The potential lipopolysaccharide (LPS)-degrading capability of three different systems was investigated. The acellular slime mould Physarum polycephalum was chosen as an example of a phagotrophic microorganism utilizing bacteria as food. Gut juice from the snail Helix pomatia was used because this animal is an example of an invertebrate ingesting materials rich in bacteria. Also the gut juice is a known source of many different enzyme activities. The fate of LPS when incubated with samples of marine mud and sand was investigated because of the abundance of gram-negative bacteria in marine environments, particularly muds. The main methods used to detect and measure LPS and to obtain evidence of its degradation were: Ketodeoxyoctonate analysis, because of the presence of this substance in almost all LPS preparations; gas-liquid chromatography (GLC) to detect long chain fatty acids; serological methods, including the ability of LPS to sensitize red cells, and haemagglutination inhibition (HAI) tests to demonstrate whether the immunospecific sugars in the oligosaccharide component were still present; and the ability of LPS to trigger complement, as an index of "endotoxic" activity. The anticomplementary activity of LPS was investigated in some detail. A range of LPS was assayed for anticomplementary (AC) activity against 5 HU50 of complement (C) from man, pig and guinea-pig. On average, levels of LPS about 200 times lower were detected with human C than with guinea-pig C and about two times lover than with pig C. There was little variation in C samples from different people in responsiveness to LPS AC activity. Different LPS varied in AC activity over a 10-fold range with each species of C. The rank order of their activities also varied with species of C. With human C, the most active LPS could be detected down to 2.5 ug. It is suggested that the AC effect of LPS is mediated principally via the Alternative Pathway. Physarum polycephalum appeared to attack only the lipid component of LPS, with a consequent reduction in AC activity and ability to sensitize red cells, while the KDO and HAI values were unaffected. GLC analyses indicated that the lauric, myristic and palmitic acids were split off leaving the ß-hydroxynprristic acid still attached to the diglucosamine backbone. Degradation of LPS by gut juice of the snail Helix pomatia similarly appeared to affect only the lipid component. When such degraded LPS was analysed on SDS-polyacrylamide gels. one of the characteristic bands of the original LPS was lost or reduced, reflecting loss of fatty acids. Marine sediments, at different depths and sites, were extracted with phenol-water or trichloroacetic acid (TCA) and yielded more LPS in areas of high organic pollution. KDO was not, detected in any of the sediments taken below a depth of 4 cm; AC activity decreased with increasing depth. When killed gram-negative bacteria were incubated with marine sediments, degradation of the LPS was observed. The oligosaccharide component was degraded at a faster rate than the lipid moiety as demonstrated by the loss of KDO and serological specificity. Both amide and ester linked fatty acids of the lipid A were lost. Most of the previous work on alteration of the LPS molecule has involved chemical treatment. The present studies offer the possibility of more specific procedures. Purification of the P serum and snail gut juice enzyme systems might afford highly selective modes of degradation which would be very useful for increasing knowledge of the structure of LPS molecules. Also it might be possible to produce altered molecules with different patterns of biological, physiological and immunological properties.

It was found that starvation of CFLP and NIH mice for six hours prior to infection was sufficient to produce a marked improvement in the level and consistency of establishment of oral infections of D. phoxini metacercariae, if the mice were starved from 6 AN, allowing stomach emptying to occur before infection. In contrast to spermatogenesis, the detection of vitellogenesis and oogenesis in D. phoxini in the NIH mouse was markedly delayed compared with that reported in the duckling. There was no difference in establishment or loss of a 200 metacercarial oral infection in male and female NIH mice. Loss began on day 6 and was complete by day 11 pi. Growth of flukes was complete by day 3. Flukes were largely confined to the anterior 10 cm of small intestine until the loss phase, when some attached to the region 10-20 cm post pylorus before being lost. The method used detected a very low rate of egg production which declined rapidly after the onset of the loss phase of infection, although at this time there was not a corresponding decline in the percentage of flukes bearing eggs* Implantation of metacercariae into different regions of the small intestine led to the following conclusions. 1. Establishment was best 30-60 % post pylorus, and very poor in the posterior 40% of the small intestine. Inconsistent establishment in the anterior duodenum could be due to lack of preincubation, combined with other factors. 2. Recovery of flukes five days after transplantation of metacercariae was best in those implanted in the anterior 30% of the small intestine and the percentage of egg bearing flukes was highest in the anterior 10 cm of small intestine. 3. Flukes that survived until day 5 were the same size if found near to the site of implantation. Those which had moved in a posterior direction were smaller.Reduction of infection size from 400 to eight metacercariae resulted in a four day delay in expulsion of primary infection, which otherwise occurred normally. Some delay in growth of flukes in a 400 metacercarial infection may have been due to changes in the gut preceding expulsion. Cortisone acetate treatment delayed the onset of main fluke loss, which occurred after day 13 pi, by which time a normal primary infection was completely removed. 11% of flukes still remained on day 25 pi in treated mice. It is suggested that this represents loss due to senescence, or to a delayed, reduced immunological response. Flukes in cortisone acetatetreated mice were large (comparable to those from low level infections) and remained in the anterior 10-15 cm of the small intestine. Serial transplantation also resulted in increased longevity of flukes but losses were greater, probably due to the trauma of recovery and transplantation. Transplantation of flukes undergoing expulsion resulted in their reestablishment in naive donors, though once again losses occurred. Transplanted flukes then had a longevity similar to that of flukes in an oral primary infection. The results indicate 1. Expulsion of a primary infection is host-mediated. 2. D. phoxini is highly immunogenic as the expulsion of light infections (eight metacercariae) is similar to that of infections initiated by 400 metacercariae. 3. Damage to flukes is reversible, as shown by the establishment and survival of flukes in the process of expulsion, upon transfer to naive hosts. The characteristics of a 200 metacercarial secondary infection (administered three weeks after a 200 inetacercarial primary infection) are described. After normal establishment, rejection occurred between two and four days post infection. Fluke development was impaired, functional vitellaria did not form and eggs were not produced. Growth stopped before day 2 pi. No waning of immunological memory occurred when the interval between primary and secondary infections was increased to seven months. Reduction of the size of the immunizing infection to as few as five metacercari ae resulted in no reduction in the effect of the immune gut on the rate of expulsion of secondary infection, although the inhibitory effect on fluke growth was less marked. Abbreviation of a 200 metacercarial to 15h duration apparently did not diminish resistance of mice to subsequent reinfection. The effects of the immune gut on the growth, development and longevity of D. phoxini were found to be reversible when flukes from a secondary infection were transplanted into a naive host. The effect of the immunized gut on transplanted, almost mature (three day old) flukes from primary infection was less marked than the effect on metacercariae surgically implanted into the duodenum, survival of the three day old flukes in immune mice was almost as long as would be expected in primary infection, and development proceeded to completion. Serum from mice infected with D. phoxini eight days previously failed to transfer immunity. Immunity transferred adoptively by IMLNC was manifested as an acceleration of expulsion, and a retardation of vitelline development and reduced growth of flukes in recipient mice compared with controls. As few as 1x107 IMLNC affected expulsion. 2x107 IMLNC affected body length and vitelline development also. IMLNC taken from donor mice between days 2 and 6 of a primary infection were most effective. After day 6, efficacy declined, however IMLNC taken from donor mice on day 21 after primary infection unexpectedly had some effect on recipient challenge infection. IMLNC taken 12 hours after secondary infection were effective but those from days 6 and 12 of secondary infection were not. I MLNC transferred less than two days before challenge of recipient mice did not transfer immunity. T. lymphoblast activity was high and cellularity of the MLN increased following primary and secondary infections, but these changes were not coastsknL correlated temporally with the efficacy of I MLNC. T-lymphoblasts were ineffective but a population of mainly non-dividing B cells was effective in transferring immunity adoptively. Histopathological changes in the mouse intestine associated with D. ph oxini infection were characterized, and the effect of adoptively transferred immunity (via IMLNC) on these parameters was studied. Infection was characterized by marked globule leukocyte proliferation and eosinophilia which preceded and accompanied the expulsion phase of infection. Both responses occurred more rapidly in secondary than in primary infection. By comparison, the response of lamina propria mast cells was delayed and very limited, and was not marked in secondary infection. The response of goblet cells to infection was minor, and irregular during normal infection, however it is possible that mucus production by individual cells may be increased during infection. Adoptive transfer of immunity led to an acceleration of all cellular responses. sIg+ve MLNC transferred immunity most effectively and generated a level of inflammation which was severe compared with normal infection, and was uncharacteristic as it involved increased goblet cell differentiation. The poor ability of T cells to transfer immunity might have been attributable to low viability and/or selective depletion during cell separation. High variability was observed in the number of plasma cells in the intestine during infection. The most marked increases occurred in lgG, and 1gM secreting plasma cells during primary infection and lgG, during secondary infection.

The generation of an effective immune response relies on the coordinated migration and interaction of cells of the immune system. Interactions between leukocytes and other cells occur within secondary lymphoid organs or at sites of inflammation. These interactions are tightly policed by a family of small chemotactic cytokines, or chemokines, that drive leukocyte migration. Chemokines and their receptors are broadly divided into two functional groups, homeostatic and inflammatory. Typically homeostatic chemokines and their receptors regulate leukocyte migration to and within secondary lymphoid organs, whereas inflammatory chemokines and their receptors control migration of cells to sites of inflammation. However, this division is now blurred, as some inflammatory chemokine receptors also possess clear homeostatic roles. One such receptor is the chemokine receptor CCR2, which binds a number of inflammatory chemokines, including CCL2, CCL7 and CCL12. Further complexity is added to the chemokine biology field by the discovery of atypical chemokine receptors, which lack the capacity to signal in manner similar to conventional chemokine receptors, and are proposed to act as chemokine scavengers. The most frequently studied atypical chemokine receptor, D6, scavenges inflammatory chemokines, with a number of ligands overlapping with those of CCR2. CCR2 has been reported to play a non-redundant role in the egress of Ly6Chi monocytes from the bone marrow (BM) during homeostasis and inflammation. However, the role of CCR2 on other cell types has not been fully characterised and it is not known how much other CCR2+ cells may contribute to the role ascribed to CCR2 in several diseases, such as collagen-induced arthritis (CIA) and atherosclerosis. In addition, there is currently limited information about the leukocytic expression of D6, which can also bind CCL2. Although more information is available about CCR2, there are several conflicts reported between surface levels of CCR2 detected by antibody and transcript levels. To gain a true understanding of the function of these receptors in diseases it is crucial to have an accurate profile of CCR2 and D6 expression in vivo and subsequently determine the function of CCR2 and/or D6 on these cells. Therefore, the primary aim of this thesis was to generate a detailed profile of CCR2 expression by leukocytes, focusing mainly on the spleen, but including lymph nodes (LNs), blood and BM. In Chapter 3, I demonstrate that currently available anti-murine CCR2 antibodies are unreliable, as they were either unable to detect CCR2 or could only detect high levels of CCR2. I describe a novel technique using fluorescently labelled CCL2 (CCL2AF647), which substantially enhanced CCR2 detection and enabled a detailed characterisation of D6 activity. Using this method, I present a comprehensive analysis of CCR2 and D6 expression on mouse leukocytes. CCR2 expression was primarily limited to myeloid cells, dendritic cells (DCs), and natural killer (NK) cells, with lymphoid cells having low levels of CCR2. Interestingly, my data illustrates that D6 expression is not as restricted as published data suggests, as several cell populations, such as plasmacytoid DCs (pDCs) and some monocyte populations were found to express both CCR2 and D6. Furthermore, I present preliminary evidence that suggests that CCR2 might exhibit cell-type specific responses to its ligands. I also systematically analysed the cellular composition of lymphoid organs and the blood of CCR2 knock-out (KO) mice and compared it to age- and gender-matched wild-type (WT) controls. These studies revealed that CCR2 deletion only affected the distribution of cells of monocytic origin during homeostasis. In contrast, the results described in Chapter 4 demonstrated that systemic inflammation induced by lipopolysaccharide (LPS) affected the frequency of several populations possessing CCR2 activity, including Ly6Chi monocytes and conventional DCs (cDCs). I also present data that suggests that LPS temporally regulates CCR2 and D6 activity in a cell type-specific manner. In Chapter 5, I detail the identification of two subsets of pDCs, present in the spleen, BM, blood, and LNs that differ in their expression of CCR2 and D6. Both subsets were able to stimulate naïve T cell proliferation and responded to toll-like receptor 7 (TLR7) and TLR9 stimulation, in terms of interferon alpha (IFNα) production and expression of activation markers. However, transcriptomics and flow cytometry did reveal some notable differences between these two subsets and deficiency in D6 or CCR2 led to changes in the surface immunophenotype of pDCs at certain anatomical locations. I also demonstrate that pDCs are able to migrate efficiently towards CCL2 in vitro, but that CCR2 was not required for the reported CCL2 dependent migration of pDCs to imiquimod-inflamed skin. Taking these results together, I have established CCL2AF647 as a novel sensitive method of CCR2 detection, which facilitated the identification of two pDC subsets. Although I was unable to find a role for CCR2 on pDCs, further study might better define the function that CCR2, and D6, plays on pDCs. In Chapter 6 I discuss my findings, relating the results to published data and forming conclusions and hypotheses that can be examined further in future experiments.

The prevalence of fungal infections is on the rise due to the increase of immune suppressed individuals. Neutrophils are key immune cells in the fight against fungal infections. The study of neutrophil biology is hampered by the short lived nature of the cells and the fact that they cannot be easily genetically modified. In this thesis, I generate and characterise myeloid precursor cell lines that can be genetically manipulated and differentiated into functional neutrophils. These in vitro generated neutrophils were adoptively transferred into live animals and tracked during inflammatory responses. Clec7a, a cell surface β-glucan receptor found on myeloid cells, and its role in immune response to fungal infections has been well characterised in macrophages and dendritic cells but less so on neutrophils. In this thesis, a model for elucidating the role of Clec7a on neutrophils was developed using primary cells and was able to show that Clec7a deficiency on neutrophils impairs recognition of zymosan and C. albicans but that this impairment was largely overcome by serum opsonisation of the particles. The in vitro generated neutrophils were comparably tested and, although the cells have their limitations, they largely supported the conclusions found using primary cells.

HCMV encodes a plethora of immune-modulating functions, many of which have yet to be assigned to specific genes. In prospect of performing high-throughput screens to identify and characterise such functions, a library of recombinant adenoviruses (RAds) each encoding a V5 epitope-tagged HCMV protein was generated. Protein expression was validated and characterised for the vast majority of RAds by western blot and immunofluorescence. HCMV has been reported to both upregulate cell surface expression of Fas, and render cells resistant to Fas-mediated killing. This thesis demonstrated that Fas levels are markedly reduced at the surface of HCMV-infected cells as an early function that persists through the late phase. Screening a panel of HCMV deletion mutants eliminated 83 genes as not required for Fas downregulation, while screening the RAd library did not identify any single HCMV gene as being sufficient for this function. Deep sequencing of the HCMV transcriptome recently led to the identification of UL150A as a novel protein-coding gene. To test this prediction, UL150A was tagged within the strain Merlin genome. UL150A was shown to encode multiple protein products, and be expressed with early and late kinetics. In a screen of the RAd library, gpUL4 was observed to be secreted from cells. To investigate this function in the context of HCMV infection, an epitope-tag was inserted at the 3’-end of the UL4 gene in the strain Merlin genome. Tagged gpUL4 was secreted from cells infected with strain Merlin. Secreted gpUL4 was more heavily glycosylated, and produced in greater abundance than its intracellular counterpart late in infection. Active secretion would be consistent with gpUL4 acting as a virokine, cytokine or cytokine/chemokine-binding protein. gpUL4 purified from supernatants of Merlin- or RAd-UL4-infected cells inhibited NK cell degranulation. Furthermore, gpUL4 did not copurify with virus particles, indicating it is unlikely to be a virion component.

Spinal cord injury (SCI) affects approximately 40,000 people in the UK; the most common type of SCI is a contusion injury and the majority of these cases are male and aged between 16 and 30 years old. The initial physical trauma to the spinal cord during injury leads to substantial damage and loss of neurones. After the primary traumatic insult has occurred a sequence of events is initiated that sets off a cascade of biochemical, cellular and inflammatory events that are massively destructive and continue for weeks to months after the initial SCI. This phenomenon is known as “secondary death” and leads to an increase in the size of the damaged area. Infiltrating monocytes and monocyte-derived macrophage have been implicated as a crucial component in the perpetration of secondary death. It was demonstrated in this thesis that staphylococcus protein A (SpA), when in complex with IgG forms homogeneous small immune complexes (SIC) that can polarize macrophages in vitro to an anti-inflammatory phenotype, resulting in increased production of the immune-suppressive cytokine IL-10 and reduced ability to produce the pro-inflammatory cytokine IL-12. SIC treatment of IFNγ-primed macrophages in conjunction with LPS also induces a down-regulation of MHC II surface expression; however, the macrophages still exhibit normal levels of co-stimulatory molecule CD86 compared to a classically-activated macrophage. In an in vivo setting it was demonstrated that SpA binds to monocytes and preferentially to the “inflammatory” Ly6Chi monocyte sub-set. The binding of SpA to this monocyte population induced the maturation of the Ly6Chi “inflammatory” monocyte into Ly6Clow “anti-inflammatory” monocytes within the steady state and in the sterile inflammatory setting of SCI. In the inflammatory environment of the damaged spinal cord, SpA treatment induced a higher percentage of Ly6Clow monocytes to produce the immune-modulatory cytokine IL-10 compared to the control treated group. These observations indicate when SpA and IgG form SIC they interact with macrophages and monocytes in vitro or in vivo polarizing them to an anti-inflammatory phenotype. In conclusion, it has been shown in this thesis that SIC has the potential to be used as a method of polarizing monocytes and macrophages to an anti-inflammatory phenotype, which in turn has the potential to modify the overt inflammatory response that is responsible for the death of neurons days to weeks after the initial injury and is responsible for reduced functional recovery in SCI patients.

The studies reported in this thesis deal with the in vivo transmission of homologous and heterologous IgG and other proteins across the proximal small intestine of the suckling rat. The uptake of these labelled macromolecules from the intestine was monitored, and the subsequent appearance of radioactive material in the serum, viscera and carcass was examined by ultrafiltration and ultracentrifugation techniques. The rate of transmission of intact IgG and IgG breakdown products across the intestinal barrier was measured with the intention of obtaining further information about the mechanisms of immunoglobulin transport. The significance of the digestive activity of the suckling rat gut to the process of antibody transmission was also assessed. This was done by gel filtration analysis of the radioactive material present in the gut wall and wash after the intraluminal injection of labelled rat IgG. The ability of trypsin inhibitor to stimulate the transmission of intact IgG was investigated, and the possibility that the IgG receptor on the surface of proximal enterocytes was trypsin sensitive was also examined. Attempts were made, using a variety of subcellular fractionation techniques, to isolate and identify the organelles involved in the receptor mediated transcellular transport of IgG.

Salmonella Typhimurium definitive type 104 (ST DT104) isolates resistant to antibiotics have been an issue since the multi-resistant clone was identified in 1985. In order to advance understanding of ST DT104 infections in Scotland, 2,796 human and 2,439 animal isolates with their corresponding antibiotic resistance patterns submitted from 1988 to 2004 were used to conduct descriptive, hierarchical, and geographical cluster studies and to construct a time series model. The analyses showed that using the 13 antibiotics used by the Scottish Salmonella Reference Laboratory, isolates could be allocated into two distinct groups. The first group containing the ApC1SpStSuTe R-type with its associated resistance patterns, mainly the ApCISpStSuTeTm and the ApCISpStSuTeNa R-types, dominated the trend throughout the study period. The second group, mainly composed of fully sensitive isolates, formed a low proportion except during the period from 1988 to 1990. Temporal analyses showed that there was an epidemic from 1993 to 1998 in human ST DT104 and from 1992 to 1999 in animals. Spatial analysis identified the southern part of Scotland as the higher relative risk area for both human and animal infections caused by multi-resistant ST DT104 strains In contrast, the central belt of Scotland was mainly the relative risk lower spatial cluster for the multi-resistant ST DT104 R-types. Of note in the spatio-temporal analysis was the stability and persistence of the chromosomally mediated multiple resistance compared to the more sporadic plasmid mediated resistance types of the second group. Although there were many similarities between the infections in humans and animals, there was no consistent temporal association between the emergence of clones in humans compared with animals suggesting that the ecological and epidemiological direction of the relationships is complex.

This thesis describes work undertaken to consider how strategies of vaccination can be manipulated to generate specific types and magnitude of immune response to prevent infection or treat established infection. The vaccination strategies employed were based on the initial delivery of DNA vaccine through intramuscular injection or ballistic delivery using a gene gun, followed by heterologous boosts, based on the same antigen delivered using an alternative route. These boost strategies included a) intramuscular delivery of purified recombinant HBcAg, b) mucosal delivery of HBcAg expressed by an attenuated strain of Salmonella typhimurium, and c) intranasal delivery of the protein accompanied by a mucosal adjuvant. Following each vaccination regime tested a number of specific immune responses, including serum and mucosal antibody production, CD4+ T helper proliferation in spleens and lymph nodes, CD8+ T cell activation and killing were measured. These experiments revealed that the character of the immune responses primed in response to DNA vaccination differed according to route of immunisation, with intramuscular vaccination inducing a rapid CTL response. CD4+ T cells generated appeared to be of the Th1 phenotype and showed the strongest localisation in the spleen. In contrast, following vaccination with the gene gun, CD4+ cells of a Th2 phenotype were generated with responses being found to be stronger in the local draining lymph nodes that the spleen. Combining DNA delivered using the intramuscular route with recombinant purified protein delivered by the same route was effective at boosting systemic antibody titres, as was boosting using attenuated Salmonella expressing the same antigen when delivered to the mucosal surface of the gut. In contrast, boosting of DNA primed animals with purified protein, even in the presence of an appropriate mucosal adjuvant, was not successful at increasing titres of systemic antibodies. Interestingly, specific mucosal immune responses were absent when either of the heterologous vaccination strategies used a mucosal boosting approach.

Epithelial cells (ECs) are key players in the modulation of immune responses in addition to its main role as a barrier against external stimuli. Upon stimulation with pathogens/allergens, airway ECs can produce a wide range of innate mediators that recruit and activate immune cells which play a key role in maintaining immune homeostasis in the lungs. One such mediator is indoleamine 2,3- dioxygenase (IDO), which is a rate-limiting enzyme involved in tryptophan (TRP) catabolism. IDO plays a role in the modulation of immune responses to antigenic challenges and also acts as an anti-microbial factor. Therefore, the main aim of this project was investigating IDO expression, immune regulatory function and regulation pattern in human airway ECs in response to clinically relevant toll-like receptor (TLR) ligands and allergen extracts. This was done in 2D cultures and in a 3D model using ECs from healthy and diseased donors. The data clearly demonstrated that ECs constitutively produce IDO which is up-regulated in response to IFN-. However, I have shown for the first time, that IDO production and activity is down-regulated in response to TLR ligands and allergen extracts. Indeed, using gene silencing, I demonstrated that ‘resting’ ECs (i.e. with high IDO expression) can suppress T cell activation in an IDO-dependent manner, but this regulatory function is lost in response to TLR agonists mimicking bacterial or viral infections. These data provide new insight into how ECs, as part of their function in maintaining immune homeostasis in airways, can influence downstream innate and adaptive immune responses through the production of IDO. In this study, I also focused on developing an immunocompetent 3D model of human airway ECs in regards to produce certain in vivo characteristics. In addition, the effect of EC differentiation state and cross-talk with other cells on IDO activity was investigated. My data showed that EC lines and primary human bronchial ECs (pHBECs) could be differentiated at the air-liquid interface (ALI) and form ZO-1 tight junctions. Moreover, ECs had higher TEER values when co-cultured with fibroblasts than when cultured alone which indicates that fibroblasts facilitate EC differentiation, highlighting the importance of paracrine interactions and cross-talk between cells in maintaining EC barrier function. Moreover, the data showed differential regulation of IDO activity on the basis of culture conditions which suggest that differentiation and co-culture status might affect the orientation and expression pattern of TLRs. Furthermore, our data in regards to IDO regulation in physiological and pathophysiological settings demonstrated the differential regulation of IDO and considerably different IDO basal levels in healthy cells compared with diseased cells, emphasizing the immunosuppressive role of IDO. In conclusion, these advances in the understanding of IDO expression and regulation in different tissue/cell types in health and disease could contribute to delineating the potential role of IDO in inflammatory responses in different pathologies and enable the development of novel IDO-targeted therapeutic strategies.