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The interaction between extracellular matrix (ECM) and hepatic stellate cells (HSC) is the major event in liver fibrosis and recovery. The integrins, as a family of cell surface adhesion molecules, mediate adhesion of HSC to ECM and regulate a variety of cellular functions. In this thesis, I have examined the hypothesis that the ECM regulated HSC phenotype in liver fibrosis and recovery is mediated by integrins; and that integrin αvβ₃ plays a role in HSC activation, proliferation and apoptosis. These were examined by 1) investigating the correlations of ECM remodelling with HSC activation, proliferation or apoptosis in-vivo and in-vitro; 2) evaluating the regulation of integrin expression and adhesion affinities by different substrata in-vitro; 3) investigating the function of integrin αvβ₃ on extracellular and intracellular signalling mechanisms that regulate cell survival or apoptosis. To do so, rat CCl₄ liver fibrosis/recovery models and human liver biopsies were employed. In-vitro substrata-HSC cell models, integrin αvβ₃ agonists (vitronectin, fibronectin, collagen I), and antagonists (matrigel, echistatin and integrin specific antibodies), and various biochemical techniques were used. In-vivo studies demonstrated that as fibrosis progressed in liver, collagen I, MMP-2, MT1-MMP, TIMP-1, CTGF, TGF-b1 expression were increased and collagenase activity was suppressed. As spontaneous recovery occurred, the expression of these fibrotic effectors declined and returned to normal, whereas collagenase activity was increased to normal. The change of a-SMA expression, a HSC activation marker, was associated with fibrogenesis or spontaneous recovery, indicating HSC as a key cell in liver fibrosis. In-vitro studies using substrata-HSC cell models demonstrated that substrata in ECM regulate HSC activation, proliferation, arrest and apoptosis, which were associated with cell morphology change and altered integrin expression. This study is first reporting that integrin αvβ₃ is expressed by activated HSC and involved in HSC proliferation and survival in a fibrotic microenvironment.

Allergic reactions involve the explosive release of inflammatory mediators from mast cells including the proteases tryptase, carboxypeptidase A3 (CPA3) and chymase; and there have been suggestions that dipeptidyl peptidase I (DPPI) could be secreted as well. Our aim has been to investigate the levels of mast cell proteases and a panel of proinflammatory cytokines in serum during allergic reactions provoked by drugs or food to identify and replicate distinct clinico-immunological endotypes of allergic reactions using topological data analysis. In addition, we have investigated factors that can modulate the release and expression of mast cell mediators and their subcellular localization. Patients who had suffered drug- or food-induced allergic reactions were recruited from two separate centers (Southampton, UK and Doha, Qatar). Healthy individuals who had no history of allergic diseases were recruited to serve as controls. Detailed clinical assessment and measurements of mast cell proteases and pro-inflammatory cytokines were performed. The clinical and biochemical parameters were analyzed using topological data analysis. In addition, cells of the LAD2 mast cell line were employed to investigate the effect of bacterial infection on the release and expression of mast cell mediators. This cell line was also employed to investigate the subcellular localization of mast cell proteases through the application of immunocytochemical analysis. There were higher levels of CPA3 in baseline serum samples from patients with more severe historical allergic reactions than in those from healthy individuals (p< 0.0001). CPA3 levels of ≥ 6.5 ng/ml were associated with severe allergic reactions in patients with drug allergies (AUC: 0.61, 95% CI: 0.51-0.71, p= 0.048), and levels of ≥ 3 ng/ml in those with food allergies (AUC: 0.74, 95% CI: 0.62-0.86, p= 0.002). Higher serum levels of CPA3 were seen also in patients with one or more concomitant atopic conditions (including atopic dermatitis, hay fever and asthma) compared to those in healthy subjects (p< 0.0001). A strong association was found between the severity of allergic reactions and concomitant atopic illnesses (p< 0.0001).Topological data analysis allowed identification of four novel clinico-immunological clusters those with: (I) high CPA3 and IL-13 levels, females, drug reactions, more severe historical reactions; (II) high CPA3, low IL-13, males, food reactions, more severe historical reactions; (III) low CPA3 and IL-13 levels, young, females, food reactions, mild historical reactions; and (IV) low CPA3, high IL-13, females, drug reactions, mild historical reactions. These clusters were replicated in both geographical cohorts. In separate studies with cultured LAD2 cells, it was found that S. aureus infection could inhibit IgE- and non-IgE-dependent mast cell degranulation and down-regulate gene expression for major cytokines involved in anti-bacterial defense mechanisms (including that for TNFα, IL-8, and IL-1β). Bacterial exposure also altered the phosphorylation of protein kinases downstream of FcRI engagement. At the subcellular level, DPPI was observed inside the granules of mast cells and colocalized with tryptase, CPA3 and chymase. Our finding that DPPI may be present in association with other mast cell proteases within the granules suggests that DPPI is secreted upon mast cell stimulation and may have extracellular roles in immune modulation. DPPI could thus represent a novel marker for mast cell activation that can enhance the diagnosis of allergic reactions when measured in combination with tryptase and CPA3. The potential for bacterial infection to interfere with mast cell responses could reduce the susceptibility to allergic reactions and as the mechanisms involved deserve consideration as a novel therapeutic approach to prevent development of severe reactions. Serum levels of CPA3 have the potential to predict the severity of allergic reactions to drugs or food. Identification of four multidimensional endotypes underlines the connection between CPA3 and IL-13 levels and their association with clinical features of patients who have drug or food allergies. Their use as biomarkers can help to identify those at particular risk of allergic reactions and allow optimal interventions to be undertaken.

Allergic drug reactions to drugs are becoming more frequent throughout the world and are among the most common and serious form of immuno-pathological process in modern clinical medicine. These reactions can lead to life-threatening anaphylaxis which can occur within minutes of receiving the drug. Symptoms provoked by the explosive release of mediators from mast cells and basophils release may include itchiness, angioedema, difficulty breathing and circulatory collapse. There are few reliable laboratory tests available to identify those with drug hypersensitivity who may be at risk of anaphylactic shock, or even to establish if such a reaction has occurred. Confirmation of drug hypersensitivity by skin testing or oral challenge can put the patient at risk of serious reaction, and as such are expensive and performed at few centres. The aim of the study was to seek to develop new tests for drug allergy based on assessment of mast cell or basophil activation either in vitro or in vivo. Patients were recruited who were suspected of having suffered an allergic reaction to medicine and who were undergoing skin testing or oral drug challenge. Blood was collected before and following the challenge, the nature of symptoms provoked were recorded, and clinical history and demographic data obtained. The activation of basophils in vitro in response to the drug was assessed by measurement of histamine release by immunoassay or up-regulation of CD63 by flow cytometry. In parallel studies, cells of a rat basophil leukaemia cell line (RBL-703-21) that had been transfected with the α chain of the human IgE receptor, were passively sensitised with serum and challenged with the drug implicated. The release of β-hexosaminidase in cell supernatants was measured to determine the degree of drug-induced cell activation. Mast cell tryptase was successfully purified from human lung tissue, and preliminary attempts made to optimise the detection of the basophil product basogranulin by dot blotting with specific antibodies BB1 and BB5, but time did not allow measurement of either of these markers of allergic reaction in the patients studied. Skin weals were provoked in some 40% of patients following skin testing, though other symptoms were frequently recorded in the presence or even absence of a weal. These included hives, post-nasal drip, abdominal pain, vomiting, cough, shortness of breath, wheeze and stridor, and there was considerable heterogeneity in clinical responses. In patients with a positive test outcome on skin or oral challenge, raised levels of serum IgE specific to that drug were commonly observed (where the test was available). Increased expression of CD63 was provoked by in vitro challenge of basophils in a minority of patients who responded on in vivo challenge, but no histamine release into cell supernatants was detected where this was examined. Drug challenge of RBL-703-21 cells passively sensitised with patient serum resulted in the release of only a small quantities of β-hexosaminidase, and no evidence was found for direct activation of cells by the drugs themselves. There was, however, significantly higher net release of β-hexosaminidase from patients with positive skin reactions to the neuromuscular blocking agent rocuronium than in those who did not respond on skin challenge. The reliable diagnosis of drug hypersensitivity remains a formidable challenge, and there is no ready replacement of skin or oral challenge of patients with drugs suspected of causing a reaction. The present study indicates some of the difficulties as well as ways forward in developing new laboratory tests. In vitro challenge of blood basophils or passively sensitised cells of mast cell or basophil-like cell lines hold some promise, but it will be crucial for the sensitivity of such procedures to be increased and optimised with a range of drugs. Assays for mast cell or basophil products in the circulation of patients experiencing reactions should also be valuable as diagnostic tools.