Dissertations / Theses on the topic 'Autoimmune disorder'

Although genetic variation is a major risk factor of neurodevelopmental disorders (NDDs) such as tics and childhood obsessive-compulsive disorder (OCD), environmental factors during pregnancy and early life are important in disease expression. Epidemiological studies and animal models indicate maternal inflammation is one potential environmental factor. In my thesis, I compared the frequency of proinflammatory states in mothers of 200 children with tics/OCD with 100 autoimmune neurological controls and 100 age- and sex-matched healthy controls. I prospectively recruited children with tics/OCD aged ≤16 years sequentially referred to the Children’s Hospital at Westmead, and used a structured interview to capture data, focusing on maternal autoimmunity. Children with tics/OCD were phenotyped to identify associations with maternal autoimmunity, and exploratory biomarker testing and transcriptome analysis were performed in a subgroup of mothers to identify immune pathways potentially relevant to neurodevelopment in offspring. A total of 61 (30.5%) mothers of children with tics/OCD had autoimmune disease compared with 20 (20%) mothers of children with autoimmune neurological conditions (p=0.054) and 12 (12%) mothers of healthy controls (p=0.0004, adj OR 2.7 (95th CI 1.28-5.68)). Other proinflammatory states were also more common in mothers of children with tics/OCD than mothers of healthy controls (p<0.0001). Maternal autoimmunity was not associated with a distinct clinical phenotype in the child. Maternal blood and published Tourette brain transcriptomes showed common enrichment of differentially expressed genes in innate immune pathways. Our results indicate dysregulation of innate immune pathways may be pivotal in the expression of NDDs and should be a focus of further research. Understanding immune mechanisms in neurodevelopment could unveil opportunities to mitigate risk to children by reducing exposure to inflammation and open new avenues for treatment.

Bakgrund: Typ 1 diabetes är en obotlig autoimmun sjukdom där immunförsvaret angriper och förstör de insulinproducerande ß-cellerna i bukspottkörteln. Detta leder tillslut till total insulinbrist vilket är ett dödligt tillstånd. Genom att öka mängden fysisk aktivitet hos individer med typ 1 diabetes minskar risken för följdsjukdomar. Syfte: Syftet med studien var att undersöka de barriärer som finns till fysisk aktivitet hos individer med typ 1 diabetes. Metod: Den metod som valdes var kvantitativ metod där enkäten “Barriers to Physical Activity in Diabetes type 1” (BAPAD1) valdes ut. Totalt svarade 66 individer med typ 1 diabetes på enkäten. Resultatet analyserades sedan med hjälp av Excel. Resultat: Den vanligaste barriären till fysisk aktivitet var “rädslan för hypoglykemi” följt av “Att du tappar kontrollen över din diabetes under fysisk aktivitet” och “att du tappar kontrollen över din diabetes efter fysisk aktivitet”. Konklusion: Sammanfattningsvis fick vi vår frågeställning besvarad. Tidigare studier visar att rädsla för hypoglykemi är den vanligaste barriären till fysisk aktivitet och det bekräftades även i denna studie.<br>Background: Type 1 diabetes is an autoimmune disorder where the immune system destroys the insulin-producing pancreatic-ß cells. This eventually leads to total insulin deficiency which is a fatal condition. By increasing the amount of physical activity in individuals with type 1 diabetes the risk of sequelae reduces. Objective: The aim of this study was to investigate the barriers to physical activity in individuals with type 1 diabetes. Method: The chosen method to this study was the questionnaire “Barriers to Physical Activity in Diabetes type 1” (BAPAD1). A total of 66 individuals with type 1 diabetes filled the questionnaire according to their own barriers to physical activity. The answers were later analysed in Excel. Result: The most common barrier to physical activity was “the fear of hypoglycemia” followed by “The loss of control over your diabetes during physical activity” and “The loss of control over your diabetes after physical activity”. Conclusion: Over all the result of the study got our question answered. Previous studies show that fear of hypoglycemia is the most common barrier to physical activity, and this was also confirmed in this study

Neuromyelitis optica (NMO) is a rare autoimmune disorder, distinct from multiple sclerosis, causing inflammatory lesions predominantly in the optic nerves and spinal cord. An autoantibody (NMO IgG) against aquaporin-4 (AQP4), a water channel expressed on astrocytes, is thought to be causative. A clinic and laboratory-based survey of neuromyelitis optica spectrum disorders (NMOSDs) in Australia and New Zealand was undertaken to establish incidence and prevalence across the region and in populations of differing ancestry. Patients with clinical and laboratory features that were suspicious for NMOSD were referred to me. Testing for NMO antibodies was undertaken in all suspected cases. From this group, cases were identified who fulfilled the 2015 Wingerchuk diagnostic criteria for NMOSD. A capture-recapture methodology was used to estimate incidence and prevalence based on additional laboratory identified cases. The capture-recapture analysis gave an adjusted incidence estimate of 0.37 (95% CI 0.35 to 0.39) per million per year and a prevalence estimate for NMOSD of 0.70 (95% CI 0.61 to 0.78) per 100,000. NMOSD was three times more common in the Asian population (1.57 (95% CI 1.15 to 1.98) per 100,000) compared with the remainder of the population (0.57 (95% CI 0.50 to 0.65) per 100,000). The latitudinal gradient evident in multiple sclerosis was not seen in NMOSD. These figures are comparable with figures from other populations of largely European ancestry. I found NMOSD to be more common in the population with Asian ancestry.Frequencies of self-determined ethnic ancestry were calculated for confirmed NMOSD, suspected NMOSD, and MS. Prevalence rates for NMOSD and MS according to ancestry were compared. NMOSD cases were more likely to have Asian, Indigenous, or Other ancestry compared to suspected NMOSD or MS. There were no differences in the clinical phenotype of NMOSD seen in Indigenous compared to European ancestry populations. Per 100,000, the prevalence estimate for NMOSD in people with Māori ancestry was 1.50 (95% CI 0.52–2.49), which was similar to those with Asian ancestry 1.57 (95% CI 1.15–1.98). NMOSD prevalence in Australian Aboriginal and Torres Strait Islander populations was 0.38 (95% CI 0.00–0.80) per 100,000. I found the prevalence of NMOSD in the Māori population was similar to South East Asian countries, reflecting their historical origins. The prevalence of MS in this group is intermediate between those with South East Asian and European ancestry living in New Zealand. Both NMOSD and particularly MS appear to be uncommon in the Indigenous populations of Australia. In collaboration with laboratory scientists, I compared five different assays for antibodies to aquaporin-4 in cases of NMOSD and controls to assess their relative utility. All aquaporin-4 antibody assays proved to be highly specific. Sensitivities ranged from 60 to 94%, with cell-based assays having the highest sensitivity. Antibodies to MOG were detected in 8/79 (10%) of the residual suspected cases of NMOSD. Under the 2015 IPND diagnostic criteria for NMOSD, cell-based assays for aquaporin-4 are sensitive and highly specific, performing better than tissue-based and ELISA assays. A fixed cell-based assay showed near-identical results to a live-cell based assay. Antibodies to MOG account for only a small number of suspected NMOSD cases (8/177 [5%]).When compared to multiple sclerosis (MS), age at onset, relapse rates and disability levels (EDSS) were significantly higher in NMOSD than in MS. Lesions and symptoms referable to the optic nerve were more common in NMOSD, whereas brainstem, cerebellar and cerebral lesions were more common in MS. Longitudinally extensive spinal cord lesions were seen in two thirds of cases with NMOSD. Elevations of CSF, white cell count and protein were more common in NMOSD. I have confirmed a clinical pattern of NMOSD that has been seen in several geographical regions. I have demonstrated the clinical utility of the current diagnostic criteria. Distinct patterns of disease are evident in NMOSD and MS, but there remains a large number of patients with NMOSD-like features who do not meet the current diagnostic criteria for NMOSD. This group remain a diagnostic challenge<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Medicine & Dentistry<br>Griffith Health<br>Full Text

Inherited and acquired disorders of the neuromuscular junction are an important cause of muscle weakness and fatigability. In this thesis I focus on the autoimmune disorders of neuromuscular transmission. Myasthenia Gravis (MG) is the most common of these diseases and is typically caused by antibodies against the post-synaptic acetylcholine receptor. Lambert Eaton Myasthenic Syndrome (LEMS) is a pre-synaptic disorder typically caused by antibodies against voltage gated calcium channels (VGCC). With regard to LEMS, my main aim was to gain a more complete understanding of the pathomechanisms of the disease. To date, the direct effect of LEMS IgG on presynaptic neurotransmitter release had not been investigated in detail. I examined how LEMS IgG affects neurotransmitter release by imaging action potential dependent vesicle exocytosis using a fluorescent dye. I found that LEMS IgG significantly inhibited the rate of synaptic vesicle release but this effect was lost in synapses from a Cacna1a knockout mouse. These data provide direct evidence that LEMS is caused by impaired neurotransmitter release due to an effect on P/Q-type VGCCs. With regard to MG, I studied the long-term outcome of patients with thymomatous and non-thymomatous MG after thymectomy and found that in general the outcome was favourable in the majority of patients with 34% of patients achieving complete stable remission. I also reviewed the long-term outcome of patients after a severe exacerbation of MG requiring ITU admission. Despite the significant mortality associated with severe exacerbations of MG, it was found that specialised neuro-intensive care was associated with a good long-term prognosis in the majority of patients. There were no significant differences in outcome in those with early or late onset MG. Overall the data presented in this thesis provide new insights into the pathomechanisms of LEMS IgG and provide new information regarding the long-term outcome of patients with MG.

Idiopathic endogenous posterior uveitis encompasses a spectrum of chronic intraocular inflammatory disorders which are thought to be autoimmune in nature. The animal model experimental autoimmune uveoretinitis (EAU) is mediated by CD4+ T-lymphocytes, and has proved invaluable in the study of the underlying immunopathogenesis of uveitis and alternative immunosuppressive therapies, for example, oral tolerance induction. This thesis describes, in a model of retinal-extract induced EAU, the effects of intranasal administration of retinal antigens prior to induction of EAU with retinal extract. The thesis has demonstrated that immunisation with emulsified retinal extract and CFA (without pertussis) induces a dose-dependent intraocular inflammation, which at high doses leads ultimately to total loss of rod photoreceptor outer segments and retinal necrosis. A course of intranasal inoculations with retinal extract (tolerance induction), prior to immunisation with antigen suppresses the histological and clinical response of EAU. Animals which were tolerised with microgram quantities of antigen showed evidence of mild inflammation of the ciliary body and inner retinal vessels (vasculitis) but no evidence of direct photoreceptor damage when compared to controls. Intranasal inoculation with retinal extract suppressed S-Ag-induced EAU but not vice versa, despite the ability of S-Ag intranasal inoculations to suppress S-Ag induced disease. Tolerised animals demonstrated normal antibody responses to S-Ag, IRBP and retinal extract, and exhibited a significantly suppressed delayed hypersensitivity response to retinal extract but normal response to a non-specific antigen, PPD. Adoptive transfer of splenocytes from tolerised animals suppressed the induction of EAU in some naive recipients. These findings suggest that active suppressor mechanisms are involved in the induction of tolerance, which concurs with other findings of CD8+ T-lymphocyte mediated suppression in oral tolerance models. In order to study the future application of 'tolerance therapy', we attempted to suppress sensitised animals by intranasal tolerance which resulted in an incomplete suppression of EAU.

Different mutations within the NALP3 gene are thought to be associated with development of several types of hereditary auto inflammatory disorders such as neonatal onset multisystem inflammatory disorder (NOMID) and muckle-wells syndrome (MWS). In this work two separate mutations E688K and G569R were supposed to be constructed by site-directed mutagenesis in the cloned wild type NALP3 genes and further expressed in bacterial and mammalian host cells for functional studies in protein -protein interaction models.

Autoimmunity and immune dysregulation are associated with a subset of movement and psychiatric disorders. This paradigm is largely supported by the discovery of autoantibodies against neuronal antigens, like the dopamine-2 receptor (D2R). T cells are a prominent cell subset in the immune system, however, their role in these diseases is unknown. Herein, we identified and characterised D2R-specific T cells in movement and psychiatric disorders in children and adults. In children with suspected autoimmune or neurodevelopmental movement and psychiatric disorders (n=24), activated D2R-specific T cells were detected in 8/24 (33%) patients when their peripheral blood was stimulated with a library of D2R peptides and assessed for CD25+CD134+CD4+ T cells via flow cytometry. The D2R-specific T cells recognised three immunodominant regions: aa121-131, aa171-181, and aa396-416. These regions were predicted with computational methods to bind with high affinity to the HLA of D2R-specific T cell positive patients and were associated with elevated levels of pro-inflammatory cytokines that characterise Th1 and Th17 cells, as quantified by a cytometric bead array and an enzyme-linked immunosorbent assay. The eight D2R-specific T cell-positive patients were seronegative for D2R antibodies, as evaluated with the flow cytometry live cell-based assay. These findings on autoreactive T cells in children formed the basis for investigating D2R-specific T cells in adults with isolated dystonia, a movement disorder that is often idiopathic and has been associated with impaired dopamine signalling. In adults with dystonia (n=20), activated D2R-specific T cells were detected in 6/20 (30%) patients via flow cytometry after stimulation with seven immunogenic regions of D2R. A subset of the D2R-specific T cell-positive patients had activated CD39+ Treg cells (2/6) and 1/6 D2R-specific T cell-positive patient concomitantly harboured activated CXCR5+ Tfh cells and D2R antibodies. In summary, this thesis offers new insights into autoreactive T cells against D2R in the movement and psychiatric disorders. Our observations encourage studies to further understand explore T cell dysregulation to better identify novel subsets of movement and psychiatric disorders and have clinical implications in improving diagnosis and treatment.

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle's, less in Huxley's/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

Die genaue Implikation natürlicher Killer(NK)-zellen und Neutrophile in Autoimmunerkrankungen des zentralen Nervensystems (ZNS) ist nach wie vor ungeklärt und wurde daher im Mausmodell der multiplen Sklerose (MS), der experimentellen Autoimmunenzephalomyelitis (EAE), sowie bei MS und Neuromyelitis optica (NMO) Patienten untersucht. Bei MS Patienten konnte eine mit der Krankheitsaktivität korrelierende, reduzierte Zahl zirkulierender CX3CR1+NK Zellen festgestellt werden. Daher wurden die NK Zell-Dynamiken und der Einfluss von CX3CR1 auf diese im EAE Mausmodell untersucht. Hierbei konnte in Wildtyp(WT) sowie auch CX3CR1-defizienten EAE Mäusen eine Rekrutierung peripherer NK Zellen in das ZNS beobachtet werden. Anders als bei WT EAE Mäusen wiesen die NK Zellen bei CX3CR1-defizienten Mäusen einen primär unreifen Phänotyp auf, der möglicherweise als ursächlich für die erhöhte Krankheitsaktivität dieser Tiere gemutmaßt werden kann. Der Transfer reifer NK Zellen vor Immunisierung CX3CR1-defizienter Tiere zeigte folglich protektive Effekte und lässt schlussfolgern, dass die CX3CR1-vermittelte Rekrutierung reifer NK Zellen die EAE Neuroinflammation limitiert. Die Diskriminierung der MS von der klinisch ähnlichen NMO stellt nach wie vor eine Herausforderung dar. Neutrophile in ZNS-Läsionen und der Cerebrospinalflüssigkeit(CSF) können bei NMO, nicht aber MS Patienten nachgewiesen werden, weshalb Neutrophile aus dem Blut von NMO und MS Patienten hier vergleichend untersucht wurden. Die Neutrophile beider Patientengruppen wiesen einen aktivierten Phänotyp im Vergleich zu gesunden Kontrollen auf. Im Gegensatz dazu zeigte sich eine von Medikation und neurologischen Defiziten der Patienten unabhängige, kompromittierte Funktionalität der NMO verglichen mit MS Neutrophilen im Hinblick auf Migration, oxidativen Burst und Degranulierung. Die Neutrophilenfunktionalität könnte entsprechend potentiell als diagnostisches Diskriminierungskriterium zwischen der MS und der NMO dienen.<br>The implication of natural killer (NK) cells and neutrophils in autoimmune disorders of the central nervous system (CNS) remains elusive, and therefore was investigated in a mouse model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), and in patients with MS and neuromyelitis optica (NMO), respectively. In MS, a decreased frequency of circulating CX3CR1+NK cells correlating with the patient disease activity has been reported. Therefore, the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in response to neuroinflammatory insult were investigated in the EAE model. Here, NK cells similarly mobilized from the periphery and accumulated in the CNS in both wild-type (WT) and CX3CR1-deficient mice during EAE. However, in mice lacking CX3CR1 the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in the WT counterparts, apparently contributing to EAE exacerbation in those animals since transfer of mature WT NK cells prior to immunization of CX3CR1-deficient mice exerted a protective effect. Together, these data suggest that the CX3CR1-mediated recruitment of mature CX3CR1+NK cells limits EAE neuroinflammation. Due to clinical similarities, the discrimination between MS and NMO is still challenging. In contrast to MS, neutrophil accumulations were found in CNS lesions and the cerebrospinal fluid (CSF) of NMO patients wherefore a comparative analysis of peripheral blood neutrophils in NMO and MS patients was performed. The results revealed an activated neutrophil phenotype in NMO and MS when compared to healthy individuals. In contrast, analysis of neutrophil migration, oxidative burst activity and degranulation showed a compromised neutrophil functionality in NMO compared to MS, which was not influenced by the treatment regime and clinical parameters of the patients. Thus, neutrophil functionality may represent a new diagnostic tool to discriminate between NMO and MS.

Background: Celiac disease (CD) is an autoimmune disorder that affects genetically susceptible individuals and is induced by dietary gluten. Treatment consists of a lifelong gluten-free diet. CD is common and affects about 1% of the general population. The classic symptoms include diarrhea and malabsorption, but many patients have only mild symptoms or no symptoms at all. The proportion of individuals presenting with atypical symptoms or discovered only when investigating an associated condition of CD is increasing. Aims: The aim of this thesis was to investigate the risk of possible associated disorders through Swedish population-based registers. The objective was to gain more information on the consequences of having CD and to identify high risk groups where screening may be considered. Materials and methods: We used the Swedish hospital discharge register to examine the risk of liver disease, autoimmune heart disease, Addison’s disease and thyroid disorders in a cohort of about 14,000 individuals with CD and an age and sex matched reference population of 70,000 individuals. In the last study we used all regional pathology registers and the cancer registry to examine the risk of hematopoietic cancer, including lymphoma in three different cohorts: I) 28,810 individuals with CD; II) 12,681 individuals with small intestinal mucosal inflammation but without villous atrophy; and III) 3552 individuals with latent CD (a positive serology test for CD with a normal small intestinal biopsy). Results: CD is statistically significantly associated with an increased risk of liver disease, Addison’s disease, thyroid disease and lymphoma. We also found an increased risk of lymphoma in individuals with small intestinal mucosal inflammation. There was no statistically significant association between autoimmune heart disease or leukemia and CD. Latent CD was not associated with any hematopoietic cancers. Conclusion: This thesis found a positive association between CD and a number of autoimmune and inflammatory disorders. Clinicians need to have a high awareness of this association and to test for these conditions when symptoms appear.

Over the last decade, multiple autoantibodies targeting brain proteins and receptors have been identified in adults and children. Autoantibody exposure in the brain can lead to transient or permanent behavioural or cognitive abnormalities. Current treatment options for autoimmune brain-reactive autoantibody-associated diseases include global immunosuppressive therapies, but these can have severe side effects, and are not always efficacious. Understanding the nature and specificity of brain autoantibodies may reveal potential therapeutic strategies alleviating or preventing the neurological pathologies and behavioural abnormalities associated with antibody-mediated brain disorders. Recently, anti-dopamine-2 receptor (D2R) antibodies have been identified in a subgroup of children with autoimmune movement and psychiatric disorders. D2R is an important brain receptor involved a variety of functions including voluntary movement, learning, memory, attention, and hormonal regulation. Currently, knowledge of the function of D2R tertiary structures, including the extracellular N-terminus, is limited, and its role in autoantibody binding is unknown. Here we report a major biological role for D2R extracellular N-terminus as a regulator of receptor surface availability, and as a major epitope targeted in brain autoimmunity. Human embryonic kidney cells were transfected with D2R mutants modified in their extracellular domains, and the level of cell surface expression and epitope specificity of 35 anti-D2R antibody-positive patient sera were analysed using a quantitative flow cytometry assay. We found that N-glycosylation at amino acids N5 and/or N17 was critical for high surface expression via interaction with the last 15 residues of extracellular D2R N-terminus. No anti-D2R antibody-positive patient sera bound to the three extracellular loops, but all patient sera (35/35) targeted the extracellular N-terminus. Overall, patient antibody binding was dependent on two main regions encompassing amino acids 20 to 29, and 23 to 37. Residues 20 to 29 contributed to the majority of binding (77 %, 27/35), among which sera from 26 % (7/27) of patients bound to amino acids R20, P21, and F22, 37 % (10/27) patient sera binding was dependent on residues at positions 26 and 29, and 30% (8/27) sera required R20, P21, F22, N23, D26, and A29. Seven patient sera bound to the region 23 to 37 independently of D26 and A29, but most sera exhibited N-glycosylation-independent epitope recognition at N23. Interestingly, no evident segregation of binding pattern according to patient clinical phenotypes was observed. Finally, we describe the optimisation of a method for single-cell isolation and RT-PCR for the generation of monoclonal antibodies. D2R N-terminus is a central epitope in autoimmune movement and psychiatric disorders, and this knowledge could help the design of novel specific immune therapies tailored to improve patient outcome.

O timo é o órgão linfóide primário responsável pelo estabelecimento inicial de um repertório funcional de células T. A via de sinalização Notch é essencial para o desenvolvimento de células T a partir de células-tronco hematopoiéticas, e a distribuição de seus receptores e ligantes no timo humano ainda é desconhecida. A expressão de AIRE é crucial para a seleção de um repertório de receptores de linfócitos T (TCR) sem autorreatividade. Neste estudo, analisamos o padrão de expressão de AIRE e a distribuição de Notch em timos pacientes com cardiopatias congênitas, parte dos quais com síndrome de Down. Descrevemos a localização intratímica e os tipos celulares capazes de expressar os diferentes receptores e ligantes Notch. A expressão de AIRE em células epiteliais medulares foi significantemente reduzida em timos de crianças com síndrome de Down, deficiência esta que pode explicar a alta incidência de doenças autoimunes nesta cromossomopatia.<br>The thymus is a primary lymphoid organ which is essential for the initial establishment of a functional repertoire of T cells. Notch signaling is crucial for T-cell lineage development from hematopoietic stem cells; however, distribution of Notch ligands and receptors in human thymus is still unknown. AIRE is crucial for the selection of a T-cell-receptor (TCR) repertoire purged of self-reactive specificities. In this study, we analyzed the expression patterns of AIRE and Notch in human thymuses from children with congenital cardiopathies that undergo heart surgery, part of whom with Down syndrome. We described the intra-thymic localization and the cell types that express Notch receptors and ligands. AIRE expression in medullary epithelial cells is significantly decreased in Down syndrome patients. This deficiency could explain higher incidence of autoimmune disease in Down syndrome.

Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Disease (MCTD) are chronic, autoimmune disorders that target overlapping autoantigens and exhibit similar clinical manifestations. Despite 40 years of research, a reliable biomarker capable of diagnosing these syndromes has yet to be identified. Previous studies have confirmed that components of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) such as U1A are 1000 fold more autoantigenic than any other nuclear component in SLE patients. Based on these findings, I hypothesize that models derived from the U1 snRNP autoantigenic properties could distinguish SLE from MCTD patients. To test this hypothesis, 30 peptides corresponding to protein regions of the U1 snRNP were tested in triplicates by indirect ELISA in sera from SLE or MCTD subjects. In addition laboratory tests and clinical manifestations data from these patients were included and analyzed in this investigation. Statistical classification methods as well as bioinformatics pattern recognition strategy were employed to determine which combination, if any, of all the variables included in this study provide the best segregation power for SLE and MCTD. The results confirmed that the IgM reactivity for U1 snRNP and U1A have the power to significantly distinguish SLE from MTCD patients as well as identify kidney and lung malfunctions for these subjects (p ≤ 0.05). Furthermore, the data analysis revealed eight novel classification rules for the segregation of SLE and MCTD which are a better classification tool than any of the currently available methods (p ≤ 0.05). Consequently, the results derived from this study support that SLE and MCTD are indeed separate disorders and pioneer the description of eight novel classification criteria capable of significantly discerning between SLE and MCTD patients (p ≤ 0.05).

The research projects described in this thesis are part of numerous collaborations with different scientific groups of italian and foreign universities but all aim at identifying, synthesising and validating new chemical structures as potential pharmacological agents for the treatment of several diseases (bacterial, cancerous, autoimmune or parasitic). All the projects are extremely multidisciplinary, and this gave me the chance to extend my knowledge towards new subjects, not strictly related to my educational background and at the same time to understand the complexity of medicinal chemistry, in all its facets. My principal task was to design and synthesise new chemical compounds with a polycyclic and hetero-polycyclic structure employing conventional and non-conventional synthetic methodologies. The high degree of diversity of these projects lead me to study and explore a considerable variety of chemical structures and functional groups, allowing me to learn and understand a very complex matter, but at the same time very fascinating, such as the organic chemistry applied to the pharmaceutical area. Beside the more theoretical knowledges related to the synthesis planning, this thesis is also marked by the strong impact and effort due to the laboratory practical for the preparation and physico-chemical characterization of the synthesised derivatives, which allowed me to deepen the understanding of fundamental spectroscopic and spectrometric techniques such as NMR and mass spectrometry. Moreover, the biological evaluation of the synthesised compounds played a crucial role in all this works and I am very grateful to the researchers with whom I had the chance to collaborate. A brief description of the major results presented in the various chapters follows. Chapter 1. Diflunisal, an old anti-inflammatory drug with a salicylic structure, has been recently repurposed for its capacity of sensitising methicillin-resistant Staphylococcus aureus to the action of methicillin. The major drawbacks related to the use of this drug in the antibacterial therapy are related to the anti-inflammatory mechanism of action, which can lead to gastrointestinal lesions, and to the poor water solubility. For this reasons we designed and synthesised some aza-analogs of diflunisal, following a synthetic route involving the obtainment of key oxazole intermediates and which exploit the Diels-Alder reaction to generate a small library of hydroxy-pyridine-carboxylic acids, variously substituted. From the biological investigation, the synthesised derivatives showed no cytotoxic activity, but some of them presented interesting anti-inflammatory and anti-bacterial properties, sensitising the bacteria S. aureus, Streptococcus pyogenes, Enterococcus faecium e Pseudomonas aeruginosa to the action of some common antibiotics. Chapter 2. In the last ten years, we have been developing 7-phenyl-pyrroloquinolinones derivatives as a class of compounds which showed interesting anticancer properties. These compounds act as tubulin polymerization inhibitors and numerous derivatives have already been synthesised allowing the comprehension of the structure-activity relationships. The major problem of these compounds is their poor metabolic stability due to multiple oxidations. For this reason, we designed and synthesised some fluorinated derivatives to explore the effect of this element on the biological activity and on the metabolic stability. The biological evaluation includes in vitro cytotoxic activity, tubulin polymerization inhibition assays, metabolic stability test and two in vivo experiment on a murine model of melanoma. Chapter 3. RORγt, an isoform of the retinoic acid-related orphan receptor gamma has been identified as the master regulator of T-helper 17 (TH17) cell function and development, making it an attractive target for the treatment of autoimmune disorders. By exploring the structures of natural and synthetic steroidal ligands and of arotinoids ligands, we designed a hybrid structure as lead compound and we generated a small library of analogs, taking advantage of the Friedel-Crafts and Suzuki reactions. Cytotoxic assays, estrogenic activity and modulation of RORγt activity were evaluated. The compounds demonstrated to be less potent than ursolic acid used as reference, but still represent a good starting point in the development of RORγt inverse agonists with a steroidal scaffold. Chapter 4. The work described in this chapter was performed during a 6-months research period spent in the molecular modelling laboratory of professor Andrea Brancale at Cardiff University (UK). The trans-membrane receptor Notch and its signalling system are key factors in the development and homeostasis maintenance of most tissues. An irregular activity of this system represents one of the principal driver in the pathogenesis and progression of cancer diseases, especially leukemia. Notch activity is highly context-dependent; indeed, it shows oncogenic and tumour-suppressor roles. Thus, its modulation represents a concrete therapeutic possibility, but still no molecules that directly interact with Notch receptor are proceeding in the clinical phases at the moment. For this reasons, we decided to take in consideration two different binding sites and to perform a molecular modelling study to identify new possible ligands. Different virtual screening study were performed, both structure-based and ligand-based and at the end, 62 molecules from different commercial database were selected and purchased for the biological evaluation. From the biological screening, it was possible to identify two new hit compounds, which will be subject to further hit-to lead optimization studies. Chapter 5. Chagas disease is a neglected tropical disorder caused by the protozoan Trypanosoma cruzi. The actual treatment consists in the administration of benznidazole or nifurtimox, two old anti-parasitic agents, which present several side effects and limited efficacy, especially in the chronic phase of the disease. An interesting molecular target for the treatment of the infection is represented by the enzyme Trypanothione reductase (TryR), which is responsible for the parasite’s defence against oxidative stress. TryR inhibition increases the susceptibility of T. cruzi towards anti-parasitic drugs, probably allowing a reduction in the dosage and thus a major success of the therapy. A virtual screening study on the structure of TryR was performed, and again the β-carboline scaffold was confirmed as a key core for the rational design of new inhibitors. The multistep synthesis, involving Pictet-Spengler and amidic coupling reactions, of 7 derivatives was successfully completed, and the biological evaluation is ongoing at the Universidad Metropolitana de Ciencias de la Educacion (UMCE), Santiago, Chile.<br>L’attività di ricerca descritta in questa tesi di dottorato si inserisce all’interno di progetti avviati con diversi ricercatori presso Università sia italiane che estere e hanno come scopo principale l’identificazione, la sintesi e la validazione di nuove piccole molecole quali potenziali agenti farmacologici per il trattamento di diverse patologie: batteriche, antiparassitarie, tumorali e autoimmuni. I progetti presentano delle caratteristiche marcatamente multidisciplinari e questo ha permesso di estendere le mie esperienze ad ambiti non strettamente inerenti alla mia formazione curriculare e conseguentemente di acquisire consapevolezza della complessità della chimica farmaceutica nelle sue varie accezioni. La mia principale attività è consistita nella progettazione e sintesi chimica di composti organici a struttura policiclica ed etero-policiclica applicando metodi di sintesi classici ed avanzati. La diversità dei progetti mi ha indotto ad affrontare e approfondire lo studio di nuove procedure di sintesi organica fondamentali per l’ottenimento di una grande varietà di strutture chimiche proposte come potenziali farmaci. Accanto allo studio per la progettazione dei percorsi sintetici, questa tesi di dottorato è contraddistinta dalla grande mole di lavoro sperimentale in laboratorio come si evince dall’elevato numero di composti intermedi e finali ottenuti e che sono stati purificati e compiutamente caratterizzati da un punto di vista chimico-fisico. Pertanto, anche le mie competenze riguardo l’uso di strumentazioni presenti e non nel Dipartimento di Scienze del Farmaco si sono notevolmente consolidate, in particolare ho potuto approfondire le tecniche di indagine spettroscopica più diffuse ed essenziali nell’ambito della chimica farmaceutica come la spettroscopia NMR e la spettrometria di massa. La maggior parte dei composti da me preparati in laboratorio sono già stati studiati anche per la loro attività biologica allo scopo di valutarne il potenziale farmacologico e per questo sono molto grato ai ricercatori con cui ho proficuamente collaborato. Segue una breve descrizione dei risultati ottenuti nei vari capitoli. Capitolo 1. Diflunisal, un noto farmaco antiinfiammatorio della categoria dei salicilati, è stato recentemente riproposto come agente in grado di sensibilizzare lo Staphylococcus aureus meticillino-resistente all’azione della meticillina. I principali problemi legati all’utilizzo di questo farmaco nella terapia antibiotica sono legati al meccanismo d’azione antiinfiammatorio, che può condurre a lesioni gastrointestinali anche gravi, e alla scarsa solubilità in acqua. Per queste ragioni abbiamo progettato e sintetizzato degli aza-analoghi del diflunisal, seguendo una via sintetica che prevede l’ottenimento di intermedi ossazolici e l’applicazione della reazione di Diels-Alder per generare così una libreria di analoghi acidi idrossi-piridin-carbossilici variamente sostituiti. A seguito della valutazione biologica i composti sintetizzati non presentano attività citotossica ed alcuni di loro presentano interessanti proprietà antinfiammatorie e soprattutto antibatteriche, sensibilizzando i batteri S. aureus, Streptococcus pyogenes, Enterococcus faecium e Pseudomonas aeruginosa all’azione di alcuni antibiotici. Capitolo 2. I 7-fenilpirrolochinolinoni (7-PPyQ) appartengono alla famiglia dei pirrolochinolinoni, una classe di composti che ha rivelato in anni recenti interessanti proprietà antitumorali. In particolare, alcuni derivati del 7-PPyQ agiscono come forti inibitori della polimerizzazione della tubulina e dimostrano grande affinità per il sito della colchicina. I dati ottenuti dallo studio di numerosi derivati hanno consentito la comprensione delle relazioni struttura-attività. Il problema principale di questi composti è dovuto alla scarsa stabilità metabolica in seguito ad ossidazioni multiple. Per questo motivo sono stati progettati e sintetizzati alcuni derivati inserendo l’atomo di fluoro in diverse posizioni allo scopo di esplorare l’influenza di questo elemento sull’attività biologica e sulla stabilità metabolica. La valutazione biologica comprende i test di citotossicità in vitro e antitumorale in vivo su modello murino di melanoma., saggi di inibizione della polimerizzazione della tubulina e ovviamente i test in vitro di stabilità metabolica. Capitolo 3. Il recettore nucleare RORγt, un’isoforma del recettore orfano correlato ai recettori dell’acido retinoico ROR, è stato identificato come principale responsabile dell’attività e dello sviluppo dei linfociti T-helper 17 (TH17), i quali sono implicati nella patogenesi di alcune malattie autoimmuni. Questo rende RORγt un bersaglio ideale per il trattamento di queste patologie. Esplorando la struttura dei ligandi steroidei naturali e sintetici di questo recettore e dei ligandi arotenoidi, è stata progettata una struttura ibrida come lead compound e generato una piccola libreria di analoghi, sfruttando in particolar modo le reazioni di Friedel-Crafts e di coupling di Suzuki. Saggi di citotossicità, attività estrogenica e modulazione dell’attività del recettore RORγt sono stati effettuati. I composti risultano essere meno potenti dell’acido ursolico, agonista inverso usato come riferimento, ma rappresentano un buon punto di partenza per lo sviluppo di nuovi agonisti inversi del recettore RORγt, a struttura steroidea. Capitolo 4. L’attività descritta in questo capitolo è stata possibile grazie ad un periodo di 6 mesi trascorso presso il laboratorio di modellistica molecolare del professor Andrea Brancale presso Cardiff University (UK). Il recettore trans-membrana Notch e il suo sistema di signalling sono fattori chiave nello sviluppo e mantenimento dell’omeostasi in molti tessuti. Un’attività irregolare di questo sistema rappresenta uno dei principali driver nella patogenesi e progressione di molte forme tumorali, in particolar modo leucemiche. L’attività di Notch è fortemente dipendente dal contesto, infatti presenta caratteristiche sia di oncogene che di oncosoppressore. La sua modulazione rappresenta quindi una possibilità terapeutica concreta. Ad oggi, non esistono molecole in clinica che agiscono direttamente sul recettore Notch, per questo motivo prendendo in esame due diversi binding sites, è stato effettuato uno studio di modellistica molecolare adiuvato da simulazioni di docking per identificare nuovi possibili ligandi. Sono stati realizzati diversi studi di virtual screening, sia con approccio structure-based che ligand-based, e 62 molecole provenienti da diversi database commerciali, sono state selezionate per la valutazione biologica. Dai risultati ottenuti è stato possibile identificare due hit compounds, i quali verranno sottoposti a ulteriore indagine in futuri studi di hit-to-lead optimization. Capitolo 5. La malattia di Chagas è una malattia tropicale negletta causata dal protozoo Tripanosoma cruzi. Attualmente, viene trattata mediante somministrazione di benznidazolo o nifurtimox, due vecchi agenti antiparassitari, che presentano molti effetti collaterali e limitata efficacia, soprattutto nella fase cronica dell’infezione. Un possibile bersaglio molecolare per il trattamento dell’infezione è rappresentato dall’enzima Tripanotione Reduttasi (TryR), responsabile della difesa del parassita contro lo stress ossidativo. L’inibizione di questo enzima incrementa la suscettibilità di T. cruzi nei confronti dei farmaci antiparassitari, probabilmente consentendo una riduzione del dosaggio e un maggiore successo della terapia. Uno structure-based virtual screening sulla struttura di TryR è stato effettuato confermando lo scaffold β-carbolinico come cruciale per la progettazione razionale di nuovi inibitori. La sintesi multi-step, comprendente reazione di Pictet-Spengler e coupling ammidico, è stata completata con successo per 7 derivati e al momento i composti sono sottoposti a valutazione biologica presso l’Universidad Metropolitana de Ciencias de la Educacion (UMCE), Santiago, Chile.

La sclérose en plaques (SEP) est une maladie auto-immune du système nerveux central générant de nombreux symptômes neurologiques, parmi lesquels la douleur chronique qui est très invalidante et fréquente. A ce jour, la SEP est une maladie incurable avec une étiologie complexe, multifactorielle et encore mal comprise. De nombreuses données suggèrent que les polyphénols végétaux pourraient avoir des bénéfices thérapeutiques en régulant le stress oxydant et la neuroprotection dans la SEP. Dans ce contexte, ce travail de thèse a pour objectif d’évaluer l'effet d’un traitement curatif chronique à l'extrait de pépins de raisin (GSE pour Grape Seed Extract) dans un modèle murin reproduisant certaines des caractéristiques cliniques et neuropathologiques de la SEP, l'encéphalomyélite auto-immune expérimentale (EAE). Dans un premier temps, la composition biochimique du GSE a été évaluée. Par la suite, le traitement des souris EAE par le GSE 10 jours après l’induction du modèle (J10) a montré une amélioration à la fois du score neurologique et des troubles sensitifs chez les souris. Des analyses biochimiques et moléculaires au niveau du cerveau et de la moelle épinière ont montré dès J20 une correction des anomalies du stress oxydant permettant une restauration des altérations de la myéline, de la prolifération astrogliale et microgliale et des niveaux d’expression des sirtuines. Enfin, une analyse protéomique a permis de confirmer ces résultats et d’envisager des mécanismes d’action bénéfiques supplémentaires du GSE, notamment la correction de la dégradation des lipides. L’ensemble des effets du GSE décrits au cours de cette thèse soutient fortement l'idée que le GSE pourrait être une approche thérapeutique efficace pour le traitement de la SEP<br>Multiple sclerosis (MS) is an autoimmune disease of the central nervous system leading to many neurological symptoms, among which chronic pain is common and very disabling. To date, MS is an incurable disease with a complex, multifactorial and still poorly understood etiology. Numerous evidence suggest that plant polyphenols may have therapeutic benefits in regulating oxidative stress and providing neuroprotection in MS. In this context, this thesis work aimed to evaluate the effect of a chronic curative treatment with grape seed extract (GSE) in a mouse model reproducing some of clinical and neuropathological features of MS, the experimental autoimmune encephalomyelitis (EAE).First, the biochemical composition of GSE was evaluated. Subsequently, the treatment with GSE initiated from day 10 post-induction (D10) showed both an improvement in the neurological score and sensory disorders in mice. Biochemical and molecular analyzes in the brain and spinal cord showed from D20 a correction of oxidative stress abnormalities allowing restoration of myelin alterations, astroglial and microglial proliferation and levels of sirtuins expression. Finally, a proteomic analysis allowed to confirm these results and to identify new additional beneficial effect of GSE, such as the correction of lipid degradation. All the effects of GSE described during this thesis strongly supports the idea that GSE could be an effective therapeutic approach for the treatment of MS

碩士<br>中山醫學大學<br>醫療產業科技管理學系碩士班<br>106<br>Background: Autoimmune diseases and psychiatric diseases are chronic diseases. Both of diseases have multiple comorbidity and more medical utilization. The complexity of the diseases made a challenge for the clinicians and health provider to manage it. The diseases makes burden to treatment and management. . We used a population-based retrospective cohort study to explore the questions on clarifying the correlation when the two diseases coexist; including the comorbidity, the medical utilization and the survival rate. The results may help the cli-nician to notice the special need for these patients and the health provider to design the better treatment strategy. Method: We identified subjects who were diagnosed with common autoimmune disease including rheumatoid arthritis, systemic lupus erythematous, Sjogren’s syndrome, ankylosing spon-dylitis and psoriasis between January 1, 2000, and December 31, 2012, in the Taiwan Na-tional Health Insurance (NHI) Research Database. A cross-sectional cohort study was con-ducted for patients with the diagnosis as mentioned above. Independent variation including common autoimmune diseases, psychiatric diseases, coomorbidity, and demographic characteristics; the independent variation were other comorbidity and medical utilization and mortality. There were patients 6,868 were included and 27,472 matched controls observed until diagnosed with psychiatric disorders or until death. This study was approved by the Institutional Review Board of Chung Shan Medical University in Taiwan (CS15134). Results: The collected data showed the prevalence of the selected autoimmune disease and psychi-atric diseases were 1.1% and 8.13%from 1997 to 2013. Under analysis of this retrospective cohort study, there was higher incidence of psychiatric disease for patients with autoim-mune disesase; the incident rate was 1.08 (95% confidential interval =0.99-1.18) and ad-justed hazard ratio was 1.42 (95% confidential interval=1.27-1.59). Systemic lupus ery-thematosus showed highest adjusted hazard ratio (aHR=1.66, 95% confidential ratio is 1.31(95% confidential incidence= 1.20-1.42). The mortality rate in patient with autoim-mune disease, the aHR=1.52(95% confidential incidence=1.38-1.67). Patients with system-ic lupus erythematosus showed the highest, aHR=3.17(95% confidential incidence=2.527-3.979). Through this study, we conducted a nested cases (cases with or psychiatric disease) and control (without psychotic diesease). Patients with autoimmune disease had more physical comorbidity and more psychiatric comorbidity, especially comorbid with depression and anxiety. Autoimmune disease combined with psychiatric disorders had highest mortality risk (OR= 2.037). The medical utilization marked increased in hospitalization and outpa-tient followed-up. Conclusion: Patients with autoimmune disease comorbid with psychiatric disease have more comor-bidity, more medical utilization and more mortality rate. The results remind clinicians to pay more attention on identify the psychiatric disease while treating patients with autoim-mune disease. To determine if the combined care with psychiatrists leading to better out-come and better cost efficiency would need further investigation.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2015<br>Background: A doença relacionada com a imunoglobulina G4 (DR-IgG4) é uma entidade sistémica caracterizada por elevação da IgG4 sérica, hipertrofia dos orgãos afectados com infiltração linfoplasmocítica maciça com plasmócitos IgG4-positivos e fibrose tecidular. A pancreatite autoimune (PAI) é uma forma de pancreatite com provável etiologia autoimune que pode apresentar envolvimento difuso ou focal do pâncreas. A PAI tipo 1 é a manifestação pancreática da DR-IgG4 e a Tipo 2 uma doença específica do pâncreas. Na forma focal a PAI partilha características com o adenocarcinoma do pâncreas. Objectivos: Realizar uma revisão do conhecimento actual da PAI quanto à patogénese, epidemiologia, características clínicas, histológicas, imagiológicas, manifestações extrapancreáticas da DR-IgG4 e tratamento. Focam-se as descobertas recentes quanto ao diagnóstico e diagnóstico diferencial com adenocarcinoma do pâncreas. Métodos: Pesquisa na base de dados da Pubmed usando as palavras DR-IgG4 e PAI. Seleccionei apenas os artigos entre 2006-2015. Conclusões: Apesar da intensa investigação, o diagnóstico diferencial entre PAI e adenocarcinoma do pâncreas ainda é desafiador para os clínicos. A sua correcta diferenciação pode evitar cirúrgia desnecessária em doentes com PAI, pois estes podem ser tratados com sucesso com corticoesteroides e rituximab. Se não for possível excluir com segurança o adenocarcinoma do pâncreas a cirurgia é mandatória.<br>Background: IgG4-Related Disease (IgG4-RD) is a systemic disease characterized by elevation of serum IgG4, enlargement of the affected organs with lymphoplasmacitic infiltration with IgG4-positive plasmocytes and tissue fibrosis. Autoimmune pancreatitis (AIP) is a form of pancreatitis with probable autoimmune etiology that can present as focal or diffuse form of involvement. Type 1 AIP is the pancreatic manifestation of IgG4-RD whereas Type 2 is a pancreas-specific disorder. The AIP focal form shares features with pancreatic cancer. Objectives: We intend to make a brief review on the current knowledge about AIP regarding patogenesis, epidemiology, histologic, clinical and imagiological features, laboratory findings, extrapancreatic involvement and treatment. We will focus the scientific breakthroughs in the last years concerning the diagnosis and differential diagnosis with pancreatic adenocarcinoma. Methods: Selective literature research in Pubmed database regarding IgG4-RD and AIP. Only those articles between 2006 and 2015 were selected. Conclusion: Although the intensive investigation in the last years, the differential diagnosis between AIP and pancreatic adenocarcinoma is still a challenge for clinicians. The correct differentiation between them can prevent unnecessary surgery in AIP patients since these can be successfully treated conservatively with steroids and rituximab. If pancreatic adenocarcinoma cannot be excluded with safety surgery is mandatory.

The risk of autoimmune disorders such as multiple sclerosis (MS) may be influenced by environmental factors early in life. This epidemiological thesis provides new knowledge on the role of early life environmental factors in MS in particular, for which the specific aetiology is unknown. Two main potential environmental determinants of MS, ultraviolet radiation (UVR) and infections, are explored through an analysis of their timing of action in the life course and a consideration of their possible protective effects. Other organ-specific autoimmune disorders whose aetiology is also unknown, including type 1 diabetes and rheumatoid arthritis (RA), are also assessed for links with UVR for comparison with MS. Two existing national Australian datasets provided the outcome data for the analyses. The 1995 Australian National Health Survey (NHS) provided summary prevalence estimates for ecological (population-level) analysis of four immune disorders other than MS. The 1981 Australian MS Survey, used for the largest part of the thesis, provided individual-level MS-case data for 1981 throughout Australia, and was further modified to construct a longitudinal MS-rates study dataset for analysis of timing of birth. Ecological analysis of the 1995 NHS data showed that geographic latitude was positively associated, and regional ambient UVR inversely associated, with the prevalence of type 1 diabetes in Australia. Ambient UVR exposure may thus be a protective factor against such disease at the population level. The association supports previous ecological findings for MS in Australia and adds to the evidence that UVR exposure might be a modifiable determinant for autoimmune disease generally. Longitudinal analysis of the reconstructed 1981 Australian MS Survey dataset showed that increased MS risk of around 30% was evident in Australians born in November to December (southern hemisphere early summer) compared with those born in May to June (early winter). This MS-risk pattern, indicating environmental factor(s) acting around the time of birth, mirrored (seasonally) that seen for MS in the northern hemisphere, suggesting globally similar perinatal environmental determinants modifying the risk of MS onset. Most importantly, this Australian pattern was found to be fully accounted for by individual, regional (state) and seasonal ambient UVR levels specific to the prenatal period seven to eight months before birth. Low ambient (maternal) UVR exposure in the first trimester of pregnancy thus appears to be associated with a higher risk of MS in the offspring. Birth-order analysis of cases in the 1981 MS Survey further showed that early birth order was independently associated with MS risk, MS cases being more likely to be one of the older siblings in their sibships. Consistent with the hygiene hypothesis, this result suggests that a lack of microbial exposure in early childhood may increase MS risk later in life. Population health implications of these findings are discussed. In particular, safe sun exposure and/or vitamin D supplementation during early pregnancy may help prevent subsequent onset of high-morbidity, long-duration and presently incurable autoimmune disorders such as MS in the offspring.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2019<br>A Esclerose Múltipla (EM) e o Síndrome Guillain-Barré (SGB) são ambos doenças autoimunes e desmielinizantes que afetam, respetivamente, o Sistema Nervoso Central (SNC) e o Sistema Nervoso Periférico (SNP), pertencendo ainda a um grupo de doenças neurodegenerativas que envolvem lesões inflamatórias associadas a desmielinização, induzindo dano no axónio e consequente neurodegeneração, o que leva a uma perda de função progressiva. A EM é uma doença inflamatória crónica do SNC sendo a causa mais frequente de distúrbios neurológicos em jovens adultos. É uma doença que consiste na inflamação, desmielinização e uma variável perda axonal. A sua etiologia ainda não é completamente conhecida, mas presume-se que envolva a interação entre fatores genéticos e ambientais, estimulando um ataque autoimune e consequentes danos na mielina e nos axónios. Clinicamente, a maior parte dos doentes tem uma fase recidivante-remitente, caracterizada pela presença de surtos seguida de recuperação. Destes doentes, a maioria progride para uma doença secundária progressiva e os restantes doentes desenvolvem uma Esclerose Múltipla primária progressiva. Alguns doentes têm ainda um síndrome clinicamente isolado que corresponde a um primeiro episódio de sintomas neurológicos no SNC, sendo que estes podem ou não evoluir para Esclerose Múltipla. Em termos de tratamento, estão aprovados os medicamentos modificadores de doença, especialmente no caso de doença recidivante-remitente. A SGB é uma doença inflamatória, mas do SNP, sendo a causa mais frequente de paralisia flácida aguda. Esta doença autoimune é antecedida por uma infeção viral ou bacteriana, como vírus Influenza ou Campilobacter jejuni, que são capazes de desencadear uma resposta imune anormal direcionada contra os componentes dos nervos periféricos, por mimetismo molecular. As formas mais frequentes são polineuropatia desmielinizante inflamatória aguda e neuropatia motora axonal aguda, existindo ainda a neuropatia motora sensorial axonal aguda e a síndrome de Miller Fisher. Os doentes com SGB têm insuficiência respiratória e disfunção autónoma como complicações associadas. O tratamento é composto por uma abordagem multidisciplinar que inclui cuidados médicos gerais e imunoterapia. As prioridades na investigação da EM e da SGB incluem o desenvolvimento de biomarcadores e um melhor conhecimento da imunopatogénese, para que haja medicina personalizada.<br>Multiple Sclerosis (MS) and Guillain-Barré Syndrome (GBS) are both demyelinating and autoimmune disorders affecting, respectively, the central nervous system (CNS) and the peripheral nervous system (PNS), which means they belong to a group of neurodegenerative diseases that involve inflammatory lesions associated with demyelination, inducing axonal damage and consequent neurodegeneration, leading to progressive loss of function. MS is a chronic inflammatory disorder of the CNS and is assumed to be the most frequent cause of neurological disability in young adults. This disorder consists in inflammation, demyelination and variable levels of axonal loss. The etiology is still unknown but it is presumed to involve interaction between genetic and environmental factors that triggers an autoimmune attack, resulting in damaged myelin and axons. Clinically, most of the patients experience a relapsing-remitting phase, characterized by relapses followed by recovery. The majority of them, late on enter in a progressive phase called secondary progressive MS. The remaining patients pursue a progressive course that is called primary progressive MS. There is also clinically isolated syndrome corresponding to a first episode of neurologic symptoms in the CNS, and people who experience it may or may not develop MS. In terms of therapeutic options, disease-modifying treatments are approved specially to treat relapsing remitting form of the disease. GBS is also an inflammatory demyelinating disease of the PNS and it is the most frequent cause of acute flaccid paralysis. This autoimmune disorder is, in most cases, preceded by viral or bacterial infections, such as Campylobacter jejuni or Influenza virus, that are capable of triggering an abnormal immune responses directed against components of the peripheral nerves by molecular mimicry. Clinically, the most frequent forms of GBS is acute inflammatory demyelinating polyradiculoneuropathy and acute motor axonal neuropathy, but there is also acute motor-sensory axonal neuropathy and Miller-Fischer syndrome. Patients with GBS commonly have respiratory insufficiency and autonomic dysfunction as associated complications. The treatment of this syndrome is composed by a multidisciplinary approach that includes general medical care and immunotherapies. The priorities for MS and GBS investigation include establishment of biomarkers and an improved knowledge of the immunopathogenesis, to go towards personalized medicine.<br>Farmácia Lisboa; Hospital de Santo António dos Capuchos

Introduction: In 2007 Ontario implemented a grade 8 quadrivalent human papillomavirus (qHPV) vaccination program targeting the virus that causes cervical cancer. Despite being 6 years post-implementation, few post-licensure studies have assessed the safety of the qHPV vaccine in this adolescent population. Since autoimmune disorders are often targeted for post-marketing surveillance by regulatory agencies, it is important to assess the risk of developing an autoimmune disorder post-qHPV vaccination. Objectives: The objectives of this thesis were to assess the risk for developing an autoimmune disorder following qHPV vaccination, assess for effect modification by the presence of predisposing risk factors, identify the period of highest risk and explore the risk for individual autoimmune disorders. Methods: A population-based retrospective cohort of girls eligible for Ontario’s qHPV vaccination program was identified using population-based databases. The risk of autoimmune disorders following qHPV vaccination was ascertained using the self-controlled case series method. Results: The risk of developing a new autoimmune disorder, adjusted for age, seasonality, concurrent vaccines and infections was 1.28 (95% CI: 0.87 – 1.89), and this association was independent of a history of immune-mediated disorders (p=0.39). The risk was not increased during days 7-24 post-vaccination (adjusted RR = 0.87, 95% CI: 0.43 – 1.74), but appeared to increase thereafter (adjusted RR = 1.36, 95% CI: 0.77 – 2.41 and RR = 1.62, 95% CI 0.94 – 2.78 respectively, for days 25 – 42 and days 43 – 60), although these differences were non-significant. The risk may be increased for certain disorders including Bell’s palsy (RR = 2.30, 95% CI: 0.67 – 7.95), systemic autoimmune rheumatic disorders (RR = 1.84, 95% CI: 0.42 – 8.02), Hashimoto’s disease (RR = 1.39, 95% CI: 0.46 – 4.22), and juvenile rheumatoid arthritis (RR = 1.31, 95% CI: 0.83 – 2.08), although none of these associations were statistically significant. Conclusion: This thesis demonstrated that no statistically significant increased risk for autoimmune disorders following qHPV vaccination was detected. However, there remains some uncertainty about the safety of the qHPV vaccine for a subset of the autoimmune disorders. The results from this analysis need to be pooled with those of other studies to confirm whether these are true safety signals.<br>Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2014-04-23 22:30:41.428

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2018<br>Em 1998, Sweedo, apercebeu-se que existiam algumas crianças que sofriam com sintomatologia obsessivo-compulsiva associada ainda a tiques. Este era um grupo que se separava das crianças com POC primária e Perturbação de tiques isolada, já que neste grupo parecia existir um início bastante abrupto da sintomatologia, estando esta associada ainda a uma infeção anterior por Streptococcus do grupo A. Intrigada, decide iniciar um estudo com 50 crianças para verificar se esta relação não seria mesmo uma associação causal e não uma mera coincidência. Assim nasce o conceito de PANDAS, um acrónimo para Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus Infections. Este artigo visa abordar de uma forma concisa e de fácil compreensão toda a evolução do conceito de PANDAS, a sua base fisiopatológica autoimune, o seu diagnóstico, o seu tratamento e ainda as suas variantes.<br>In 1998, Sweedo realized that there were some children suffering with obsessive-compulsive symptomatology and also with tics. This was a group of kids completely different from the ones that had the primary diagnose of OCD and Tics Disorders, since in this group there seemed to be a rather abrupt onset of symptoms, which was still associated with a previous infection by group A Streptococcus. Intrigued with this, Sweedo decides to start a study to see if this was just a coincidence or if the infections led to the beginning of the symptoms. That’s how PANDAS was born, an acronym for Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus Infections. This article aims to approach in a concise and easily way all the evolution of PANDAS’ concept, the autoimmune pathophysiological basis, the diagnosis, treatment and also PANDAS’ variants.

Indiana University-Purdue University Indianapolis (IUPUI)<br>Multiple sclerosis (MS) is a disabling disease that afflicts an estimated two million people worldwide. The disease is characterized by degradation of the myelin sheath that insulates neurons of the central nervous system manifesting as a heterogeneous collection of symptoms. Two enzymes, protein arginine deaminases type 2 and 4 (PAD2 and PAD4) have been implicated to play an etiologic role in demyelination and neurodegeneration by catalyzing a post-translational modification of arginine peptide residues to citrulline. The pathogenesis of MS is poorly understood, though vitamin D deficiency is a well-associated risk factor for developing the disorder. Using the experimental autoimmune encephalomyelitis (EAE) model of MS we demonstrate vitamin D treatment to attenuate over-expression of PAD 2 and 4 in the brain and spine during EAE. In addition, we identify two molecules produced by peripheral immune cells, IFNɣ and IL-6, as candidate signaling molecules that induce PAD expression in the brain. We demonstrate vitamin D treatment to inhibit IFNɣ mediated up regulation of PAD2 and PAD4 both directly within the brain and by modulating PAD-inducing cytokine production by infiltrating immune cells. These results provide neuroprotective rational for the supplementation of vitamin D in MS patients. More importantly, these results imply an epigenetic link between vitamin D deficiency and the pathogenesis of MS that merits further investigation.

Tese de Doutoramento em Biologia Experimental e Biomedicina, no ramo de Neurociências e Doença, apresentada Instituto de Investigação Interdisciplinar da Universidade de Coimbra<br>The human brain is distinctively unique due to its remarkable ability for complex language, higher cognition, emotion regulation and executive control of behaviour. Yet, these are precisely the brain functions impaired in several neurological and neuropsychiatric disorders that afflict a large percentage of the population worldwide. Emerging evidence in the field suggests that one potential functional process thought to be compromised in pathological conditions is homeostatic synaptic plasticity. This form of plasticity is able to modulate the overall activity of neuronal networks so that it never goes off-balance, even in face of constant changes, such as occurring during development, sensory experience or even during learning-related adaptations. At the molecular level, bidirectional compensatory changes in the postsynaptic accumulation of glutamate receptors of the AMPA-type are thought to be one cellular substrate to achieve neuronal homeostasis. However, full comprehension of the AMPAR regulatory mechanisms that underlie this form of plasticity, and of how such mechanisms can fail in the context of disease, is still elusive. Contactin-associated proteins 1 and 2 (Caspr1 and Caspr2) are integral transmembrane cell-adhesion molecules that have received considerable attention in the past few years due to emerging roles in the regulation of synaptic, cellular and functional processes in the brain. Previous evidence from our laboratory identified Caspr1 as an AMPAR-interacting protein necessary to regulate the trafficking of surface AMPARs into synapses. However, the molecular mechanisms underlying this role of Caspr1 are still uncharacterized, and no link has yet been established between its role in AMPAR regulation and synaptic plasticity mechanisms. Herein, we describe a posttranscriptional mechanism orchestrated by Caspr1 and the RNA-binding protein ZBP1 in the regulation of AMPAR. We found that ZBP1 binds to the mRNA of the GluA1 subunit of AMPARs in an activity-dependent manner, and regulates synaptic levels of cell surface GluA1-containing AMPARs. During periods of prolonged activity inhibition, when homeostatic responses are elicited to upscale surface AMPARs, endogenous expression of Caspr1 is upregulated, and the phosphorylation of ZBP1 increases, resulting in a significant decrease in GluA1 mRNA molecules bound to ZBP1, suggesting an activity-dependent release of GluA1 transcripts to undergo translation on demand. Importantly, when the expression of either Caspr1 or ZBP1 is lost, synaptic upscaling of AMPARs is compromised, indicating a specific requirement for both Caspr1 and ZBP1 in the regulation of postsynaptic AMPARs during homeostatic synaptic plasticity. Mutations in the CASPR2-encoding gene CNTNAP2 have been recurrently implicated in several neuropsychiatric disorders including autism, schizophrenia and intellectual disability. Recent findings have attributed an important function for Caspr2 in synapse regulation, but the full-spectrum of mechanisms mediated by CASPR2 remains elusive. Importantly, it is still unclear how perturbations in CASPR2 function become pathogenic and drive the severe cognitive and psychiatric symptoms presented by patients. Herein, we find that Caspr2 is expressed in cortical excitatory synapses, and identify Caspr2 as a novel AMPAR-interacting protein capable of regulating the trafficking of AMPARs to synapses. Moreover, we demonstrate that loss of Caspr2 impairs AMPAR function and in vivo excitatory synaptic transmission in the cortex, and reveal a requirement for Caspr2 in the regulation of homeostatic mechanisms necessary for the expression of visually-driven experience-dependent plasticity. Finally, autoantibodies targeting CASPR2 have been recently discovered in patients with autoimmune synaptic encephalitis that can manifest with severe memory deficits, cognitive impairments and psychosis. However, it is not clear to date if CASPR2 autoantibodies (CASPR2-Abs) perturb CASPR2 functions, and whether they can mediate a direct pathogenic effect that can drive the disease symptoms. Taken into consideration the role we describe for Caspr2 in the regulation of AMPARs, we hypothesized that CASPR2-Abs exert their pathogenic effect by disrupting this Caspr2 function. Using human immunoglobulin (IgGs) preparations from a patient with CASPR2 encephalitis, we found that patient IgGs significantly alter the synaptic distribution of Caspr2 and cell surface AMPARs. Moreover, patient IgGs hamper Caspr2 function in vivo and perturb basal glutamatergic synaptic transmission in the visual cortex of mice. Additionally, patient IgGs prevent the triggering of long-term potentiation, whilst sparing homeostatic synaptic scaling mechanisms. Finally, we reveal that patient IgGs bound to the neuronal surface can undergo time-dependent internalization, thus underpinning a likely mechanism of pathogenesis elicited by CASPR2-Abs. Altogether, our findings identify Contactin-associated proteins as crucial regulators of glutamatergic synaptic transmission and synaptic plasticity mechanisms, and suggest that these processes are likely targets for the pathogenesis of several neurological and neuropsychiatric disorders, including those ensuing from genetic- or antibody-mediated disruptions of CASPR2.<br>O cérebro humano é único, capaz de processos altamente complexos tal como cognição, formas de linguagem superior, regulação de emoções e controlo executivo do comportamento, que nos distinguem de outros mamíferos. No entanto, são precisamente estas as funções cerebrais afectadas em doenças neurológicas e do foro psiquiátrico, que atingem uma larga percentagem da população em todo o mundo. Evidências recentes sugerem que um dos processos cerebrais que possa estar comprometido em condições patológicas é a plasticidade homeostática. Esta forma de plasticidade é capaz de modular a actividade de redes neuronais de forma a mantê-la dentro de limites funcionais necessários para um adequado funcionamento do cérebro, mesmo face a constantes alterações potencialmente desestabilizadoras, como as que ocorrem durante o desenvolvimento neuronal, em resposta ao ambiente sensorial ou até durante processos de aprendizagem. Ao nível molecular, a homeostase neuronal é conseguida por exemplo através de alterações compensatórias e bidirecionais na acumulação pós-sináptica de receptores de glutamato do tipo AMPA (AMPAR). No entanto, os mecanismos regulatórios de AMPAR que estão na base deste tipo de plasticidade ainda não são completamente conhecidos, e não se compreende ainda de que forma estes mecanismos podem falhar num contexto de doença. As proteínas 1 e 2 associadas à Contactina (Caspr1 e Caspr2) são moléculas de adesão celular que têm recebido considerável atenção nos últimos anos devido a novas funções que lhes foram atribuídas na regulação de processos sinápticos, celulares e funcionais do cérebro. Resultados anteriores obtidos no nosso laboratório identificaram a Caspr1 como uma nova interactora de receptores AMPA necessária para regular o seu tráfego para a sinapse. No entanto, os mecanismos moleculares que estão na base da função da Caspr1 ainda não foram caracterizados, e nenhuma relação foi ainda estabelecida entre a sua função na regulação de AMPAR e mecanismos de plasticidade sináptica. Neste estudo, descrevemos um novo mecanismo pós-transcripcional regulado pela Caspr1 e pela proteína de ligação a RNA, ZBP1. Descobrimos que a ZBP1 é capaz de se ligar ao RNA mensageiro da subunidade GluA1 dos AMPARs de uma forma dependente de actividade, e que regula os níveis sinápticos basais de AMPARs à superfície da célula. Durante períodos de bloqueio crónico de actividade, quando processos homeostáticos são activados para induzir o escalamento sináptico de AMPARs, a expressão endógena da Caspr1 é aumentada, bem como os níveis de fosforilação da ZBP1, o que reduz significativamente o número de moléculas de RNAm de GluA1 ligado à ZBP1, e sugere que a actividade neuronal poderá induzir uma libertação de transcriptos de GluA1 para que possam ser traduzidos quando necessário. Finalmente, inibição da expressão endógena da Caspr1 ou da ZBP1 compromete o escalamento sináptico de AMPARs, o que indica que a Caspr1 e a ZBP1 são necessárias para a regulação de AMPARs durante mecanismos de plasticidade homeostática. Mutações no gene codificante da Caspr2, CNTNAP2, foram já implicadas em várias doenças neuropsiquiátricas, incluindo autismo, esquizofrenia e défice intelectual. Evidências recentes sugerem que a Caspr2 tem um importante papel na sinapse, no entanto não se conhecem ainda os mecanismos através dos quais a Caspr2 regula a função sináptica. Além disso, é ainda incerto de que forma perturbações na função da CASPR2 se podem tornar patogénicas e causar o desenvolvimento de sintomas cognitivos e psiquiátricos nos pacientes. Neste estudo vimos que a Caspr2 é bastante expressa em sinapses excitatórias do cortéx, e identificámos a Caspr2 como uma nova proteína interactora de AMPARs capaz de regular o seu tráfego para a sinapse. Adicionalmente, demonstrámos que inibição da expressão endógena da Caspr2 perturba a função de AMPARs e a transmissão sináptica excitatória in vivo, e revelámos que a Caspr2 é necessária para mecanismos de escalamento sináptico que regulam a expressão de plasticidade sináptica induzida in vivo por experiência visual. Autoanticorpos contra a CASPR2 foram recentemente identificados em pacientes com encefalite autoimune, que podem apresentar graves défices de memória, perturbações cognitivas e psicose. No entanto, não se sabe ainda se os autoanticorpos contra a CASPR2 perturbam a sua função, e se poderão mediar um efeito patogénico directo que possa ser causal para o desenvolvimento dos sintomas da doença. Tendo em consideração o papel que descrevemos para a Caspr2 na regulação de AMPARs, colocamos a hipótese de os autoanticorpos exercerem o seu efeito patogénico por disrupção desta função da Caspr2. Usando amostras de imunoglobulinas humanas purificadas a partir do plasma de um paciente com encefalite anti-CASPR2, descobrimos que estas alteram significativamente a distribuição sináptica da Caspr2 endógena e de AMPAR superficiais. Adicionalmente, vimos que as IgGs do paciente bloqueiam a função da Caspr2 in vivo e perturbam a transmissão glutamatérgica no córtex visual de ratinhos. Vimos também que as IgGs do paciente inibem a indução de mecanismos de potenciação de longo-termo, embora o escalamento sináptico se mantenha intacto. Finalmente, revelamos que IgGs do paciente que se ligam à superfície neuronal são internalizadas ao longo do tempo, sugerindo assim um potencial mecanismo patogénico induzido por estes anticorpos. Em conclusão, os nossos resultados identificam as proteínas Casprs como reguladoras fundamentais da transmissão glutamatérgica e de mecanismos de plasticidade homeostática, e sugere que estes processos sejam facilmente perturbados no contexto de doença e contribuam assim para a patogénese de doenças neuropsiquiátricas, nomeadamente para doenças associadas à Caspr2.<br>NARSAD Independent Investigator Grant<br>Fondation Jerôme Lejeune