The innate immune system is a generic response to infection or injury. Evidence shows the innate response has immunological memory capable of altering subsequent responses to stimuli. Fibroblasts are ubiquitous stromal cells capable of responding to inflammatory triggers, and of orchestrating endothelial cell and leukocyte behaviour during inflammation. Repeated challenge with cytokines (such as tumour necrosis factor (TNF) a) induced an augmented second response to stimulation. Fibroblasts from multiple anatomical locales significantly increased cytokine secretion upon second challenge with TNFa. The precise mediators augmented depended on fibroblast site of origin. Depending on site, memory was inherent, or only present in fibroblasts from chronically-inflamed tissue. This suggests a phenomenon intrinsic to some sites but pathological in others. The secreted mediators from the fibroblast initial or memory responses exerted differing effects on leukocytes, dependent upon fibroblast site of origin. Finally, examination of intracellular signalling showed the augmented response was at least partly due to prolonged activity of nuclear factor (NF) KB during the memory response. Innate immune memory exists in fibroblasts from multiple tissues, but may be pathologically acquired in some. The altered response to second challenge may represent a fibroblast mechanism for altering the recruitment and behaviour of the inflammatory infiltrate.

The liver is a unique tolerogenic organ with dual blood supply. Both regulatory lymphocytes and effector lymphocytes are present in the normal and inflamed liver along with innate immune cells. The balance between these two subsets of lymphocyte is crucial in maintaining immune homeostasis by adjusting either hepatic tolerance or mounting immunity against invading pathogens. Thus, it is important to understand the intrahepatic regulatory T cells phenotype and role played by distinct chemokine receptors expressed on regulatory T cells as they are major player in controlling hepatic tolerance. At the same time, it would be crucial to explore the role of new subset of Th17/Tc17 effector lymphocytes characteristic and their positioning in inflamed liver. This thesis demonstrates the crucial role of chemokine receptors in recruitment and positioning of both intrahepatic regulatory T lymphocytes and IL-17 secreting Th17/Tc17 effector lymphocyte in both normal and inflamed human liver.

The liver contains a number of tissue-associated lymphocyte populations, of which many have been implicated in the pathogenesis of chronic liver diseases. γδ T cells, particularly the Vδ2^neg subset, are known to comprise a substantial proportion of tissue-associated lymphocytes, although their immunobiology remains poorly understood. Here, the localisation, TCR diversity, immunophenotype and function of human intrahepatic γδ T cells was explored with an emphasis on highlighting any potential role in chronic liver disease and also to further understanding of tissue-associated γδ T cells, using the liver as a model tissue. Intrahepatic γδ T cells were predominantly localised in the sinusoids and did not increase in frequency with chronic inflammation. Vδ2^neg cells exhibited private TCR clonal focussing, with complex CDR3 regions suggestive of antigen-driven expansions, concordant with a loss of naive-like CD27^hi cells present in the periphery. Expanded clonotypes were phenotypically T_EM- or T_EMRA-like, with T_EMRA-like clonotypes shared between liver and blood and resembling vasculature-associated virus-specific CD8⁺ T cells while T_EM clonotypes were identified only in the liver and resembled tissue-resident CD8⁺ T cells. These findings suggest that disease has minimal impact on intrahepatic γδ T cells, while supporting an adaptive paradigm for these cells in the formation of tissue-associated subsets.

RhoJ is an endothelial expressed Rho GTPase, and its knock-down impairs endothelial cell (EC) migration and tubulogenesis, increases stress fibre (SF) and focal adhesion (FA) numbers. This work aimed to determine the intracellular localisation of RhoJ, identify its binding partners, test how it is activated and further explore its function in ECs. Endogenous RhoJ localised to FAs and overexpression of its active mutant (daRhoJ) promoted EC migration, and diminished FA and SF numbers. In addition to FAs, overexpressed RhoJ localised also to endosomes and RhoJ knock-down slightly delayed transferrin recycling. Vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2) and thrombin activated RhoJ in ECs. PAK-interacting exchange factor β (βPIX) and G protein-coupled receptor kinase-interacting target 1 (GIT1), which promote FA disassembly, were identified as RhoJ-binding partners. RhoJ co-localised with these proteins in ECs, and βPIX knock-down and to a lesser extent GIT1 knock-down reduced RhoJ localisation to FAs. Overexpression of daRhoJ increased the amount of GIT1 and βPIX in FAs, and increased the total amount of the βPIX protein in ECs. In conclusion, RhoJ localises to FAs, promotes EC migration, regulates FA and SF numbers, interacts with βPIX and GIT1 and is activated by pro-angiogenic factors.

We have established a murine model of pneumococcal pneumonia in order to characterise the host inflammatory response. Intranasal infection with Streptococcus pneumoniae resulted in constant bacterial loads within airways of susceptible mouse strains whilst the pathogen burden increased in both the lung tissues and bloodstream. The bacteria induced inflammation that was evident as perivascular cell recruitment in histological sections. Mast cell granule staining indicated that this population degranulated early in the inflammatory response, releasing TNFa into the lung environment. Levels of TNFa and IL-1b increased during mid infection with release of IL-6 and the anti-inflammatory cytokine IL-10 not occurring until late infection. Kinetics of IL-10 were too slow to prevent inflammation causing damage to the host tissues. The total protein levels in bronchoalveolar lavage fluid (a marker of the integrity of the alveolar/capillary barrier) increased significantly during the experiment. Dose response results indicate that there is likely to be a threshold number of bacteria required to induce this inflammatory response both in the lungs and bloodstream. Inoculation of bacteria into a mouse strain resistant to the above infection resulted in bacterial clearance from both the pulmonary airways and tissues, with few more becoming bacteraemic. The location and timing of the inflammatory response in this mouse strain was significantly different. Inflammatory cell influx occurred mainly within the airways, with perivascular areas unaffected. Mast cell degranulation occurred rapidly following infection. TNF activity and IL-1b levels within lung airways were induced earlier and to a greater extent than in susceptible mice. IN contrast, the levels of TNF activity and IL-6 within lung tissues were lower in resistant mice. Although damage to lung integrity still occurred, this was only transient and evident during early/mid infection.

In the first part of the present study the immune responses in NIH mice against asexual blood stages of either avirulent DK or virulent DS strains of P. chabaudi adami, in single or mixed infections, were examined using the ELISA test for the detection and measurement of cytokine and antibody production. The present results showed that the profile of the immune response in all the above infections suggests a sequential Thl/Th2 CD4+ T cell response. Previous studies have shown that there is a sequential Thl/Th2 response in Plasmodium chabaudi AS infection which is reflected in the activation of both cell- and antibody-mediated responses. These findings, therefore, indicate that vaccines which induce both arms of the protective immune response against malaria parasites could be most effective. The sequential Th1/Th2 response was supported by detecting early high levels of IFNγ and IgG2a during the acute phase of the infection and later by elevation of IL-4 and IgGl levels during the course of infection compared to controls. The levels of IgG2a were at highest levels at or immediately after the peak parasitaemia while the levels of IgG1 increased in later stages in the course of infection. However, a higher level IFNγ early in the infection indicated a stronger Thl response in the avirulent DK strain infection compared to the virulent DS strain or mixed infections. On the other hand, in the virulent DS infection a stronger Th2 response with higher IL-4 levels compared to the DK strain and mixed infections was observed in mice treated with chloroquine. In the mixed infection, an infective dose consisting of 8x103 pRBCs of the avirulent DK strain and 2x103 pRBCs of the virulent DS strain was used. Despite a relatively low number of pRBCs of the DS strain in the infective dose the peak parasitaemia was significantly higher than that in the single-infection of 1x104 pRBCs of the DK strain. The mixed infection also showed a significantly lower peak parasitaemia compared to that in mice given 1x104 pRBCs of the DS strain single-infection in untreated mice. So, it may be concluded that a higher peak parasitaemia in the mixed infection compared to the DK single-infection is reflected in a higher replication rate of the DS strain compared to the DK strain during the course of the mixed infection.

Selective delivery of drugs to parasites via plasma membrane transporters offers an effective approach to specifically target pathogens. Using biochemical techniques, we have identified and characterised a number of purine nucleobase and nucleoside transporters in protozoan parasites such as Leishmania major, Leishmania mexicana, Trypanosoma brucei and Toxoplasma gondii. In one study, the uptake of [3H]adenine, [3H]hypoxanthine, and [3H]allopurinol, and antileishmanial hypoxanthine analogue, by Leishmania major promastigotes was investigated. The results showed that these compounds were all taken up by a single high-affinity transporter, LmajNBT1, with Km values of 4.6±0.9, 0.71±0.07, and 54±3 μM and Vmax­ values of 3.2±0.3, 2.8±0.6, and 0.24±0.06 pmol (107 cells)-1s-1, respectively. [3H]adenine transport was fully inhibited by the natural purines guanine and xanthine, with Ki values of 2.8±0.7 and 23±8 μM, respectively. Using purine analogues, an extensive inhibitor profile for LmajNBT1 was obtained, which allowed the construction of a quantitative model for the interactions between the transporter binding site and the permeant. The model predicts that hypoxanthine was bound through hydrogen bonds to N(1)H, N3, N7, and N(9)H of the purine ring, with a total Gibbs free energy of -39.5 kJ/mol. The interactions with adenine were similar, except for a weak hydrogen bond to N1 (unprotonated in adenine). The predicted model of substrate binding for LmajNBT1 was almost identical to that for the Trypanosoma brucei H2 (TbH2) transporter. It is proposed that the architecture of their respective binding sites is very similar and that LmajNBT1 can be named a functional homolog of TbH2. This thesis also reports the first identification and characterization of a purine nucleobase transporter in Leishmania amastigotes. Uptake of [3H]hypoxanthine by Leishmania mexicana amastigotes was mediated by a single high-affinity transporter, LmexNBT1, with Km and Vmax values of 1.6±0.4μM and 0.092 ± 0.057 pmol (107 cells)-1s-1 respectively, with high affinity for adenine, guanine, and xanthine, with Ki values of 4.2±0.8, 1.7±0.1, and 13±2 μM respectively, but low affinity for nucleosides and pyrimidine nucleobases. Allopurinol was apparently taken up by the same transporter (Km of 33.6±6.0 μM). All evidence was compatible with a model of a single purine nucleobase transporter being expressed in amastigotes. Using various purine nucleobase analogues, a model for the interactions between hypoxanthine and the transporter's permeant binding site was constructed and compared with previously obtained models for substrate recognition by nucleobase transporters, and found to be very similar to the models of the LmajNBT1 and TbH2 transporters, but markedly different from the human FNT1 transporter.

The primary mission of the immune system is to defend against invading pathogens. The normal healthy body can distinguish self from non-self antigens. When a new antigen is encountered, such discriminatory capacity would generate a productive immune response against invasive pathogens or exert antigen-specific tolerance, the latter to prevent harmful immune responses against self-components or non-dangerous food or environmental antigens. Peripheral tolerance plays an important role in preventing T cells response to self or harmless antigens. A breakdown in tolerance within an individual can result in the development of a variety of autoimmune disorders. Full T cell activation requires at least two signals. The first one is provided by the TcR recognizing cognate peptides derived from antigen in the context of appropriate MHC molecules expressed by antigen presenting cells (APC). The second is mediated by “co-stimulation” via interaction of CD28 on the T cell with CD80/86 on the APC. The clonal anergy is induced when the TcR is ligated in the absence of co-stimulation, one of the proposed mechanisms of peripheral tolerance, describes a state of long lasting unresponsiveness to antigen, in the T cell. Despite widely studies in this area, however, the mechanisms of induction of anergy and the efficient markers for diagnosis of anergy are still not clear. One of the mechanisms which contributes to forming tolerance is anergy, which can be defined as defect in cellular proliferation and IL-2 production. Furthermore, GTPase Rap1 has been reported to inhibit the generation of pERK signals and to accumulate in tolerant cells. However, most of previous studies have done by biochemical assessment of signaling in T cell lines or clones upon polyclonal stimulations in vitro, and thus has generated some conflicting data. For solving this problem, our lab has developed the technique, laser scanning cytometry (LSC), for observation of responses in individual antigen-specific T cells within their environmental niche within primary or secondary lymphoid tissue. By LSC, it has reported that there are significant differences in the amplitude and cellular localization of phosphorylated ERK signals when naïve and in vitro-primed and tolerized T cells respond to Ag. To further investigate the role of Rap1 by LSC, it revealed that counter regulation in Rap1 and phosphor-ERK expression during the maintenance phase of tolerance and priming of antigen-specific CD4+ T cells in vitro and in vivo. In T cells, the maintenance phase of anergy has been reported to reflect defective activation of transcription factor, such as c-Jun/c-Fos, that are involved in formation of the AP-1 complex, which is required for inducing transcription of the IL-2 gene and optimal activation and effector function of T cells. In turn, this appears to be determined by the lack of recruitment of the ERK, JNK and p38 MAPK signaling cascades. The small GTPase, Rap1, has long been implicated in such desensitisation of ERK, and the consequent reduced IL-2 production, observed in tolerised T cells. However, the most of these studies were processed with T cell lines or clones in vitro and as such are not necessarily representative of physiological responses of primary antigen-specific T cells. Consistent with the previous finding, we have extended these studies to investigate whether Rap1 plays a role in determining commitment to anergy and priming during induction and maintenance phases. As expected, analysis in the DNA synthesis during maintenance phase reported that the primed T cells exhibited a higher response than either naïve or anergic T cells, whilst the anergic T cells displayed an even lower DNA synthesis than naïve T cells undergoing a primary response. To further investigation in cytokine production of IL-2 and IFNγ at 24, 48 and 96 hour during the maintenance phase, consistent with previous studies, the primed T cells produced the highest levels of IL-2, relative to anergic cells with the lowest levels, at the first 24 hours after challenge with antigen. However, the IL-2 production from primed and anergic T cells both drop down from 48 hours and to very low level at 96 hours but accompanying with gradual increase of IFNγ production. This implicates both anergic and primed cells consumed IL-2 secreted in the early stage of maintenance phase for supporting following cellular differentiation. The assessment of cellular proliferation also indicates that both primed and anergic cells had undergone several rounds of division. Whereas the primed cells proliferated more and faster than anergic cells over the first two days, after that anergic cells were able to catch up with primed cells. Consistent with above proliferative responses, the primed T cells showed higher levels of ERK activation than anergic cells at day 1 but lower levels of ERK activation than anergic cells at day 3. Surprisingly, there is no difference in Rap1 activation between primed and anergic T cells during maintenance phase. The additional finding from cellular proliferation during maintenance phase revealed that both primed and anergic cells undergo clonal expansion during induction of priming and tolerance, which leads the further investigation in functional outcomes, MAPK signaling and mTOR pathways studies during induction phase. The primed cells exhibited higher levels of DNA synthesis than anergic cells at 48 hours whereas they had similar levels of DNA synthesis at 96 hours. The IL-2 and IFNγ production were only detectable within the first 48 hours but not 96 hour. Collaborating with the data from cellular proliferation indicates the IL-2 were consumed for promoting the cells survival and proliferation since both populations showed clear peaks representing differential numbers of cell division from day 2 (48 hour) onwards, whereas the primed cells proliferated more and faster than anergic cells during whole induction phase. Moreover, cyclic activation of ERK was seen in the primed T cells and at higher levels of activation than in the anergic population, which did not exhibit these kinetics in western blotting. Interestingly, the primed T cells exhibited slightly higher levels of Rap1 than anergic cells from 48 hour until 96 hour during induction phase. Consistent with data from in vitro, the proliferation response in mimicking physiological model also can be replicated. Additionally, the counter regulation in ERK and Rap1 activation also occurred during the induction of priming and tolerance, which is investigated by adenoviral gene transfer of Ad Rap1 S17N, an inactive mutant of Rap1. Furthermore, modulation of Rap1 expression with Ad Rap1 S17N in cells during induction of anergy, revealed that Rap1 activity acts to limit cellular proliferation and thus switching off Rap1 activity upregulates cellular proliferation to generate a phenotype more resembling priming of normal (or GFP-) T cells by antiCD3+anti-CD28, which showed higher proliferation that GFP- cells stimulated with anti-CD3 only. However, when these adenoviral transfer experiments were repeated in the more physiological model, the higher proliferation exhibited in anergic Ad Rap1 S17N transduced cells were not replicated, suggesting that the enhancing effect of Ad Rap1 S17N might be substituted by signals generated under these more physiological conditions. There did not appear any difference between anergic and primed cells in terms of ERK/Rap1 signalling during the induction phase and introduction of Ad Rap1 S17N did not modulate ERK activity in transduced cells treated with anti-CD3 or anti-CD3+anti-CD28, suggesting that Rap may target some other effector during the induction phase. To sum up these data, Rap signaling in anergy and priming as well as the use of the dominant negative construct suggested that Rap was not acting to suppress ERK activation during induction of anergy.

No description available.

The principal aim of this project was to investigate mechanisms of drug resistance in Trypanosoma brucei, the causative agent of disease in humans (sleeping sickness) and livestock (nagana), which affects large areas of sub-Saharan Africa. By understanding the mechanisms of resistance, the useful life of current therapies (of which there are only a few) may be extended, diagnostics to identify resistant parasites could be developed and the design of novel therapies aided. We therefore developed parasites with high levels of resistance to the clinically important drug pentamidine, which is the first-line treatment for early stage West African sleeping sickness and is closely related to the main veterinary treatment diminazene aceturate (Berenil). The characterisation of this strain revealed that the resistance phenotype was at least in part due to the loss of the previously characterised high affinity pentamidine transporter (HAPT). To investigate the protein(s) responsible for HAPT activity, and to identify any other proteins contributing to the resistance phenotype, we employed a proteomic approach. The plasma membrane sub-proteome (TbPM) of long slender bloodstream form trypanosomes was defined. A number of interesting observations were made from TbPM, and it will no doubt be of benefit to the greater scientific community. One example is the positive identification of many proteins hitherto designated as putative. A quantitative approach was then employed to analyse the resistant parasites using isotope-coded affinity tagging (I-CAT) and difference gel electrophoresis (DiGE), including a novel combination of DiGE and 16-BAC protein separation technologies. Both the plasma membrane subproteome and the soluble proteome were investigated, and a number of regulated proteins identified. The role of some proteins, with potentially relevant functions, such as a kinase, adenylate cyclase and a protein involved in kinetoplast stability should be further investigated.