BALB/c mice infected with P. chabaudi AS develop immunity to erythrocytic-stage infection with early Th1 responses followed by a switch towards Th2 responses later to mediate protection during chronic disease. In order to determine the importance of the Th2 cytokines, IL-4/IL-13, in inducing protective immunity, the course of P. chabaudi infection was monitored in IL-4Rα-deficient mice. Interestingly, an early delay in the onset of peak parasitaemia in IL-4Rα-/- compared with WT control BALB/c mice was evident. Consequently, we demonstrated that IL-4Rα deficiency resulted in mice becoming more susceptible to chronic P. chabaudi infection with increased recrudescence, mortality and an impaired Th2 immune response compared with WT control mice. Similar results in the overall disease and immunological profiles between IL-4Rα-/- and wild-type mice were obtained whether male or female mice or the AJ or AS strains of P. chabaudi were used to infect mice. Thus, the protective role of IL-4Rα signalling during chronic disease was not parasite strain-specific or host gender dependent. However, males were significantly more susceptible than female mice and consequently further studies involving cell-type IL-4Rα-/- mice, utilized female mice to identify functional targets of IL-4/IL-13 protection. Abrogated IL-4Rα expression on macrophages/neutrophils (LysMcreIL-4Rα-/lox) mice had minimal effect on the outcome of P. chabaudi AS chronic infection and was comparable to WT mice implicating no major role for alternatively activated macrophages during chronic infection. In contrast, CD4+ T-cell-specific IL-4Rα-/- (LckcreIL-4Rα-/lox) mice infected with P. chabaudi AS developed increased recrudescence, increased mortality and impairment of Th2 immunity during the chronic infection similar to that of the global IL-4Rα-/- mice. This highlights the importance of signalling via CD4+ T-cells signalling via IL-4Rα for protective immunity during chronic infection. Paradoxically, CD4+CD8+ T-cell-specific IL-4Rα-/- (iLckcre IL-4Rα-/lox) mice displayed a similar disease profile to WT control mice but manifested a delayed Th2 phenotype during the latter stage of the disease with enhanced splenomegaly in comparison to the WT and IL-4Rα-/- mice. Thus while protection during chronic infection with P. chabaudi AS appears dependent on CD4+ T-cells responsive to IL-4, CD8+ T cells responsive to IL-4 have a more complex and more difficult role to interpret.

Hepatitis C virus (HCV) is a hepatotropic blood-borne virus which causes chronic hepatitis in the majority of cases and represents a global health burden. In order for HCV to enter cells, proteins on the surface of the virus must interact and bind to receptors on target cells. HCV surface molecules involved with receptor binding, and cellular entry, as well as immune escape, are the glycoproteins E1 and E2. The cellular receptors SRBI, CD81, CLDNs and most recently occludin have been shown to facilitate HCV entry into hepatocytes. Several conservative regions on E1E2 have, through substitution mutagenesis, proven to be important for receptor binding and antibody neutralisation. We aimed to characterise one discontinuous region, amino acid residues 611, 613-619 and 621, and its role in the interaction with CD81 by single alanine substitution mutagenesis. Mutant plasmids were transfected into HEK 293FT cells and assessed for protein expression and binding by conformation-sensitive, CD81-inhibiting antibodies. Also, to investigate whether a conformational change of the E1E2 occurs upon SRB1 binding, rendering the CD81 binding domains accessible, two assays have been compared. A plate based experiment, exclusive of SRBI and a cell based assay, including SRBI was designed to examine the antigenic exposure of the CD81 binding regions to targeting monoclonal antibodies. Additionally, Huh-7 cells expressing different levels of SRBI were used to investigate whether some HCVpp isolates rely on high SRB1 levels for infectivity and sensitivity to neutralising antibodies. These studies were performed to help elucidate the regions and residues important in HCV E1E2: receptor interaction and their interplay with each other and with neutralising antibodies.

No description available.

Spondyloarthropathies (or Spondyloarthritides; SpAs) are a group of heterogeneous but genetically related inflammatory disorders in which ankylosing spondylitis (AS) is considered the prototypic form. Among the genes associated with AS, HLA-B27 allele has the strongest association although the cause is still not clear. Rats transgenic for the human HLA-B27 gene (B27 rats) develop a systemic inflammation mirroring the human SpA symptoms and thus provide a useful model to study the contribution of this MHC class I molecule in the disease development. Of particular interest was the observation of absence of arthritis in B27 rats grown in germ-free conditions and a recent theory suggests that microbial dysbiosis and gut inflammation might play a key role in initiating the HLA-B27-associated diseases. Studies in our laboratory have previously demonstrated that HLA-B27 expression alters the development of the myeloid compartment within the bone marrow (BM) in B27 rat and causes loss of a specific dendritic cell (DC) population involved in self-tolerance mechanisms within the gut. The aim of this thesis was to further analyse the myeloid compartment in B27 rats with a particular focus on the osteoclast progenitors and the bone phenotype and to link this to the gut inflammation. In addition, translational studies analysed peripheral monocyte/pre-osteoclasts in AS patients and teased apart the role of cytokines in in vitro human osteoclast differentiation. To understand the dynamics of the myeloid/monocyte compartment within the B27-associated inflammation, monocytes within the bloodstream and BM of B27 rats were characterised via flow cytometry and their ability to differentiate into osteoclast was assessed in vitro. Moreover, an antibiotic regime was used to reduce the B27 ileitis and to evaluate whether this could affect the migration, the phenotype, and the osteoclastogenic potential of B27 monocytes. B27 animals display a systemic and central increase of “inflammatory” CD43low MOs, which are the main contributors to osteoclastogenesis in vitro. Antibiotic treatment reduced ileitis and also reverted the B27 monocyte phenotype. This was also associated with the reduction of the previous described TNFα-enhancement of osteoclast differentiation from B27 BM precursors. These evidences support the idea that in genetically susceptible individuals inflammation in the gut might influence the myeloid compartment within the BM; in other terms, pre-emptively educate precursor cells to acquire specific phenotype end functions after being recruited into the tissue. This might explain the enhanced differentiation of osteoclast from B27 BM progenitors and thus the HLA-B27-associated bone loss. The data shown in this thesis suggest a link between the immunity within the gut and BM haematopoiesis. This provides an attractive and novel research prospective that could help not only to increase the understanding of the HLA-B27-associated aetiopathogenesis but also to unravel the cellular crosstalk that allows the mucosal immunity to program central cell differentiation. Human translational studies on monocyte subsets, cytokines and cytokine network in AS osteoclastogenesis evidenced altered osteoclast differentiation in the presence of IL-22 although no differences in the phenotype and functions of circulating CD14+ monocytes were observed. In addition, studies on the role of TNFα and TNFRs showed a dual role of this inflammatory cytokine in the human OC differentiation. In particular, the activation of TNFR1 in monocytes in early osteoclastogenesis inhibits OC differentiation while TNFα-biasing for TNFR2 on osteoclast precursors mediates the osteoclastogenic effect. Whether similar mechanisms are involved in the TNFα-mediated joint destruction in human rheumatic diseases needs further investigations. This could contribute to the development of novel and more specific anti-TNFα agents for the treatment of bone erosion. In conclusion, taken together my studies support the idea of a crosstalk between the periphery and the central system during the inflammatory response and provide new insights to the mechanisms behind the enhancement of osteoclastogenesis in B27-associated disorders.

No description available.

No description available.

Critical metabolic steps of the central carbon metabolism pathways have been of primary focus for the discovery of novel drug targets against Leishmaniasis, a neglected tropical disease caused by Leishmania. It is known that this particular protozoan parasite also relies on exogenous amino acids; however, comparative studies between exogenous amino acids utilisation and their essentiality for parasite growth and metabolism were often less emphasised. In this thesis, a novel approach was used to apply metabolomics techniques in a newly developed amino acids rich chemically defined medium for in vitro culture of Leishmania promastigotes. The chemically defined medium allowed for controlled customisable environment for deeper understanding of amino acids metabolism and parasite growth. A previously reported semi-defined growth medium (REIX (Steiger and Black, 1980)) was selected as a starting point. Undefined components of REIX were omitted and the composition of the resulting defined medium was varied through the addition, individually or in groups of amino acids and trace components (including magnesium, calcium, zinc, iron salts, biopterin, folate, riboflavin and lipoic acid). Growth assays were performed to identify a medium composition, referred to as Nayak medium, that supported robust growth over serial passages, at a similar rate to a conventional, serum supplemented rich growth medium (HOMEM (Berens et al., 1976)). Subsequently, the importance of individual amino acids was studied by systematic analysis in media containing equimolar quantities of each proteogenic amino acid, bar one (termed “amino acid knock out media”). Growth rate, concentration dependent growth, cell body length, total cell protein and acetate secretion were all recorded. Based on these results, it was found that exogenous supply of L-Tryptophan, L-Phenylalanine, L-Arginine, L-Lysine, L-Leucine and L-Valine as most critical for viability of promastigotes. Hence, it was of interest to further investigate the intracellular composition of the other 14 amino acids and their anabolic and catabolic pathways within L. mexicana promastigotes. Comparative investigation of intracellular amino acid metabolism was conducted using a metabolomic finger-printing technique in log phase L. mexicana promastigotes cultured in defined medium. Previous studies on amino acid pathways were mostly based on genome annotation or reductionist experimental approaches, however, network mapping of metabolomics data as shown in Chapter 5 allowed to understand new insights about compensatory amino acid metabolic pathways, not predicted before. The 45 known components of the defined medium allowed for easier distinction from ~400 metabolites of the intracellular samples. The free amino acids pool showed increased abundance of L-Alanine, L-Glycine, L-Proline, L-Histidine, L-Asparagine, L-Threonine and L-Tyrosine in the intracellular metabolome which partly explains the dispensability of these amino acids in culture media as observed in single amino acid knock out experiments. Approximately 64 metabolites, putatively identified as related to amino acid metabolism were analysed by pathway mapping. This showed a unique utilisation pattern amongst all amino acids following a two-step transamination and a decarboxylation with significant NADH production. The downstream intermediates were secreted into the environment, rather than elaborate degradation pathways as followed within the mammalian systems. Validation of the observed secretion pattern of amino acid degradation was conducted using metabolic foot-printing technique at six different time points, which provided data on relative quantification of metabolite uptake and secretion from the culture media at different growth stages. About ~200 metabolites were putatively identified from across the replicates in the dataset collected over a growth period of 9 days in both the positive and negative ionisation mode and only ~70 metabolites could be identified with authentic standards for individual mass/charge ratio and retention time comparison. Depletion profiling of the culture media showed that L-Tryptophan, L-Aspartate, L-Glutamate, L-Arginine, L-Methionine and L-serine were continuously utilised with more than 50% consumed over the 9-day growth period. Amino acids such as L-Leucine, L-Threonine, L-Valine, L-Phenylalanine, L-Tyrosine and L-Lysine were partially utilised, while L-Glycine, L-Glutamine, L-Asparagine, L-Proline and L-Alanine were continuously secreted during the growth period under conditions tested. Overall, ~25 % of the total metabolites were found depleted from the medium, ~15% not changed significantly, 20% un-identified metabolites and interestingly, about ~40% of metabolites were significantly enriched in the medium. Systematic analysis of the 40% secreted small molecules secreted in the culture media constituted the exometabolome. The proportion of keto acids exceeded the other constituents of the exometabolome, confirming the partial degradation and overflow metabolism. Hence, the possibility that keto acids could also contribute to virulence was speculated. Early results from various biochemical analysis allowed new insights about amino acid derived enol lactones, especially the aromatic pyruvates to support the establishment of parasitism, with macrophage cells as in vitro experimental model. However, targeted molecular biology studies are required to further understand the role of exometabolome in parasitism; especially by inhibition of the Leishmania enzymes L-Tryptophan transaminase or phenyl pyruvate dehydrogenase as they are non-homologous to mammalian systems. In summary, this study has shown simple, customisable platform for in vitro culture of Leishmania promastigotes in defined medium, intracellular and exometabolome of Leishmania adaptable for various studies. L-Tryptophan and L-Phenylalanine were found as the most critical exogenous amino acids for parasite viability and their analogues inhibited parasite growth. Hence, warrants further studies for their effectiveness as drugs in the field. Also, Leishmania exometabolome could serve as diagnostic signature for species identification, taxonomical comparisons, virulence patterns, physiological comparisons within varied nutrient systems.

Hypoxia has been linked to the pathogenesis of hepatic steatosis in murine and human models. There is an abundance of data suggesting that HIFs play a central role in regulating hepatic lipid metabolism. This study suggested that hypoxia-induced hepatic lipid accumulation is through de novo lipogenesis and free fatty acid uptake, and is dependent on hypoxia inducible factors la and 2a. On the contrary, hepatitis C infection reduced de novo lipogenesis and free fatty acid uptake in both normoxic and hypoxic conditions in vitro, and this inhibition is viral strain-dependent. In the clinical setting, chronic hepatitis C {CHC) and non-alcoholic fatty liver diseases {NAFLD) are associated with hepatic steatosis and insulin resistance. Using an integrative physiological approach that measures lipid and carbohydrate flux in vivo we demonstrated that patients with CHC had modest increase in insulin resistance and that the relative contribution of tissue specific insulin sensitivity in patients with CHC and NASH varied. Furthermore, curing HCV infection improved hepatic and subcutaneous adipose tissue insulin resistance. The improvement in hepatic and adipose tissue insulin resistance was more pronounced in patients infected with genotype 3 HCV, whilst the improvement in skeletal muscle insulin resistance was more evident in genotype 1 infection, demonstrating a genotype-specific effect in the metabolic perturbation in CHC. Further studies are required to confirm that genotype specific effect of HCV on insulin resistance and its link with NASH.

Liver cirrhosis is the fifth leading cause of death worldwide and the definitive treatment for liver cirrhosis is liver transplantation although there are limitations such as organ availability and surgical risks. Therefore, alternative therapies have been studied extensively and stem cell therapies have shown some promising results although most studies are small and not randomised. The aim of this thesis was to explore the effectiveness of stem cell therapy in patients with chronic liver disease as well as explore the mechanism behind fibrosis resolution achieved with cell therapy. There were three parts to the thesis: firstly, I examined the mechanistic actions behind fibrosis reduction by the infusion of bone marrow derived haematopoietic stem cells (HSC) in mice chronic fibrosis liver injury model. I worked on both immune-histochemical staining and qPCR to measure the effect oval cell response, matrix metalloproteinases and macrophage subsets within the liver with HSC therapies. Secondly, I recruited patients with chronic liver diseases for a multicentre, randomised, controlled trial to assess the clinical effectiveness of either subcutaneous granulocyte-colony stimulating factor (GCSF) or GCSF with repeated HSC infusions. The co-primary outcomes were improvement in severity of liver disease measured by model for end stage live disease (MELD) at 3 months and the trend of MELD change over time. The results showed that neither of the treatments improved the clinical outcomes. Lastly, I performed a systematic review of current published studies of stem cells therapies in liver diseases. The results showed that stem cells improved patients’ clinical parameters in the short term (< 6 months) but had no benefit on long term outcomes. In conclusion, bone marrow derived stem cell therapy did not seem to be effective in liver cirrhosis.

Mycobacterium tuberculosis is ostensibly an intracellular pathogen, which may form pellicle-like biofilms in the peripheries of tuberculosis cavities. Environment-induced cell wall modifications and extracellular polymeric substance production may alter host-pathogen interactions. Specifically, expectorated mycobacteria from cavities, which establish infection in new hosts, may have distinct phenotypic adaptations to impair early clearance by the innate immune system. M. tuberculosis H37Rv biofilm extracellular polymeric substance was identified using scanning electron microscopy. Biofilm phenotype non-covalently-bound extracts of cell wall lipids and carbohydrates were compared to planktonic phenotype and a relative reduction in the proportion of constituent glucose in biofilm carbohydrate extracts was discovered, indicative of a reduction in α-glucan prevalence. Comparison of carbohydrate extracts’ potency in stimulating cytokine and chemokine secretion in whole blood and complement activation elucidated reduced C3b/iC3b deposition onto biofilm carbohydrate extracts. Labelling live dispersed M. tuberculosis planktonic and biofilm samples with fluorescent antibodies showed C3b/iC3b, C5b-9, MBL and C1q deposition was reduced on biofilm phenotype cells, using flow cytometry. The relative contribution of each major pathway of complement activation was investigated and greater dependence on classical pathway activation by M. tuberculosis biofilm cells compared to planktonic cells was observed. Implications of these findings in M. tuberculosis pathogenesis are discussed.