Complement plays an important role in the humoral response to protein antigens, but its role in anti-capsular antibody responses is unclear. The immunogenicity of a panel of twelve CPSs from S. pneumoniae was investigated in mice deficient in complement receptors 1 and 2 (CD35/CD21, respectively). With the exception of serotype 4, IgM anti-polysaccharide responses were not dependent on the presence of CD35/CD21. In contrast IgG anti-polysaccharide responses, which were infrequently and variably elicited in C57BL/6 mice, were critically dependent on the presence of CD21/CD35. Although classified as T-independent type 2 (TI-2) antigens, antibody responses to pneumococcal CPSs, except serotype 4, were different from those to dinitrophenol (DNP)-Ficoll, a model TI-2 antigen. Our results indicated that complement plays a critical role in IgG anti-polysaccharide and anti-hapten responses. However IgM anti-pneumococcal CPS responses, with the only one exception, were surprisingly complement-independent. Our findings establish a differential role of complement in humoral responses to a model TI-2 antigen and clinically relevant polysaccharide antigens. In addition to the studies of S. pneumoniae CPSs, the adjuvant properties of CPSs from K. pneumoniae were also invesitgated. Several K. pneumoniae CPS preparations, in particular K1 and K3, have been reported to possess potent immunomodulatory properties. However the purity of CPSs used was not investigated adequately. We modified previously established methods of CPS purification in order to obtain highly purified CPSs for our studies. Pure CPSs with molecular weights of 1-2 x 106 daltons failed to augment the antibody response to chicken gamma globulin (CGG) when used as an adjuvant in mice. In vitro studies also demonstrated that crude but not highly purified CPSs induced the proliferation of or cytokine production from splenocytes from normal and LPS-hyporesponsive mice. The active component (s) in crude CPSs has not been identified, but it is resistant to heat, protease, nuclease and alkaline treatments. We have also ruled out the possibility that the immunomodulatory effects were due to bacterial DNA or LPS, two common contaminants in these preparations. Since not all the components in CPS extracts from K. pneumoniae have been characterised, further investigations may identify novel adjuvant candidate(s).

Dendritic cells (DC) sense infection in their microenvironment and undergo a dynamic process of changes in phagocytic capacity, morphology and migratory activity in order to induce optimum T cell immunity. The acquisition of these properties is termed "DC maturation". In this study, key selected aspects of this DC maturation process have been explored. One aspect of DC maturation is the signalling pathways that DC use to respond to micro-environmental signals. DC respond to conserved microbial structures in their micro-environment via pattern recognition receptors including Toll-like receptors (TLRs). Generally it is thought that this leads to activation of the mitogen-activated protein kinase (MAPK) pathways, p38, ERK, and JNK. JNK and p38 MAPK are also activated by reactive oxygen species, including hydrogen peroxide (H202), known to be present in the inflammatory DC milieu. Therefore the ability of H2O 2 to stimulate DC, or modulate their response to TLR ligands such as lipopolysaccharide (LPS) was examined. Activation of JNK, associated with inhibition of tyrosine phosphatases, is linked to induction of DC apoptosis. JNK inhibition partially protected the DC from the pro-apoptotic effects of H2O2. Furthermore, H2O2 and LPS synergise in inducing JNK activation, and DC apoptosis. These studies suggest that excessive DC maturation, which may induce pathogenic T cell activation may be limited by DC apoptosis. The second section of the thesis explores the mechanisms leading to MAPK activation upon TLR ligation. Biochemical studies demonstrated that mixed lineage kinase 3 (MLK3), a kinase not previously implicated in DC maturation, was phosphorylated upon TLR3 and 4, but not TLR2 ligation. Studies using a selective MLK inhibitor supported a role for the mixed lineage kinase (MLK) family of MAPK kinase kinase (MAP3K) proteins in regulation of DC signalling, phenotype and cytokine secretion, in response to TLR3 and 4, but not TLR2 ligation. Selective activation of MAP3K may therefore contribute to the heterogeneity and flexibility of DC/pathogen interaction. A further feature of the DC sensory role is antigen uptake, but the relationship between morphology and phagocytic capacity has not been defined. Therefore a simple and flexible assay to examine this relationship was developed. DC with long dendritic processes were less efficient at antigen internalisation than round DCs.

The aim of this project was to investigate the effects of HIV infection on class II biosynthesis and surface expression on professional and non-professional APC. To investigate the mechanism by which HIV controls class II expression in non-professional APC, a transfection model was designed for the expression of the HIV <I>env</I> gene, either singly, or co-transfected with an HIVΔ<I>env</I> plasmid, in HLA-DR4+ Chinese hamster ovary (CHO-DR) cells. It was shown that the env glycoproteins produced in this model were processed correctly in both single and double transfectants, and could be incorporated into virus particles released from <I>env</I>/HIVΔ<I>env</I> transfected cells. Env production in CHO-DR cells led to an increase in the surface expression of conformationally immature class II. This increase was not due to higher levels of class II biosynthesis, or more rapid intracellular processing. This shows that, in the presence of the HIV env glycoprotein, a greater proportion of cell-associated class II reaches the cell surface, and that env may act as a chaperone, stabilising immature class II molecules. Using Western blotting, it was also shown that class II, in the form of αβ dimers, could be incorporated into virus particles released from <I>env</I>/HIVΔ<I>env</I>-transfected CHO-DR cells. It is possible that this is dependent on association with env glycoproteins, suggested by the effect of env expression on surface class II expression. The results indicate that HIV has different effects, depending on the cell type under investigation. In professional APC, class II surface expression remains unchanged despite increased biosynthesis, whilst in non-professional APC there is increased surface class II on infected cells. Data from the single, and double transfection models indicate that these effects may be mediated by the HIV env glycoprotein.

More than 40% of the world's population still live under the threat of malaria. Present drug and vaccine strategies are failing to eradicate or even control the disease. A greater understanding of the molecular biology of the intracellular parasite is necessary in order to develop better chemotherapy and vaccine strategies. One aspect of the parasite's biology which may yield new drug targets, are novel trafficking pathways which are established when it invades a red blood cell. In order to study the mechanisms involved in such pathways, specific proteins found in defined locations in the host cell must be studied. One group of proteins that may prove useful in such studies are exported integral membrane proteins because they may act as markers for the polarity of certain membranes. Previous work in our laboratory has focused on the integral membrane protein exp-1. In order to test the validity of a model proposed for its transport to the cytoplasm other exported, parasite encoded, integral membrane proteins need to be identified. This thesis has focused on other parasite encoded proteins which appear to be exported by the parasite to the parasitophorous vacuole membrane (PVM). The initial approach adopted was to study a protein recognised by a specific monoclonal antibody, 7.7. Using a double labelling indirect immunofluorescence technique this antigen, termed exp-2, was shown to be co-localised with exp-1 in the parasitophorous vacuole membrane (PVM) and in vesicle-like structures in the erythrocyte cytoplasm. In the course of attempting to isolate the exp-2 gene another antigen, exp-3, was isolated. Further characterization of exp-3 indicated that it was a member of a two gene family. Both genes were isolated and found to be located on chromosome 13. Sequence analysis of the two genes showed that the genes appear to be highly conserved at the amino terminus and diverge in a repeat region towards their C-terminus.

The original hygiene hypothesis has been revised, suggesting that deficient regulatory mechanisms are responsible for the rising trends in inflammatory disorders. Helminths establish long-lived chronic infections in their definitive host, with avoidance and manipulation of host effector response. Throughout helminth infections, immunoregulatory networks are generated, limiting immunopathology and permitting survival of parasites. I propose that helminth induced regulatory networks extend to regulate damaging allergic and autoimmune inflammatory responses. This thesis aimed to experimentally dissect the relationship between helminth infections and allergic diseases. We show here that cellular populations generated during a helminth infection can control both Th1-mediated autoimmune, and Th2-associated allergic inflammation. CD4⁺CD25⁺ T cells or CD19⁺ B-cells from mesenteric lymph nodes (MLN) of mice chronically infected with <i>Heligmosomoides polygyrus</i> were transferred to allergen sensitive or myelin oliogodenodrocyte glycoprotein (MOG) _(p35-55) immunised recipients. Allergen-sensitive mice receiving cells from infected donors had significantly reduced airway eosinophilia, broncho-alveolar lavage (BAL) fluid IL-5 and eotaxin secretions upon airway challenge in a model of allergic airway inflammation. Similarly, MOG_(p35-55) immunised mice receiving CD19⁺ B-cells from infected mice, had a significantly delayed onset and reduced severity of disease with fewer CD4⁺IFN-γ⁺ cells in the central nervous system (CNS) during experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). I propose a testable regulatory model encompassing multidimensional infectious tolerance with an array of regulatory cells. Taken together, these data highlight the immunoregulatory potential of chronic helminth infections, explaining the inverse relationship between helminth infections and dysregulated inflammatory events.

Our laboratory uses a murine model of lymphatic filariasis that involves the peritoneal implant of the filarial nematode<i> Brugia malayi</i> to obtain a source of macrophages activated <i>in vivo</i> in chronic Th2 inflammatory conditions. These ‘nematode-elicited’ macrophages (NeMф) show a highly IL-4 dependent phenotype including the ability to suppress the proliferation of a wide range of cells and the expression of Arginase1, a characteristic marker of alternative activation. We decided to take a genomic approach to define what genes determine the IL-4 dependent phenotype of NeMф. This analysis revealed the striking expression of Fizz1 and Ym1; genes of uncertain function. We have shown that Fizzl and Ym1 expression is a characteristic feature of nematode infection and that expression is induced at the site of parasite migration or residence. Both genes are also directly responsive to IL-4 and IL-13, and are therefore reliable markers of Th2-driven immune responses. Finally, the study of their expression profile in haematopoietic cells showed restriction to antigen presenting cells, pointing to a role in shaping the immune response by regulating antigen presentation. Having originally characterised NeMф in C57BL/6 mice, we wanted to see whether the NeMф function was similar in BALB/c mice, the other commonly used mouse strain in our laboratory. In BALB/c mice, we found that both the function and gene expression of NeMф was not IL-4 dependent, and showed that this was due to compensation by the Th2 cytokine IL-13. The dependence on Th2 cytokines and the high expression of Arginase1 and Ym1 suggests that NeMф may mediate wound repair; one of the hypothesized functions of alternatively activated macrophages.

No description available.

No description available.

Malaria is one of the most widespread parasitic diseases. It is caused by <i>Plasmodium</i> protozoa. <i>Plasmodium falciparum</i> infection leads to the most virulent symptoms in human. Researchers have been devoted to develop a malaria vaccine for malaria eradication, especially against <i>P.falciparum</i>. Rhoptry associated protein-1 (RAP-1) is one of the <i>P.falciparum</i> vaccine candidates because it protected monkeys in an immunization experiment and some monoclonal antibodies raised against this protein can inhibit parasite growth <i>in vitro</i>. This thesis describes work in which the epitopes of the inhibitory monoclonal antibodies were mapped by using a combination of recombinant DNA technology and peptide synthesis. The part of the <i>rap-1</i> gene, which contains epitope of inhibitory monoclonal antibodies, was subcloned and expressed in <i>Escherichia coli</i> in a β-galactosidase fusion form. The use of restriction enzymes and exonuclease III to generate different fragements of <i>rap-1</i> gene suggested that the epitopes of these monoclonal antibodies cluster near a proteolytic cleavage site on the protein (between A_190 and D_191). This was confirmed when the epitope of all the inhibitory monoclonal antibodies were located on a TLTPLEELYP_210 peptide generated as one of a series of overlapping decapeptides. The study of RAP-1 protein immunogenicity in rabbit and humans showed that both species are also able to recognize this 'inhibitory epitope'. The epitope mapping of another set of monoclonal antibodies raised against a recombinant RAP-1 protein lacking its 'native' conformation was carried out. The results suggested that this expressed protein can stimulate immune response against the 'inhibitory epitope' but two additional epitopes are also recognized by these monoclonal antibodies. Sequence analysis of the <i>rap-1</i> gene from a number of isolates and clones demonstrated that the RAP-1 protein is highly conserved in <i>P. falciparum</i> from several parts of the world. This finding verified the potential of the RAP-1 protein as a malaria vaccine candidate.

A murine model for filarial infection has been used as an <i>in vivo </i>source of nematode-elicited Macrophages (NeMs). Mice are surgically implanted into the peritoneal cavity with adult <i>Brugia malayi.</i> By one week post-infection, the PEC population is dominated by macrophages that display IL-4 dependent features such as the expression of Arginase1, RELM-α and Ym1. In light of the increasing evidence that macrophages show functional adaptivity, it was decided to study the NeMs response to pro-inflammatory Th1 activating signals as a model to investigate whether the switch between alternative and classical activation can occur in macrophages differentiated in an <i>in vivo</i> infection setting. Despite the long-term exposure to Th2 cytokines and anti-inflammatory signals <i>in vivo</i>, we found that NeMs were not terminally differentiated but could switch from alternative activation to a more classically activated phenotype in response to LPS/IFN-γ. This was reflected by a switch in the enzymatic pathway for arginine metabolism from arginase to iNOS and the reduced expression of <i>RELM-α </i>and <i>Ym1.</i> To ask whether these Ms could be induced to be antimicrobial, we also carried out infections with “type 1”-inducing pathogens. It was found that LPS/IFN-γ treated NeMs were able to control infection with <i>Leishmania mexicana</i> as effectively as LPS/IFN-γ activated thioglycollate-elicited macrophages (ThioMs) and parasite killing was mediated by nitric oxide production. NeMs were also infected with <i>Mycobacterium bovis</i> BCG. It was found that NeMs responded to low doses of BCG by controlling it for the entire timeframe of the study, i.e. 6 days. NeMs responded to high doses of BCG infection with early control of infection and high levels of apoptosis, and that this phenotype is independent of IL-4.