No description available.

Wiskott Aldrich Syndrome (WAS) is an X-linked immunodeficiency characterised by low numbers of low volume platelets, eczema, severe immunodeficiency and increased susceptibility to malignant and autoimmune diseases. Wiskott Aldrich Syndrome protein (WASp), the product of the gene mutated in WAS is a multidomain cytosolic protein which acts by integrating downstream signals from cell surface receptor signalling cascades and transduces these signals into remodelling of the actin cytoskeleton. WASp is involved in cellular processes as diverse as cell polarisation, migration, phagocytosis and T cell synapse formation. Dendritic cell function and migration is known to be impaired in WAS. Specifically, dendritic cells lacking WASp in both mice and humans show an impaired ability to form transient actin rich adhesive structures called podosomes. In this thesis, I am presenting the characterisation of a panel of domain deletion WASp proteins and a series of point mutations which, in children give rise to a range of disease phenotypes. I have expressed these constructs as GST tagged proteins and developed a bead based in vitro assay to asses the protein activity. I have compared the ability of these mutants to bind key regulators of WASp function, WIP and Cdc42, using a variety of immunoprecipitation techniques. Finally I have expressed EGFP tagged WASp proteins in myeloid cell lines to assess expression and stability of these constructs and in WASp null murine dendritic cells to assess their ability reconstitute podosomes. Point mutations which cause WAS have slightly increased actin polymerisation activity compared to wild type WASp (WT) and have a normal affinity for Cdc42. Although they have a reduced affinity for WIP compared to WT, there is still significant association of WIP to mutant WASp in cells. These mutants have reduced stability and different patterns of phosphorylation compared to WT, when expressed in myeloid cells lines. The EVH1, Polyproline and VCA domains of WASp are all essential for optimal actin polymerisation activity, whereas the Basic domain and the point mutation H246D (which inhibits Cdc42 binding) could be deleted with no loss of molecular activity. The EVH1 domain is essential for WIP binding and the Basic domain is essential for Cdc42 binding. Deletion of the EVH1 domain resulted in protein which had greater stability than WT WASp, suggesting that residues within the EVH1 domain are essential for the physiological degradation of WASp. This work demonstrates that mutant WASp which causes WAS in patients has normal molecular activity, but the regulation of mutant WASp degradation is impaired in myeloid cells. Results from this project also suggest that Cdc42 is not essential for normal WASp functioning, but does appear to be essential for the phosphorylation of the Y291 residue, which is known to be critical for the regulation of WASp activity. Further work will aim to elucidate the functional (in vivo) significance of these findings.

Autoimmunity results from a breakdown in tolerance to self-antigens. Interleukin-17A (IL-17A) is a cytokine that has been implicated in the development of certain autoimmune disorders, notably multiple sclerosis and its mouse model experimental autoimmune encephalomyelitis (EAE). In order to further understand mechanisms that lead to the development of autoimmunity, the objectives of this study were to investigate the sequence of immunological events that lead to the development of an autoimmune response and to generate and characterise a reporter mouse for IL-17A. EAE is a well-established model of an autoimmune response directed against selfantigens in the central nervous system and mimics many aspects of the human disease multiple sclerosis. EAE is a CD4 T cell-mediated disease, in that these cells can be used to transfer disease to naïve recipient mice. Following EAE induction, IL-17A-expressing cells were increased in frequency within the CD4 and γδ T cell populations in the draining lymph nodes, with a simultaneous increase in the number of these cell populations in the blood. Disease development was associated with the appearance of IL-17A and IFN-γ-expressing CD4 T cells, as well as IL-17Aexpressing γδ T cells in the spinal cord. EAE induction requires the systemic administration of pertussis toxin for disease development. It was found that pertussis toxin enhanced antigen-specific IL-17A and IFN-γ production in the periphery. An IL-17A reporter mouse was generated in which activation of the IL-17A promoter is reported by expression of Enhanced Yellow Fluorescence Protein (EYFP). In order to generate the mouse, a strain was first generated in which Cre recombinase expression is driven by the IL-17A promoter. This mouse was then crossed with a ROSA-26_EYFP strain in which expression of EYFP in the ubiquitously expressed ROSA-26 locus is usually inhibited by the presence of a LoxP-flanked-transcriptional stop sequence. Expression of Cre recombinase would remove the transcriptional stop sequence, leading to irreversible expression of EYFP in all cells that had activated IL-17A and their progeny. The results from this study suggest that pertussis toxin can amplify antigen-specific cytokine responses in EAE, an effect which could be attributed to enhancing disease pathogenesis. The IL-17A reporter mouse will be an invaluable tool to investigate the generation, lifespan and function of IL-17A-expressing cells in the development of immune responses.

No description available.

Autoimmunity, a term from the life-sciences, refers to that strange behaviour where an organism’s defences turn on and against its own tissues. Autoimmune disease marks this process as a painful, suicidal, and terrifying relation to one’s own body. Taking an autobiographical approach to autoimmune illness, this thesis examines the autoimmunity of the autos ‘itself’ in order to deconstruct a paradigm of immunity that paralyses one within process of self-destructive- defence. With Jacques Derrida’s appropriation of autoimmunity the term enters a deconstructive philosophical discourse. For Derrida, autoimmunity attacks not (only) the body but immune defences themselves and names the opening of the body to the future ‘to-come.’ And yet, the use of this term is couched in a rhetoric of terror that emphasises the threat of even worse events ‘to-come.’ This thesis explores whether the trauma of autoimmunity need necessarily emphasise an exponential threat that only refers to the worst. Or, whether this trauma might be ‘treated’ in a manner that affirms autoimmunity. Amongst the treatments employed are various narratives of science and science fiction. Chapter One explores the relations between biomedical and deconstructive autoimmunity and treats their traumatic symptoms psychoanalytically. Chapter Two employs the immunitary logic of systems theory to comfort my dis-ease, a dis-ease I share with the laboratory animal. While Chapter Three turns to the biopolitics of immunity and Roberto Esposito’s account of an affirmative biopolitics. However, while each of these ‘treatments’ attest to the possibility of opening the terror of the trauma to-come to the promise of better, they also repress, suppress, and ignore the possibility of a radical finitude, which, for Derrida, is essential for any ethical relation. Yet, if a radical finitude here takes on the character of the worst, it must be emphasised that it is an autoimmune finitude, for better or worse.

X-linked Severe Combined Immunodeficiency (SCID-X1) is an immunopathy caused by a mutation of the common gamma chain (γc) gene, IL2RG, which results in a lack of T cells, NK cells and with dysfunctional B cells. Current gene therapy methods involve the addition of a correct γc gene via integrating viral vectors. However, these current non-targeting gene addition strategies can result in transformation of the cell. A novel solution to this problem is met by targeted gene correction via homologous recombination stimulated by a site specific cleavage event caused by zinc finger nucleases (ZFN) within the disease gene. A γc deficient mouse has been created by replacing the murine il2rg locus with a mutated human IL2RG containing a point mutation frequently seen in SCID-X1 patients. The mutant human IL2RG is transcribed and initial analysis of this new SCID-X1 model has revealed a phenotype mirroring γc gene knockout mice. Lineage negative bone marrow cells from these mice, transduced with integrating lentiviral vector encoding functional IL2RG can reconstitute the immune cells in the Rag2-/-γc-/- double knockout SCID mouse model. Therefore the humanised mouse model of SCID-X1 can be corrected and is an appropriate platform to assess the efficiency of various gene targeting and correction strategies for the human mutation including ZFN induced homologous recombination. We have successfully achieved targeted homologous recombination in both a human T cell SCID-X1 cell line model and the humanised mouse embryonic stem cells with IL2RG specific ZFN.

Drug hypersensitivity reactions represent a significant clinical problem and an impediment to drug development. It is currently impossible develop drugs with no immunological liability; furthermore, it is very difficult to predict which individuals will develop hypersensitivity when exposed to a therapeutic treatment regimen. This is because susceptibility is a function of the chemistry of the drug, the genetic background of the patient and environmental factors such as patient demographics, disease and concomitant medications. In this study, we focused on two drugs: piperacillin and telaprevir as examples of idiosyncratic hypersensitivity reactions. Regarding piperacillin, we characterized the piperacillin reactive T-cells response of piperacillin hypersensitive patients with cystic fibrosis in terms of proliferation, cytokine secretion in addition to TCR-Vβ and chemokine receptor expression. Piperacillin-responsive CD4+ clones expressing a diverse TCR-Vβs proliferated and secreted Th1/Th2 cytokines in a dose-dependent manner. Clones expressed chemokine receptors consistent with a mixed Th1/Th2 response. Piperacillin responsive T-cells displaying a similar phenotype were also generated from naïve healthy volunteers using a dendritic cell-T-cell priming technique. However, TCR-Vβ expression was more restricted; high expression of TCR-Vβ 9, 13.2, 18 and 4 was detected. Piperacillin is β-lactam antibiotic that is known to covalently modify lysine residues on proteins such as human serum albumin. Therefore, we characterized the absolute levels of piperacillin protein binding in patients and in vitro culture and whether piperacillin albumin adducts activate patient T-cells. With the aid of mass spectrometry, we identified the main lysine residues that piperacillin modifies and synthesized a piperacillin modified peptide incorporating amino acid residues of albumin to establish a standard curve for quantification of binding. The level of modified Lys541 ranged from 2.7-4.7% in patients. Analysis of incubation medium from piperacillin-responsive clones revealed that a similar level of piperacillin-modified Lys541 in albumin was required for the stimulation of T-cells. Antigen presenting cells cultured with piperacillin for 24h also activated the clones with 2.8% Lys541 modification at this time-point. Piperacillin-albumin conjugates that had levels and epitopes identical to those detected in patients were synthesized and purified, and were shown to stimulate T cells in an antigen processing dependent manner. Piperacillin-albumin conjugate clones expressed a restricted pattern of TCR-Vβ with high expression of TCR-Vβ 9 in many clones. Telaprevir is antiviral drug used for the management of hepatitis C. We succeeded in generating clones responsive to a telaprevir metabolite (VRT-127394) using PBMCs priming technique and these clones were found to be 100% cross reactive with telaprevir. These clones were found to be activated via the direct binding of the drug (metabolite) to MHC molecules on antigen presenting cells. Most clones were CD4 T-cells and they displayed a restricted pattern of TCR-Vβ expression. Together, our results define in immunological, chemical and quantitative terms the drug immune receptor interactions that can drive a T-cell response. For β-lactam antibiotics, the levels of modification that activated T-cells in vitro are equivalent to the ones formed in patients.

Mortality from cardiovascular disease (CVD) is predicted to surpass that of infectious disease in sub-Saharan Africa (SSA) by 2030. HIV doubles the risk of CVD in high resource settings, but the contribution of HIV and immune activation to the risk of CVD in SSA is unknown. HIV-1-infected adults with CD4 < 100 cells/ul were recruited 2 weeks following initiation of anti-retroviral therapy (ART) within the REALITY trial (NCT01825031), along with healthy HIV-uninfected adults and followed for 44-weeks. Acute infections (malaria, TB, cryptococcal meningitis, pneumonia, gastroenteritis) were recorded. Pulse wave velocity (PWV) was assessed using the Vicorder system. Flow cytometry identified T-cell activation (HLA-DR/CD38+), exhaustion(PD1+) and senescence(CD57+) in all participants and circulating microparticles(CMPs) in 72 participants. Independent predictors of PWV were identified using linear regression. Backwards elimination was performed with an exit of p > 0.1 Variables with univariable p < 0.2 were included (spearman-rho or Wilcoxon ranksum). 279 HIV-infected adults had similar median(IQR) age [36(31-43) vs 35(3-41) years, p=0.4], but lower systolic BP [120(108-128) vs 128(114-134) mmHg, p < 0.01], BMI [20(18-21) vs 22(20-25) kg/m2, p < 0.01] and proportion of women [122(44%) vs 66(60%), p < 0.01] than 110 HIV uninfected adults. Following adjustment for confounders, HIV infection was associated with a 12%-increase in PWV (p < 0.01) at baseline, which remained at week 10 (14%-increase, p=0.02) but resolved by week 24. %CD4-PD1 and %CD8-PD1 were independently associated with PWV at baseline (fold change 2% and 3% per 10%increase, p=0.06 and 0.05 respectively). A decrease in %CD4-PD1 was associated with improvement in PWV by week 44 (rho 0.20, p=0.02). At baseline, median (IQR) CMPs were increased in HIV infection [5.1(2.0-18.0) x 10⁶ versus 0.4(0.2-6.0) x 10⁶, p < 0.00001)] and in high versus low immune activation [4.0(2.3-5.6) x 10⁶ versus 0.3(0.1-0.5) x 10⁶, p < 0.0001)]; and were strongly related to PWV (rho 0.42, p < 0.001). An acute infection during the study carried a 51% adjusted increase in %CD8 activated T cells at week 44 (p=0.02) and an increase in PWV at week 44 of 0.80m/s [versus -0.10m/s (p=0.01)] for HIV uninfected participants. These results strongly implicate HIV and immune activation in increased endothelial damage during the first 12 weeks of ART therapy. Improvement in PWV on ART and cotrimoxazole is associated with decreases in immune activation. HIV and co-infections may present modifiable CVD risk factors in low resource SSA setting.

Drug hypersensitivity reactions are an important problem for pharmaceutical industry, especially when reactions are observed in late phase clinical trials. Furthermore, management of patients with reactions leads to personal suffering and financial burden on the NHS. Reactions are almost impossible to predict as there is no simple relationship between the dose of drug administered and the development of hypersensitivity. In recent years, pharmacogenetic studies identified strong associations between the expression of specific HLA alleles and susceptibility to different forms of hypersensitivity, which would explain why only a small number of drug-exposed patients develop hypersensitivity. Studies utilizing peripheral blood mononuclear cells have detected drug-specific T-cells in patients with hypersensitivity, but not drug-exposed tolerant controls, suggesting that the adaptive immune system plays an important role in the disease pathogenesis. Despite this, there remains a need to further understand mechanisms as more detailed knowledge will assist the development of diagnostic and predictive assays. Data described herein utilized hypersensitivity reactions to the β-lactam antibiotic piperacillin as a model to investigate the phenotype and function of drug-specific T-cells in blood and skin, focusing specifically on the profile of cytokines secreted. PBMC from hypersensitive patients were activated to proliferate in vitro with piperacillin. T-cell clones responsive to the drug were generated from blood of all patients studied. CD4+ clones were stimulated to proliferate with piperacillin in a concentration-dependent manner and the proliferative response was associated with secretion of Th1 and Th2 cytokines alongside IL-22. In contrast, IL-17 was not secreted from piperacillin-specific clones. Piperacillin-specific CD4+ clones were also isolated from inflamed skins of 2 piperacillin hypersensitive patients. Activation of these clones was associated with secretion of Th1, Th2 cytokines and IL-22, in the absence of IL-17. Finally, CD4+ nitroso sulfamethoxazole (SMX-NO)-responsive clones were isolated from sulfamethoxazole hypersensitive patients and a similar cytokine secretion profile was observed, which suggests that IL-22 secretion might be a common feature of drug hypersensitivity. Evolution of T-cell culture methods means it is now possible to prime naïve T-cells from healthy donors to antigens, including drugs, which they have not previously been exposed to. Piperacillin and SMX-NO were found to prime naïve CD4+ and CD8+ T-cells from healthy donors, when the drug-derived antigens were presented in the context of autologous dendritic cells. Cloned drug-specific T-cells secreted a similar panel of cytokines to that observed with patient cells. Of particular importance was the detection of IL-22 in the absence of IL-17. The final component of the project utilized cloned T-cells with specificity for SMX-NO, piperacillin and flucloxacillin to explore mechanisms of drug-specific T-cell activation and potential cross-reactivity. Clones responsive against all 3 drugs were activated via a hapten mechanism involving (1) formation of protein adducts, (2) antigen processing and (3) presentation of derived peptides in an MHC-restricted manner. No cross-reactivity was observed with the 3 drugs. Collectively, these data showed that drug-haptens activate T-cells from patients with clinically divergent forms of hypersensitivity. T-cells secrete a similar profile of cytokines including the tissue-specific cytokine IL-22 following stimulation through the T-cell receptor. Furthermore, it is possible to prime naïve T-cells with a similar function against drugs using peripheral blood mononuclear cells from healthy donors.

Fish and shellfish allergy is a leading cause of anaphylaxis. There is limited data providing accurate information on the prevalence, clinical characteristics and management guidelines for this type of food allergy. Furthermore, it is recognised that food hypersensitivity negatively impacts on the health-related quality of life (HRQL) of sufferers when compared to healthy controls, as well as those suffering from other chronic diseases. However, little is known about the HRQL of adults with a fish and or shellfish allergy and how this may differ compared with other allergies. As this is a food allergy with an often later onset and one which is persistent throughout an individual’s life, it is of interest to examine the associated effect on HRQL in order to build upon the existing knowledge of this type of food allergy. A programme of research set out to first determine the prevalence as this underpins the knowledge base for food allergy. Next, in order to diagnose food allergy appropriately, an in-depth knowledge of the mechanisms and clinical presentations is needed. Once diagnosis is made, the best ways of managing the food allergy, taking into account the health-related quality of life of an individual, need to be known. This research was guided by a quantitative methodology and consisted of a systematic review of the prevalence of fish and shellfish allergy worldwide and a cross-sectional study of adult patients (≥ 16 years of age) with a record of fish and or shellfish allergy, from three NHS allergy outpatient clinics, as well as members of a patient support group (Anaphylaxis Campaign), which sought to describe the clinical characteristics and measure the HRQL of this sample. The main findings of this research were that very few studies have established the prevalence of fish and shellfish allergy using the gold standard, double-blind, placebo-controlled challenge criteria, with the majority instead relying on self-reported questionnaire-based methods. Where food challenges were used, the prevalence for fish allergy was found to be 0-0.3% and for shellfish allergy was 0-0.9%. It was shown that fish and shellfish allergy often co-exist, fish and shellfish allergic individuals frequently have other atopic conditions, and the clinical phenotype with regards to reactivity to vapours and tolerance of tinned fish varies between individuals. In addition, the associated HRQL of fish and shellfish allergic adults was found to be negatively impaired. This research has identified some novel findings, which have both clinical and research implications. There is a need for the development of clinical guidelines for the diagnosis of fish and shellfish allergy, to ensure consistent dietary and avoidance advice as well as provide management strategies to reduce the associated effects on the individual’s HRQL. A promising new treatment for food allergy, oral immunotherapy, needs to be investigated further for its effectiveness in treating fish and shellfish allergy as this would improve HRQL further.