Periodontal disease is associated with an inflammatory response to a pathogenic biofilm. The host response may cause gingival inflammation, which can progress to irreversible gingival recession, alveolar bone destruction and tooth loss. Enhanced understanding of the host-biofilm relationship may inform novel therapeutic approaches. A key molecule involved in inducing and mediating pro-inflammatory responses are the IL-17 cytokine family. An in vitro model system potentially provides a platform to investigate biofilm interaction with epithelial cells. The aim of this study was to develop in vitro mono-species and multi-species biofilms and investigate the survival of biofilms in cell culture conditions, and simultaneously assess the epithelial response to the bacterial biofilms and planktonic cells with respect to viability, apoptosis and inflammatory mediators. This study also looked to determine whether IL-17A is expressed within and released from periodontal tissues and to investigate its role in the regulation of epithelial cell cytokine and chemokine production. Mono- and multi-species biofilms of P. gingivalis, F. nucleatum, A. actinomycetemcomitans and S. mitis were developed, which were assessed for survival in cell culture conditions, recovery from biofilms and morphology. Gingival tissue from patients with chronic periodontitis or healthy controls were analysed for IL-17A gene expression by qPCR. Protein expression and cellular localization was determined by immunofluorescence. Single cell suspensions of gingival tissue were stimulated in vitro and IL-17A release assessed. Epithelial response after bacterial and IL-17A co-culture was assessed. The individual bacteria survived preferentially in multi-species biofilm compared with mono-species biofilm in cell culture conditions. The viability, apoptosis and inflammatory mediator response depended on the type (pathogen or commensal) and form (planktonic or biofilm) of bacteria. Diseased gingival tissues expressed significantly higher levels of IL-17A mRNA than healthy samples. IL-17A localised to mast cells in the inflamed gingival tissue, and was released in cell culture supernatants following stimulation. Stimulation of epithelial cells with IL-17A resulted in the transcriptional regulation and release of numerous cytokines and chemokines. The initial component of the entire investigation has provided a quantitative and qualitative assessment of both mono- and multi-species biofilms that can be used to investigate how oral biofilms interact with the host epithelium. The epithelial-biofilm co-culture model has demonstrated clear differences between (i) planktonic and biofilms, (ii) pathogens and commensals, and (iii) live and dead bacterial challenge. These observations and the utility of the model will provide a platform to investigate key questions relating to pathogen and host within the oral cavity and beyond. From this study, it appears that IL-17A plays an important role in the protective periodontal immune response to bacterial pathogens. The upregulation of acute inflammatory mediators (such as IL-8) will promote neutrophil recruitment and potentiate the removal of any invading microbial threat. Therefore it is important to understand the benefits of this cytokine, before systemic therapeutic agents are used to antagonise its actions. The hope for the future is to unravel the details of the mechanisms involved and thereby identify novel therapeutic targets for inflammatory and infectious disease.

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Despite current improvement in newborn care, infection is still a common cause of neonatal morbidity and mortality. Innate immunity is the first line of defence against pathogens particularly in newborn infants. Quantitative and functional deficit in non-cellular (complement system) and cellular (neutrophils) arms of innate immune system is believed to contribute to neonatal susceptibility to infection. Neutrophil granule subsets contain a variety of proteases, including elastase, cathepsins, MMP-9 and proteinase 3 along with different granule markers and receptors. This thesis demonstrated that normal term neonatal neutrophils express more proteinase 3 and CD177 on their surface while no differences were found in expression of markers CD35, CD66b and CD63 (representing the secretory, secondary and primary granule subsets, respectively). Cord neutrophils contain more PR3 than adult cells but the proportion of PR3 released by cord and adult neutrophils was similar. In contrast, neonatal neutrophils contained only half of the cathepsin G and elastase functional activity of adult neutrophils. Bronchoalveolar lavage fluid studied from preterm infants ventilated for respiratory distress demonstrated higher proteinase 3 concentrations in lavage samples from infants who went on to develop chronic lung disease than in infants with resolved respiratory distress syndrome. Concentration of proteinase 3 in lavage samples was significantly higher than MMP-9 and elastase levels, suggesting that it may have an important role in disease pathogenesis. Complement is an equally important component of the innate immune system that plays a central role in recruiting and activating neutrophils, as well as being directly bactericidal through the terminal lytic pathway. Analysis of neonatal complement system revealed that Complement function and terminal components levels particularly C9 are deficient (except C7) in healthy term newborn infants compared to normal adults. Bactericidal capacity of a selection of neonatal sera was tested along with adult sera against four serovars of Ureaplasma parvum, a potentially important perinatal pathogen. Results showed impaired bactericidal capacity of neonatal serum compared to adult serum especially against SV1. Ureaplasma SV3 was the most serum sensitive serovar whereas killing of the resistant serovars SV6 and 14 could not be induced by supplementation of the deficient components C6, C8 and C9.

Staphylococcus aureus is the most common cause of hospital-acquired infection and contributes significantly to patient morbidity and mortality. The ability of S. aureus to switch to an alternative phenotype in the presence of antimicrobial agents is clearly favourable. One of these alternatives are small colony variants (SCVs). The novel phenotypes include changes to colony morphology, antibiotic susceptibility, haemolytic activity and many other physiological activities. It is now recognised that SCVs have a deficiency in electron transport, owing to mutations affecting its efficacy. This study investigated SCVs in various ways. In the evolution component changes (mutations) occurring sequentially in successive cycling (15 cycles), were identified. In this experiment selection was made for sequentially SCV mutants and wild type revertants. Two sequenced clinical MRSA strains COL and N315 were chosen so changes in sequence in SCVs and wild type revertants could be compared. Selection for SCVs was made independently for triclosan and gentamicin for both strains. The final SCV and WT strains isolated were compared physiologically and genetically and showed differences in frequency, biochemical profiles, pigment production, haemolysis, catalase, coagulase, levels of intracellular ATP and phage yield. The genomic sequence of the final 4 cycle isolates (SCV15) showed numerous and diverse mutations occurred COL and N315 SCVs. Over 70 mutations were found and 33 were determined as historic mutations and the rest were termed novel mutations. The novel mutations occurred during the cycling process. The historic mutations occurred prior to the experiment and these mutations were acquired during growth in laboratory culture. Only one mutation was found to be common between COL and N315 and this was in the fabI gene. These data indicate mutations occurring in ~1.3% of the genome (~ 40 Kb) can generate mutants with the SCV phenotype. Susceptibility to phage 80α and transduction of S. aureus wild type and their SCVs 1-3 was studied. Wild type strain of S. aureus and SCV3 both yielded a high number of lysogens (~68%) the remaining being resistant mutants. SCV 1 and SCV 2 provided a much lower proportion of lysogens (4-10%). There was no obvious relationship between cellular ATP levels and lysogen formation. Consequently the frequency of lysogen formation (or that of resistance mutants) cannot be related to energy status. Transduction of ciprofloxacin resistance (grlA) was observed into COL wild type at a 5-10-fold higher frequency than into SCV1. Transduction of rifampicin resistance (rpoB) into SCVs was reduced almost 10-fold. As transduction was significantly decreased into SCVs it is hypothesised this process was influenced by ATP levels. The data thus suggests that SCV strains will be less efficient in gene exchange by transduction in vivo. Three SCVs previously isolated from S. aureus COL on the basis of different growth rate were further studied. Results clearly support the hypothesis that there is a physiological diversity in SCV populations. Sensitivity of S. aureus wild type and SCVs strains to various antimicrobial was determined. The SCV strains were more sensitive to some antibiotics and heavy metals than the wild type strain.

Investigations have been made into the genetic control of immunity to the nematode Trichuris muris. Both background genes and genes within the mouse major histocompatibility complex (MHC), H-2, were shown to influence the expulsion of T. muris with the former having the stronger influence. At least two genes within the H-2 complex determined response phenotypes, the effects of "resistance" or "susceptibility" alleles at I-A being modulated by resistance or susceptibility alleles at aD end locus/loci. Differential responsiveness within slowly responding mouse strains suggested that parasite-dependent effects were also important. The primary antibody response to T. muris excretory/secretory (E/S) antigen, predominantly an IgG response, was also shown to be controlled by background and H-2-linked genes. In general, mouse strains less resistant to infection developed higher levels of IgG than- more resistant strains of mice. However strains of mice possessing the H-2q haplotype, irrespective of their genetic background, rapidly developed higher levels of IgG1 antibodies than strains of other haplotypes, H-2q haplotype mice tending to be more resistant to infection. Recognition of two high molecular weight (MW) E/S antigens by IgG as revealed by immunoprecipitation was also found to be almost exclusively H-2q restricted. This restriction may be partly quantitative but as such would operate in vivo due to the restriction on the ability to produce high levels of specific IgG. Both H-2q restricted phenomena may be part of, but not absolute requirements for, protective immunity. Parasite-induced effects on host immunity were also studied. Later larval and adult stages of T. muris were shown to be immunosuppressive, immunosuppression being long lasting and preventing the expulsion of subsequent infections. Vaccination with E/S antigen was shown to protect strains of mice which are slow to expel worms (poor-responder) or totally unable to expel worms (non-responder) from a primary infection with T. muris. However protection was slow to be expressed. Antigen recognition profiles of vaccinated strains of mice differed from their primary infection recognition profiles and included the recognition of the two high MW antigens shown to be H-2q restricted in a primary infection. Thus altering the mode or route of E/S antigen presentation may lead to shifts in responsiveness of H-2 genotypes to specific determinants and/or boost specific antibody levels sufficiently to reveal recognition of these antigens. Prior experience of a patent primary infection prevented vaccination protecting non-responder mice against subsequent infections. This inability was correlated with suppressed IgG1 antibody levels and failure to recognise three high MW antigens including the IL-2q restricted antigens. Using a panel of monoclonal antibodies raised against E/S antigen it was shown that E/S antigens, apparently including both immunogenic and immunosuppressive molecules, were localised to granules within the stichocyte cytoplasm of the adult T. muris stichosome.

The development of the gastrointestinal nematode Heligmosomoides polygyrus (syn. Nematospiroides dubius) in the mouse was studied. The stage specific production of acetylcholinesterase was measured in both excretory/ secretory products and in worm homogenates and found to be maximal between days 4-6 post infection, corresponding to the fourth larval stage of the parasite's life cycle. Analysis of the proteolytic enzymes found in the same preparations of the parasite again revealed a stage specific release. Quantitative examination showed a maximum concentration of proteolytic enzymes in the early third larval stage, whilst qualitative analysis revealed a number of molecules at 96, 76, 42, 33, 18, 16, and 13 kDa in the early stages, which gradually disappeared as the parasite aged until only those at 76, 18, 16, 13 kDa remained by day 120. The molecules present on the surface of the various stages of the parasite were extracted using a number of procedures. Various stage specific surface molecules were identified as were two possible sex specific molecules at 76 and 145 kDa. The immune response to a primary infection of the parasite was characterised in three strains of mice with different degrees of susceptibility to infection (SJL, BALB/c and CBA). It was noted that the better the strain was at expelling the parasite, the greater and swifter was the response as assessed through the use of a number of criteria. These included white blood cell counts, differential cell counts, the Band T cellularity of the secondary lymphoid organs, the response of these cells to mitogens, the mucosal mast cell response, quantitative antibody response (Mancini and ELISA) and qualitative antibody response to parasite antigens (immunoblot). In each case SJL responded better than BALB/c which, in turn responded to higher degree than CBA. Functional host protective immunity was stimulated in the same three strains of mice using a challenge infection following a 9-day anthelmintic abbreviated infection. The same criteria were used to measure the immune response to the parasite as for the primary infection and, as for the primary infection, it was found that the high responder strains gave a more rapid and more intense reaction to the parasite than the low responder strain. Immunisation prevented the establishment of a proportion of the challenge infection and also resulted in the premature expulsion of parasites. The parasite surface molecules which were recognised by mice undergoing either a primary infection or an immunising infection were identified. It was revealed that molecules at 208, 145, 92, 76 and 62 kDa on adult parasites were recognised by mice which had expelled a primary infection. Mice which were immune to a challenge infection recognised molecules at 62 and 20-15 kDa on larval parasites. A molecule at 30.5 kDa was also recognised by immune mice and corresponded to the molecular weight of acetylcholinesterase in the ES.

The objective. of this project was to determine the protein specificity of the cytotoxic T- lymphocyte (CTL) response to rotavirus infection in mice and to assess the rotavirus serotype/strain independent nature of this response. Previous work, involving the rotavirus group at Warwick, had shown that the outer shell glycoprotein VP7 is a major target antigen for a CTL response that is virus serotype-independent. However, that work did not cover all twelve rotavirus proteins, was confined to one strain of adult mice (C57BL/6, H- 2b) and covered only two of the fourteen VP7 serotypes (serotypes 3 and 6) (Offit et al., 1994). Recombinant vaccinia viruses expressing individual rotavirus UKtc proteins VP2, VP3, NS26 and NS12 were constructed to complete a set of recombinant vaccinia viruses covering the full complement of rotavirus proteins from the bovine UKtc strain. These were used to define rotavirus proteins eliciting a CTL response in three different mouse haplotypes. UKtc NS53 and UKtc VP7 stimulated a strong CTL response only in the H-2b MHC class I haplotype (UKtc NS53 and UKtc VP7 were restricted at H-2Db and H-2K b, respectively). Conversely, UKtc VP3 stimulated a strong CTL response in the H-2d and H-2k (but not in H-2b) MHC class I haplotypes. Work using congenic mouse strains was used to verify that the VP7 protein specific CTL response is restricted solely by the MHC class I antigens. Rotavirus RRV NS53 was not only found to elicit a CTL response in the H-2b MHC class I haplotype, similar to UKtc NS53, but also in the H-2d MHC class I haplotype. Thus, the individual rotavirus protein that elicits a CTL response not only depends on the MHC class I haplotype, but also on the actual rotavirus strain being tested. Many of the previous studies looking at the CTL response-to individual rotavirus proteins have, unlike this study, used several different rotavirus strains and, therefore, may have given an inaccurate representation of the rotavirus proteins that elicit a CTL response. Recombinant vaccinia viruses were also used to examine the serotype/strain independent nature of the CTL response against the major target antigens. The analysis was extended to cover VP3 from two different strains, NS53 from three different strains and VP7 from seven of the fourteen serotypes. VP3 and NS53 were found to elicit a strain- dependent response whereas the serotype-independent nature of the CTL response to VP7 was confirmed. Since the serotype-independent nature of the rotavirus VP7-specific CTL response was found to cross-protect between half of the VP7 serotypes, irrespective of the immunising. serotype, it would be reasonable to speculate that the CTL response is serotype-independent between all the VP7 serotypes. Finally, recombinant vaccinia viruses were used to locate CTL epitopes on NS53 and VP7. Recombinant vaccinia virus expressing a UKtc NS53 deletant mutant (P9DM5) showed there to be at least one strain specific epitope in the first 150 amino acids of the UKtc NS53 protein. Recombinant vaccinia viruses expressing four different UKtc VP7 fragments spanning 46% of this protein were examined. It was found that the fragment spanning the restriction enzyme sites at nucleotide 90 (CIaI) and nucleotide 196 (Hhal), i. e. between amino acids 13 and 48 of the mature UKtc VP7 protein, contained a serotype- dependent CTL epitope. The finding suggests that the immunodominant epitope identified in the same region of VP7 by Franco et al. (1993) was not the serotype-independent epitope.

The autotransporters are a family of extracellular proteins, found in various Gram-negative bacteria, that have many different functions but appear to have a similar mechanism of export. In B. pertussis, the virulence-regulated proteins Pertactin, BrkA, Tcf, and Vag8 have structural homology at their C-termini (30-kDa) and the N-terminal of the mature proteins share structural characteristics such as RGD and SGXG motifs. Recently, another member of the B. pertussis autotransporter family, Bap-5 (Blackburn, 2000) (GenBank accession number AF081494) or BapC (GenBank accession number AJ277634.1) was identified. The present work has suggested that BapC, like BrkA, is a serum-resistance factor. B. pertussis brkA, bapC double and bapC single mutants were created, and showed greater sensitivity to killing by normal human serum than their wild-type strains but they were not as sensitive as a bvg mutant strain. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis strains after intranasal infection in the mouse. Moreover, the brkA, bapC double and bapC single mutants were found to be more sensitive to the antimicrobial peptide, cecropin P1, than the parent strain. Nucleotide and amino acid analyses of the bapC region spanning the poly(C) and poly(G) tracts of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms in some strains but it appeared that all had an ORF that would be able to produce some form of BapC.

The human bacterial pathogen, Streptococcus pneumoniae (the pneumococcus), has been shown to modulate different parts of the innate immune response of its host, however its ability to modulate the adaptive immune response remains largely uninvestigated. Furthermore, the importance of the adaptive arm of the immune system in responding to Streptococcus pneumoniae has only recently begun to be elucidated. I therefore investigated a potentially novel pneumococcal immunomodulatory mechanism involving the effect of the pneumococcal toxin, pneumolysin, upon the cells at the heart of the adaptive immune response; the CD4 T cell. I generated purified pneumolysin and a purified pneumolysin mutant called F433 to allow me to examine this potential effect. I found that pneumolysin inhibits in vitro antigen specific murine CD4 T cell proliferation and cytokine production and that this effect is not observed with the F433 mutant pneumolysin. Furthermore, I demonstrated that pneumolysin accomplished this inhibitory activity by inducing apoptosis of activated CD4 T cells and suggest that lipid rafts may be involved in this process since we also demonstrated that pneumolysin preferentially binds to lipid rafts. Finally I demonstrated that pneumolysin is able to inhibit the in vivo accumulation of T cells and also inhibits in vivo antibody production. I propose that the immunomodulatory mechanism I have described may play an important role during pneumococcal infection and that this warrants further investigation. I propose that detailed in vivo studies are required to demonstrate that this mechanism functions during infection and to elucidate the effect this has upon the course of infection.

Resistance to infection is essential to ensure survival and thus maximise offspring potential. However, resistance is not ubiquitous across the animal kingdom, or even within a population from the same species. It is thought that this is due, in part, to the costs involved in producing and maintaining a competent immune system and a corresponding decrease in other fitness-related characteristics. The focus of this research was to determine how immune resistance can impact upon mechanisms relating to ageing, resistance to oxidative stress and motor function. In order to do this a Drosophila melanogaster model system was implemented, selected for resistance to larval parasitism by the parasitoid wasp, Asobara tabida. Firstly, it was necessary to gain a greater understanding of the immune mechanisms within the Drosophila model. This included how aspects of the immune system changed over time, in order to determine how these might act upon other processes at different stages of the ageing system. Resistance to larval parasitism corresponded to an increased number of circulating immune cells during the larval phase. This difference was no longer apparent in the adult Drosophila. Young resistant adult females revealed increased levels of overall cell metabolism, measured by the production of intracellular reactive oxygen species (ROS), a finding not seen in males or in the developing larvae. Lifespan was reduced in the resistant female, but not male, Drosophila. It was hypothesised that this reduction may in part be due to the augmented production of intracellular ROS in the young adult female, which at high concentrations can cause oxidative stress with known cytotoxic effects. However, differences in resistance did not translate to altered survival under acute oxidative stress, induced by the consumption of the toxin paraquat. Other factors may regulate these changes in longevity in the resistant females, such as genetic or resource-based trade-offs. Functional assays were performed to assess motor function in the larvae and adult Drosophila. Resistant larvae showed less turning behaviour on a non-food background than their control counterparts, a trait generally linked to more proficient motor function. Differences in motor function continued into the adult females, where increased climbing velocities were found irrespective of age. This implies that changes in motor function may be determined during development, thus variations in resistance during this phase can cause life-long impacts on the individual, presumably by altering the development of other physiological systems.