I have explored the infection of human cells by the gammaretroviruses XMLV (xenotropic murine leukaemia virus) and FeLV (feline leukaemia virus). For XMLV the main aim was to assess the risks and consequences of contamination of human-mouse xenografts. For FeLV, the aim was to explore the susceptibility of human cells in vitro to assess the risks and identify the barriers to zoonotic infection in vivo. Xenografting of human cells to mice is used commonly in many disciplines of biomedical science. Infection of xenograft-derived cell lines has been reported as a common observation but it was unclear how many of these lines were infected due to in vitro cross-contamination rather than de novo infection in vivo. I conducted a prospective study and demonstrated that more than 40% xenografts passaged through BALB/c nude mice acquired XMLV. Xenografts passaged through NSG, another commonly used mouse strain for engraftment studies, did not yield replication-competent XMLV, although there was some evidence of activation of related replication-defective viruses. The source of XMLV in BALB/c mice appeared to be Bxv1, a locus encoding a replication competent virus which I showed was absent from NSG mice. I also showed that de novo isolated XMLV replicates to high copy number in MCF7 breast cancer and induces subtle changes in growth properties. Transcriptome analysis suggested that up regulation of EGR1 (early growth response gene 1) may be responsible for these growth effects. I also analysed susceptibility to XMLV infection in human cells and showed that while primary PBMCs are highly resistant, many cell lines can be infected. The Raji Burkitt’s lymphoma cell line was found to be highly susceptible to XMLV and displayed a much larger transcriptional response compared to MCF7, with marked up regulation of a series of markers of innate immunity. I examined the susceptibility of human cells to infection with FeLV B, the viral subtype which appears to be the most likely zoonotic agent. Cell lines of human origin were found to vary in susceptibility with no clear relationship to cell lineage. While some cell lines were completely susceptible for FeLV B, most showed limited virus replication and G to A hypermutation that correlated with the expression of APOBEC family members. Primary PBMCs and some leukaemia cell lines showed profound resistance to FeLV B infection at an early stage of replication and accumulated proviral DNA with only a few mutations. The mediator of this early block has not yet been identified but appears likely to be important in preventing cross-species spread of gammaretroviruses to the human population.

Apoptotic cells (AC) are able to modulate the immune system, dampening inflammation and triggering anti-inflammatory responses by various immune cells as a consequence of interaction and uptake. Rituximab (RTX) is an anti-CD20 monoclonal antibody used as a treatment in several autoimmune diseases, including rheumatoid arthritis (RA). Treatment results in B cell depletion, with B cell apoptosis known to contribute to RTX-mediated B cell death. However the simple removal of B cells from the system does not seem to account for all the beneficial effects of this biologic. We propose that RTX treatment in RA results in the re-establishment of temporary tolerance to the system, through an apoptotic B cell-dependent mechanism. Initial in vitro and in vivo investigations were undertaken to explore the validity of this hypothesis. The present work sought to examine the immunomodulatory capacity of apoptotic B cells and to determine whether the potential anti-inflammatory effects of apoptotic B cells are modulated by RTX, with both in vitro methods and an in vivo model of autoimmunity utilized in these studies. The results presented in this thesis demonstrate that apoptotic B cells have comparable effects on bone marrow derived macrophage (BMDM) phenotype and function in vitro as previously described AC from other cellular sources. Surprisingly, in the in vitro assay system used, viable cells had the same immunomodulatory effects on BMDM as AC, for all criteria investigated. Preliminary studies indicate this may be a promising avenue of inquiry, however further work is needed before a conclusion can be reached as to the relative level of involvement of apoptotic B cell-mediated tolerance in the improvement seen on RTX treatment in RA.

Neutrophils are efficient phagocytic cells that form the body’s first line defence against entry of foreign infectious microorganisms, but also contribute to tissue damage and non-infectious, chronic inflammation. Neutrophils contain a variety of separate classes of proteinase containing granules: primary (azurophilic) granules [containing serine proteinases elastase (NE), proteinase 3 (Pr3), and cathepsin G (Cat G)], secondary granules [containing metalloproteinase 8 (MMP-8)] and tertiary granules [containing metalloproteinase (MMP-9)]. These proteases are important molecules in immune and inflammatory processes. Sustained inflammation is associated with accumulation of these proteinases and is assumed to contribute to normal parenchymal damage and pathology. Regulation of proteolysis induced by these proteases is crucial to avoid self-induced damage. In the first part of my PhD thesis, I sought to examine the surface expression of Pr3 and CD177 (a surface receptor of Pr3) on neutrophils, in presence of physiological inhibitors of proteases (alpha-1-antitrypsin; AAT). I have demonstrated that membrane-bound Pr3 (mPr3) is still detectable on the surface of neutrophils in the presence of purified inhibitors and autologous serum as a source of physiological inhibitors. The interaction between CD177 and Pr3 was also examined by expressing CD177 cDNA on the surface of non-neutrophil cells (CHO cells), as was the ability of purified AAT and serum to interfere with these interactions. AAT was able to remove Pr3 from the surface of CD177-CHO cells, and similar results were observed for AAT removal of Pr3 binding to CD177-expressing neutrophils. In the second part of this thesis, I examined the change in neutrophil proteinases (Pr3, MMP-8 and -9) expression following neutrophil transmigration. In vitro transmigrated neutrophils showed no significant change in mPr3 expression compared to un-migrated neutrophils and both CD177-positive and CD177-negative subsets were able to migrate across HUVEC cells. In addition, intracellular Pr3 and MMP-8 also showed no change after in vitro transmigration. For comparison I also examined the CD177 and proteinase expression in salivary neutrophils (in vivo low inflammation transmigration) relative to matched volunteer blood neutrophils. In contrast to the in vitro data, I found only CD177-positive (with Pr3 bound to the surface) neutrophils present in the saliva of healthy individuals. I also found that levels of MMP-8 and MMP-9 were completely depleted in salivary neutrophils, relative to the matched levels in blood neutrophils from the same donor. In the third part of this thesis I used confocal microscopy to examine the intracellular distribution of neutrophil proteinases within different granule subsets. It was found that selected neutrophil proteinases co-localised with two different granule markers; showing their location in either azurophilic granules (CD63) or secondary granules (CD66b). However, the relationship with Pr3 revealed some discrepancies compared to previous reports. Some neutrophil proteins were co-localized both before and after neutrophil stimulation; whereas others were co-localisation before but not after stimulation. This suggested that degranulation of subsets of granules had occurred.

Magnetic Resonance Imaging (MRI) is a medical imaging technique which is especially sensitive to different soft tissues, producing a good contrast between them. It allows for in vivo visualisation of internal structures in detail and became an indispensable tool in diagnosing and monitoring the brain related diseases and pathologies. Amongst others, MRI can be used to measure random incoherent motion of water molecules, which in turn allows to infer structural information. One of the main challenges in processing and analysing four dimensional diffusion MRI images is low signal quality. To improve the signal quality, either denoising algorithm or angular and spatial regularisations are utilised. Regularisation method based on Laplace--Beltrami smoothing operator was successfully applied to diffusion signal. In this thesis, a new regularisation strength selection scheme for diffusion signal regularisation is introduced. A mathematical model of diffusion signal is used in Monte--Carlo simulations, and a regularisation strength that optimally reconstructs the diffusion signal is sought. The regularisation values found in this research show a different trend than the currently used L-curve analysis, and further improve reconstruction accuracy. Additionally, as an alternative to regularisation methods a backward elimination regression for spherical harmonics is proposed. Instead of using the regularisation term as a low-pass filter, the statistical t-test is classifying regression terms into reliable and corrupted. Four algorithms that use this information are further introduced. As the result, a selective filtering is constructed that retains the angular sharpness of the signal, while at the same time reducing corruptive effect of measurement noise. Finally, a statistical approach for estimating diffusion signal is investigated. Based on the physical properties of water diffusion a prior knowledge for the diffusion signal is constructed. The spherical harmonic transform is then formulated as a Bayesian regression problem. Diffusion signal reconstructed with the addition of such prior knowledge is accurate, noise resilient, and of high quality.

Neutrophil apoptosis is an important process contributing to the resolution of inflammation. This is because it allows the neutrophil membrane to remain intact preventing it’s potentially histotoxic contents from being released into the extra-cellular milieu, a process that can contribute to the exacerbation of many inflammatory disorders such as rheumatoid arthritis. When considering the life-span of a neutrophil and how it can be augmented by various inflammatory mediators to allow it to carry out its essential protective role in the body’s innate immune defences it is also important to consider how to terminate this process when the inflammatory insult has been dealt with or when the system goes awry. It is this information that we believe may hold the key to developing novel anti-inflammatory therapies. Through exploitation of the mechanisms controlling neutrophil apoptosis, it may be possible to selectively target these cells to enter apoptosis, and therefore help aid the process of resolution, especially if used in conjunction with treatments that up-regulate phagocytosis of apoptotic cells. This is important given that the main treatment for disorders of the inflammatory response are glucocorticoids, which whilst proven to be a powerful treatment for eosinophil based diseases such as asthma where they increase eosinophil apoptosis in conjunction with enhancing phagocytic clearance of apoptotic cells, glucocorticoids have been found to have the converse affect on neutrophils, actually serving to prolong their life-span potentially exacerbating the condition. Furthermore, it has been previously shown that the transcription factor nuclear factor kappa B (NF-κB) plays a pivotal role in neutrophil apoptosis, becoming activated by inflammatory agents such as lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-α). NF-κB activation results in the transcription of many pro-inflammatory agents and anti-apoptotic proteins such as X-linked inhibitor of apoptosis (X-IAP) increasing the life-span of the neutrophil. Interestingly, it has also been demonstrated that key neutrophil survival proteins such as myeloid cell leukemia-1 (Mcl-1) are not directly regulated by NF-κB activation. Therefore it is because of the aforementioned reasons that I have chosen to investigate further neutrophil apoptosis including the role played by NF-κB. Thus, I have investigated the hypothesis that NF-κB-dependent and independent survival proteins critically regulate the rates of neutrophil apoptosis and that newly identified pro-apoptotic agents such as the cyclin-dependent kinase (CDK) inhibitor, R-roscovitine interferes with the expression of such survival proteins. It has been previously found by myself and others in our laboratory during the course of this thesis that cyclin dependent kinase inhibitor (CDKi) drugs such as R-roscovitine are powerful novel anti-inflammatory agents with the ability to up-regulate rates of neutrophil apoptosis in vitro and influence the resolution of neutrophilic inflammation in vivo. Whilst the exact mechanism of CDK inhibitor drugs on neutrophil apoptosis remains elusive, work shown in this thesis demonstrates that R-roscovitine has the ability to over-ride powerful anti-apoptotic signals from pro-inflammatory agents such as granulocyte-macrophage colony stimulating factor (GM-CSF) and LPS causing the neutrophils to enter apoptosis. Furthermore, it has been found that R-roscovitine causes a decrease in levels of the antiapoptotic protein Mcl-1 in as little as 2h and that it prevents the maintenance / protective effect that GM-CSF has on the Mcl-1 protein levels. In addition R-roscovitine may also reduce levels of the NF-κB regulated protein X-IAP. The effect of R-roscovitine on X-IAP was investigated further using an X-IAP HIV-tat construct, though results from this remain inconclusive. This is because although the X-IAP construct appeared to be extending neutrophil longevity, it was discovered that LPS contamination of the construct had occurred which could therefore pose an alternative explanation for the increase in neutrophil life-span. As X-IAP, TNF-α and LPS are all regulated by NF-κB and given that NF-κB is already known to be a key player in neutrophil biology, the effects of R-roscovitine on this important transcription factor were investigated. It was discovered that R-roscovitine does not directly activate NF-κB, since this CDK inhibitor drug does not cause degradation and loss of the cytoplasmic inhibitor of NF-κB, IκBα. This lack of NF-κB activation was confirmed since R-roscovitine did not mobilize the NF-κB subunit, p65, from the cytoplasm to the nucleus. Furthermore, R-roscovitine (unlike the NF-κB inhibitor gliotoxin) does not interfere with the ability of LPS or TNF-α to activate NF-κB. Therefore by R-roscovitine to induce apoptosis, although this does not rule out the involvement of NF-κB at a later stage. When considering a reagent for possible use as a novel anti-inflammatory agent I think it is important to assess what effects it has on the activation state of the neutrophil. Therefore the effects of R-roscovitine on the activation markers CD62L, CD11b and shape change were assessed. It was found that R-roscovitine alone did not cause any significant neutrophil activation as measured using the parameters stated above. Importantly, it was also found that R-roscovitine did not interfere with the activation states induced by the inflammatory mediators GM-CSF, LPS, TNF-α or leukotriene B4 (LTB4). Another important consideration is the effect of R-roscovitine on the removal of apoptotic cells by macrophage phagocytosis. Results demonstrated that pre-treatment of macrophages with R-roscovitine did not augment their uptake of apoptotic neutrophils. In addition Rroscovitine did not detrimentally affect the increase in phagocytosis that results from macrophage treatment with the synthetic glucocorticoid dexamethasone. The data presented in this thesis suggest that CDK inhibitor drugs such as R-roscovitine are novel powerful pro-apoptotic agents for neutrophils with the ability to over-ride antiapoptotic signals from multiple pro-inflammatory mediators. It has been discovered that Rroscovitine causes a reduction in one of the neutrophil’s most prominent anti-apoptotic proteins (Mcl-1) whilst not altering the activation state of the neutrophil and furthermore it does not interfere with the uptake of apoptotic neutrophils by macrophages or result in any alteration to the increase in phagocytosis caused by treatment with dexamethasone. In conclusion, CDK inhibitor drugs such as R-roscovitine have the potential to be promising candidates for novel anti-inflammatory agents with the ability to selectively target neutrophil apoptosis whilst not interfering with steroid induced up-regulation of phagocytosis, therefore allowing a two pronged attack to help treat neutrophil based inflammatory disorders.

No description available.

No description available.

No description available.

Having passed all pre-clinical safety testing, the superagonistic anti -CD28 therapeutic monoclonal antibody (mAb ) TGN 1412, intended for the treatment of rheumatoid arthritis and B-cell chronic lymphocytic leukaemia, was approved by German and UK regulatory authorities for first-in-man Phase One clinical trial. Shortly after infusion, all six healthy trial volunteers suffered unexpected and profound systemic pro-inflammatory cytokine release, later termed a 'cytokine storm,' causing multi-organ failure. This unexpected and near fatal cytokine release syndrome (CRS) publically highlighted the failure of current pre-clinical safety testing procedures, emphasising an urgent need for novel cytokine release assays (CRAs) capable of predicting adverse properties of therapeutic mAbs. A wet coat mAb immobilisation approach, developed here, has proven predictive of clinical outcome and would have anticipated TGN1412 immunotoxicity in man. This approach IS now being widely applied by the pharmaceutical industry and contract research organisations (CROs). Comparative studies, testing TGN 1412 against a panel , of therapeutic mAbs, identified a unique mechanism of TGNl412-driven cytokine release. Substantial concentrations of the cytokine lL-2 were subsequently found to be a hallmark for the cytokine storm observed in-vivo, signifYing IL-2 as a TGN1412-like response biomarker. Multiple pro-inflammatory cytokine release was also shown to be principally effector memory T cell (T EM) derived in man. Human and macaque comparative immunophenotyping crucially identified macaque T EM cells as lacking CD28 expression, explaining pre-clinical animal model testing failures. A more physiologically relevant aqueous phase co-culture assay using monocyte-derived dendritic cells is also shown capable of eliciting a TGN1412-like cytokine release profile equivalent to that detected in-vivo and in-vitro using wet coat immobilisation; implying presentation in-vivo likely involved dendritic cells. This thesis describes the most likely mechanisms of action responsible for TGN1412 immunotoxicology in man and provides a plausible explanation for the pre-clinical safety testing failures, findings vital to the fields of immunomodulatory therapeutic mAb development and immunotoxicology.

Background: Interleukin (IL)-33 is a newly identified member of the IL-1 cytokine family. Multiple cell types are able to produce or respond to IL-33, including non-haematopoietic structural cells, innate and adaptive immune cells. The biological activity of IL-33 was initially described as being associated with the promotion of type 2 immune responses which were characterized by the induction of CD4+ T helper (Th) 2 cells. For example, exogenous administration of IL-33 in experimental models caused pathological changes in mucosal tissues such as the lung and gastrointestinal tracts; early studies reported that IL-33 can activate Th2 cells, mast cells, eosinophils or basophils to produce type 2 cytokines such as IL-4, IL-5, and IL-13. This was associated with pathological changes reminiscent of asthma, fibrosis and ulcerative colitis. Recently, a newly recognised cell population which was inducible by IL-33 and referred to as ‘type 2 innate lymphoid cells’ was identified and these were thought to be important for initiating type 2 immunity. However, the underlying mechanism by which IL-33 was involved in the inflammation and remodelling of diseases of the respiratory and gastrointestinal tracts remains to be fully understood. Hypothesis: My hypothesis is that IL-33 is induced in the gut and lung mucosa by inflammatory signals and mediates both early inflammation and late fibrosis by amplifying the innate immune response. Aims: To address this hypothesis I set out the following aims: i) to investigate the induction and effect of IL-33 via its receptor ST2 on cellular pathogenic pathways in the development of lung fibrosis (chapter 3); ii) to unravel the mechanism by which IL-33 promotes lung fibrosis (chapter 4); iii) to understand the involvement of the IL-33/ST2 pathway in ulcerative colitis (chapter 5). Methods: To address these aims I used two experimental murine models. To investigate the effect of IL-33 in the fibrosis phase of airway mucosal inflammation I used the bleomycin (BLM)-induced lung fibrosis (chapters 3 and 4). To investigate the effect of IL-33 in the acute phase of mucosal inflammation in the gut, I used dextran sulphate sodium (DSS)-induced colitis (chapter 5). These disease models are widely accepted for laboratory investigation and I acknowledge that they do not reflect the full complexity of the human conditions. However they are extremely useful for hypothesis generation. Results: My results showed i) that IL-33 promotes the pathogenesis of bleomycin-induced lung fibrosis. This was indicated by IL-33 being constitutively expressed in lung epithelial cells but induced in macrophages by bleomycin. The specificity of this response was confirmed by using either ST2-deficient mice, or neutralising anti-IL-33 antibody treatment, which both attenuated lung fibrosis (chapter 3). ii) that IL-33 promotes the initiation and progression of pulmonary fibrosis by recruiting and directing inflammatory cell function, and enhancing the production of pro-fibrogenic cytokines IL-13 and TGF-β in an ILC2- and M2-macrophages (chapter 4). iii) that IL-33 signalling via ST2 induces an IL-4-dependent immune response that is pathogenic in the early stage of ulcerative colitis. I found that the clinical indices of DSS-induced experimental UC, diarrhoea and colon inflammation, were respectively impaired in ST2 knockout mice and exacerbated in WT mice by treatment with exogenous recombinant IL-33. These were associated respectively with reduced and enhanced expression of inflammatory chemokines and angiogenic cytokines in vivo. The exacerbation effect of treatment with recombinant IL-33 on DSS-induced acute colitis was abolished in IL-4 knockout mice (chapter 5). Conclusion and prospect: Together, my results demonstrated that IL-33 expression was up-regulated in the lung and colon epithelium/endothelium in experimental BLM-induced fibrosis and DSS-induced colitis respectively. Furthermore, IL-33 exacerbated both diseases through recruiting and activating inflammatory cells and increasing the production of type 2 cytokines. Finally, I discussed the pathological mechanisms of IL-33 in mucosal tissue based on my results and the current literature. I concluded that this insight into IL-33 biology is informative of a new potential pathogenic pathway and might be a useful biomarker of disease and that targeting IL-33 may provide a new biological therapeutic approach in these disorders (chapter 6).