Cultured allogeneic keratinocyte transplantation in the large white pig

Introduction: Banked cultured Langerhans cell (LC) free allogeneic keratinocytes, could be a readily available resource for the treatment of burns. The key questions that have not been resolved regarding the natural history of allotransplanted cultured keratinocytes, are when, how and why they are eliminated. Previous work at the Blond McIndoe Centre in a Large White pig model has demonstrated the survival of allotransplanted keratinocytes at three weeks in a ''chimeric" context (transplanting mixtures of autologous and allogeneic keratinocytes). The cultured cells were labelled by retroviral gene transfer to introduce a lacZnIs histochemically detectable marker. This work however, involved grafting onto de-epidermalised dermis that was shown to be capable of regenerating an epidermis without the aid of transplanted keratinocytes. Additionally, the keratinocyte labelling efficiency was low. The purpose of this study was to investigate the fate of cultured allogeneic transplanted keratinocytes, when they are the only source of epidermal tissue in a wound incapable of epidermal regeneration. To accomplish this, the aim was to develop a more efficient means of porcine keratinocyte labelling with the lacZnIs marker and to immunohistochemically characterise the cellular infiltrate with regard to allogeneic split skin graft rejection and compare this with the immune cell infiltrate associated with lacZnIs labelled LC-free cultured allogeneic keratinocytes grafted onto Integra(TM) (a non-regenerative dermal template). Methods: The Large White pig was used in view of the similarity of its skin architecture to humans. Retroviral gene transduction of cultured porcine keratinocytes was conducted with a MFGIacZnIs construct in the PT67amphotropic packaging cell line. Leucocyte phenotypes infiltrating porcine skin were characterised immunohistochemically using a panel of monoclonal antibodies specific to porcine antigens: CD1, CD3, CD4, CD5, CDS, CDS, CD25, CD45, SUKC3, SH/C6, SLA DP, SLA DP, SLA DR and igXLC with ABC staining. Porcine Jejunum was employed as a positive control. LC depletion with Anti-MHC II and complement vyas monitored using flow cytometry. MFGIacZnIs labelled LC-depleted cultured keratinocytes sheets and keratinocyte-dermal composites were auto and allografted onto Integra and onto deep fascia respectively. LacZnIs j3-galactosidase expression was detected using X-Gal staining of tissue biopsies at different time points up to 21 days following grafting and a reduced panel of monoclonal antibodies (omitting CDS, SLA DP and SLA DQ) was used to immunophenotype the infiltrate at these time points. Results: The new PTS7 producer line succeeded in achieving close to 100% of keratinocyte transduction with the MFG lacZ construct following three passages in a cell culture system containing irradiated retroviral producer cells and 3T3 fibroblasts as feeder cells. Stability was demonstrated with no apparent reduction in lacZnis expression by porcine keratinocytes following a further seven passages on irradiated STS fibroblasts. A reduction was demonstrated however, both in the number and size of colonies formed by keratinocytes following transduction. LCs were shown to be successfully eliminated from disaggregated epidermal cells using antibody and complement. We also demonstrated that overnight incubation results in up regulation of MHC class II antigen and increases the efficiency of LC elimination. As with retrovirally transduced keratinocytes a reduction was demonstrated however, both in the number and size of colonies formed by keratinocytes that had undergone treatment with antibody and complement. The differential distribution of leucocyte phenotypes as determined by the panel of antibodies within normal porcine skin was shown for the first time

Strategic Planning in a Turbulent and Uncertain Context`

A report for Group Strategy, Policy Coordination and Relations, City of JohannesburgUncertainty and turbulence is a perennial feature of the context within which cities must plan but there are time periods in which a sense of uncertainty is heightened. This has been the case in the period since the Global Financial Crisis of 2007/08 where the experience of uncertainty has reached levels not known since the Great Depression of the 1930s. The Financial Crisis was an ‘uncertainty shock’ but it has left tremors in its aftermath. As one commentator put it, the recovery from the crisis has been “anaemic, brittle and fraught with uncertainty” (Tyson 2015). The combination of economic uncertainty in the contemporary period with political turbulence, global terrorism, and anxieties over climate change has created a ‘perfect storm’ of uncertainty. Wiltbank et al. (2006) write that “unknowability (true unpredictability) can be a disquieting and disruptive phenomenon.” This is especially so for the activity we call ‘planning’ which, almost by definition, requires some degree of confidence in our expectations for the future. The legitimacy of planning depends on the extent to which is can handle radical uncertainty. While the more traditional forms of planning may be helpless in the face of this uncertainty, there are, fortunately, approaches to planning within contexts of uncertainty that have evolved in both corporate and public sector planning practice since at least the late 1960s. In this paper we begin by outlining some of the dimensions of uncertainty, both globally and for the context of Johannesburg, focussing on aspects relating to demography, environmental threats, economy, society and politics, acknowledging, of course, that these are all profoundly interrelated. We then explore the multifaceted literature on planning for uncertainty, using the framework provided by Wiltbank et al. (2006) which distinguishes between approaches which are concerned with the better positioning of an institution within an uncertain environment, and those which actively attempt to shape environments. We apply the useful elements of the varying approaches to a brief analysis of Johannesburg’s current Growth and Development Strategy (GDS) exploring the extent to which the GDS has successfully accommodated uncertainty. We conclude with recommendations on how a new or revised GDS may better respond to uncertainty. Our approach to this study is broadly informed by a key insight offered by Peter Drucker in his seminal piece, ‘Planning for Uncertainty’ published in The Wall Street Journal in 1992. Drucker asked the question “What must we do––in fact, what must we become––if we are to successfully navigate the treacherous waters of unpredictability?” He argues, with reference to corporate planning but with relevance to public sector planning, that traditional approaches to dealing with uncertainty are not helpful: Uncertainty––in the economy, society, politics––has become so great as to render futile, if not counterproductive, the kind of planning most companies still practice: forecasting based on probabilities. (Drucker 1992) Drucker suggests an alternative approach to thinking about the future. Instead of asking “what is most likely to happen?" planning for uncertainty asks, instead, “what has already happened that will create the future?” This is the approach we take below in outlining dimensions of uncertainty. Our analysis is not futuristic and speculative but rather asks what the existing or emergent trends, and identified risks, are and what this may mean for the future. The reality, of course, is that the future is thoroughly unpredictable and we can only present these dimensions as illustrative of the scope of uncertainty and not as the basis of any form of projection. More important, appreciating these sorts of uncertainties, is how we can proceed with non-predictive forms of planning. A critical question that will address further in the text is whether our planning should focus only on adapting to uncertainty, or whether it should actively seek to shape the nature of the uncertainty. We do not assume that uncertainty is necessarily negative. In the corporate literature, at least, there is a strong recognition that uncertainty brings opportunity. This is arguably also the case in planning for the future of large and complex cities. Kaplan (2008) writes that, with uncertainty “often the basic meaning of a situation is up for grabs”. With uncertainty comes the opportunity to rewrite scripts in more desirable ways. However, there are massive risks if trends are misread or ignored.A report for Group Strategy, Policy Coordination and Relations, City of JohannesburgAA201

SPATIAL FUTURES: ASPIRATIONS AND ACTIONS REGARDING FORM AND SPATIAL CHANGE IN JOHANNESBURG

A report for Group Strategy, Policy Coordination and Relations, City of JohannesburgAddressing the racially divided, sprawling and socially inequitable spatial form of South African cities has been key to strategic spatial planning and urban spatial frameworks in South African cities, including in Johannesburg. These ideas were included in the Johannesburg 2006 Growth and Development Strategy (GDS), and in the 2011 GDS, which focused more strongly on resilience, but making strong links to spatial form. They have also been a consistent element of various rounds of Johannesburg Spatial Development Frameworks (SDFs). However, despite several of these concerns being embodied in national urban and city policies, objectives to restructure cities spatially have proven to be very difficult to achieve, and there is a growing frustration and questioning of whether some of these objectives are still appropriate. At the same time, the urban restructuring agenda, and the areas that spatial policy addresses have been constrained in practice, and there are several gaps and silences in the issues that are addressed. This paper provides a discussion of the choices, tensions, and trade-offs facing spatial policy in Johannesburg. It considers whether the policy objectives expressed in existing spatial policies (including the Johannesburg GDS and SDF) are still relevant, and address key spatial dynamics and issues. It does this by exploring several key areas of debate around the spatial form of cities and spatial policy internationally, examining how they manifest in Johannesburg, and highlighting these choices, tensions and trade-offs. It recognises, as a starting point, that while urban spatial policies have some power to shape spatial change, spatial trends and dynamics occur in a complex environment, where there are many drivers and shapers of spatial change. As emphasised in the position paper on ‘Strategic Planning in a Turbulent and Uncertain Context’, spatial policies that hope to influence spatial change need to understand the (shifting) key trends and drivers that affect space, including demographic, economic and social patterns that influence the demand for space. There are many examples of spatial plans which missed key trends, vastly over- or under- estimated population growth, and consequently planned for spatial forms which proved to be inappropriate. The spatial form of cities is also shaped by markets of various forms. Planning may attempt to engage with and regulate or direct these markets in the interests of its social and spatial goals and objectives, but it does not have completely free reign. Further, there are frequently disjunctures between strategic spatial planning and implementation, reflecting limits in terms of capacity, political will, institutional cooperation/integration and other factors. Finally, city spatial policies do not occur in isolation, nor do spatial policies necessarily have the power desired by planners. Spatial change and spatial form is critically affected by infrastructural investments, particularly in relation to transport (roads, transit systems), which are frequently follow a different planning process and logic (UN-Habitat, 2009). Likewise, differences between spheres of government and sectoral departments with power to invest in the built environment are also key to the disjunctures between spatial plans and outcomes. The emphasis on housing delivery on scale, along with cheaper land on the periphery, has undermined spatial policies towards urban compaction both internationally (Buckley et al., 2016) and in South Africa (Charlton, 2014). The recent international emphasis on ‘mega- projects’ is often driven by the private sector (such as major gated estates), but also by parts of the public sector (for example eThekwini’s airport). It is also influencing spatial change, bypassing spatial plans or forcing their adaptation (Shatkin, 2008; Robbins et al, 2015; Todes, 2014). This paper explores several key points of focus and debate affecting the spatial futures of cities, particularly in relation to Johannesburg. It draws out the key choices, tensions and trade-offs in these areas, and their implications for future spatial planning in Johannesburg. These include: • The debate over the creation of a more compact urban form, versus expanding and sprawling cities, including the discussion of new cities and satellite cities. Sustainability and resilience as key discourses and their implications for urban spatial form, and the role of transport and mobility will be considered in this context. Understandings of densification, how it is encouraged and managed will also be discussed. • Trends towards social exclusion versus arguments for spatial justice and the right to the city. This discussion considers trends towards privatised and splintered urbanism, gated communities, gentrification, and safety and security as a driver. It also discusses other dimensions of exclusion/inclusion—race, gender and the question of migrant spaces, and policies on socio-spatial integration. • Processes of spatial change in poor neighbourhoods, and initiatives to improve conditions there, including upgrading informal settlements, the growth of informal trade, addressing backyard housing. • Relationships between space and economic development, including the dynamics of growth and decline across the city, debates over promoting development on the periphery versus existing areas of agglomeration, and initiatives to promote economic development in townships. • City-region and multi-scalar governance, including the extent to which metropolitan governance addresses competing tensions and interests across the city, cross-border issues, and disjunctures and tensions between spheres of government.A report for Group Strategy, Policy Coordination and Relations, City of JohannesburgAA201

Component Neural Systems for the Creation of Emotional Memories during Free Viewing of a Complex, Real-World Event

To investigate the neural systems that contribute to the formation of complex, self-relevant emotional memories, dedicated fans of rival college basketball teams watched a competitive game while undergoing functional magnetic resonance imaging (fMRI). During a subsequent recognition memory task, participants were shown video clips depicting plays of the game, stemming either from previously-viewed game segments (targets) or from non-viewed portions of the same game (foils). After an old–new judgment, participants provided emotional valence and intensity ratings of the clips. A data driven approach was first used to decompose the fMRI signal acquired during free viewing of the game into spatially independent components. Correlations were then calculated between the identified components and post-scanning emotion ratings for successfully encoded targets. Two components were correlated with intensity ratings, including temporal lobe regions implicated in memory and emotional functions, such as the hippocampus and amygdala, as well as a midline fronto-cingulo-parietal network implicated in social cognition and self-relevant processing. These data were supported by a general linear model analysis, which revealed additional valence effects in fronto-striatal-insular regions when plays were divided into positive and negative events according to the fan's perspective. Overall, these findings contribute to our understanding of how emotional factors impact distributed neural systems to successfully encode dynamic, personally-relevant event sequences