Typescript. / Digitized by Kansas Correctional Industries

O presente trabalho tem como finalidade estudar a resposta imunológica em escolares do sexo masculino, de 14 a 20 anos de idade, vacinados há 2 e 12 meses com BCG intradérmico, pertencentes a três Escolas do SENAI, nos municípios de Osasco, São Bernardo do Campo e São Paulo, Brasil, no período de agosto a dezembro de 1979. A população de estudo, constituída de 900 escolares, foi distribuída em três grupos: vacinados há 2 meses, vacinados há 12 meses e não vacinados, segundo as variáveis: idade, cor, sintomático respiratório, comunicante e presença de cicatriz pós-vacinal. Foram feitos 270 testes imunológicos, de transformação blástica de linfócitos para avaliar a resposta celular, e teste de hemaglutinação passiva para verificar a resposta humoral, pela pesquisa de anticorpos específicos. Simultaneamente foi realizado o teste tuberculínico com PPD Rt23 2UT,para avaliar a conversão tuberculínica pós-vacinal e a correlação da mesma com as respostas imunológicas. A resposta imunológica celular apresentou maior porcentagem de positividade do que a resposta humoral e se manteve praticamente inalterada no decurso de 2 para 12 meses. Não houve associação estatisticamente significativa entre a resposta imunológica celular e reatividade tuberculínica, nas pessoas vacinadas há 2 e 12 meses. Não houve relação entre reatividade tuberculínica, presença de cicatriz pós-vacinal e resposta imunológica. / This survey is to study the immune response of male students, between 14 and 20 years old, who had received intradermal BCG 2 and 12 months ago. They studied at SENAI schools in Osasco, São Bernardo do Campo and São Paulo, Brazil, during august to december of 1979. The population of this study was 900 students separated in three groups: one who had received vaccine 2 months ago; other after 12 months and the last one who did not receive vaccine. The variables age, color, respiratory symptom, contact and presence of post vaccinal cicatrix were analysed. They were realised 270 immunologic tests of blastic transformation of lymphocyte in order to evaluate the cellular response and the hemagglutination test to find out the specific antibody to verify the humoral response. The tuberculin test was realized with PPD Rt23 2UT to evaluate the post-vaccinal tuberculin conversion and the correlation with the immune response. The cellular immune response demonstrated that has major positive percentage than the humoral response and was unaltered between 2 and 12 months groups. There were no statistical significant correlation between cellular immune response and tuberculin reativity in boys who received vaccine 2 and 12 months ago. There was not relation among tuberculin reativity, post vaccinal cicatrix and immune response.

Leung Pui-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 226-266). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.viii / 撮要 --- p.xii / TABLE OF CONTENTS --- p.xvi / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis & Leukemia --- p.1 / Chapter 1.1.1 --- Introduction to Hematopoiesis and its Regulation --- p.1 / Chapter 1.1.2 --- Leukemia --- p.7 / Chapter 1.1.2.1 --- Classification and Epidemiology of Leukemia --- p.7 / Chapter 1.1.2.2 --- Conventional Approaches to Leukemia Therapy --- p.13 / Chapter 1.1.2.3 --- New Approaches to Leukemia Therapy --- p.16 / Chapter 1.2 --- Coumarins --- p.18 / Chapter 1.2.1 --- Introduction to Coumarins --- p.18 / Chapter 1.2.1.1 --- "Historical Development, Occurrence of Coumarins and their Functions in Plants" --- p.18 / Chapter 1.2.1.2 --- Beneficial Effects of Coumarins for Human Use --- p.20 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Coumarins --- p.22 / Chapter 1.2.2.1 --- Chemical Structures of Coumarins --- p.22 / Chapter 1.2.2.2 --- Biosynthesis of Coumarins --- p.24 / Chapter 1.2.2.3 --- Toxicology of Coumarins --- p.24 / Chapter 1.2.2.4 --- Mode of Entry of Coumarins --- p.25 / Chapter 1.2.2.5 --- Metabolic Pathways of Coumarins --- p.27 / Chapter 1.2.3 --- Pharmacological Activities of Coumarins --- p.30 / Chapter 1.2.3.1 --- Anti-edema and Anti-inflammatory Activities --- p.30 / Chapter 1.2.3.2 --- Cardiovascular Protective Function --- p.32 / Chapter 1.2.3.3 --- Anti-tumor Activity --- p.33 / Chapter 1.2.3.3.1 --- Anti-tumor Activity In Vitro --- p.33 / Chapter 1.2.3.3.2 --- Anti-tumor Activity In Vivo and Clinical Applications --- p.34 / Chapter 1.2.3.4 --- Immunomodulatory Activity --- p.36 / Chapter 1.2.3.4.1 --- Immunological Considerations --- p.36 / Chapter 1.2.3.4.2 --- Immunomodulation In Vitro --- p.37 / Chapter 1.2.3.4.3 --- Immunomodulation In Vivo --- p.38 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.41 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.43 / Chapter 2.1.1 --- Animals --- p.43 / Chapter 2.1.2 --- Cell Lines --- p.43 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.44 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.49 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.49 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.52 / Chapter 2.1.7 --- Cell Death Detection ELISAplus Kit --- p.54 / Chapter 2.1.8 --- Reagents for Total RNA Isolation --- p.55 / Chapter 2.1.9 --- Reagents and Buffers for RT-PCR --- p.56 / Chapter 2.1.10 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Aicds --- p.60 / Chapter 2.1.11 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.61 / Chapter 2.1.12 --- Reagents for Measuring Caspase Activity --- p.67 / Chapter 2.1.13 --- Reagents for Neutral Red Assay --- p.70 / Chapter 2.2 --- Methods --- p.71 / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.11 / Chapter 2.2.2 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.71 / Chapter 2.2.3 --- Determination of Cell Viability --- p.72 / Chapter 2.2.4 --- In Vivo Anti-tumor Study --- p.73 / Chapter 2.2.5 --- Analysis of Cell Cycle Profile/DNA Content by Flow Cytometry --- p.74 / Chapter 2.2.6 --- Measurement of Apoptosis --- p.74 / Chapter 2.2.7 --- "Islation ,Preparation and Culture of Mouse Peritoneal Macrophages" --- p.76 / Chapter 2.2.8 --- Gene Expression Study --- p.77 / Chapter 2.2.9 --- Protein Expression Study --- p.80 / Chapter 2.2.10 --- Determination of the Mitochondrial Membrane Potential --- p.84 / Chapter 2.2.11 --- Measurement of Caspase Activity --- p.84 / Chapter 2.2.12 --- In Vivo Macrophage Migration Assay --- p.85 / Chapter 2.2.13 --- Study of Endocytic Activity of Macrophages --- p.85 / Chapter 2.2.14 --- Determination of Nitric Oxide Production by Macrophages --- p.86 / Chapter 2.2.15 --- Study of Mitogenesis of Murine Splenocytes --- p.87 / Chapter 2.2.16 --- Analysis of T Cell Population and its Sub-populations --- p.88 / Chapter 2.2.17 --- Measurement of Lymphokine Activated Killer (LAK) Cell Activity by Neutral Red Assay --- p.90 / Chapter 2.2.18 --- Statistical Analysis --- p.91 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI PROLIFERATIVE EFFECT OF COUMARINS ON MACROPHAGE-LIKE LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Results --- p.94 / Chapter 3.2.1 --- Anti-proliferative Effect of Coumarins on Various Macrophage-like Leukemia Cell Lines In Vitro --- p.94 / Chapter 3.2.2 --- Kinetic and Reversibility Studies of the Anti-proliferative Effect of Coumarins on Macrophage-like Leukemia PU5-1.8 Cells --- p.105 / Chapter 3.2.3 --- Cytotoxic Effect of Coumarins on Macrophage-like Leukemia PU5-1.8 Cells In Vitro --- p.110 / Chapter 3.2.4 --- Effect of Coumarins on the Cell Cycle Kinetics of the Macrophage-like Leukemia PU5-1.8 Cells In Vitro --- p.113 / Chapter 3.2.5 --- Effect of Coumarins on the Expression of Cell Cycle- and Growth-regulatory Genes in the Macrophage-like Leukemia PU5-1.8 Cells --- p.119 / Chapter 3.2.6 --- Effect of Coumarins on the Expression of Cell Cycle-regulatory Proteins in the Macrophage-like Leukemia PU5-1.8 Cells --- p.123 / Chapter 3.2.7 --- Effect of Coumarins on the In Vivo Tumorigenicity of the Macrophage-like Leukemia PU5-1.8 Cells --- p.127 / Chapter 3.2.8 --- Effect of Coumarins on the In Vivo Growth of the Macrophage- like Leukemia PU5-1.8 Cells in Syngeneic BALB/c Mice --- p.131 / Chapter 3.3 --- Discussion --- p.133 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING EFFECT OF COUMARINS ON MACROPHAGE-LIKE LEUKEMIA PU5- CELLS --- p.1.8 / Chapter 4.1 --- Introduction --- p.143 / Chapter 4.2 --- Results --- p.152 / Chapter 4.2.1 --- Induction of Apoptosis in the Murine Macrophage-like Leukemia PU5-1.8 Cells by Coumarins --- p.152 / Chapter 4.2.2 --- Modulatory Effect of Coumarins on the Expression of Apoptosis-regulatory Genes in the Macrophage-like Leukemia PU5-1.8 Cells --- p.159 / Chapter 4.2.3 --- Modulatory Effect of Coumarins on the Expression of Apoptosis-regulatory Proteins in the Macrophage-like Leukemia PU5-1.8 Cells --- p.166 / Chapter 4.2.4 --- Effect of Coumarins on the Mitochondrial Membrane Depolarization of the Macrophage-like Leukemia PU5-1.8 Cells --- p.169 / Chapter 4.2.5 --- Effect of Coumarins on the Induction of Caspase Activity in the Macrophage-like Leukemia PU5-1.8 Cells --- p.172 / Chapter 4.2 --- Discussion --- p.177 / Chapter CHAPTER 5: --- STUDIES ON THE IMMUNOMODULATORY EFFECT OF COUMARINS ON MURINE MACROPHAGES AND OTHER IMMUNE CELLS / Chapter 5.1 --- Introduction --- p.184 / Chapter 5.2 --- Results --- p.187 / Chapter 5.2.1 --- Effect of Coumarins on the Viability of Macrophages In Vitro --- p.187 / Chapter 5.2.2 --- Effect of Coumarins on the In Vivo Migration of Macrophages --- p.190 / Chapter 5.2.3 --- Effect of Coumarins on the Endocytic Ability of Murine Macrophages --- p.192 / Chapter 5.2.4 --- Effect of Coumarins on the Nitric Oxide Production by Murine Macrophages --- p.194 / Chapter 5.2.5 --- Effect of Coumarins on the Viability of Murine Splenocytes --- p.199 / Chapter 5.2.6 --- Effect of Coumarins on the Mitogenesis of Murine Splenocytes --- p.201 / Chapter 5.2.7 --- Effect of Coumarins on T-cell Population and its Sub-populations --- p.205 / Chapter 5.2.8 --- Effect of Coumarins on the Induction of Murine LAK Activity --- p.208 / Chapter 5.3 --- Discussion --- p.210 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.218 / REFERENCES --- p.226

Systemic lupus erythematosus is an autoinflammatory disorder driven by the development of autoantibodies to self-nucleic acids, in particular to DNA and to chromatin. Loss-of-function mutations of the secreted deoxyribonuclease DNASE1L3 have been implicated in the development of aggressive familial lupus. In addition, recent genome-wide association studies have linked a hypomorphic variant of DNASE1L3 to sporadic lupus. Studies in the lab determined that Dnase1l3-deficient mice develop rapid autoantibody responses against dsDNA and chromatin, and at older ages this leads to a lupus-like inflammatory disease. These disease manifestations were completely independent of the intracellular DNA sensor STING, which has been implicated in other examples of self-DNA driven autoinflammatory diseases. My project focused on developing assays to track the activity of DNASE1L3, as well as identifying the endogenous source of self-DNA normally processed by DNASE1L3. Using mouse models that allow the depletion of specific cell populations, we found that circulating DNASE1L3 is produced by hematopoietic cells, in particular by CD11c+ dendritic cells and by tissue macrophages. Taking into account the unique properties of DNASE1L3, we discovered that this enzyme is uniquely able to digest chromatin contained within and on the surface of apoptotic microparticles. Loss of DNASE1L3 activity in circulation results in elevated levels of DNA in plasma, in particular within microparticles. Microparticles are extensively bound by anti-chromatin autoantibodies isolated from both murine models of lupus as well as prototypical human clones. In addition, Dnase1l3-deficient mice have high levels of circulating IgG that bind to microparticles from young ages, and these titers increased as disease progressed in aged animals. Pretreatment of microparticles with DNASE1L3 largely abrogated this binding, demonstrating that DNASE1L3 directly reduces the immunogenicity of microparticles. We also studied two human patients with null mutations in DNASE1L3, and observed increased DNA circulating in plasma and, in particular, in their microparticles, demonstrating a conserved role for DNASE1L3 in mice and humans. Finally, we obtained plasma samples from a cohort of patients with sporadic SLE, and found that roughly 80% had circulating IgG that avidly bound microparticles. Roughly half of this group failed to bind to microparticles that had been pretreated with DNASE1L3, and this DNASE1L3-sensitive group also presented with lower levels of DNASE1L3 activity. We conclude that extracellular chromatin associated with microparticles acts as a potential self-antigen capable of causing loss of tolerance to self-DNA and inflammatory disease in both mice and humans. The secretion of a DNA-processing enzyme thus represents a novel, conserved tolerogenic mechanism by which dendritic cells restrict autoimmunity.

Abstract not available

Abstract not available

Includes a list of publications co-authored by the author during the preparation of this thesis. Thesis amendments in back leaf. Includes bibliographical references (leaves 123-168). The studies described concentrate on the cloning and characterisation of the two Drosophila caspases, DRONC and DECAY

Aim: Subunit vaccines have received increasing attention due to their good safety profile. However, subunit vaccines feature low immunogenicity, and soluble antigen is largely ignored by the immune system due to its lack of danger signals. To stimulate an appropriate immune response, subunit antigen vaccines require the addition of an adjuvant and multiple administrations. This study aimed to formulate biodegradable lipid implants, containing a suitable adjuvant, which delivers antigen in a sustained manner. The physico-chemical characteristics of the implants and their ability to stimulate immune responses towards a model antigen in vivo were investigated. Methods: Lipid implants were prepared from phospholipid and cholesterol. Different adjuvants were added, and their potential to induce an immune response to the model antigen ovalbumin (OVA) was investigated. The adjuvants and immunomodulators assessed were Quil-A (QA), imiquimod, and an α-Galactosylceramide (α-GalCer) analogue. Liposomal dispersions were prepared using the lipid film hydration method. These were freeze-dried, and the powder compressed into matrices (diameter of 2 mm). Physico-chemical characterisation was undertaken by transmission electron microscopy (TEM) to investigate the release of colloidal structures (liposomes, immunostimulating complexes [ISCOMs]) upon hydration with release media. Surface changes of the implant matrices were analysed using scanning electron microscopy (SEM). The release of the fluorescently-labelled antigen ovalbumin (FITC-OVA) and its entrapment into the colloidal particles was investigated using spectrofluorophotometry. Additionally, incorporation of the cationic cholesterol derivative DC-cholesterol (DCCHOL) into implants to allow for charge-charge interactions with the negatively-charged OVA, and replacement of the phospholipid with a phospholipid having a higher transition temperature to facilitate the manufacturing process, were attempted and assessed. The immune response stimulated towards OVA released from the implants was analysed in vivo using a C57Bl/6 mouse model. Expansion of CD8⁺ T cells and CD8 T cells specific for the CD8 epitope of OVA (SIINFEKL), as well as expansion of CD4⁺ T cells, were assessed. The ability of implants to stimulate T cell proliferation and interferon-γ production after in vitro restimulation with OVA was analysed. Serum samples were analysed for OVA-specific IgG antibodies. Results: Lipid implants containing Quil-A released colloidal structures upon hydration with buffer. The type of colloids observed by TEM depended on the ratio of QA:cholesterol:phospholipid. Release of OVA was sustained over ten days in implants prepared with egg yolk PC. However, the release kinetics depended strongly on the choice of phospholipid. In vivo, lipid implants containing Quil-A evoked expansion of CD8⁺ T cells. The immune response to one implant was comparable to that obtained by two equivalent injection immunisations. Therefore, the implants obviated the need for multiple immunisations in the vaccination regime tested here. Expansion of CD8⁺ T cells towards the Quil-A-containing implant was greater than that achieved by the immunomodulators imiquimod and the α-GalCer analogue. Quil-A-containing implants produced OVA-specific IgG antibodies to a greater extent than the implants containing imiquimod or α-GalCer. Incorporation of the cationic DCCHOL did not increase the entrapment efficiency of OVA into liposomes. However, the in vivo investigation of DCCHOL-containirig implants showed an adjuvant effect of DCCHOL on antibody responses, but not on cell-mediated immunity. Conclusion: Lipid implants offer great potential as sustained release vaccine delivery systems. The lipid components in the implant formulation were well-tolerated and biodegradable. Lipid implants combine the advantages of sustained release of antigen and particulate delivery by the formation of colloidal particles.

Aim of thesis: Immuno-stimulating complexes (ISCOMs) are particulate vaccine delivery systems composed of Quillaja saponins, cholesterol and phospholipid. ISCOMs are typically spherical cage-like structures with a diameter of 40 nm and carry a negative charge. Incorporation of the respective vaccine antigen into the particles generates more potent vaccines than a simple mixture of both vaccine components. This requires the antigen to display either hydrophobic domains or positive charges, which allow interaction with the ISCOM particles. However, not all antigens fulfil this requirement and modification of these becomes necessary. Hence, the aim of this study was to design novel preparations of ISCOMs with a positive charge, suitable for adsorption of native hydrophilic antigens and poly-nucleotides, and test their potential as a novel vaccine carrier platform. Methods: Two cationic lipids, DC-cholesterol and DOTAP, were selected to prepare the cationic modifications of ISCOMs. DC-cholesterol substituted for cholesterol in classical ISCOMs, whereas DOTAP substituted for their phospholipid component. The phase behaviour of colloidal systems containing Quil-A, phosphatidylcholine (PC) and DC-cholesterol and of colloidal systems comprised of Quil-A, cholesterol and DOTAP was studied by transmission electron microscopy (TEM). Lipid-film hydration was utilised as the first method to prepare these colloidal systems. Selected compositions containing either DC-cholesterol or DOTAP were also prepared by dialysis as second method. A novel third method for preparing homogenous dispersions of classical ISCOMs was developed utilising ethanol injection. This method was also applied in an attempt to prepare cationic modifications of ISCOMs including DC-cholesterol and DOTAP. As in the colloidal systems comprising Quil-A, PC and DC-cholesterol transformations of structures were observed upon dilution with aqueous solutions, these transitions were also studied on classical ISCOMs using TEM and dynamic light scattering techniques. Loading of cationic colloidal structures composed of Quil-A, PC and DC-cholesterol was performed with the model protein antigen ovalbumin (OVA) and a model plasmid, and the resulting structures were analysed by fluorescence spectroscopy, TEM and gel electrophoresis. The immunological properties of non-loaded and OVA-loaded structures were studied in terms of their ability to activate murine bone marrow derived dendritic cells (mBMDC) as antigen presenting cells (APC) and OVA-specific CD8+ T cells in vitro. Results: Substitution of cholesterol in classical ISCOMs with DC-cholesterol resulted in the formation of cationic cage-like structures similar to the classical particles. These were observed in pseudo-ternary Quil-A:PC:DC-cholesterol systems and even in pseudo-binary Quil-A:DC-cholesterol systems prepared by lipid-film hydration. Compositions at which cage-like structures were observed included high weight proportions of DC-cholesterol (> 60%). However, samples were relatively heterogeneous, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DC-cholesterol. The ionic strength, pH and composition of the hydration buffer were demonstrated to be important variables influencing the formation of cage-like structures. Morphological changes of pre-formed cationic cage-like structures were observed upon dilution. However, classical anionic ISCOMs showed a similar behaviour. The numbers of cationic cage-like structures appeared to increase upon prolonged storage of samples. Purification of structures and longitudinal analysis of their composition suggested an increased formation of stoichiometrically defined DC-cholesterol:Quil-A:PC complexes over time, rather than a change in composition. The substitution of phospholipid in classical ISCOMs with DOTAP also resulted in heterogeneous dispersions, and aggregation of colloidal structures was observed at equimolar ratios of Quil-A and DOTAP. Phase separation phenomena were proposed based on TEM observations. However, the formation of cage-like particles with a positive [zeta]-potential was not observed. Although ethanol injection was introduced as a novel method to prepare classical ISCOMs, its application did not result in more homogenous dispersions of cationic colloidal structures containing DC-cholesterol or DOTAP. Dialysis also failed to produce higher numbers of well-defined cationic particles, although using this method homogeneous, anionic ISCOM-like particles containing DOTAP were obtained. The efficient adsorption of OVA and plasmid DNA onto cationic structures containing Quil-A, PC and DC-cholesterol was demonstrated. The adsorption process was accompanied with a decrease in [zeta]-potential, aggregation of structures and changes in the ultra-structure, particularly at high protein:lipid ratios. The in vitro immunogenicity of dispersions containing Quil-A, PC and DC-cholesterol was equivalent to that of classical ISCOMs in terms of activation of mBMDC and OVA-specific CD8+ T cells, even though smaller amounts of Quillaja saponins and total lipid were co-delivered with OVA. Furthermore, the uptake of OVA by BMDC appeared to be more efficient in conjunction with the novel cationic dispersions. Conclusions: Cationic colloidal structures containing Quillaja saponins offer great potential as vaccine delivery systems. Their advantages thus far include simple and efficient adsorption of antigen, efficient uptake by APC and immunological activity in vitro. With further development, cationic carriers containing Quillaja saponins may constitute a very potent vaccine delivery platform suitable for a variety of subunit antigens, and suffice both pharmaceutical and immunological requirements.

The autoimmune diseases embody a diverse range of common human conditions that are caused by a loss of self-tolerance in the host immune system to a specific organ or tissue type. Approximately 5% of the general population are affected by autoimmune diseases which include type 1 diabetes (T1D), rheumatoid arthritis (RA) and Graves disease (GD). The majority of the autoimmune diseases are multifactorial in origin, brought about by a combination of both environmental and genetic factors. Numerous susceptibility loci have been identified for each autoimmune disease and a number of these loci have been shown to be shared amongst the autoimmune diseases. The fine-mapping of susceptibility loci to the underlying disease genes remains the current challenge facing complex disease genetics. This project aimed to further characterise the autoimmune disease susceptibility locus IDDM6 on chromosome 18q12-21. This was achieved by using a comparative mapping approach that incorporates the study of genetic association in human autoimmune disease alongside the consomic mapping of the orthologous region in the non-obese diabetic (NOD) mouse model of autoimmunity. Deleted in colorectal carcinomas (DCC) provided a strong candidate gene at IDDM6 and the resident R201G polymorphism was identified as a functional candidate A potential mechanism for the R201G polymorphism involvement in T1D aetiology was identified where the polymorphism may affect the ability of DCC to induce apoptosis in vitro. However, no evidence for R201G association could be detected in autoimmune disease case-control datasets from the New Zealand (NZ) population (T1D n = 428, RA n = 730, autoimmune thyroid disease n = 192 (AITD); versus n = 1246 healthy controls). In addition, no evidence for R201G involvement in T1D could be provided in a transmission disequilibrium test (TDT) incorporating 382 affected sib-pair families (54.2% transmission; P = 0.15). Significant association of R201G with GD was detected in a United Kingdom (UK) dataset (P = 0.002) from the Newcastle population (423 cases vs. 393 controls) but this was not replicated in an additional dataset from the UK Birmingham population (731 cases vs 668 controls; P = 0.81). It was concluded that the R201G polymorphism may encode susceptibility to GD but is unlikely to be the sole aetiological variant that accounts for the linkage previously observed at IDDM6 in autoimmune disease. To further investigate DCC as a positional candidate at IDDM6, five SNPs were selected from a 100 kb window surrounding a DCC-resident microsatellite that had previously been associated with T1D, called "88,21". The five SNPs were genotyped in the NZ T1D dataset, and the ascertainment of estimated haplotypes in this dataset revealed association of a rare haplotype with T1D, called haplotype H (3.31% cases vs 1.17% controls; P = 0.0044), in addition to global association of all haplotypes (P = 0.018). Haplotype H was also associated in an independent case-control dataset from the UK comprised of 400 T1D subjects and 443 healthy controls (P = 0.038). Maximum support for association of haplotype H was extended when both the UK and NZ T1D datasets were combined (P = 0.0017). Association of haplotype H could not be verified in a family-based test for association using the 382 UK T1D families (P = 0.40). However, the inclusion of the DCC SNPs in a TDT analysis of the published DCC-resident microsatellites "88,21" and "55,26", that had been used to identify IDDM6, extends support for the previously-associated 2-10 haplotype (2-10 refers to the published allele nomenclature at "88,21" and "55,26" respectively; 2-10-haplotype A; 59.6% T; P = 0.0058). There was no evidence for association of the five SNPs with RA or AITD when using either individual SNP analyses or estimated haplotypes in the NZ datasets. A similar lack of association was reported for the UK Newcastle GD dataset. Taken together, these data further support DCC, or a nearby gene, as conferring susceptibility to T1D. The human genetic data that supports IDDM6 involvement in autoimmune disease is further strengthened by consomic mapping of the orthologous region in mouse, using the non-obese diabetic mouse (NOD) model of autoimmune disease. In this thesis, the first evidence for a diabetes and thyroiditis susceptibility locus on mouse chromosome 18 is presented, which have been designated Idd21 and Sat1 respectively. This was achieved by using a chromosome-replacement strain with chromosome 18 derived from the diabetes-resistant Biozzi ABH strain on a diabetes-susceptible NOD genome, called NOD.ABH[Chr�⁸]. Mouse chromosome 18 contains orthology to both IDDM6 and the rat diabetes-susceptibility locus Iddm3. The NOD.ABH[Chr�⁸] mice showed a dramatic and significant reduction in diabetes incidence (30% of females were affected by 7 months of age versus 85% in NOD; P <0.0001) and that of thyroiditis (15.5% at 12 months compared to 37.4% in NOD; P <0.002). The comparative mapping of the chromosome 18 autoimmune susceptibility locus IDDM6 in human and mouse presented in this thesis provides further support for this locus. This research also clearly defines the next steps required to fine-map IDDM6 to the underlying disease genes, especially in regard to the DCC gene.