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No description available.

No description available.

No description available.

Macrophages as professional phagocytes have been considered as prominent participants in the response to acute infection and play a significant role in preventing infecting bacteria multiplying and damaging the host environment. Macrophages have complex mechanisms equipped with specialised receptors to recognise their targets. Upon internalisation of receptor-bacterial complex, macrophages initiate killing of the ingested microbes partly through the generation of free radicals species. However, the excessive production of reactive oxygen species (ROS) and nitric oxide (NO) during phagocytic activity may also contribute to the inflammatory process which although is beneficial in killing bacteria may result in deleterious consequences, especially following an interaction of NO with superoxide radicals to form reactive nitrogen species such as peroxynitrite. Probiotics are beneficial bacteria which have been shown to activate host innate immune response by influencing phagocytic activity of macrophages. This immunomodulatory effect has also been shown due to release of a number of soluble proteins by probiotic bacteria in the culture medium. However, the mechanism of regulation is not clear. In this study, we have therefore evaluated the influence of cell free Lactobacillus rhamnosus GG conditioned medium (LGG-CM) on macrophage phagocytosis and also on ROS and NO production by the J774 macrophage cell line, and further evaluated the acute effect of LGG-CM on NO and ROS production during the process of macrophage phagocytosis of E.coli in real time. Murine macrophage J774 cells were used in the study as these cells are readily activated by bacterial lipopolysaccharide (LPS) and produce NO. Gentamicin protection assay was employed in order to investigate the effect of LGG-CM on the ingestion and digestion phases of macrophages phagocytosis of E. coli HfrC. The cells were exposed to different dilutions of LGG-CM in the absence and presence of bacterial LPS, E. coli and the inhibitors or scavengers for ROS and NO. The J774 macrophages were loaded with free radicals sensitive fluorescent dyes, namely, H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Acute free radical production was measured using a fluorescence microplate reader and changes were analysed by cumulative sum (CuSum) calculations. The fluorescence measurements were taken every two minutes for the first 60 minutes to monitor free radicals production during the ingestion period and from 60 minutes to 280 minutes to monitor free radicals production during the bacterial digestion period. The fluorescence was measured at 485 nm excitation and 528 nm emissions. The LGG-CM did not have any toxic effect on E. coli or macrophage viability, as determined by antibacterial studies and cell proliferation assays. The LGG-CM treated macrophages ingested significantly less bacteria both at 30 minutes and 60 minutes of incubation (p< 0.05). However, only higher concentrations of LGG-CM (75 % LGG-CM and 100 % LGG-CM) significantly increased the bacterial digestion rate of E. coli by the macrophages (p< 0.01). The LPS on its own did not have any effect on macrophage phagocytosis. The LGG-CM mediated enhanced bacterial digestion was inhibited by ROS and NO inhibitors and ROS scavengers. The LGG-CM at higher concentration induced increase expression of NADPH oxidase subunits (p47 phox and gp91 phox) at 6 hours (p< 0.01). A low concentration of LGG-CM (10 % LGG-CM) or LPS did not cause any significant change in the basal levels of ROS or NO production. In contrast, a high concentration of ii LGG-CM significantly enhanced ROS generation but also significantly reduced the NO level in both the ingestion and digestion phases of phagocytosis. These findings are novel and suggest for the first time that probiotics may release factors in culture medium which enhances macrophage digestion capability. The LGG-CM also has the capability to increase ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing the production of NO. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance especially in several pathological conditions associated with gut inflammation or bacterial infection and may prove useful in improving intestinal homeostasis.

As part of the diagnosis of liver disease, a Computerised Tomography (CT) scan is taken of the patient, which the clinician then uses for assistance in determining the presence and extent of the disease. This thesis presents the background, methodology, results and future work of a project that employs automated methods to segment liver tissue. The clinical motivation behind this work is the desire to facilitate the diagnosis of liver disease such as cirrhosis or cancer, assist in volume determination for liver transplantation, and possibly assist in measuring the effect of any treatment given to the liver. Previous attempts at automatic segmentation of liver tissue have relied on 2D, low-level segmentation techniques, such as thresholding and mathematical morphology, to obtain the basic liver structure. The derived boundary can then be smoothed or refined using more advanced methods. The 2D results presented in this thesis improve greatly on this previous work by using a topology adaptive active contour model to accurately segment liver tissue from CT images. The use of conventional snakes for liver segmentation is difficult due to the presence of other organs closely surrounding the liver this new technique avoids this problem by adding an inflationary force to the basic snake equation, and initialising the snake inside the liver. The concepts underlying the 2D technique are extended to 3D, and results of full 3D segmentation of the liver are presented. The 3D technique makes use of an inflationary active surface model which is adaptively reparameterised, according to its size and local curvature, in order that it may more accurately segment the organ. Statistical analysis of the accuracy of the segmentation is presented for 18 healthy liver datasets, and results of the segmentation of unhealthy livers are also shown. The novel work developed during the course of this project has possibilities for use in other areas of medical imaging research, for example the segmentation of internal liver structures, and the segmentation and classification of unhealthy tissue. The possibilities of this future work are discussed towards the end of the report.

Recognition of membrane-bound ligands is a fundamental event that determines the fate of lymphocytes during immune responses. Extensive studies have been performed in order to understand the recognition of soluble ligands, however very little is known of the parameters that determine the interaction of membrane-bound ligand/receptors. In my PhD thesis 1 have studied how lymphocytes interact with membrane-bound ligands. I initially characterise how B cells recognise membrane-ligands by expressing CFP-tagged antigens on the surface of target cells. To further dissect this process, I have set-up a system of artificial lipid bilayers that allows a quantitative analysis. Fluorescently labelled antigen molecules of varying affinities are displayed at a range of densities on glass-supported bilayers. The interaction of transgenic B cells with them can then be followed by different microscopy techniques in real time. 1 have described a dynamic cellular response in the early stages of the recognition process in which B cells spread and contract on the antigen bearing membranes to collect the bound molecules in a central cluster to later extract it. This response is triggered by signals delivered through the BCR and is an active process guided by actin polymerisation. The extent of the spreading response is dependent on both the antigen density and its affinity for the BCR and it determines the amount of antigen accumulated. I have also set-up a technique to analyse at the molecular level the differential dynamics of reorganisation of key co-receptor molecules involved in triggering B cell activation. Finally, I extended the bilayers system to characterise the interaction of human NK T cells with a set of specific ligands of different affinities for the T cell receptor (TCR). I have determined the thresholds for the formation of the immunological synapse. In summary, 1 have characterised the early events triggered upon membrane-antigen recognition and elucidated a novel mechanism by which B cells are able to gather antigen and therefore perform their biological function.

Neutrophil chemotaxis to IL-8 and Gro-α was reduced in patients with acute and chronic liver failure either due to alcoholic liver disease or hepatitis C compared with controls. This impairment was correlated with the severity of the disease. A partial correction in chemotaxis of patients’ neutrophils was observed after cross incubation with the control sera and vice versa using control neutrophils and patients’ sera. Neutrophil chemotaxis in patients with chronic liver failure is further reduced 2 hours after oral administration of an amino acid solution, which simulates human blood. Neutrophils isolated from the portal venous blood had similar chemotactic defects to neutrophils isolated from peripheral venous blood. However, chemotaxis was significantly reduced in neutrophils isolated from hepatic venous blood compared to neutrophils isolated from portal or peripheral blood. In cross over studies, portal neutrophil chemotaxis was significantly reduced after incubation with hepatic venous serum and vice versa. The CXC chemokines IL-8, IFN-γ-inducible protein (IP-10), and Monokine Induced by Interferon-γg (mig) were significantly elevated in patients with both acute and chronic liver failure compared with controls. There were no significant difference in neutrophil expression of both CXCR1 and CXCR2 chemokine receptors in patients with either acute or chronic liver failure compared with the controls. Neutrophil chemotaxis to CXC chemokines is impaired in patients with either acute or chronic liver failure. There is a functional defect in both CXCR1 and CXCR2 chemokine receptors. Impaired neutrophil chemotaxis may combine to the increased risk of infection in these patients. This impairment in chemotaxis may be due to CXC receptor desensitization caused by circulating humeral factor/s plus the intrinsic defect of the neutrophils.

Aim: when imaging patients for x-ray examinations Diagnostic Radiographers should position the patient so that bones, joints and soft tissues can be clearly visualised. In order to achieve this a widely accepted set of positioning criteria have been developed for each anatomical region. In the trauma setting the radiographer must either move the injured body part sufficiently to meet the criteria or manipulate the imaging equipment to achieve a similar representation of the anatomy. This difference between the presenting position of the patient and the imaging position required presents the radiographer with an ill-defined problem which employs careful management to minimise patient discomfort, avoid risk of injury and optimise image quality for diagnosis. Little is known of radiographer problem solving in the clinical setting. This research uses focussed ethnography to investigate how the radiographer achieves appropriate positioning of the patient through the application of problem solving. Method: a focussed ethnographic study was undertaken in the clinical setting at two hospital sites. Sixty three observations of trauma imaging examinations were undertaken followed by semi structured interviews with the practitioners. The data were analysed thematically following a structure recommended for focussed ethnography. Results: the findings of this unique study demonstrated a multi-stage assessment process used to evaluate the patients’ injury and ability to co-operate with the examination. In light of the assessment the conduct of the examination varied with the degree of complexity of the examination and a measure of this complexity was developed to illustrate this. Findings demonstrated that in agreement with known models of practice the level of cognition required moved from subconscious to conscious as the complexity of the examination increased it was also found that radiographers recognised the importance of experience in managing imaging examinations. Opportunities for re-design of the examination request card were also identified to aid communication between the referrer and the radiographer and assist in the radiographers’ assessment of the patient. Areas for further research are also suggested.

The αβT cell receptor (TCR) orchestrates immunity through the recognition of peptides, derived from degraded proteins, presented on major histocompatibility complex (MHC) molecules. The remarkable ability of the receptor to respond to a vast plethora of antigens is driven by V(D)J recombination, a process which generates a highly diverse TCR repertoire by somatic gene rearrangement of coding DNA. TCR diversity is confined to three short hairpin loops on each TCR chain, called the complementarity determining region (CDR), which form the antigen-binding site. The germline-encoded CDR1 and CDR2 loops predominantly contact MHC, whereas the hypervariable CDR3 are non-germline and primarily bind to the MHC-bound peptide. In this study, we developed a novel in vivo mutagenesis approach which redirects somatic gene rearrangement using V(D)J recombination machinery to diversify and optimise TCR binding. This approach involves embedding a gene recombination cassette into the peptide-binding CDR3β region of established TCRs. A retrogenic system was employed to facilitate the in vivo processes necessary for gene rearrangement and thymic selection. We demonstrate that the recombination cassette can successfully induce gene rearrangement and introduce variation to the targeted CDR3β site. Thymocytes expressing the diversified TCRs can be selected on MHC and develop into functional peripheral T cells. Subsequent exposure to cognate ligands also allowed us to identify optimised and 'immunodominant' TCRs. In addition, we produced a novel chimeric TCR chain which comprises Vα and Cβ domains. This TCR chain forms a heterodimer with endogenous TCRα chains to form a unique Vα-Vα antigen-binding surface. Thymocytes expressing this novel form of αβTCR were able to engage efficiently with both MHC classes and develop normally into functional T cells typical of a conventional repertoire. Collectively, these findings suggest that the germline CDR loops are not essential for mediating MHC recognition during MHC-restricted T cell development and function.