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ItemInvestigating the influence of BCG and hepatitis B vaccine on neonatal immune responsesCox, Lianne ( 2020)Neonatal infectious diseases result in an estimated 40% of neonatal deaths worldwide and contribute significantly to chronic morbidity. Childhood disease-specific immunisation is irrefutably linked to the decline in deaths from targeted infections over the last century. However, neonatal immunisation strategies are limited, in part, due to impairments in their adaptive immune function. Vaccine-induced protection from severe forms of tuberculosis (TB) with the Bacille Calmette-Guerin vaccine (BCG) and perinatally-acquired hepatitis B infection with the hepatitis B vaccine (HBV) are two exceptions, with these vaccines commonly administered to neonates worldwide. Evidence for heterologous (‘non-specific’) effects (NSEs) of various vaccines used in childhood, most notably for BCG, is increasing. This refers to the immunomodulatory capabilities of vaccines to influence immune outcomes beyond inducing protective immunity against the vaccine’s specific targeted disease. There is limited evidence for these effects in neonates, particularly for HBV. The research reported in this thesis aimed to explore the influence of BCG, HBV and concurrent administration of both vaccines at birth on the neonatal immune responses to unrelated antigens compared with unvaccinated babies in a randomised control trial (RCT): The Early Life Vaccine and Immunity Study (ELVIS). Neonatal blood samples from 128 participants, collected seven days after randomisation, were stimulated with various unrelated antigens for 20 hours. Cytokine responses, measured in the supernatants by an enzyme-linked immunosorbent assay (ELISA) method, were analysed using non-parametric statistical tests to determine differences in median responses between the four groups: BCG alone, HBV alone, concurrent BCG and HBV and the unvaccinated control group. I found minimal differences in the median in vitro cytokine responses to all stimulants between the four groups. However, both vaccines independently influenced cytokine-stimulant responses. Effects on responses were strongest for BCG-vaccinated babies, but only decreased interferon gamma (IFN-gamma) responses to the Toll-like receptor (TLR) ligand resiquimod (R848) and monocyte chemoattractant protein-1 (MCP-1) responses to heat-inactivated E. coli were significantly different from controls. Combined vaccines tended to induce similar cytokine-stimulant response patterns as BCG alone, although for some cytokine-stimulant pairs, the BCG-induced effects were mitigated by HBV and vice versa. This study adds to the evidence for NSEs of vaccines in neonates. It is the first study to investigate the influence of HBV immunisation on immune responses to unrelated antigens, finding no statistically significant differences in median cytokine responses compared with controls. The finding that concurrent vaccination with HBV and BCG at birth induced the same cytokine-stimulant response pattern as BCG alone, suggested that cytokine responses to unrelated stimulants are driven by BCG. This is also the first study to show that in neonates concurrent HBV and BCG vaccination at birth weakens the NSEs of BCG for certain cytokine-stimulant pairs. Further research into neonatal vaccine NSEs are warranted. Future studies should explore and further investigate the clinical relevance of certain cytokine-stimulant response signatures identified in my thesis and the mechanism for these observations in neonates. These results will direct research on how we could potentially exploit any beneficial vaccine NSEs to provide protection against infection in the very young.
ItemExploring Efficient Generation of InsulinProducing Cells from iPSCs, to Enhance Cell Therapy for Type I DiabetesWang, Tianyu ( 2020)Type I Diabetes Mellitus (TIDM) is one of the immune disorders and results from the loss of cells that make insulin (INS), a hormone that regulates the level and availability of glucose within the body. The most common treatment is exogenous insulin therapy which involves regular insulin injections or the use of an insulin pump. However, this treatment is imperfect and cumbersome, driving researchers to find more effective methods to control blood glucose levels. One potential treatment would involve making INS producing beta cells using pluripotent stem cells and then using these manufactured cells as a beta cell replacement therapy. Making insulin-producing cells in the laboratory involves several stages from stem cells to definitive endoderm, pancreatic endoderm, endocrine precursor, and the last step to mature beta cells. Any improvements in specific steps for the production of PSC derived pancreatic beta cells has the potential to lower the cost of an eventual treatment, making such therapy more widely available. In this project, we examined variations in the differentiation protocols with the ultimate aim of increasing the yield and functionality of the final cell product. One of the significant findings to emerge from this thesis is, in Chapter 4, we show that SCF addition has the potential to enhance cell proliferation during the stages of early differentiation. A surface receptor for Stem Cell Factor, cKIT, is widely applied to determine endoderm formation in the differentiation process of in vitro pluripotent stem cells. Despite its long-standing relationship with endoderm formation, a potential role for SCF itself during this process has not been systematically addressed. Using a suspension-based differentiation system, we show that the addition of SCF to differentiating cultures of PSCs enhanced the proliferation of early definitive endoderm, marked by the expression of CXCR4 and EPCAM. This effect of SCF was discernable within 2 days of differentiation initiation and coincided with the down-regulation of its receptor, cKIT. SCF treatment appeared not to affect the kinetics of differentiation, with key markers such as MIXL1, SOX17, CXCR4, and EPCAM showing identical expression patterns in untreated and treated cultures. Taken together, our results indicate that SCF addition at an early stage of differentiation will potentially reduce the number of input PSCs required to yield a given amount of endodermal products for the downstream application. This thesis is organized into six main chapters. Chapter 1 is a literature review concerning the introduction of pluripotent stem cells, Type I diabetes, pancreas development, normal molecules used in vivo differentiation protocol for beta cells generation. Chapter 2 describes the materials and methods of our experiments. Chapter 3 investigates the role of Activin A in definitive endoderm formation, which attaches great importance to adding Activin A from the beginning of differentiation. Chapter 4 shows that SCF promotes the proliferation during definitive endoderm differentiation. Chapter 5 presents images of the final cell product, which successfully show the signals of insulin. Chapter 6 is related to the limitations of previous research and the suggestions for future work.
ItemImproving the diagnosis of scabies in low-resource settingsOsti, Millicent ( 2020)Scabies is a parasitic disease and a global health problem that predominantly affects disadvantaged communities in low-resource settings. Scabies significantly impacts the health and quality of life of those with the disease. To accurately assess the global burden of disease and to compare data across regions, standardised diagnosis with consistent disease definitions is necessary. In low-resource settings, diagnosis by clinical assessment is the principal diagnostic method. In the absence of available experts, non-expert health workers are likely to play critical roles in diagnosis, including for prevalence mapping. Currently, standardised processes for clinical diagnosis for scabies do not exist. This thesis explores the diagnosis of scabies in low-resource settings and the role and utility of non-expert health workers in the diagnosis of scabies. Chapter 3 describes the evaluation of non-expert health workers in the diagnosis of scabies and impetigo using clinical criteria. The diagnosis of four briefly-trained nurses was compared to the consensus diagnosis of two experienced doctors. The sensitivity of the nurses’ diagnosis compared to the reference standard was 55.3% for scabies with a specificity of 89.9% Sensitivity for moderate to severe scabies was 93.5% with a specificity of 74%. The accuracy of diagnosis by non-expert health workers is promising and may be acceptable for scabies and impetigo disease mapping in low-resource settings. Chapter 4 describes the development and evaluation of a training protocol for the diagnosis of scabies and impetigo for non-expert health workers. The aim of this study was to measure the change in knowledge and confidence of the participants and explore their experience and perceptions of the training. Training was evaluated using a case-based test, a questionnaire and semi-structured interviews. The overall results of the case-based test were 90% for scabies and 75.5% for impetigo. The mean score for both self-reported knowledge and confidence increased from 2.5 to 4.5 following training and scores increased for all nurses (mean difference 2, 95%CI 1.1-2.9, P=0.005). The study showed that training local health staff in scabies diagnosis was enjoyable for participants and led to improvements in self-reported knowledge and confidence. Chapter 5 investigates the prevalence of scabies and impetigo using a cross-sectional study in a primary school in Gizo in the Solomon Islands. Using the International Alliance for the Control of Scabies (IACS) diagnostic criteria the classified the diagnosis of scabies. The prevalence of scabies was 54.3% and prevalence of impetigo was 32.1%. 63.5% of those with impetigo had scabies, corresponding to a population attributable risk of 11.8%. The study highlighted the extremely high burden of these diseases supporting the need for interventions for scabies in this community. Chapter 6 evaluates the methods of data collection, analysis and display for describing in detail the distribution of scabies lesions in a pilot study. The study used a novel technique of representation of dermatological lesions in the form of a choropleth map. The study found that the methods used were feasible for a larger population and would describe valuable detailed information on specific lesion location in scabies. The study will provide information on lesions at specific body sites to determine if simplified examinations are appropriate for prevalence surveys. The public health control of scabies requires identification of high-prevalence communities to target interventions, as well as methods to monitor the effectiveness of interventions. Such programs would be dependent on accurate and standardised diagnosis for population mapping. This thesis suggests methods to improve the diagnosis of scabies in low-resource settings. Modifications to training and diagnostic methods are likely to improve diagnostic accuracy. Improvements to scabies diagnosis will contribute to efficient collection methods and reliability of prevalence data.