No description available.

No description available.

No description available.

No description available.

Whether to assess the functionality of equipment or as a determinate for the accuracy of assays, reference standards are essential for the purposes of standardisation and validation. The ELISPOT assay, developed over thirty years ago, has emerged as a leading immunological assay in the development of novel vaccines for the assessment of efficacy. However, with its widespread use, there is a growing demand for a greater level of standardisation across different laboratories. One of the major difficulties in achieving this goal has been the lack of definitive reference standards. This is partly due to the ex vivo nature of the assay, which relies on cells being placed directly into the wells. Thus, the aim of this thesis was to produce an artificial reference standard using liposomes, for use within the assay. Liposomes are spherical bilayer vesicles with an enclosed aqueous compartment and therefore are models for biological membranes. Initial work examined pre-design considerations in order to produce an optimal formulation that would closely mimic the action of the cells ordinarily placed on the assay. Recognition of the structural differences between liposomes and cells led to the formulation of liposomes with increased density. This was achieved by using a synthesised cholesterol analogue. By incorporating this cholesterol analogue in liposomes, increased sedimentation rates were observed within the first few hours. The optimal liposome formulation from these studies was composed of 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and brominated cholesterol (Brchol) at a 16:4:12 µMol ratio, based on a significantly higher (p<0.01) sedimentation (as determined by a percentage transmission of 59 ± 5.9 % compared to the control formulation at 29 ± 12 % after four hours). By considering a range of liposome formulations ‘proof of principle’ for using liposomes as ELISPOT reference standards was shown; recombinant IFN? cytokine was successfully entrapped within vesicles of different lipid compositions, which were able to promote spot formation within the ELISPOT assay. Using optimised liposome formulations composed of phosphatidylcholine with or without cholesterol (16 µMol total lipid) further development was undertaken to produce an optimised, scalable protocol for the production of liposomes as reference standards. A linear increase in spot number by the manipulation of cytokine concentration and/or lipid concentrations was not possible, potentially due to the saturation that occurred within the base of wells. Investigations into storage of the formulations demonstrated the feasibility of freezing and lyophilisation with disaccharide cryoprotectants, but also highlighted the need for further protocol optimisation to achieve a robust reference standard upon storage. Finally, the transfer of small-scale production to a medium lab-scale batch (40 mL) demonstrated this was feasible within the laboratory using the optimised protocol.

Removal of dead or diseased cells is crucial feature of apoptosis for managing many biological processes such as tissue remodelling, tissue homeostasis and resolution and control of immune responses throughout life. Tissue transglutaminase (TG2) is a protein crosslinking enzyme that has been implicated in apoptotic cell clearance but also mediates many important cell functions including cell adhesion, migration and monocyte-macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4, ß1 and ß3 integrin. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise extracellular role of TG2 in apoptotic cell clearance remains ill-defined. This thesis addresses macrophage TG2 in cell corpse clearance. TG2 expression (cytosolic and cell surface) in human macrophages was revealed and data demonstrate that loss of TG2 activity through the use of inhibitors of function, including cellimpermeable inhibitors significantly inhibit the ability of macrophages to clear apoptotic cells (AC). This includes reduced macrophage recruitment to and binding of apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus it defines for the first time a role for TG2 activity at the cell surface of human macrophages in multiple stages of AC clearance and proposed that TG2, in association with heparan sulphates, may exert its effect on AC clearance via crosslinking of CD44.

Many key immune pathway genes in vertebrates have conserved Drosophila counterparts; therefore, the fly has become an influential model for the study of innate immunity. The Drosophila immune response consists of a humoral response; characterised by the production of anti-microbial peptides and cytokines and a cellular response; mediated by plasmatocytes, the fly analogue cell to the mammalian macrophage. Plasmatocytes have been studied in detail during the embryonic and larval stages of development however, plasmatocyte homeostasis and function in adult flies remains poorly understood. This thesis firstly characterises plasmatocytes in adult Drosophila in terms of their numbers, expression markers and proliferative potential. Secondly the plasmatocyte response to dietary lipids is studied in adult flies, as a model for the mammalian macrophage response to dietary lipids. Excessive dietary fat consumption in humans is associated with an increased incidence of high fat diet related diseases, including type II diabetes and atherosclerosis. Numerous aspects of high fat diet induced disease pathology in mammals are attributed to lipid induced inflammation, in which macrophage mediated lipid scavenging and/or sensing is believed to be of central importance. We found that as dietary fat content increased; flies exhibited a rise in triglyceride and glucose levels, whilst simultaneously up regulating expression of the IL-6-like JAK-STAT cytokine unpaired3 (upd3), in addition, plasmatocytes adopted a lipid laden foam cell-like morphology. These phenotypes are similar to those observed in humans during high fat diet induced diseases. Interestingly plasmatocyte depletion or plasmatocyte specific knock down of the CD36-like scavenger receptor croquemort (crq) or cytokine upd3 in flies was sufficient to rescue various high fat diet induced phenotypes. Findings in this thesis identify a plasmatocyte specific and therefore macrophage specific role in the progression of high fat diet disease pathophysiology, which could prove significant in allowing for a better understanding of high fat diet disease aetiology in mammals.

Stomata are microscopic pores located in the epidermis of most terrestrial plants. As well as serving as gateways for gas exchange and transpiration, stomata have recently become known to have increasingly complex relationships with pathogens, with many pathogens utilising stomata as entry portals. However, the research so far has largely focussed on bacterial and fungal pathogens, leaving a gap in the knowledge about the interactions between plant viruses and stomata. This study aims to investigate the interactions and relations between stomata and viruses by investigating potential for stomata to act as entry portals for viruses and the developmental changes which occur under virus infection. In order to test the hypothesis that stomata can act as entry portals for plant viruses, a series of experiments was performed which altered stomatal apertures before applying purified virus suspended in solution in an aerosol. It was found that Nicotiana tabacum plants were more likely to become infected when TMV virus solution was applied when stomata were open. Natural and chemical factors were used to manipulate stomatal apertures prior to virus application. Stomatal development was investigated following a virus infection. A range of host-virus systems was used and it was found that susceptible host types had a general reduction in stomatal index and density when infected with a virus. Transcripts of genes involved in stomatal development were also tested for changes in healthy and infected plants and were found to vary upon infection. Knock-out mutants of various stomatal developmental or functional genes showed varying developmental responses to a virus infection, with notable changes in rin4 which resulted in an increase in stomatal development post infection. The results presented in this project provide an insight into a relatively new field of research which has so far been neglected in the field of plant pathology.

Two major components of the common carp Cyprinus carpio innate response, i.e. the C-reactive protein (CRP) and the complement system, have been analysed in common carp either orally immunostimulated with β-glucan or infected with a pathogen or both. Immune response was examined at different levels, from serum circulating protein levels or activity to related gene expression in a wide range of immune related tissues such as liver, head kidney, mid-gut, gills and spleen. In addition, recombinant technologies were investigated in order to generate and produce subsequent amounts of carp CRP. Oral administration of β-glucan significantly stimulated carp CRP and complement responses with elevated serum CRP levels and complement activity and up- and down regulation of related genes in immune related tissues from 7 days of treatment. A subsequent intraperitoneal injection of LPS or Poly(I:C) as mimicries of bacterial and virus infection respectively also did have significant effects on the production (gene expression) and the circulation (scrum levels or activities) of these acute phase proteins. In addition, results from a challenge conducted on common carp with Aeromonas salmonicida, the causative agent of furunculosis, have shown that β-glucan enhanced and stimulated carp CRP and complement responses to the bacterial infection. These findings arc of importance in respect to the use of β-glucan as an alternative low cost strategy to the use of vaccines or antibiotics to enhance and stimulate the fish immune system. Innate response was analysed in carp infected with the Koi Herpes Virus (KHV), the etiological agent of a virulent and lethal disease. Results have shown a serum CRP level 10 fold increase and a serum complement activity 5 fold increase in less than 72 hours, revealing that CRP and complement act as acute phase reactants in response to KHV infection in common carp.

No description available.