Ex vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

Innovation adoption and fit, linking R&D and industry

Significant sums of money are invested in developing technological innovations that have low levels and rates of adoption. Several approaches have been put forward in an effort to improve rates of adoption. This paper presents the results of study that examined the innovation fit of key technological innovations in the beef industry. Findings indicate that be assessing the innovation fit throughout the R&D process researchers and end users can collaborate to improve the innovation fit and the rate of adoption. The paper also put forward a model that demonstrates the linkages between R&D, adoption and innovation fit.

(-)-CGP12177 increases contractile force through beta1l-adrenoceptors but not through beta3-adrenoceptors in human right atrial myocardium

(-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. As verified with recombinant receptors, the cardiostimulant effect of (-)-CGP12177 is mediated through a site at the beta1-adrenoceptor with lower affinity (beta1LAR) compared to the site through which (-)-CGP12177 antagonizes the effects of catecholamines (beta1HAR). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through beta3-adrenoceptors (Skeberdis et al 2008). We therefore investigated whether the effects of (-)-CGP12177 on human atrial trabeculae are antagonized by the beta3-adrenoceptor-selective antagonist L-748,337 (1 microM). (-)-CGP12177 (200 nM) caused a stable increase in force which was significantly reduced by the addition of (-)-bupranolol (1 microM), P = 0.002, (basal 4.45 ± 0.78 mN, IBMX (PDE inhibitor) 5.47 ± 1.01 mN, (-)-CGP12177 9.34 ± 1.33 mN, (-)-bupranolol 5.79 ± 1.08 mN, n = 6) but not affected by the addition of L-748,337 (1 microM), P = 0.12, (basal 4.48 ± 1.32 mN, IBMX 7.15 ± 2.28 mN, (-)-CGP12177 12.51 ± 3.71 mN, L-748,337 10.90 ± 3.49 mN, n = 6). Cumulative concentration-effect curves for (-)-CGP12177 were not shifted to the right by L-748,337 (1 microM). The –logEC50M values of (-)-CGP12177 in the absence and presence of L-748,337 were 7.21±0.09 and 7.41±0.13, respectively (data from 25 trabeculae from 8 patients, P=0.2) The positive inotropic effects of (-)-CGP12177 (IBMX present) were not antagonized by L-748,337 but were blunted by (-)-bupranolol (1 microM). The results rule out an involvement of beta3-adrenoceptors in the positive inotropic effects (-)-CGP12177 in human right atrial myocardium and are consistent with mediation through beta1LAR. Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227

3-D reconstruction of an ancient Egyptian mummy using X-ray computer tomography

Computer tomography has been used to image and reconstruct in 3-D an Egyptian mummy from the collection of the British Museum. This study of Tjentmutengebtiu, a priestess from the 22nd dynasty (945-715 BC) revealed invaluable information of a scientific, Egyptological and palaeopathological nature without mutilation and destruction of the painted cartonnage case or linen wrappings. Precise details on the removal of the brain through the nasal cavity and the viscera from the abdominal cavity were obtained. The nature and composition of the false eyes were investigated. The detailed analysis of the teeth provided a much closer approximation of age at death. The identification of materials used for the various amulets including that of the figures placed in the viscera was graphically demonstrated using this technique.

Vitamin D in health and disease : an insight into traditional functions and new roles for the 'sunshine vitamin'

Vitamin D is unique among the vitamins in that humans can synthesize it via the action of UV radiation upon the skin. This combined with its ability to act on specific target tissues via Vitamin D Receptor’s (VDR) make its classification as a steroid hormone more appropriate. While Vitamin D deficiency is a recognized problem in some northern latitude countries, recent studies have shown even in sunny countries such as Australia, vitamin D deficiency may be more prevalent than first thought. Vitamin D is most well known for its role in bone health, however, the discovery of VDR’s on a wide variety of tissue types has also opened up roles for vitamin D far beyond traditional bone health. These include possible associations with autoimmune diseases such as multiple sclerosis and inflammatory bowel diseases, cancer, cardiovascular diseases and muscle strength. Firstly, this paper presents an overview of the two sources of vitamin D: exposure to ultraviolet-B radiation and food sources of vitamin D, with particular focus on both Australian and international studies on dietary vitamin D intake and national fortification strategies. Secondly, the paper reviews recent epidemiological and experimental evidence linking vitamin D and its role in health and disease for the major conditions linked to suboptimal vitamin D, while identifying significant gaps in the research and possible future directions for research.

Vitamin D and sun protection : the impact of mixed public health messages in Australia

Exposure of the skin to sunlight can cause skin cancer and is also necessary for cutaneous vitamin D production. Media reports have highlighted the purported health benefits of vitamin D. Our aim was to examine attitudes and behaviours related to sun protection and vitamin D. A cross-sectional study of 2,001 residents in Queensland, Australia aged 20-70 years was undertaken. Information collected included: skin cancer risk factors; perceptions about levels of sun exposure required to maintain vitamin D; belief that sun protection increases risk of vitamin D deficiency; intention, and actual change in sun protection practices for adults and children. Multivariate models examined predictors of attitudinal and behavioural change. One-third (32%) believed a fair-skinned adult, and 31% thought a child required at least 30 minutes per day in summer sun to maintain vitamin D levels. Reductions in sun protection were reported by 21% of adults and 14% of children. Factors associated with belief that sun protection may result in not obtaining enough vitamin D included aged ≥ 60 years (OR=1.35, 95% CI 1.09-1.66) and having skin that tanned easily (OR=1.96, 95% CI 1.38-2.78). Participants from low income households, and those who frequently used sun protective clothing were more likely to have reduced sun protection practices (OR=1.33, 95% CI 1.10-1.73 and OR=1.73, 95% CI 1.36-2.20, respectively). This study provides evidence of reductions in sun protection practices in a population living in a high UV environment. There is an urgent need to re-focus messages regarding sun exposure and for continued sun protection practices.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

The impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25-hydroxyvitamin D concentrations

Studies have examined the associations between cancers and circulating 25-hydroxyvitamin D [25(OH)D], but little is known about the impact of different laboratory practices on 25(OH)D concentrations. We examined the potential impact of delayed blood centrifuging, choice of collection tube, and type of assay on 25(OH)D concentrations. Blood samples from 20 healthy volunteers underwent alternative laboratory procedures: four centrifuging times (2, 24, 72, and 96 h after blood draw); three types of collection tubes (red top serum tube, two different plasma anticoagulant tubes containing heparin or EDTA); and two types of assays (DiaSorin radioimmunoassay [RIA] and chemiluminescence immunoassay [CLIA/LIAISON®]). Log-transformed 25(OH)D concentrations were analyzed using the generalized estimating equations (GEE) linear regression models. We found no difference in 25(OH)D concentrations by centrifuging times or type of assay. There was some indication of a difference in 25(OH)D concentrations by tube type in CLIA/LIAISON®-assayed samples, with concentrations in heparinized plasma (geometric mean, 16.1 ng ml−1) higher than those in serum (geometric mean, 15.3 ng ml−1) (p = 0.01), but the difference was significant only after substantial centrifuging delays (96 h). Our study suggests no necessity for requiring immediate processing of blood samples after collection or for the choice of a tube type or assay.

Public health and clinical dilemmas resulting from imprecise vitamin D tests

Objective: To determine whether there are clinical and public health dilemmas resulting from the reproducibility of routine vitamin D assays. Methods: Blinded agreement studies were conducted in eight clinical laboratories using two commonly used assays to measure serum 25-hydroxyvitamin D (25(OH)D) levels in Australasia and Canada (DiaSorin Radioimmunoassay (RIA) and DiaSorin LIAISON® one). Results: Only one laboratory measured 25(OH)D with excellent precision. Replicate 25(OH)D measurements varied by up to 97% and 15% of paired results differed by more than 50%. Thirteen percent of subjects received one result indicating insufficiency [25-50 nmol/l] and another suggesting adequacy [>50 nmol/l]). Agreement ranged from poor to excellent for laboratories using the manual RIA, while the precision of the semi-automated Liaison® system was consistently poor. Conclusions: Recent interest in the relevance of vitamin D to human health has increased demand for 25(OH)D testing and associated costs. Our results suggest clinicians and public health authorities are making decisions about treatment or changes to public health policy based on imprecise data. Clinicians, researchers and policy makers should be made aware of the imprecision of current 25(OH)D testing so that they exercise caution when using these assays for clinical practice, and when interpreting the findings of epidemiological studies based on vitamin D levels measured using these assays. Development of a rapid, reproducible, accurate and robust assay should be a priority due to interest in populationbased screening programs and research to inform public health policy about the amount of sun exposure required for human health. In the interim, 25(OH)D results should routinely include a statement of measurement uncertainty.

Elucidating the links between UV radiation and vitamin D synthesis : using an in vitro model

Ultraviolet radiation (UV) is the carcinogen that causes the most common malignancy in humans – skin cancer. However, moderate UV exposure is essential for producing vitaminDin our skin. VitaminDincreases the absorption of calcium from the diet, and adequate calcium is necessary for the building and maintenance of bones. Thus, low levels of vitamin D can cause osteomalacia and rickets and contribute to osteoporosis. Emerging evidence also suggests vitamin D may protect against falls, internal cancers, psychiatric conditions, autoimmune diseases and cardiovascular diseases. Since the dominant source of vitamin D is sunlight exposure, there is a need to understand what is a “balanced” level of sun exposure to maintain an adequate level of vitamin D but minimise the risks of eye damage, skin damage and skin cancer resulting from excessive UV exposure. There are many steps in the pathway from incoming solar UV to the eventual vitamin D status of humans (measured as 25-hydroxyvitamin D in the blood), and our knowledge about many of these steps is currently incomplete. This project begins by investigating the levels of UV available for synthesising vitamin D, and how these levels vary across seasons, latitudes and times of the day. The thesis then covers experiments conducted with an in vitro model, which was developed to study several aspects of vitamin D synthesis. Results from the model suggest the relationship between UV dose and vitamin D is not linear. This is an important input into public health messages regarding ‘safe’ UV exposure: larger doses of UV, beyond a certain limit, may not continue to produce vitamin D; however, they will increase the risk of skin cancers and eye damage. The model also showed that, when given identical doses of UV, the amount of vitamin D produced was impacted by temperature. In humans, a temperature-dependent reaction must occur in the top layers of human skin, prior to vitamin D entering the bloodstream. The hypothesis will be raised that cooler temperatures (occurring in winter and at high latitudes) may reduce vitamin D production in humans. Finally, the model has also been used to study the wavelengths of UV thought to be responsible for producing vitamin D. It appears that vitamin D production is limited to a small range of UV wavelengths, which may be narrower than previously thought. Together, these results suggest that further research is needed into the ability of humans to synthesise vitamin D from sunlight. In particular, more information is needed about the dose-response relationship in humans and to investigate the proposed impact of temperature. Having an accurate action spectrum will also be essential for measuring the available levels of vitamin D-effective UV. As this research continues, it will contribute to the scientific evidence-base needed for devising a public health message that will balance the risks of excessive UV exposure with maintaining adequate vitamin D.

Devising a scenographic approach in Jonzi D's Hip Hop theatre

Jonzi D, one of the leading Hip Hop voices in the UK, creates contemporary theatrical works that merge dance, street art, original scored music and contemporary rap poetry, to create theatrical events that expand a thriving sense of a Hip Hop nation with citizens in the UK, throughout southern Africa and the rest of the world. In recent years Hip Hop has evolved as a performance genre in and of itself that not only borrows from other forms but vitally now contributes back to the body of contemporary practice in the performing arts. As part of this work Jonzi’s company Jonzi D Productions is committed to creating and touring original Hip Hop theatre that promotes the continuing development and awareness of a nation with its own language, culture and currency that exists without borders. Through the deployment of a universal voice from the local streets of Johannesburg and the East End of London, Jonzi D creates a form of highly energized performance that elevates Hip Hop as great democratiser between the highly developed global and under resourced local in the world. It is the staging of this democratised and technologised future (and present), that poses the greatest challenge for the scenographer working with Jonzi and his company, and the associated deprogramming and translation of the artists particular filmic vision to the stage, that this discussion will explore. This paper interrogates not only how a scenographic strategy can support the existence of this work but also how the scenographer as outsider can enter and influence this nation.