The Evidence-Based Medicine (EBM) movement endorses a hierarchy of evidence that places randomized controlled trials at the top. More specifically, double-blind, placebo- controlled trials are often considered to be the 'best of the best'. This view leads to the paradox that treatments that seem to be most strongly supported by evidence, ranging from tracheotomies to rabies vaccines, have never been tested in randomized trials of any description and are hence supported by (allegedly) sub-optimal evidence. Moreover many of these treatments do not seem supportable by best evidence - how, for example do we keep the surgeons who perform tracheotomies 'blind'. After a brief introduction (chapter 1), and review of the literature (chapter 2), I argue that criticisms of the EBM hierarchy can be launched from the simple basis that best evidence rules out the most plausible rival hypothesis (chapter 3). To examine the relative evidential weight of placebo controlled trials compared to 'active' controlled trials (in which the control treatment is an existing established treatment) requires a good deal of conceptual work. I defend a modified version of Grunbaum's (1981/1986) definition of placebos (chapter 4), then provide constraints on what can count as a 'legitimate' placebo control (chapter 5). Next, I explain why double-blinding does not always rule out additional rival hypotheses. I then argue that the arguments for the superiority of placebo controls are flawed. The 'assay sensitivity' argument is limited in scope and based on a misconception about the nature of placebo controls (chapter 7), while the claim that only placebo controlled trials measure the absolute effect size relies on the questionable assumption that placebo and non-placebo effects add rather than interact (chapter 8). I conclude that the evidence hierarchy endorsed by EBM does not stand on solid foundations.

Type I diabetes (T1D) is an autoimmune disease in which the insulin producing β cells of the pancreas are selectively targeted for destruction by autoimmune effector T cells. The aberrant effects of these effector T cells may in part be due to a dysfunction in the regulatory T cell (TREG) compartment and currently there is great interest in developing methods to strengthen the immunoregulation of patients with T1D. A potential way to boost immunoregulation in these patients would be the use of adoptive TREG therapy whereby expanded polyclonal TREG are transferred into patients. Although this treatment in mouse models of disease has shown promise it has been found that antigen specific TREG cells are much more efficacious of preventing disease, and can even reverse disease. The translation of these murine experiments into the human setting is however complex, since the generation of large numbers of antigen specific TREG from human patients is currently a major hurdle. One way to remove this barrier is to utilise lentiviral gene transfer technology, which can allow for the transfer of antigen specific T cell receptor (TCR) genes into a desired cell population. Specifically for T1D, it is hypothesised that TREG engineered to express a MHC Class I restricted (MHCI) TCR, although unconventional, would selectively function at the site of inflammation i.e. within the islets. This project, therefore, aims to generate MHCI islet antigen specific TREG with the hypothesis that these would confer islet antigen specific suppression. To test this hypothesis we engineered human CD4+ TREG to express two MHCI islet antigen specific TCRs whilst using a third high affinity pathogenic MHCI TCR as a control. As others have shown, we demonstrate that the control TCR was effectively able to re-direct the antigen specificity of TREG cells through signalling and function. However, we discovered that transfer of autoimmune MHCI TCRs were unable to yield the same results as the control TCR due in part to their natural low affinity for antigen. To circumvent this, we engineered CD4+ TREG to express an MHCI autoimmune TCR along with the CD8αβ co-receptor or a CD8αβ high affinity variant. Using this system, human TREG could be successfully re-directed towards an islet specific peptide and exhibit antigen specific suppression. Thus, this study is the first of its kind to use an autoimmune disease relevant, MHCI TCR to successfully re-direct the Ag specificity of TREG cells.

B cells recognize a wide range of antigens by their specific surface B cell receptor. BCRs are immunoglobulin polypeptides and made up of heavy chain (IGH) and light chain. Light chains are of two types: kappa (IGK) or lambda (IGL). The variable regions of immunoglobulin chains are generated by somatic rearrangement of V, D and J segments to generate a diverse repertoire of BCRs. The aim of this thesis is to analyze features of immunoglobulin gene rearrangements in the context of heavy and light chain pairings and light chain gene expression. Mature naïve B cells generated in blood or tissues have a ratio of IGK:IGL expression of approximately 2:1. In contrast IgA plasma cells exhibit IGK:IGL ratio of approximately 1:1. Such a bias could be acquired by antigen selection or by changes in light chain gene rearrangements such as light chain revision. In order to better understand this, IGL genes were PCR amplified, cloned and sequenced from the DNA of transitional, naïve and IgA expressing B cells. Unusual biases in genetic reading frame were observed in the IgA expressing cells of the most commonly used IGL families. This was further investigated using a high throughput sequencing method that identified biases in the IGL genes of IgA with preferential selection of IGLV2-14 in IgA subsets (Chapter 3). In the bone marrow, IGL gene rearrangement starts only if the rearrangement at IGK locus is non-functional. However, approximately 25% to 30% of IGL expressing cells have previously undergone apparently functional IGK gene rearrangement, but for some reasons these productive rearrangements were not expressed. In order to better understand the regulation of the IGK locus, IGK gene rearrangements were sequenced from naïve B cells expressing either IGK or IGL. Sequences were divided into productive and non-productive according to the characteristics of the junction. The actual expressed genes in cDNA were also amplified from IGK expressing B cells. Overall results identified selection and expression biases operating at the time of establishment of IGK repertoire (Chapter 4). During B cell development, IGH rearrangements that pair better with surrogate light chains are selected at the pro-B cell stage. Therefore, it was decided to investigate if there is any possibility that later during B cell development, rearranged light chains could show preferences for particular IGH gene segments. If so, this will help in identifying additional factors shaping the IGH repertoire of naïve B cells. Rearranged IGH were sequenced from naïve B cells expressing either IGK or IGL. The relative usage of IGHV, IGHD and IGHJ and CDR-3 characteristics were compared. It was found that there is no preference of IGH to be associated with either IGK or IGL (Chapter 5). This thesis has identified some unusual features of light chain gene rearrangements. The relevance this data to B cell immunology will be discussed.

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The Histamine H4 receptor (H4R) is a classic G-protein coupled receptor (GPCR) and is expressed on a range of cell types in the body. To determine the roles of the H4R in the peripheral and central nervous system, four hypotheses have been addressed. (1) The H4R are expressed on sensory neurons in the peripheral and central nervous system; (2) The H4R is differentially expressed during acute inflammatory and neuropathic pain states; (3) Antagonism of the H4R with the antagonist JNJ 7777120 ameliorates central inflammation observed during experimental autoimmune encephalomyelitis (EAE); (4) Antagonism of the central H4R with JNJ 777120 has anxiolytic effects and improves memory performance in mice. Based on immunofluorescence approaches we have demonstrated for the first time the presence of the H4R on both putative A-delta (Aδ) and C-fibre sensory neurons in the skin and dorsal root ganglia. Using the novel agonist selective H4R agonist, VUF 8430 revealed functional expression of H4R on primary rodent dorsal root ganglia neurons. Using quantitative immunoblotting, we provide preliminary evidence for the rodent H4 receptor bilateral and unilateral upregulation in the skin, but not the spinal cord in the acute phase of Complete Freund’s adjuvant induced inflammatory pain and nerve constriction acute neuropathic pain, respectively. The H4R is an attractive target for the treatment of many inflammatory disorders. However, in contrast to the beneficial anti-inflammatory effects of H4R antagonism observed in literature, blockade of the receptor using JNJ 7777120 in acute EAE (autoimmune disease) appeared to exacerbate the pathology, and increased spinal cord inflammation, demyelination, microglia activation and functional deficits. Antagonism of the central H4R with JNJ 7777120 at 10 and 20 mg/kg (i.p.) appears to affect the behaviour of Balb/c mice but there is no indication of altering anxiety or non-spatial memory performance. However, the study did provide preliminary evidence for central behavioural effects with regard to time spent in the central part of the platform. Overall, this thesis provides new evidence for multiple roles of the H4R in the mammalian central and sensory neurons.

Clinical evidence suggests that a negative expectancy of pain decreases the effectiveness of interventions aimed to reduce pain. Experimental pain models provide a method to investigate neurophysiological and psychological correlates of sensory processing and decision-making of clinically observed phenomena in the context of pain. The purpose of this thesis was to develop an experimental paradigm to investigate the neural and psychological mechanisms of the distorting effect of negative expectancy on pain-reducing predictive cues, the latter of which is thought to be the underling mechanism of all effective treatments for pain. Chapter One lays the foundation required to understand the thesis: it introduces the current direction as well as the expanding research in the field of physiological, psychological, and imaging pain research. The reviewed literature and identified knowledge gaps sculpt the aims, objectives, and justification of this PhD research. Chapter Two discusses the technical aspects that allow translating the theoretical concepts addressed in Chapter One into experimental settings; in this chapter, commonly used methods to induce and image pain, as well as behavioural assessment measures, are discussed. A brief preview on the most recent state-of-the-art technology in neuroscience imaging is also provided. Chapter Three provides a thorough description of the two pilot studies that were conducted to develop a suitable experimental paradigm to investigate the impact of prior negative expectations on pain-reducing predictive cues, using a laser-heat as a pain model. The pilot work provides experimental justification for the final paradigm that was implemented in subsequent studies. Electroencephalography (EEG) was used to probe the neural and cognitive mechanisms underlying the expectancy modulation of pain. In Chapter Four, the main hypothesis that the experimental induction of negative expectations indeed reduces the pain-alleviating effect of predictors of a non-painful outcome was examined. As hypothesised, participants reported laser stimuli as more painful in the context of prior negative expectations. Furthermore, participants had higher amplitudes of the N2 peak of the laser evoked potential in this context. This N2 difference was correlated with the activation of the midcingulate cortex (MCC). Results also revealed a positive correlation between participants’ self-rated scores on the fear-of-pain questionnaire (FPQ) and the enhanced N2. This suggests a priming effect may have occurred through the increased recruitment of saliency mechanisms at pre-evaluative stages of sensory processing, to which fearful individuals were more prone to. Chapter Five takes this finding a step further by modulating pain catastrophising in the anticipation that individuals who are classified as high-pain catastrophisers would be more prone to the prior negative expectancy effect, as well as more responsive to an intervention designed to minimise it, which, in the study, was a sensory-focusing task designed to take attention away from catastrophic thinking. Results, however, showed that both groups reported equally higher intensity ratings in the prior negative expectancy condition, which were equivalently attenuated by the localisation task. Interestingly, the localisation task selectively increased the amplitude of P2 peak under prior negative expectancy trials. This effect was associated with the activation of the right orbitofrontal cortex (OFC) and secondary somatosensory cortex (SII), highlighting these regions’ role in the integration of motivational and cognitive components of pain to shape subsequent behavioural responses. The final chapter presents a critical analysis of the thesis findings and highlights its limitations, the clinical and research significance, future work, and draws final conclusions. The findings from this PhD thesis stress the importance of considering the distorting effect of negative expectancies on pain in planning effective pain intervention protocols. It also sheds a light on the possible mechanisms that cause this effect.

Regulation of mtDNA content is critical to normal human health and is often abnormal in mitochondrial diseases. Only a proportion of a female’s mtDNA is used to populate the oocytes she forms during embryogenesis. This mtDNA bottleneck can cause rapid shifts in heteroplasmy levels between generations in families with mtDNA mutations. The heteroplasmy levels of several mtDNA mutations were analysed from mother-child pairs using previously validated pyrosequencing assays. Analysis of the shifts in heteroplasmy caused by the mtDNA bottleneck reveals that the inheritance of the pathogenic tRNA mutations m.3243A>G and m.8344A>G do not show selection bias. However, the distribution of m.8993T>G (in MT-ATP6) in offspring suggests this mutation is selected for during the mtDNA bottleneck. This finding is in agreement with meta-analysis performed on previously published data, which also reveals biased inheritance of the LHON mutations m.11778G>A and m.3460G>A, in Complex I genes. Selection for these mutations explains the higher prevalence of homoplasmy, as fixation can occur within fewer generations. Crucial to formation of the mtDNA bottleneck is the dramatic increase in mtDNA replication rate that allows primordial germ cells (PGCs) to repopulate their mitochondrial genomes. Gene expression analysis during pre-implantation embryonic development has not revealed any other biological processes associated with regulation of mtDNA copy number. Upregulation of Lrpprc correlates with the expression of transcription factors as pre-implantation development progresses at this stage. The limited number of PGCs in the early stages of post-implantation development prevented sufficient quantity of high-quality RNA to be isolated for use with gene expression analysis. The reduction of mtDNA copy number observed during the mtDNA bottleneck was modelled in myoblasts and fibroblasts, using ddC, a reverse transcriptase inhibitor. Although some gene expression changes were induced during repopulation of mtDNA, these were limited to below 3-fold change. Forcing reliance on oxidative phosphorylation through culture with galactose media could not increase the rate of mtDNA replication or induce greater gene expression responses. Ragged-red fibres (RRFs) are a common hallmark of many mitochondrial diseases, caused by proliferation of mitochondria in the subsarcolemmal region of muscle tissue. Multiplex immunohistochemistry allowed fibre typing and identification of RRFs in muscle tissue from a patient with the m.8344A>G mutation. A novel technique was iii established to ensure extraction of intact high-quality RNA from laser-microdissected muscle fibres. The RNA was used in a pilot microarray experiment to study the differences not only between control and patient tissue, but specifically within RRF, and has identified a multitude of potential signalling pathways involved in mitochondrial biogenesis and formation of RRFs. This thesis reveals the complexity of mtDNA copy number regulation and, the critical involvement it has to human health and the inheritance and pathogenicity of mitochondrial diseases.

Spinal cord stimulation (SCS) appears to be an effective treatment for neuropathic pains, but long-term benefit of more than one year is only found in a proportion of patients treated. This thesis hypothesised that psychological factors may be important as determinants of outcome. A literature review in this field, whilst demonstrating lack of reliable psychological predictors of SCS treatment, suggested that those thought to be predictive such as depression were more complex. Whilst depression was associated with lower efficacy of treatment by SCS, the treatment itself improved depression. Therefore, depression should not necessarily be seen as a contra indicator, especially when pain and depression interact. A prospective study with one year follow up of patients implanted with spinal cord stimulator was conducted. Forty patients were included in the final analysis. Functional pain and psychological measures were recorded at six and 12 months, psychological predictors were not significant at six months but significant predictors were found at 12 months. Greater catastrophising, paired with greater anxiety and less perceived control were associated with a < 30 % reduction in pain. A qualitative study of the experience of SCS using semi-structured interviews one year following SCS implantation revealed similar findings. Thirteen patients reported coping, lack of control and helplessness as impacting upon pain experience. A demand for clearer information systems was discussed in relation to SCS preparation. Information is needed to reduce unexpected experiences including potentially painful trial and body image concerns related to the implantable SCS device. Implications for practice included preparation with expert patients and a tailored preparatory CBT course. The findings from the two studies demonstrate the necessity to improve the preparation process for patients prior to SCS. Results from both studies conclude that perception of control over pain is important for SCS efficacy and support with anxiety and catastrophic thoughts and behaviours may be advantageous. The predictive equation generated from this study needs to be tested prospectively on further cohorts of SCS patients in order to test reliability. In addition, evaluation of the impact of a tailored CBT course upon outcome needs investigation.

The liver provides a highly immunotolerant environment, that is exploited by hepatotropic viruses such as Hepatitis B virus (HBV), which establishes persistent infection in more than 350 million people worldwide. In this thesis the potential for myeloid-derived suppressor cells (MDSC) to exert metabolic regulation in this setting has been investigated. We found a mean approximate 9-fold expansion of granulocytic MDSC (gMDSC) in patients with chronic HBV infection (CHB) compared to uninfected, healthy controls (p<0.001). The most striking increases were seen in patients replicating HBV in the absence of immunopathology (p<0.01). gMDSC expressed high levels of the chemokine receptor, CXCR1, providing the potential for them to be chemoattracted by liver-derived interleukin-8 (IL-8); consistent with this, they were further enriched in the intrahepatic compartment. gMDSC from patients with CHB expressed increased amounts of arginase I, correlating with an increase in serum levels of this enzyme (p<0.01). Arginase I metabolises the conditionally essential amino acid L-arginine that is required for proliferating T cells; in line with this was an observed decrease in circulating L-arginine, particularly in those patients without liver inflammation. Liver pathology in CHB is ampli ed by the recruitment and activation of bystander (non-HBV-speci c) T cells; therefore the potential of gMDSC to down-regulate such responses was explored. Puri ed gMDSC from patients with CHB potently inhibit the expansion of bystander T cells capable of producing pro-inflammatory cytokines or mediating cytotoxicity. This inhibition was blocked using an arginase I-specific inhibitor, N-hydroxy-nor-arginine (nor-NOHA). Taken together, these data demonstrate the capacity for expanded arginase I expressing gMDSC to regulate liver immunopathology in CHB by depriving T cells of L-arginine.