Theses - Medicine

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Open Access
The molecular epidemiology of Salmonella Typhi and applications for policy
Carey, Megan
Salmonella Typhi (S. Typhi) is a Gram-negative bacterium and the etiologic agent of typhoid fever. High rates of typhoid were historically associated with urban slums in South Asia with poor sanitation, but recent multicentre surveillance studies have demonstrated that typhoid is also a major problem of urban and rural areas in sub-Saharan Africa. Typhoid necessitates antimicrobial therapy; however, antimicrobial resistance (AMR) poses a serious threat to the effective clinical management of typhoid, particularly in South Asia, where resistance to all oral antimicrobials used to treat typhoid has been reported. New typhoid conjugate vaccines (TCVs) are highly efficacious and two have been prequalified by the World Health Organization (WHO); a small number of countries have introduced TCVs into their national immunization programs. However, many countries lack primary surveillance data to inform decision-making for TCV introduction. Here, aiming to aggregate and analyse globally representative whole genome sequencing (WGS) data to inform public health action, I conducted a genomic investigation of the global distribution and transmission dynamics of AMR S. Typhi. With colleagues from Pakistan and India, I conducted phylogenetic analyses of the first molecularly confirmed azithromycin-resistant S. Typhi isolated in Pakistan and India and put them into context with contemporaneous azithromycin-resistant isolates. I found that single point mutations in acrB (efflux pump) were emerging independently in these settings, potentially associated with selective pressure. AMR in S. Typhi is associated with the H58 lineage, which arose recently before becoming globally dominant within a relatively short time. Given this lineage’s association with AMR, I sought to investigate how, when, and where it emerged. Working with collaborators from the United Kingdom Health Security Agency (UKHSA), we performed phylogenetic and phylodynamic analyses using S. Typhi from returning travellers to the UK between 1980 and 1995. This dataset, which contained the earliest described H58 S. Typhi, indicates that the prototype H58 organisms were MDR, and that they emerged spontaneously in India in 1987 and became radially distributed throughout South Asia. These early organisms were associated with a single long branch and possessed mutations associated with increased bile tolerance, suggesting that the first H58 organism was generated during chronic carriage. The subsequent increased use of fluoroquinolones led to several independent mutations in gyrA, leading to decreased fluoroquinolone susceptibility. The apparent ability of H58 to acquire and maintain AMR genes continues to pose a threat, suggesting that TCVs should be deployed across South Asia to minimise the potential emergence of new drug-resistant variants. I worked with a broad range of collaborators to establish the Global Typhoid Genomics Consortium (GTGC) to encourage sharing of S. Typhi genomic data and standardised metadata, and to enable analysis and visualisation of these data in context to support public health decision-making. Here, I engaged with groups sequencing S. Typhi genomes to analyse and draft a global update paper; the final dataset contained >13,000 sequences. This work provides an updated overview of AMR and genotype distribution and illustrates key international transmission events, with the aim of informing TCV introduction decision-making and treatment guidelines. To create a translational link between laboratory scientists, genomics experts, clinicians, and policy makers, I outlined use cases for WGS in surveillance, diagnostic development, clinical management, and informing vaccine introduction as well as typhoid control. I hope to advance this work by fostering broader participation in the GTGC, and by encouraging funding support for the generation and analysis of S. Typhi WGS data. I also intend to create greater visibility for, and advocate for the value of, such data in conversations with country decision-makers and other policymakers via the WHO.
Open Access
FAMIN in dendritic cells biochemically restrains T cell priming
Ramshorn, Katharina
FAMIN is a purine nucleoside enzyme that was first identified in genome-wide association studies (GWAS) as increasing risk for Crohn’s disease (CD). Defects in the enzyme that result in loss of catalytic activity are the only known monogenic cause of Still’s disease, a severe autoinflammatory condition that predisposes for the risk of developing macrophage activation syndrome (MAS). MAS can be triggered by common viral infections and result in hyperactivation of T lymphocytes. The work presented in this thesis builds on the discovery that FAMIN-sufficient mice are protected against influenza A infection, with dendritic cell (DC)- dependent excessive activation of CD8⁺ T cell responses in their FAMIN-deficient counterparts. Loss of FAMIN activity in DCs was shown to result in increased priming of both CD4⁺ and CD8⁺ T cells both in vitro and in vivo, resulting in enhanced antigen-specific cytotoxicity, IFNg secretion, and T cell expansion. Here, we discovered that FAMIN controls the pace of membrane trafficking and DC antigen processing to restrain T cell priming via a cytosolic NADH/ NAD⁺-dependent mechanism. We describe wide-reaching metabolic defects resulting from loss of FAMIN activity, including in the core pathways of glycolysis and TCA cycle activity, as well as upstream changes in glucose, fatty acid and amino acid metabolism. Notably, depressed rates of glycolysis and oxidative phosphorylation DCs lacking FAMIN function were not responsible for increased T cell priming capacity. Instead, we find that FAMIN balances flux through adenine-guanine nucleotide interconversion cycles to maintain the cytosolic NADH/ NAD⁺ ratio. We found that FAMIN additionally regulates T cell priming by DCs directly through the enzyme’s purine nucleoside phosphorylase (PNP) activity, converting exogenous hypoxanthine to inosine that acts on the T cell adenosine 2A receptor (A_2AR). In summary, FAMIN is a multifunctional purine nucleoside enzyme enabling flux through purine interconversion pathways highly integrated with cytoplasmic and mitochondrial metabolism. In DCs, FAMIN acts as a biochemical checkpoint to restrain T cell priming and prevent autoimmunity and autoinflammation.
Open Access
Elucidating the roles of endothelial PTBP1 and PKM2 in Pulmonary Arterial Hypertension: implications for therapy
Cuthbertson, Iona
Pulmonary Arterial Hypertension (PAH) is a rare, life-limiting disease with no cure, and is regarded the most severe form of pulmonary hypertension (PH). The disease is characterised by remodelling of peripheral pulmonary arteries, leading to increased mean pulmonary arterial pressure, right ventricular afterload, and heart failure. Pulmonary artery endothelial cell (PAEC) dysfunction is regarded as a key driver of the disease and is characterised by heightened proliferation and development of apoptosis resistance. Dysfunctional metabolism also observed within the PAH pulmonary vasculature and in systemic tissues. PAECs display a hyperglycolytic metabolic signature, fuelled by increased expression of splicing factor Poly-pyrimidine tract binding protein 1 (PTBP1) and its gene product, the glycolytic enzyme Pyruvate Kinase M2 (PKM2). Moreover, normal EC function and metabolism is restored by PTBP1 silencing, highlighting the therapeutic potential of targeting PTBP1 and PKM2. In this thesis, I have explored the therapeutic potential of pharmacological and genetic inhibition of PTBP1 and PKM2 in BOECs, a progenitor EC subset isolated from the peripheral blood, with high transcriptome homology to PAECs. Crucially, I have explored their influences in BOECs isolated from non-carriers, and PAH patient carriers of causal missense or truncating BMPR2 mutations. In doing so, my work sheds light on the causal and therapeutic roles of PTBP1 and PKM2 in several patient contexts. In this thesis, I have shown that treatment with the anti-tumorigenic compound apigenin, inhibits PTBP1 and PKM2 expression, in addition to lactate production, and functional parameters including cell proliferation and apoptosis susceptibility. I also confirm the therapeutic relevance of dimeric PKM2 to BOEC function, by demonstrating inhibition of proliferation and apoptosis susceptibility following TEPP-46 treatment. In the Sugen-Hypoxia rodent model of PAH I also demonstrated amelioration of right ventricular systolic pressure following oral gavage TEPP-46 treatment, further supporting the therapeutic potential of PTBP1 and PKM2 inhibition in PAH. Therapeutic benefit was not observed following apigenin treatment, however exploration of alternative routes of delivery and formulation strategies are needed. Ultimately, the results presented in this thesis improve our understanding of the molecular mechanisms orchestrating EC dysfunction in PAH and may guide the development of novel metabolically-targeted therapeutic strategies.
Embargo
Investigating pseudohypoxia in renal cells deficient in fumarate hydratase.
Maddalena, Lucas
The discovery that inactivating mutations in various metabolic enzymes leads to tumorigenesis has provided strong support for a primary role of altered metabolism in driving cancer. One of these enzymes, fumarate hydratase (FH), is found mutated in hereditary and leiomyomatosis and renal cell cancer syndrome, whereby affected cells are characterized by high subcellular accumulation of the metabolite fumarate. Fumarate has been shown to inhibit the prolyl hydroxylase-dependent degradation of hypoxia inducible factor-α (HIFα), leading to stabilization and transcriptional activation of HIF-1 under abundant oxygen conditions. This phenomenon, known as 'pseudohypoxia', is considered an important driver of tumorigenesis. However, the role of HIF activation and pseudohypoxia in FH-deficient renal cells is unclear. This aim of this thesis was to delineate the role of hypoxia biology in the phenotypic consequences of loss of fumarate hydratase in renal cells, by taking a holistic, systems-based approach, particularly with regards to transcriptional and metabolic alterations. Using a range of biochemical, molecular, and cell biology techniques, this thesis performed a comprehensive characterisation of the pseudohypoxic signature of Fh1-deficient mouse renal cells. Initial analyses of transcriptomic data sets showed a significant enrichment of generic hallmark hypoxic and HIF-1 target gene expression in Fh1-deficient cells under normoxic conditions. These features are abolished when Fh1 expression is re-constituted. To elucidate the role of Hif in the biology of Fh1-deficient cells, Hif1b (Arnt) was stably knocked out in Fh1-deficient and Fh1-proficient renal cells via CRISPR/Cas9 technology. These cells were then used in RNA-sequencing and metabolomics experiments to systematically delineate the oxygen-dependent/-independent and Hif-dependent/-independent metabolic and transcriptional signatures of Fh1-deficient renal cells. The resulting findings from this holistic, unbiased transcriptional and metabolic profiling have challenged the notion of pseudohypoxia in Fh1-deficient cells. Importantly, a novel link between purine metabolites, hypoxia, and Hif was discovered in Fh1-deficient cells, which may have important ramifications for tumorigenesis, driven by FH loss.
Open Access
Magnetic resonance imaging and serum biomarkers of traumatic brain injury – methodological and clinical considerations
Richter, Sophie; Richter, Sophie [0000-0003-3272-484X]
Traumatic brain injury (TBI) constitutes a major public health problem. The advent of collaborative neuro-imaging studies, advances in magnetic resonance imaging (MRI) and development of serum protein biomarkers have created an opportunity to gain new insights into the recovery after TBI. This thesis aimed to understand the role of MRI and serum biomarkers for outcome prediction after TBI. Before this clinical question could be answered, two methodological challenges had to be resolved. First, outcome data is often missing when patients are lost to follow up. We conducted a systematic review and created a novel framework for the statistical handling of missing outcome data in TBI. Second, MRI data collected at different sites may require harmonization. We examined the effect of different harmonization methods on a variety of imaging parameters, to inform our choice of method for subsequent chapters. The next two chapters concentrated on patients with mild TBI, as they represent the largest proportion of TBI patients and are in great need for better prognostic models. We demonstrated that structural white matter changes are detectable with advanced MRI, including diffusion tensor imaging (DTI), acutely after mild TBI even when conventional MRI is normal, and that these changes correlate with symptoms and recovery. We therefore assessed whether the association between DTI and outcome withstands the scrutiny of a formal prognostic study design and found that DTI substantially improves exiting prognostic models. We next focused on patients with moderate-severe TBI in whom the low level of consciousness is incompletely explained by CT appearances but might be explained by injury detectable with MRI or serum biomarkers. Unlike in mild TBI patients an MRI transfer in such critically ill patients constitutes a clinical risk. We show that protein biomarkers can help triage patients with moderate-severe TBI for MRI. We proceeded to investigate whether serum biomarkers could be used for prognosis directly, without the need for MRI. We found that serum biomarkers did significantly improve established prognostic models in this patient group with discordant CT appearances and conscious level. To conclude, this thesis presents methodological advances in the study of TBI, shows how DTI can transform outcome prediction after mild TBI and how serum biomarkers can improve MRI triage and prognosis in certain patients with moderate-severe TBI.
Open Access
Characterisation of Novel Secreted Antiviral Factors to Human Cytomegalovirus
Potts, Martin
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that infects a majority of the human population. Like other herpesviruses, HCMV readily establishes a latent infection that persists for the lifetime of the host and undergoes periodic lytic reactivation events. In healthy individuals, HCMV infection is typically asymptomatic, while in immunocompromised individuals, such as immunosuppressed transplant recipients, HCMV infection or reactivation of latent infection results in substantial end-organ disease and mortality. HCMV is also the most common congenital viral infection and can lead to neurological sequelae such as hearing loss and developmental delay. HCMV infection induces a multi-faceted immune response that controls but never clears the virus, due to a multitude of virally-encoded immune evasion factors. The broad immune response raised against the virus includes the action of intrinsic antiviral restriction factors, NK cells, neutralising antibodies and polyfunctional CD4+ and CD8+ T-cells. Although critical roles have been established for the antiviral cytokines IFNγ and TNFα during HCMV infection, the contribution of soluble factors to viral control remains poorly understood. In this thesis I demonstrate that the complement of secreted factors (secretomes) produced by immune cells co-cultured with HCMV-infected fibroblasts are capable of restricting HCMV in an in vitro viral dissemination assay. This activity was only partially mediated by IFNγ and TNFα and did not result from type I or type III interferon action. To determine the composition of immune cell antiviral secretomes using an unbiased quantitative approach, I developed a multiplexed proteomic method using a combination of SILAC and tandem mass tag (TMT) labelling. Application of this technology enabled detailed analysis of the secretomes produced by peripheral blood mononuclear cells (PBMC) derived from four HCMV-seropositive donors when co-cultured with HCMV-infected fibroblasts, including determination of the cellular origin of each secreted protein. This analysis identified critical components of the PBMC secretory response to HCMV, including secretion of the cytokines and chemokines IFNγ, IL-6, CXCL10 and CCL8 alongside potentially novel antiviral factors. Identification of the factors responsible for direct restriction of HCMV from this dataset proved challenging, however. Subsequent proteomic characterisation of the cellular pathways stimulated by the unidentified secreted antiviral factors implicated signalling via the NFκB pathway, providing avenues of investigation that may enable identification of these factors. Application of the novel secretomics methodology described here to isolated immune cell populations will facilitate further dissection of secreted antiviral immunity to HCMV in the future.
Open Access
SARS-CoV-2 Variants and Vaccines
Ferreira, Isabella
SARS-CoV-2 is a novel betacoronovirus that first appeared in late 2019 in Wuhan, China. It presented with pneumonia-like symptoms and infections ranged from mild to severe, requiring hospitalisation and medical intervention to prevent death. Due to a lack of previous immunity, SARS-CoV-2 spread around the globe at a rapid rate requiring the declaration of a pandemic and for countries to enforce national responses, such as widespread lockdowns and preventative measures including mask wearing and social distancing. Several vaccines were rapidly developed. These included BioNTech Pfizer’s BNT162b2 mRNA-based vaccine, Moderna’s mRNA-1273 mRNA-based vaccine, and Oxford AstraZeneca’s viral vectored AZD1222 vaccine. These were administered to the public in mass vaccination drives in two doses followed by a booster dose approximately six months later. Initially, the vaccines were administered to prevent infection but due to novel variants that arose, this later changed to preventing severe infection which may require hospitalisation. Several novel variants arose with substitutions occurring in the spike glycoprotein. As the spike protein is used by the virus for viral entry, these substitutions may have evolved to increase entry efficiency of the virus into the host cells. These novel variants included B.1.1.7 (alpha), B.1.351 (beta), P1 (gamma), B.1.617.1 (kappa), B.1.617.2 (delta), and B.1.1.529 (omicron). These variants had mutations in the receptor binding domain (RBD) of the spike glycoprotein which altered its conformation, thereby evading previous immunity from natural infection or vaccination, and allowing for increased binding to the SARS-CoV-2 receptor ACE2 and increased entry efficiency. This was hypothesised to cause increased transmission of the novel variants, requiring increased public health measures to prevent the spread of SARS-CoV-2, particularly in vulnerable populations, such as the elderly. The elderly population (≥80 vaccinated with BNT162b2 and an mRNA booster, and ≥70 vaccinated with two doses of AZD1222 and an mRNA booster) were determined to be a vulnerable population due to immunosenescence, which resulted in lower spike-specific antibody binding titres, lower neutralising antibody titres against spike pseudotyped viruses, lower T cell interferon gamma (IFNγ) and interleukin-2 (IL-2) responses, and reduced spike-specific memory B cell populations. In the population vaccinated with BNT162b2, these effects were reduced with the second vaccination dose and whilst responses waned over six months post vaccination, they were rescued with the booster vaccination dose, indicating a durable T and memory B cell response. Individuals primed with two doses of AZD1222 and boosted with an mRNA vaccine had lower neutralising antibody titres after two doses compared with individuals vaccinated with BNT162b2. However, this was rescued with the mRNA booster. Interestingly, a subset of atypical memory B cells which have been associated with aging were identified in the ≥70 population, indicating a mechanism for persistent immunity to form. This also conferred protection against novel variants of concern, such as B.1.1.529, which individuals were not able to sufficiently neutralise after two doses of AZD1222 coupled with waning immunity over six months post vaccination. These findings contribute to the understanding of entry efficiency of SARS-CoV-2 variants, which may be a reason for increased transmission and how this impacts immunity, particularly in the elderly. This will inform vaccination policies, particularly in the vulnerable and elderly populations, leading to greater immune protection against SARS-CoV-2 and possible future variants of concern that may arise.
Embargo
Mapping the Gut Microbiota and its Protective Functions Against Bacterial Enterocolitis
Beresford-Jones, Benjamin; Beresford.Jones, Benjamin [0000-0002-4699-8975]
The human gastrointestinal tract is colonised by trillions of microbes that actively impact the health of the host. This ecosystem, known as the gut microbiota, varies in composition between individuals and is associated with outcomes of many diseases. However, the underlying physiology of these associations remains largely unknown, preventing development of targeted microbiota therapeutics and clinical interventions. Animal models are essential for studying these host-microbiota interactions and for causally linking them to health outcomes; mice are the most widely used models for achieving this. Despite this, the mouse gut microbiota is poorly characterised compared to humans, limiting translation of research between these hosts. This thesis describes the generation of the largest and most comprehensive catalogue of high-quality mouse gut-derived genomes to date: the Mouse Gastrointestinal Bacteria Catalogue (MGBC). By facilitating the comparison of the human and mouse gut microbiotas, the MGBC demonstrates that only 2.58% of species are shared between these biomes, although over 80% of predicted functions are conserved. Application of species-level taxonomic mapping of these functions to predict the closest functionally-related species between the gut microbiotas of humans and mice demonstrates that these taxa are not necessarily the same as the closest genetically-related species. These analyses were implemented as a bioinformatic toolkit to enable other researchers to identify functionally equivalent species according to their functions of interest, with the aim of improving translation of gut microbiome research between the clinical and laboratory contexts. The MGBC improves coverage of the mouse gut microbiota, yielding metagenome classification rates of over 95%, and enables better resolution for mouse gut metagenomic studies. Nearly 2,500 mouse gut-derived shotgun metagenomes were used to characterise the taxonomic structure of the global mouse gut microbiota and identify factors associated with variation between studies. These analyses found that virtually every species of the mouse gut microbiota varied significantly between study institutes, therefore potentially representing an underlying basis for the irreproducibility crisis observed between mouse studies. This thesis further investigates the role of the gut microbiota in determining outcomes of bacterial enterocolitis caused by Salmonella Typhimurium and Clostridioides difficile. Through this work, Enterocloster clostridioformis was identified as a novel resistance-inducing microbe against Salmonella infection. This phenotype is potentially mediated through induction of protective epithelial responses and expansion of regulatory T cells in the caecal mucosa. Furthermore, by applying the MGBC to explore the association of intra-institutional microbiota variation with outcomes of C. difficile infection, this work identifies taxonomic and functional correlates of resistance and susceptibility. These findings validate the importance of the MGBC for facilitating shotgun metagenomic analyses in mice and highlight the impact of the gut microbiota on outcomes of health and disease. Mouse models will continue to be essential for developing microbiota-directed diagnostic and therapeutic interventions as well as enabling their introduction to clinical practice. This thesis therefore represents multiple advances towards understanding and tackling the obstacles posed by host-specific microbiotas. To this end, I have provided a starting point for efficient and informed translation of gut microbiota research between humans and mice, as well as the means for experimental validation of these analyses.
Open Access
Biomechanical Analysis of Human Coronary Atherosclerotic Plaques in Response to Pharmacotherapy, and in Predicting Adverse Clinical Events
Gu, Zhaotao Sophie; Gu, Sophie [0000-0001-7698-0373]
Myocardial infarction, a leading cause of mortality and morbidity worldwide, is often the result of coronary plaque rupture. Previous histological work has demonstrated that ruptured plaques are associated with higher stress, with in vivo intravascular studies linking higher plaque structural stress (PSS) with the presentation of acute coronary syndrome. Endothelial shear stress (ESS) on the other hand has been implicated in early plaque development and plaque growth, suggesting that both PSS and ESS can influence future plaque behaviour. The work presented in this thesis first demonstrates that PSS changes with pharmacotherapy, in particular, high-intensity statin (HIS) treatment prevented the rise in PSS in most advanced lesions (those with plaque burden [PB]>60%). The change in PSS was both dependent on drug therapy and baseline disease severity, and the protective effect of HIS seemed to be mediated by remodelling plaque microstructure and lumen surface. Second, it shows that plaque geometric parameters, especially the longitudinal variation measured by heterogeneity index of lumen roughness, curvature, and irregularity, can identify plaques that subsequently developed adverse clinical events. Lumen roughness heterogeneity showed a weak correlation with PSS and was an independent predictor of adverse clinical events. Finally, the role of combined PSS and ESS in plaque risk prediction was assessed through a subgroup analysis of the PROSPECT (A Prospective Natural-History Study of Coronary Atherosclerosis) study, with results suggesting that incorporation of ESS, ESS gradient, and PSS heterogeneity can improve the capability of VH-IVUS to identify plaques that lead to such events.
Embargo
Investigating the Mechanisms Underlying the Heterogeneous VSMC Contribution to Vascular Disease
Worssam, Matthew; Worssam, Matthew [0000-0003-4391-5377]
In healthy blood vessels, vascular smooth muscle cells (VSMCs) exist in a contractile, quiescent state but upon vascular insult can switch phenotype to activate proliferation, migration and remodelling of the extracellular matrix. Phenotypically switched VSMCs contribute most cells within neointimal lesions, characteristic of atherosclerosis and in-stent restenosis, diseases that underlie heart attack and stroke. Using multicolour “Confetti” VSMC-specific lineage tracing in animal models of vascular disease, our lab and others have shown that the extensive VSMC contribution to these lesions results from the clonal expansion of few cells. To understand how oligoclonal VSMC lesion contribution arises and to identify the signals activating VSMC proliferation in vivo, I used quantified VSMC clonal development over time in two models of vascular disease. Following acute vascular injury, the number and sizes of patches of clonally expanded VSMCs steadily increased before reaching a plateau, suggesting rare activation of VSMC proliferation in few cells, rather than clonal competition following widespread VSMC activation. Interestingly, only a subset of medial patches gave rise to neointimal patches, suggesting that VSMC lesion invasion represents a second selective event underlying mature lesion oligoclonality. Infrequent activation of VSMC proliferation in atherosclerosis was evidenced by the absence of plaques with high numbers of colours at any stage of plaque development. Tamoxifen-inducible, VSMC-specific deletion of contractile master regulator Myocd in adult mice had modest phenotypic effects on baseline VSMC contractile marker expression, and on injury-induced VSMC transcriptional response and clonality. In both vascular disease models, VSMC activation was greatly enriched in vascular regions displaying elastic lamina alterations, medial acellularity and immune cell recruitment, implicating these as potential proliferation-inducing cues. However, not all VSMCs in these regions formed patches, suggesting that VSMCs must be primed to respond proliferatively. Consistent with the hypothesis that VSMCs marked by stem cell antigen-1 (SCA1) may represent such a primed population, profiling of chromatin accessibility in SCA1+ VSMCs revealed substantial opening of chromatin at genes showing increased expression in injury-activated compared to healthy VSMCs. Manipulation of RUNX1 and CEBP, transcription factors whose motifs were enriched at activation-specific open chromatin regions, could allow for control of VSMC priming and proliferation.
Embargo
Systems immunology frameworks link multicellular immune perturbation phenotypes and setpoints to response outcomes
Mulé, Matthew
This thesis develops frameworks for using top-down systems biology approaches with multiomic single cell technology to understand variation in human immune system response outcomes. We integrate human population, cell subset and single cell variations in molecular phenotypes which give rise to baseline setpoints, are perturbed by vaccination or drug treatment, and are linked to emergent response / clinical outcomes. In Chapter 2, we dissect noise sources in data derived from new methods which simultaneously measure protein and mRNA in single cells (e.g., CITE-seq). After identifying two main sources of noise, we develop an open source software method for normalizing and denoising CITE-seq protein data. We then develop a computational framework for analyzing the effects of timed immune system perturbations applied to human cohorts profiled with multimodal single cell technology. Chapter 3 applies these methods on a human vaccination cohort profiled using CITE-seq. We first define highly interpretable immune cell subsets using unsupervised clustering based on the denoised protein data, then contrast the transcriptome pathways within these subsets that are differentially induced by vaccines formulated with and without an adjuvant. These robust phenotypes are further interpreted using single cell computational reconstructions of cell states. Using these comparative analyses, along with unbiased analysis of baseline phenotypes linked to antibody response, we identify a multicellular “naturally adjuvanted” human immune system setpoint more poised to respond to vaccination. Chapter 4 applies the methods developed above to a cohort of cancer patients treated with immune checkpoint inhibitors. In this work, we identify multicellular baseline setpoints linked to development of immune related adverse events after treatment which are uncoupled from the phenotypes induced by treatment. Together, these approaches help advance a quantitative, predictive understanding of human immune system variation, and pave the way for further human perturbation cohort studies across biological disciplines.
Embargo
Tissue-resident immunity in the human kidney
Stewart, Benjamin
The human kidney maintains whole organism homeostasis through tightly regulated filtration, transport, and endocrine functions that are subserved by a wide array of specialised cell types with distinct developmental origins and anatomical organisation. Amongst these cells lies a network of tissue-resident immune cells with incompletely characterised roles in tissue homeostasis, repair, and defence. Furthermore, different regions of the kidney are affected by distinct immunologically relevant insults - ascending bacterial infection in the medullary-pelvic region, and immune complex-mediated inflammation within the cortex. We hypothesised that the kidney-resident immune network is adapted to respond to and counter these immunological challenges. Recently the advent of high-throughput single-cell genomics methods has permitted the atlasing of human organs in unprecedented molecular detail. These techniques permit the unbiased characterisation of cell types and cell states, the prediction of functional programmes, and the mapping of intercellular interaction networks underpinning tissue organisation. In this thesis, we used single-cell RNA sequencing to generate the first detailed and spatio-temporally resolved atlas of the human kidney in development and maturity. This study has identified the transcriptional signatures of mature kidney parenchymal cell types from nephron epithelial, vascular, and stromal compartments, and allowed the reconstruction of the developmental trajectories of early nephrogenesis. Within the immune compartment, we charted the landscape of immune cells, and the seeding of these cells through early development, identifying a macrophage subset in mature kidneys with transcriptional similarity to macrophages seeded early in fetal life, and likely representing a long-lived self-renewing subset. This work also identified a transcriptional signature mediating antimicrobial defence in the pelvis of the kidney. Here pelvic epithelial cells express antimicrobial peptides, and neutrophil- and monocyte-recruiting chemokines, orienting the immune response towards the dominant infectious threat. We later optimised and refined the single-cell genomics approach to the human kidney, generating high quality data on a larger panel of donors in a compartmentally-resolved fashion. In this work we were able to assess the immune composition of glomerular and tubulointerstitial compartments, finding a strikingly divergent immune landscape, with glomeruli occupied by monocytes, dendritic cells, and NK cells, and the tubulointerstitial compartment housing populations of resident macrophages, T cells, and B cells. Finally we have begun to explore the dynamic transcriptional responses of kidney immune and parenchymal cells to IgG immune-complex stimulation using an ex vivo normothermic normoxic perfusion model. These experiments have uncovered cell type-specific signalling responses in stimulated monocytes, NK cells, and glomerular endothelial cells, with evidence for abundant chemokine and cytokine signalling coordinating the early response to this highly disease-relevant perturbation.
Embargo
Evaluation of metabolic and cardiovascular actions of [Pyr1]apelin-13 in patients with Type 2 diabetes mellitus
Sulentic, Petra
Name: Dr Petra Sulentic Thesis title: Evaluation of metabolic and cardiovascular actions of [Pyr1]apelin-13 in patients with Type 2 diabetes mellitus Type 2 diabetes mellitus (T2DM) is common, long term metabolic disorder characterised by hyperglycaemia (high blood glucose) resulting from insulin resistance (IR) and insulin insufficiency. Type 2 diabetes mellitus and related complications present a significant cause of morbidity and mortality and there is an urgent need to identify novel pathways that may ultimately lead to the development of new therapies to improve blood glucose control and prevent development of diabetic complications. Apelin is a naturally occurring peptide investigated in animal models and humans showing beneficial cardiovascular and metabolic properties. Apelin reduces peripheral vascular resistance (PVR) and increases cardiac index (CI) in healthy volunteers and heart failure patients, and also increases insulin sensitivity in overweight participants. Besides health risks connected to hyperglycaemia, T2DM patients have increased cardiovascular risk, therefore modulation of the apelin signalling pathway may provide a novel cardiometabolic therapeutic approach. Systemic studies were planned to investigate the effects of prolonged intravenous apelin infusions on CI, blood glucose and IR for the first time in patients with T2DM, following hypothesis that apelin would induce beneficial cardiovascular and metabolic effects in that patient group. Firstly, the tolerability and safety of apelin in increasing doses were tested in a pilot study including healthy volunteers. Subsequently, eighteen participants with increased body mass index (BMI) 25–34.9 kg/m2 who served as a model of IR and nine T2DM patients underwent a series of randomised, double blind, saline controlled, prolonged infusion studies. [Pyr1]apelin-13 was infused systemically for two hours with measurements of cardiovascular and metabolic parameters. Results showed that in participants with increased BMI, compared to saline control, [Pyr1]apelin-13 in dose 30 nmol/min caused a significant rise in CI and stroke volume index (SVI), whilst reducing peripheral vascular resistance (PVR) and mean arterial pressure (MAP). After a mixed meal, [Pyr1]apelin-13 also significantly reduced plasma C peptide without inducing significant changes in glucose or insulin plasma level. In patients with T2DM, [Pyr1]apelin-13 in the equivalent [Pyr1]apelin-13 dose of 30 nmol/min and compared to saline control, also increased SVI and CI and reduced PVR without affecting MAP and levels of insulin, glucose or C-peptide. In T2DM group a possible delay in gastric emptying was observed, representing a novel finding in this condition and requiring further investigations. In summary, following these pilot studies results, there is a reason to believe that patients with T2DM have the potential to benefit from apelin mediated vasodilation, increased CI and better glucose homeostasis combined with possible delay in gastric emptying. Therefore, targeting apelin signalling represents novel pathways yet to be explored and developed therapeutically with further studies required to investigate those effects.
Open Access
A Characterisation and Understanding of the Germinal Centre B Cell Response to Atherosclerosis in Mice using Lineage Tracing
Francis, Anna
Atherosclerosis is the most common cause of ischaemic heart disease and stroke which represent a global health concern responsible for significant levels of morbidity and mortality worldwide. It is a chronic inflammatory disease characterised by plaque build-up within arterial walls initiated by low density lipoprotein cholesterol. Elevated plasma LDL is a major risk factor for atherosclerosis and its immunogenic oxidation creates neo-epitopes that drive inflammatory immune responses underlying the pathogenesis of atherosclerosis. The germinal centre is found within secondary lymphoid organs and is responsible for the production of high affinity antibody-producing plasma cells and long-lived memory B cells against antigens such as epitopes of oxidised LDL. Class-switched plasma cells and anti-oxLDL IgG antibodies have been detected within atherosclerotic plaques and sera of human and mice suggesting that the germinal centre response is pathogenically dysregulated in atherosclerosis. The tamoxifen-inducible AID-CreERT2-Rosa-EYFP lineage tracing mouse model was used and crossed with the atherosclerosis-prone LDLr-deficient mouse model. This model inducibly and specifically labels germinal centre B cells and their progeny using tamoxifen allowing for tracking of atherosclerosis-specific B cell clones comprising germinal centre, memory, and plasma cells. LDLr-/- and LDLr+/- (‘WT’) mice were fed chow or high fat diet for up to 8 weeks and upon tamoxifen dosing (timing varied throughout studies) via intra-peritoneal injection or oral gavage, AID-expressing cells (germinal centre B cells) were fluorescently labelled with EYFP. Cells were analysed using flow cytometry and FlowJo software. All graphs and statistics were created using the GraphPad Prism 7.0. In this thesis, I sought to characterise and understand the germinal centre B cell response in atherosclerosis using a lineage tracing mouse model. Firstly, the model was validated as EYFP cells were only detected after tamoxifen administration and were only present in germinal centre-derived cell populations. Furthermore, labelling efficiency was independent of time, diet, or genotype. The germinal centre response was characterised in both WT and LDLr-/- mice demonstrating an time-dependent increase in germinal centre responses. Germinal centre reactions were exacerbated, greater in magnitude and degree of class-switching, by increased plasma cholesterol levels consequent of both HFD and knock out of the LDL receptor. The combination of HFD and LDLr-/- genotype, replicating atherosclerotic conditions, synergistically exacerbated germinal centre responses biased towards pathogenic cellular output due to elevated levels of class switching. Due to the permanence of EYFP labelling, it was possible to track EYFP labelled cells over time and throughout the course of atherosclerosis. Tracking studies revealed that hyperlipidaemic conditions induced by HFD resulted in longer-lived EYFP clones which had increased propensity to undergo class-switching. Furthermore, there was a greater output of EYFP labelled memory and plasma cells. To investigate the mechanism by which HFD induced greater germinal centre responses, interventions were conducted to block the impact of cholesterol and inflammation separately. Use of a cholesterol inhibitor drug and cholesterol free diet revealed that dietary cholesterol is key in driving the exacerbated germinal centre responses observed at early stages i.e., 4 weeks of atherosclerosis. Use of a cytokine inhibitor to dampen inflammation limited germinal centre responses at later stages of atherosclerosis i.e., 8 weeks, suggesting that inflammation plays an important role in germinal centre responses at this later timepoint. Thus, the model proposed is that dietary cholesterol is critical early on in atherosclerosis as disrupted lipid homeostasis results in autoimmune B cell responses with germinal centres primarily reacting to accumulated oxidised LDL while at later stages, the inflammation associated with atherosclerosis fuels exacerbated and pathogenic germinal centre responses. To examine the role of IgG2c and its receptor FcgRIV in atherosclerosis, the impact of IgG2c antibody complexes was tested in vitro. IgG2c significantly enhanced TNF secretion, a marker of inflammation, in FcgRIV-expressing myeloid-derived monocytes. Thus, IgG2c has the potential to exacerbate inflammatory responses from plaque macrophages and dendritic cells. In conclusion, the AID lineage tracing mouse model has been validated for use in the atherosclerosis setting. It has been characterised showing that germinal centre responses are exacerbated during the course of atherosclerosis and are skewed towards production of class- switched longer-lived B cell clones. Dietary cholesterol is the main driver of this pathogenic response in tandem with the inflammatory conditions caused by atherosclerosis. Germinal centre-derived IgG2c antibodies could play an important role in exacerbating inflammation within the plaque through plaque macrophages and dendritic cells. This thesis presents a model whereby atherogenic dyslipidaemia, as a result of elevated serum LDL levels, breaks down B cell tolerance. This results in autoimmune pathology characterised by exacerbated germinal centre responses skewed towards pathogenic antibody isotype clone production in response to LDL-induced inflammation. This provides an opportunity to target B cell-related atherosclerosis-specific responses therapeutically.
Open Access
Gaucher Disease Protects Against Tuberculosis
Fan, Jingwen
Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. This and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, I find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe from the Middle Ages through the 19th century.
Open Access
Evaluation of immunomodulatory mediators in chronic obstructive pulmonary disease and cardiovascular disease
Sun, Yuan
Chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD) are among the leading causes of mortality and poor health burden in the world. Both COPD and CVD often co-exist, due to shared risk factors and common pathophysiological pathways. There is an unmet need for effective therapeutics in COPD, given that there has been a paucity of therapeutic progress over the last decades and currently very few therapeutics have been proven to improve survival in COPD. To this end, addressing cardiovascular risk and evaluating cardiovascular therapeutics in people with COPD is an attractive prospect to reduce mortality and morbidity in this patient group. Furthermore, there is an urgent need to improve understanding of immunomodulatory mediators that may have potential as therapeutics targets in both COPD and CVD, and thus health outcomes in COPD and CVD. This thesis evaluated two distinct immunomodulatory pathways, which are of interest therapeutically in both COPD and CVD. Firstly, the Interleukin-33 (IL-33)/ST2 axis and secondly, specialised pro-resolving mediators (SPMs), specifically the specialised pro-resolving mediator Resolvin D1. Additionally, data pertaining to the anti-platelet drug aspirin in people with COPD was evaluated. The reason for this was to evaluate if aspirin may have therapeutic benefit in COPD. Given aspirin’s mechanism of actions not only as an anti-inflammatory anti-platelet therapeutics but also possible enhancement of endogenous production of specialised resolution mediators, it is helpful to evaluate patient level data relating to aspirin use and outcomes. Firstly, a systematic review and meta-analysis of published human studies of the IL-33/ST2 axis across the spectrum of human cardiovascular disease was undertaken. The role of IL-33 in CVD is unclear with both ameliorative and harmful effects reported, and circulating ST2 is a biomarker in CVD. The review included 77 studies and 62075 participants. The main findings were that incremental increases in circulating ST2 levels were associated with increased risk of all-cause mortality and cardiovascular events in populations with cardiovascular disease. IL-33 levels were found to be lower in heart failure, coronary artery disease and acute coronary syndrome patient groups, compared to controls, with the opposite being observed in stroke patients. The results of this analysis have been published in PLOS One. Secondly, levels of immunomodulatory mediators were measured in serum samples COPD patients and controls, specifically the specialised pro-resolving mediator Resolvin D1. Specialised pro-resolving mediators (SPMs) are metabolites of polyunsaturated fatty acids and are responsible for resolution after acute inflammatory events. There are several families of SPMs, including lipoxins, protectins, maresins and resolvins which are derived from different polyunsaturated fatty acids and are released at different times during the course of inflammation resolution. Of these SPM families, Resolvin D1 has been most extensively investigated in COPD pathogenesis and is thought to be dysregulated in COPD. Resolvin D1 levels were measured using enzyme linked immunosorbent assays in serum samples from 86 stable COPD patients, 140 COPD patients with recorded exacerbations at baseline and 146 healthy controls, to evaluate the differences in circulating levels between the groups. Resolvin D1 levels were significantly lower in stable COPD patients compared with COPD patients with exacerbations at baseline, which suggests potentially dysregulated resolution pathways in COPD. The finding that samples from patients who had reported acute exacerbations at baseline had higher Resolvin D1 levels than in stable COPD patients suggests that it plays an essential role in the natural resolution of inflammation in COPD. Lastly, aspirin use has in some observational studies showed encouraging data that it was associated with reduced exacerbation rates. Therefore, work was undertaken to evaluate aspirin use at baseline for its association with health outcomes in COPD populations, in a comprehensive statistical analysis of two large high quality clinical trial datasets (SUMMIT trial n=16485, IMPACT trial n=10355), which included patients with both moderate (SUMMIT) and severe COPD (IMPACT) respectively, and those with a history/risk of cardiovascular disease (SUMMIT). The patient level data from both trials were obtained and patient level analysis performed. The analysis showed that aspirin use was associated with increased risk of mortality [SUMMIT hazard ratio 1.15 (1.00-1.33), p=0.048] fully adjusted for known and possible confounders, exacerbations and cardiovascular events (including myocardial infarction and stroke) over follow up of 52 weeks (IMPACT) and median 1.8 years (SUMMIT) respectively. In summary, the IL-33 has potential as a therapeutic target and biomarker across the range of human cardiovascular disease although further studies with larger sample sizes are needed. Whilst IL-33 levels were lower in patients than in controls for several cardiovascular diseases, further investigation is needed to explain the contradictory findings in stroke patients. Resolvin D1 is dysregulated in stable COPD patients and has potential in future treatment regimens. The results of the Resolvin D1 study led to an ongoing clinical study which measures changes in patient Resolvin D1 levels over time, following a COPD exacerbation. While previous observational studies had suggested aspirin use was associated with reduced mortality risk and exacerbations in COPD patients, the findings of this extensive analysis suggest that aspirin use is associated with increased mortality risk and exacerbations, and should not presently be indicated for treating COPD patients. Future work would include assessing IL-33 and ST2 receptor expression in tissue samples from CVD patients which could identify any disruptions to this pathway. Additionally, levels of SPMs including resolvins need to be measured in COPD patients at set time points after an exacerbation, to identify changes in the trajectory of SPM levels. Finally, a randomised control trial of aspirin in COPD patients would be useful to evaluate the effect of aspirin use on patient exacerbations and all-cause mortality.
Embargo
Exploring the therapeutic potential of the apelin receptor signalling axis, and angiotensin-converting enzyme 2 (ACE2) as the SARS-CoV-2 viral entry receptor, in the cardiovascular system
Williams, Thomas; Williams, Thomas [0000-0002-1051-0595]
The apelin receptor is a class A GPCR that binds two endogenous peptide ligands, apelin and elabela/Toddler (ELA), to induce positive cardiac inotropy and vasodilatation in the cardiovascular system. The apelin receptor provides a tractable therapeutic target for multiple cardiovascular diseases, such as pulmonary arterial hypertension, heart failure, and bleeding disorders, where the receptor also mediates antithrombotic responses. Here, four novel fluorescent ligands, designed as functional analogues of endogenous apelin and ELA peptides, were validated and used as a versatile tool for the qualitative and quantitative determination of pharmacological parameters of the apelin receptor. The fluorescent ligands were also used, with other techniques, to study apelin receptor variants identified in the genomes of human patients with rare cardiovascular diseases recruited to the NIHR BRIDGE project. The V38L, T89M, and R168H variants showed distinct effects on apelin receptor pharmacology. V38L reduced receptor localisation at the membrane, but was able to bind fluorescent apelin and showed little internalisation. T89M, uniquely, was able to bind fluorescent apelin but not fluorescent ELA. R168H was unable to bind apelin receptor ligands, and was consequently inactive, pointing to loss of pharmacological function. ACE2 is a zinc-metalloproteinase type 1 transmembrane protein that cleaves angiotensin peptides to counter-regulate the renin-angiotensin-aldosterone system in the cardiovasculature and mediate hypotensive responses. The protein also acts as the membrane receptor for human coronaviruses, including SARS-CoV-2, the cause of the COVID-19 pandemic. Here, ACE2 was identified in human left ventricle tissue, and in a human embryonic stem cell-derived cardiomyocyte model that was used to screen for novel inhibitors of viral infection. Benztropine (an inhibitor targeting B0AT1 that complexes with ACE2) and DX600 (a peptide antagonist of ACE2), were shown to exhibit antiviral properties for the first time. Additionally, the tissue expression and distribution of a recently identified novel short ACE2 isoform that does not bind SARS-CoV-2, revealed enrichment of the short isoform in respiratory and bile duct epithelia. The work in this thesis advancement our understanding of both the apelin receptor and ACE2 as regulators human cardiovascular health and disease.
Embargo
Investigating the role of mitochondria in the hypoxia response using in vitro and in vivo models
Al-Habib, Alhasan
Human coiled-coil helix coiled-coil helix domain-containing protein 4 (CHCHD4) catalyses disulfide bond formation in substrate proteins, mediating their import from the cytoplasm into the mitochondria. CHCHD4 is critical for maintaining intracellular oxygenation and metabolism in human cells, partly by controlling the expression of respiratory chain complex subunits. In cancer, CHCHD4 is required for tumour growth in vivo, with overexpression in a range of cancers correlating with increased tumour progression, the hypoxia gene signature, and poorer patient survival. CHCHD4 (MIA40) is highly evolutionarily conserved and essential in yeast, mice and zebrafish. This thesis explores the role of CHCHD4 in regulating mitochondrial function and hypoxia signalling in vivo, using CRISPR/Cas9 chchd4¬-targeted zebrafish, and in vitro, using human cell systems including patient-derived cells. Zebrafish Chchd4 exists as two paralogues, Chchd4a and Chchd4b; both contain the evolutionarily conserved cysteine motifs required for mitochondrial localisation and function of CHCHD4. I confirmed that chchd4a is essential in zebrafish, with chchd4a-/- fish not surviving to adulthood. In embryos, chchd4a was shown to be required for the expression of a panel of respiratory chain proteins known to be regulated by CHCHD4, unlike chchd4b. In response to hypoxia, compared to wild type (wt), chchd4b-/- embryos exhibited a time-dependent greater induction of Hif-1α protein, yet also a correlative reduction in expression of the Hif-1α target gene egln3. Chchd4b-/- embryos also hatched faster than wt, a process that has been shown by others to coincide with increased Hif-α expression. The accelerated hatching rate of chchd4b-/- embryos was found to be rescued by chchd4a loss in normoxia. To further investigate the functions of the zebrafish Chchd4 paralogues in a cellular context, human U2OS clonal cell lines stably expressing either vector control, chchd4a, or chchd4b cDNA were generated. U2OS-Chchd4a cells exhibited a greater upregulation of HIF-1α and EGLN3 expression in hypoxia compared to the vector control, whereas U2OS-Chchd4b cells showed the opposite. Notably, expression of Chchd4b appeared to be less well tolerated in U2OS cells, whilst also appearing to localise predominantly in the cytoplasm. Taken together, these data indicate that Chchd4a and Chchd4b have distinct functions. Skin fibroblasts isolated from a patient identified to exhibit compound heterozygous mutations in CHCHD4 were also evaluated. Compared to skin fibroblasts from a healthy control patient, the patient-derived mutant CHCHD4 cells exhibited decreased CHCHD4 levels, significantly reduced basal cellular oxygen consumption rate (OCR) and increased extracellular acidification rate (ECAR), as well as reduced expression of MTCO2, a complex IV subunit. Collectively, these data suggest altered mitochondrial function in the CHCHD4 mutant patient-derived cells. In summary, this thesis explores the link between CHCHD4 and HIF signalling in vitro and in vivo, providing novel insight into CHCHD4 function using zebrafish models and human cell systems. The data presented in this thesis show unique and overlapping functions of the Chchd4 paralogues in zebrafish and, for the first time, describe the functional consequences of CHCHD4 mutations identified in a patient.
Embargo
Developing novel therapies for Mycobacterium abscessus
Evans, Ieuan
Mycobacterium abscessus (M. abscessus) is a rapid growing non-tuberculous mycobacteria (NTM) that is increasingly recognised as a major pathogen owing to its inherent resistance to a wide range of the currently available antibiotic therapies. In particular, it causes problems in patients with Cystic Fibrosis (CF) with evidence of marked inflammation, frequent infective exacerbations and an accelerated decline in lung function. Current treatment outcomes are extremely poor. There is therefore an urgent need to understand how M. abscessus resists killing by existing antibiotics and to find new treatments. I have attempted to address these issues in this thesis, particularly relating to the development of novel treatment regimens and antimicrobial agents. I first analysed the treatment outcomes for the CF patients infected with M. abscessus at the Royal Papworth Hospital to examine whether specific antibiotic combinations were associated with better treatment outcomes and could therefore form the basis for a novel treatment regimen. I showed that, despite expert care, the rates of achieving a sustained culture conversion following often prolonged treatment was around 30%, matching the results available from the limited published data. I could find no strong evidence that particular antibiotic combinations were better, further motivating my search for new treatment strategies. I then designed a novel regimen of existing antibiotics (based on synchronous targeting of cell wall, protein synthesis and cellular energetics) to use in CF patients with either treatment-naïve or refractory disease. I demonstrated good efficacy in vitro against a range of clinical isolates of M. abscessus. Subsequent GWAS analysis revealed several potential resistance pathways, including a variety of metabolic pathways and membrane transport systems, which could be used to stratify patients in a future clinical trial. I also developed and evaluated a novel highly effective formulation of acidified nitrite as a new nebulised antimicrobial therapy for M. abscessus which has been shown to have in vivo activity and is currently being evaluated in a clinical trial. Finally, I have developed a method using ultra-performance liquid chromatography to detect intracellular permeability of compounds to support fragment-based antibiotic discovery. I screened a library of 960 fragments to assess for permeation into M. abscessus. Machine learning was then used to generate a predictive algorithm for bacterial permeation that can be used in future fragment-based campaigns.
Open Access
B cell therapy in ANCA-associated vasculitis
McClure, Mark
Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a rare autoimmune disease with a UK prevalence of 250 per million. The immunopathogenesis includes neutrophil activation by B cell-derived ANCAs, activation of the alternative complement pathway, and dysregulated T cell responses that result in a necrotizing vasculitis of small blood vessels. The central role of B cells is highlighted by the presence of activated B cells in inflammatory lesions, the association of B cell activation status with disease activity and the efficacy of B cell depletion therapy with rituximab as both an induction and maintenance agent. Rituximab, the only licenced therapy for AAV, is associated with a remission failure rate of 10-30%, and for those who achieve remission, repeated doses are needed to prevent relapses, increasing the risk of hypogammaglobulinaemia and susceptibility to infections. Thus, there is an unmet need to develop a treatment regimen that induces prolonged remission without the need for maintenance therapy. This thesis investigates some of the mechanisms, shortfalls and clinical implications of B cell targeted therapies for the treatment of AAV. As our understanding of rituximab’s mechanism of action has evolved, the concept of rituximab resistance has emerged. In the first section, a combination of flow cytometry and single cell RNA sequencing (scRNA-seq) is used to demonstrate the dissociation between peripheral B cell depletion and the persistence of B cells in the tissue after rituximab using sequential nasal mucosal biopsies in patients with active AAV. Persistent nasal B cells had expression profiles suggesting the capacity for antigen presentation and production of proinflammatory cytokines. Quantification of predicted cell-cell interactions based on receptor-ligand expression revealed several significant predicted interactions between B and T cell ligand and receptor gene expression, highlighting the complex interplay of stimulatory and inhibitory signalling pathways within the tissue microenvironment, and also revealing some potential mechanisms of rituximab resistance. In the second section, experiments were conducted using COMBIVAS trial (a mechanistic study of rituximab plus belimumab versus rituximab plus placebo in AAV) samples to evaluate the early pharmacodynamic effects of BAFF antagonism with belimumab on B cells and B cell subsets in patients with active AAV. A combination of flow cytometry and scRNA-seq was used with the primary aim of understanding the mechanisms driving the early rise in circulating B cell numbers after belimumab. We report variable elevations in circulating memory B cells after belimumab that were potentially influenced by prior therapies and/or subtherapeutic drug concentrations. Evidence to support the hypothesis of belimumab-induced tissue mobilisation of B cells was supported by the finding of blood B cells from belimumab-treated samples with expression profiles that were compatible with recent tissue residency. Unfortunately, the BCR-seq analysis, which was limited by poor BCR coverage and small sample size, did not corroborate these findings. In the final section, data from a cohort of rituximab-treated AAV patients was analysed to test whether patient-specific and/or disease-specific risk factors for relapse and infection could be used in combination to generate risk prediction models to help guide decision making regarding extended rituximab maintenance therapy in AAV. While the models had insufficient power to discriminate risk between individual patients, they were able to assign patients into risk groups for both relapse and infection. The ability to identify risk groups may help in decisions regarding the potential benefit of ongoing rituximab treatment.