Genome Wide Association Studies (GWAS) have uncovered many genetic regions which are associated with autoimmune disease risk. In this thesis, I present methods which I have developed to build upon these studies and enable the analysis of the causal variants of these diseases. Colocalization methods disentangle whether potential causal variants are shared or distinct in related diseases, and enable the discovery of novel associations below the single-trait significance threshold. However, existing approaches require independent datasets to accomplish this. I extended two methods to allow for the shared-control design; one of these extensions also enables fine mapping in the case of shared variants. My analysis of four autoimmune diseases identified 90 regions associated with at least one disease, 33 of which were associated with 2 or more disorders; 14 of these had evidence of distinct causal variants. Once associated variants have been identified, we may wish to test some aggregate property, such as enrichment within an annotation of interest. However, the null distribution of GWAS signals showing association with a trait and preserving expected correlation due to linkage disequilibrium is complicated. I present an algorithm which computes the expected output of a GWAS, given any arbitrary definition of "null", and hence can be used to simulate the null distribution required for such a test. Commonly, GWAS report only summary data, and determining which genetic variants are causal is more difficult; the strongest signal may merely be correlated with the true causal variant. I have developed a statistical method for fine mapping a region, requiring only GWAS p-values and publicly available reference datasets. I sample from the space of potential causal models, rejecting those leading to expected summary data excessively different from that observed. This removes the need for the assumption of a single causal variant. In contrast to other summary statistic methods which allow for multiple causal variants, it does not depend upon availability of effect size estimates, or the allelic direction of effect and it can infer whether the pattern of association is likely caused by a non-genotyped SNP without requiring imputation. I discuss the effect of choice of reference dataset, and the implications for other summary statistics techniques.

Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...

It is well known that too much sugar uptake causes many health problems, including diabetes and obesity (Lustig et al. in Nature 482:27-29, 2012). However, a team of researchers led by Dr. Wanjun Chen of the National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH), USA, have recently shown that d-mannose, a naturally occurring C-2 epimer of glucose is likely beneficial to human health. Their studies have revealed that supraphysiological levels of d-mannose that are safely achievable via drinking-water supplementation can be preventive and therapeutic to experimental autoimmune diabetes and asthmatic lung inflammation (Zhang et al. in Nat Med 23:1036-1045, 2017).

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24329号 / 生博第488号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 杉田 昌彦, 教授 垣塚 彰 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Plasmin has been suggested to be involved in degradation of extracellular matrix (ECM) and tissue remodeling during a number of physiological and pathological processes. The aims of this thesis were to study the functional roles of plasmin during pathological inflammation in autoimmune and nonautoimmune disease models of rheumatoid arthritis (RA), multiple sclerosis (MS), wound healing and infection. In order to explain the obtained results in our functional studies as well as some previous results on the functional roles of plasmin during different tissue remodeling processes, I propose that there is a functional correlation between absence of plasmin and an inability to activate complement. The role of plasminogen during autoimmune collagen type II-induced arthritis (CIA) was studied first. The data revealed that whereas 83% of wild-type (plg+/+) mice developed CIA, none of the plasminogendeficient (plg-/-) mice got arthritis within a 40-day period. When plg+/+ mice were injected with a mixture of monoclonal antibodies against collagen type II they developed arthritis within a 5-day period, whereas no arthritis could be seen in plg-/- mice, although these mice had normal binding of antibody to the cartilage surface. These data suggest that plasmin plays an essential role in the step between antibody binding and inflammatory cell infiltration during CIA, probably during the step of complement activation. When plg+/+ and plg-/- mice were injected intra-articularly with collagen type II or 0.9% NaCl following CIA induction, plg-/- mice developed typical CIA, but the disease was less severe than in the plg+/+ mice and restricted to the injected joints. Sustained tissue necrosis was found only in the plg-/- mice after the local injection. When the antigen-induced arthritis (AIA) model was used, plg-/- mice developed a much more severe arthritis than the plg+/+ mice. These results indicate that different forms of pathogenesis exist for CIA and AIA, and further emphasize the importance of trauma in the induction of CIA in plg-/- mice. We further investigated the role of plasmin in experimental autoimmune encephalomyelitis (EAE), which is an autoimmune disease model for MS. During a 2-month period, the severity, incidence, mean onset day, mean maximal score and mean accumulative score of EAE were essentially identical in plg-/- and plg+/+ mice of B10.Q background. Histopathological studies revealed similar levels of inflammation and demyelination in plg-/- and plg+/+ mice. These data indicate that plasmin does not play an essential role in the development of EAE. The findings that plasmin is essential for the development of CIA but not needed for the development of EAE suggest that plasmin may play a pivotal role in autoimmune diseases where complement activation is critically involved in the pathogenesis. The role of plasmin was also studied in a tympanic membrane (TM) wound healing model. After TM perforations were performed, the plg+/+ TMs had all healed by day 11, whereas TM healing was completely arrested in plg-/- mice even as late as day 143. Immunohistochemical studies revealed a disturbed inflammation and tissue remodeling pattern in plg-/- mice. These data indicate that plasmin plays a central role in the healing of TM perforations. The involvement of plasminogen in ear infections was also investigated in plg-/- mice. During an 18-week experimental period, spontaneous otitis media (OM) was essentially developed in all of the plg-/- mice, whereas all of the plg+/+ mice kept a normal TM status. Positive bacterial growth was found in 5 out of 6 plg-/- mice, but only in 1 out of 6 plg+/+ mice. Immunohistochemical studies showed an accumulation of inflammatory cells, fibrin and also other extracellular matrix in the middle-ear cavity and the external-ear canal of plg-/- mice. These results show a spontaneous development of OM in plg-/- mice, but not in plg+/+ controls, suggesting that plasmin plays a critical role in the defense mechanisms during ear infections. Taken together, plasmin appears to play essential roles during autoimmune and non-autoimmune diseases in which complement activation is critical in the pathogenesis.

Intravenous immunoglobulin (IVIG) is a common treatment for the neurological autoimmune diseases multiple sclerosis, multifocal motor neuropathy, myasthenia gravis, and chronic inflammatory demyelinating polyneuropathy. However, there is scant literature regarding the psychological effects of this treatment on quality of life (QOL). Using illness intrusiveness theory and personal control theory, this correlational, cross-sectional study examined the relationship between the IVIG treatment experience and QOL in neurological autoimmune patients. Surveys were employed to collect data from 79 patients at a neurological infusion center in Phoenix, AZ. Quantitative analyses included correlation, multiple regression, and mediation analyses to determine whether (a) IVIG treatment experience predicted QOL measured by 10 Neuro-QOL scales, (b) illness intrusiveness mediated the relationship between IVIG treatment experience and QOL, and (c) personal control mediated the relationship between illness intrusiveness and QOL. IVIG treatment experience predicted QOL in 1 Neuro-QOL subscale; illness intrusiveness mediated 9 of the Neuro-QOL subscales using bias-corrected bootstrapping for statistical significance; and personal control did not mediate the relationship between illness intrusiveness and QOL. These results may affect social change by increasing the understanding of physicians, nurses, and patients regarding the psychosocial impact of IVIG treatment. Results from the study may provide insight for interventions to assist patients in adjusting to this form of treatment.

No description available.

Inflammation has been implicated in the development of cardiovascular disease and a potential underlying mechanism in the pathogenesis of impaired vascular function. Two different but complementary approaches were utilized to determine the role of inflammation on vascular function. First, to evaluate the effect of acute inflammation, we induced muscle damage to both small and large muscle mass and measured vascular function every 24 hours for up to 5 days of recovery. Eccentric exercise-induced muscle damage, in both small and large muscle mass, resulted in a transient increase in central arterial stiffness. Next, patients with systemic lupus erythematosus (SLE) were studied as a model of chronic inflammation. Measurements of vascular function were compared in habitually-exercising and sedentary SLE patients, and age-matched healthy controls. Individuals with SLE demonstrated lower vascular function than healthy controls. When SLE patients were grouped by exercise status, habitually-exercising SLE patients exhibited similar vascular function to healthy controls, and lower overall disease activity compared with sedentary SLE patients, supporting the beneficial effect of regular exercise in this population. Inflammatory biomarkers were associated with measures of macro- and microvascular function. In conclusion, acute muscle damage and chronic disease-related inflammation have a potent effect on measures of vascular function, suggesting that inflammation plays a role in the pathogenesis of vascular dysfunction and is an important biomarker for cardiovascular risk. / text

Despite individual autoimmune diseases being relatively rare, collectively these diseases afflict 8 % of the population according to the American Autoimmune Related Diseases Association. With over 75 % of those affected being women, autoimmune disease has been recognised, by the World Health Organisation and the US National Institutes of Health, as a major global women's health issue. One third of autoimmune sufferers have a rheumatological disorder, which commonly affect the joints, muscle, skin, salivary glands and kidneys. Antibodies against nuclear antigens are a serological hallmark of these diseases. Detection of these antibodies is used in the diagnosis and prognosis of the disease. The sensitivity and specificity of the test, of which the antigen is a key component, is pivotal to correct disease diagnosis and management. The relationship between circulating autoantibodies and the target antigen is complex. Improving the effectiveness of a test to assist in diagnosis and prognosis comes from characterisation and understanding these complex relationships. This thesis compares bovine spliceosomal U1 small nuclear ribonucleoprotein particle (U1 snRNP) complex with its human equivalent, and examines the validity of using this bovine derived autoantigen in the diagnosis of the human autoimmune diseases, systemic lupus erythematosus and mixed connective tissue disease. Differences between bovine and human U1 snRNP composition were characterised using a combination of electrophoretic, immunoassay and mass spectrometry techniques. Although the U1C protein could not be identified in bovine U1 snRNP, all other specificities were present. U1A remained intact, whilst the U1 snRNP specific 68K protein was dephosphorylated and a large C-terminal domain was removed, such that 68K migrated as a 30-36 kDa cluster on SDS-PAGE. Bovine SmD proteins, present in U1 and non-U1 snRNPs, were unaffected, whereas, SmB'/B was truncated to a 12 kDa peptide, which interestingly, was no longer reactive with anti-RNP sera in western blot. The recognition of human SmB'/B protein by anti-RNP sera in western blot was further examined. A technique was developed to immunoaffinity purify tryptic digests of SmB'/B which could then be analysed by mass spectrometry. Interestingly, the human replication element protein (HREP) was tentatively identified, rather than SmB'/B as expected. It may be possible, therefore, that anti-RNP sera may be reacting with a protein other than SmB'/B. To examine the contribution of the individual U1 snRNP proteins to anti-RNP and anti-Sm sera reactivities, a method was developed to dissociate bovine U1 snRNP and to purify the individual component antigens. It was demonstrated both empirically and through anecdotal feedback from a commercial diagnostic kit producer that patient sera respond better to purified Sm-free 68K than the recombinant 68K antigen. The effect of commercial processing of bovine thymus, the source for U1 snRNP antigen, was determined. In this study, variables that may be controlled during processing, such as temperature, protease activity and pH, were investigated. Hydrolysis of the intact human 68K protein with the necrotic protease, cathepsin L, produced 38 and 25 kDa fragments, whereas exposure to ambient temperature and low pH produced 32 kDa peptide fragments similar to those observed in purified bovine 68K. It was therefore proposed that 68K protein may undergo autocatalytic hydrolysis during necrotic cell death. Thorough characterisation of the bovine spliceosomal U1 snRNP proteins has not only validated their use as diagnostic reagents in autoimmune disease but also provided some insight into the inactivation of U1 snRNP function during early cell death.

<p>Droplet microfluidics is a powerful platform for both fundamental and applied biomedical research. The droplets are small in size with a diameter of 1-300 um. Thus, they could function as a miniaturized environment for quantitative and qualitative analysis. Each droplet composes of water shielded by an immiscible organic shell which enables independent control over different droplets. The large surface to volume ratio of spherical structure allows rapid mass and heat transfer, which could enable more homogeneous chemical reactions. Moreover, since multiple identical droplets could be generated simultaneously, parallel analysis for large amount of samples are possible. The use of microfluidics brings more power to droplet technology. The precise control over the flow allows droplet with preferable size and structure to be generated, which is critical for quantitative analysis, homogeneous chemical reaction as well as some in vivo applications. </p><p>Nonetheless, generation of stable, monodispersed and well controlled emulsions to meet specific biological functions are still challenging. First of all, to form more biocompatible W/O/W DE, the microfluidics devices must be patterned with desired surface wettability. W/O emulsion could only form in hydrophobic environment and the O/W emulsions could only form in hydrophilic environment. Differential patterning of the surface wettability to meet the needs are challenging. Second, DE are stabilized by two amphiphilic surfactants, one for the oil phase and the other for the water phase. Selection of appropriate surfactants should hook with specific biological application to ensure stability and biocompatibility. Third, the choice of fluid and contents in the fluid will affect the viscosity and capillary number of interfacial interaction, and eventually influences the droplet formation. The choice of biocompatible medium and buffer must take this into consideration. Fourth, the adoption of emulsions for the specific application requires optimization of the processing techniques in order to meet the needs for final analysis. For instance, control of droplet rupture for content release, modulation of oil phase permeability, quantitative analysis of content with flow cytometry, etc. </p><p>In this thesis, we will first demonstrate the design and fabrication of PDMS-based devices for automatic and high-throughput DE formation in Chapter 2. In the following chapters, we will demonstrate the successful adoption of the microfluidics generated DE for different biological applications. In chapter 3, we will illustrate the application of DE as a micro-incubator for cellular studies such genetic circuit behavior and performance in bacterial cells cultured in DE droplets and formation of 3D mammalian cell spheroid. In chapter 4, we will show the successful application of DE as drug carriers for intranasal drug delivery. In chapter 5, we showed the application of microfluidics generated DE as template for microparticle synthesis and the use of these microparticles as therapeutic agents in nucleic acid induced inflammations in autoimmune diseases.</p> / Dissertation