact that the BDNF immunoassay is non-invasive, and that NT2.N/A cells retain their functionality for a period of months, may make this system useful for repeated-dose toxicity testing, which is of particular relevance to cosmetics testing without the use of laboratory animals. In addition, the production of NT2.N/A cells without the use of animal products, such as fetal bovine serum, is being explored, to produce a fully-humanised cellular model.

Development of a multicellular co-culture model of normal and cystic fibrosis human airways in vitro

Cystic fibrosis (CF) is the most common lethal inherited disease among Caucasians and arises due to mutations in a chloride channel, called cystic fibrosis transmembrane conductance regulator. A hallmark of this disease is the chronic bacterial infection of the airways, which is usually, associated with pathogens such as Pseudomonas aeruginosa, S. aureus and recently becoming more prominent, B. cepacia. The excessive inflammatory response, which leads to irreversible lung damage, will in the long term lead to mortality of the patient at around the age of 40 years. Understanding the pathogenesis of CF currently relies on animal models, such as those employing genetically-modified mice, and on single cell culture models, which are grown either as polarised or non-polarised epithelium in vitro. Whilst these approaches partially enable the study of disease progression in CF, both types of models have inherent limitations. The overall aim of this thesis was to establish a multicellular co-culture model of normal and CF human airways in vitro, which helps to partially overcome these limitations and permits analysis of cell-to-cell communication in the airways. These models could then be used to examine the co-ordinated response of the airways to infection with relevant pathogens in order to validate this approach over animals/single cell models. Therefore epithelial cell lines of non-CF and CF background were employed in a co-culture model together with human pulmonary fibroblasts. Co-cultures were grown on collagen-coated permeable supports at air-liquid interface to promote epithelial cell differentiation. The models were characterised and essential features for investigating CF infections and inflammatory responses were investigated and analysed. A pseudostratified like epithelial cell layer was established at air liquid interface (ALI) of mono-and co-cultures and cell layer integrity was verified by tight junction (TJ) staining and transepithelial resistance measurements (TER). Mono- and co-cultures were also found to secrete the airway mucin MUC5AC. Influence of bacterial infections was found to be most challenging when intact S. aureus, B. cepacia and P. aeruginosa were used. CF mono- and co-cultures were found to mimic the hyperinflammatory state found in CF, which was confirmed by analysing IL-8 secretions of these models. These co-culture models will help to elucidate the role fibroblasts play in the inflammatory response to bacteria and will provide a useful testing platform to further investigate the dysregulated airway responses seen in CF.

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.

Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure

A key factor in the use of assisted reproductive technologies (ART) for diverse species is the safety of procedures for long-term health. By using a mouse model, we have investigated the effect of in vitro culture and embryo transfer (ET) of superovulated embryos on postnatal growth and physiological activity compared with that of embryos developing in vivo. Embryo culture from two-cell to blastocyst stages in T6 medium either with or without a protein source reduced blastocyst trophectoderm and inner cell mass cell number compared with that of embryos developing in vivo. Embryo culture and ET had minimal effects on postnatal growth when compared with in vivo development with an equivalent litter size. However, embryo culture, and to a lesser extent ET, led to an enhanced systolic blood pressure at 21 weeks compared with in vivo development independent of litter size, maternal origin, or body weight. Moreover, activity of enzymatic regulators of cardiovascular and metabolic physiology, namely, serum angiotensin-converting enzyme and the gluconeogenesis controller, hepatic phosphoeno/pyruvate carboxykinase, were significantly elevated in response to embryo culture and/or ET in female offspring at 27 weeks, independent of maternal factors and postnatal growth. These animal data indicate that postnatal physiological criteria important in cardiovascular and metabolic health may be more sensitive to routine ART procedures than growth. © 2007 by The National Academy of Sciences of the USA.

What's new pussycat? A genealogy of animal celebrity

Animal celebrity is a human creation informing us about our socially constructed natural world. It is relational, expressive of cultural proclivities, political power plays and the quotidian everyday, as well as serious philosophical reflections on the meaning of being human. This article attempts to outline some key contours in the genealogy of animal celebrity, showing how popular culture, including fairground attractions, public relations, Hollywood movies, documentary films, zoo attractions, commercial sport and mediatised moral panics - particularly those accompanying scientific developments such as cloning - help to order, categorise and license aspects of human understanding and feelings. The nature of [animal] charisma and celebrity are explored with assistance from Jumbo the Elephant, Guy the Gorilla, Paul the clairvoyant octopus, Uggie the film star, Nénette the orang-utan and Dolly the sheep. It argues that the issue of what it is to be human lies beneath the celebritised surface or, as Donna Haraway noted, the issue 'of having to face oneself'. © 2013 Taylor & Francis.