Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Resumo: A conexão pele-tireoide tornou-se uma fronteira de interesse da dermatoendocrinologia. Neste contexto, a relação entre urticária crônica idiopática (UCI) e autoimunidade tireóidea tem sido estudada nos seus diferentes aspectos. Além disso, de 30% a 40% dos casos de UCI apresentam etiologia autoimune. A tireoidite de Hashimoto (TH) representa a mais prevalente doença autoimune da tireoide (DAT), acarretando, frequentemente, hipotireoidismo. Os indícios que o quadro de UCI é mais grave e menos responsivo aos tratamentos convencionais quando associado à DAT, sugerem um papel da tireotrofina (TSH) no desenvolvimento das lesões urticariformes. O teste do soro autólogo da pele (TSSA) é um teste intradérmico in vivo utilizado para avaliar a autorreatividade, a presença de autoanticorpos funcionais ou de urticária de etiologia autoimune. Apesar dos indícios de associação entre DAT e UCI, os relatos são controversos. Os objetivos do presente estudo foram avaliar: a associação entre UCI e autoimunidade tireóidea; a frequência de alterações tireóideas em pacientes com UCI, com ênfase nas de origem autoimune; a frequência de angioedema em relação às alterações tireóideas; a associação entre TH, em pacientes com UCI, e positividade do TSSA; a expressão gênica do receptor de TSH (RTSH) na pele sã e na pápula do TSSA, de pacientes com UCI, com e sem TH. O estudo foi dividido em dois capítulos. No Capítulo 1, foi realizada uma análise retrospectiva dos prontuários médicos de 223 pacientes com urticária crônica (UC), acompanhados no ambulatório de alergia dermatológica do Hospital das Clínicas da Faculdade de Medicina de Botucatu. Foram selecionados 115 casos com UCI que dispunham dos resultados das dosagens plasmáticas de TSH, tiroxina livre (T4-L) e anticorpos antitireoperoxidase (antiTPO) e antitireoglobulina (antiTG). Tratava-se de 91 mulheres (79,1%) e ... (Resumo completo, clicar acessso eletrônico abaixo) / Abstract: The thyroid-skin connection has become a hot frontier in dermatoendocrinology. In that context, the relation between chronic idiopathic urticaria (CIU) and thyroid autoimmunity has been studied in its different aspects. Besides, from 30% to 40% of CIU cases present autoimmune etiology. Hashimoto's thyroiditis (HT) represents the most prevalent autoimmune thyroid disease (ATD), frequently causing hypothyroidism. The signs that the CIU picture is more severe and less responsive to conventional treatments when associated with ATD suggest a role of thyrotropin (TSH) in developing urticariform lesions. The autologous serum skin test (ASST) is an in vivo intradermal test to evaluate autoreactivity, presence of functional antibodies or urticaria from autoimmune etiology. Despite the signs of association between ATD and CIU, reports are controversial. The present study aimed to evaluate: the association between CIU and thyroid autoimmunity; the frequency of thyroid alterations in patients with CIU emphasizing on the autoimmune originated ones; the frequency of angioedema in relation to thyroid alterations; the association between HT, in patients with CIU, and the ASST positivity; the TSH receptor (RTSH) gene expression in healthy skin and in the papules of ASST, of patients with CIU, with or without HT. The study was divided into two chapters. In Chapter 1, a retrospective analysis was performed in the medical report of 223 patients with chronic urticaria (CU), assisted at the outpatient of dermatologic allergy of the Clinic Hospital of the Botucatu Medical School. One hundred fifteen cases with CIU and available results of plasmatic dosage of TSH, free thyroxine (FT4), and antithyroperoxidase (antiTPO) and antithyroglobulin (antiTG) antibodies were selected. There were 91 women (79.1%) and 24 men (20.9%), mean age 36.5 years, who started the follow-up between 1984 and 2006. These case ... (Complete abstract click electronic access below) / Orientador: Gláucia Maria Ferreira da Silva Mazeto / Coorientador: Célia Regina Nogueira / Banca: Luciana Patricia Fernandes Abbade / Banca: Paula de Oliveira Montandon Hokama / Banca: Laura Sterian Ward / Banca: José Antonio Sanches Junior / Doutor

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Rheumatoid arthritis (RA) is a complex autoimmune disease affecting approximately 1% of the population. Multiple factors contribute to the development of RA, with genetic factors accounting for around 60% of the disease risk. Over the last few years, genome-wide association studies (GWAS) have successfully been used to identify regions of the genome predisposing to complex disease. There are now 101 confirmed RA risk loci, but for the vast majority of these loci the causal gene and causal variant remain unidentified and therefore, their function in disease is unexplored. The majority of genetic variants, or single nucleotide polymorphisms (SNPs), associated with disease map to non-coding enhancer regions, which may regulate transcription through long-range interactions with their target genes. The aims of this project were to identify the causal genes within an RA locus, pinpoint the causal variants and elucidate the mechanisms by which the variants modify gene function. Capture Hi-C (CHi-C) was carried out with the aim of identifying long range interactions between disease-associated SNPs and genes in four related autoimmune diseases. Many long-range interactions were identified which implicated novel candidate genes, interactions involving multiple genetic loci which had a common target, and interactions with loci which had previously been implicated in disease. Complex interaction patterns were observed in many of the disease associated loci, particularly in the 6q23 locus which is associated with a number of autoimmune diseases and is the focus of the present thesis. Within the 6q23 locus, associated SNPs lie a large distance from any gene (>180kb) making it difficult to pinpoint the exact causal gene. Results from CHi-C and chromosome conformation capture (3C-qPCR) experiments indicated that restriction fragments containing disease associated intergenic SNPs could display genotype-specific interactions with genes associated with autoimmunity (IL20RA and IFNGR1). Interactions could also be detected with long non-coding RNAs (lncRNAs), The lead SNP in the 6q23 region is in tight LD with eight other SNPs which are equally likely to be causal. Bioinformatics analysis suggested that the most plausible causal SNP in the 6q23 intergenic region was rs6927172, as it maps to an enhancer in both B-cells and T-cells, is in a DNaseI hypersensitivity cluster, shows transcription factor binding and is in a conserved region. Chromatin immunoprecipitation (ChIP) demonstrated binding of chromatin marks of active enhancers (H3K4me1 and H3K27ac) and the transcription factors BCL3 and NF-κB to the rs6927172 SNP target site in Jurkat T-cells and GM12878 B-cells, suggesting the risk allele could be associated with increased regulatory activity. In conclusion, these results show that CHi-C can help identify novel GWAS causal genes with the potential to suggest novel therapeutic targets. For example IL20RA is already a target for a monoclonal antibody which has been shown to be effective in treating RA in clinical trials. This project has also provided compelling evidence that the autoimmune risk variant in the 6q23 locus, rs6927172, is within a complex gene regulatory region, involving multiple immune genes and regulatory elements, such as lncRNAs.

Autoimmunity is a modern age medical dilemma which is inextricably linked with emotional stress. Based on semi-structured interviews and participant Adverse Childhood Experiences (ACE) survey results, this study confirms that the autoimmune process may be initiated via psychosocial factors like emotional stress and childhood trauma. Ninety-three percent of participants experienced adversity or trauma in childhood, and 50% of participants talked about a period of prolonged stress that preceded the onset of their condition. This study also confirms the intimate and satisfactory relationship developed between patients and complementary and alternative (CAM) practitioners, who invite patients to be co-producers of health and holistically address patients' minds, bodies, and souls. Finally, this study demonstrates the incredible resiliency of people diagnosed with autoimmune conditions and how they find healing and meaning post-diagnosis.

Circulating monocytes have a limited life span and will undergo apoptosis in the absence of specific stimuli. Recent studies have demonstrated that monocytes can be rescued from apoptosis via lipopolysaccharide (LPS) activation or stimulation with interleukin-1 or tumor necrosis factor-α. Based on previous studies from our laboratory, we hypothesized that, in nonseptic (e.g., autoimmune) inflammation, the presence of activated T cells may enhance monocyte longevity through T cell contact-dependent signaling. Plasma membranes prepared from 6 h activated (Tm(A)) and resting (Tm(R)) purified CD4+ T cells were added to resting elutriation-purified monocytes cultured in serum-free medium. Cells were assayed for degree of apoptosis occurring over a 72-h incubation using both agarose gel electrophoresis and flow cytometry. The addition of Tm(A) (but not Tm(R)) was capable of blocking monocyte apoptosis and the ability of Tm(A) to rescue monocytes was abrogated by the addition of anti-CD40L antibodies. Rescue of monocytes from apoptosis could also be mediated by direct cross-linking of monocyte CD40. Inhibitors of tyrosine kinase activity blocked both Tm(A) and anti-CD40-mediated rescue of monocytes from apoptosis, suggesting a primary role of a tyrosine kinase signaling pathway in the events controlling monocyte longevity.

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease characterized by autoantibody production, inflammation and end organ damage resulting from an overactivation of the immune system through mechanisms that are still not completely understood. One critical component of this pernicious cycle is a loss of tolerance to self-antigen that precedes disease manifestations. Importantly, there has only been one FDA approved treatment for SLE in the last 60 years, though an additional treatment has recently been approved for lupus nephritis. Therefore, there is a need to understand how pathogenic pathways are regulated in the context of SLE. This thesis focused on not only further understanding how interferon regulatory factor 5 (IRF5) drives pathogenicity in a mouse of model of lupus but also discerning the molecular pathways important for blood dendritic cell antigen 2 (BDCA2) inhibition of type I interferon (IFN-I) release from plasmacytoid dendritic cells (pDCs). Gain-of-function polymorphisms in the transcription factor IRF5 are associated with an increased risk of developing systemic lupus erythematosus. However, the IRF5-expressing cell type(s) responsible for lupus pathogenesis in vivo is not known. This work shows that monoallelic IRF5 deficiency in B cells markedly reduces disease in the FcγRIIB−/−Yaa mouse model of lupus. Mechanistically, B cell receptor and TLR7 signaling synergize to promote IRF5 phosphorylation through IRAK4, and also increase IRF5 protein expression, with these processes being independently regulated. This synergy increases B cell-intrinsic IL-6 and TNF-𝛂 production, both key requirements for germinal center responses, with IL-6 and TNF-𝛂 production in vitro and in vivo being substantially lower with loss of one allele of IRF5. Moreover, this thesis further strengthens the notion of IRF5 as a target for the treatment of SLE by therapeutically targeting IRF5 in the FcγRIIB−/−Yaa mouse lupus model. This work shows that tamoxifen driven deletion of IRF5 after disease associated manifestations have already developed confers protection in the FcγRIIB−/−Yaa mouse model. IFN-I is a family of pleotropic cytokines that are thought to be pathogenic in the context of SLE. PDCs are a major source of IFN-I. As such, they have been shown to play a major role in anti-viral immunity and are thought to be pathogenic in context of SLE. I used the anti-BDCA2 antibody 24F4A to ligate the receptor in a human pDC cell line (Gen 2.2) and applied a variety of proteomics methods, including profiling of post-translational modifications, to evaluate signaling downstream of BDCA2. I found that phosphorylation of phosphoinositide 3-kinase adapter protein 1 (PIK3AP1)/ B cell adaptor protein (BCAP) was increased after BDCA2 engagement by 24F4A. Deletion of BCAP from Gen2.2 cells reversed BDCA2 mediated AKT phosphorylation and IFN inhibition, suggesting that PI3 kinase (PI3K) may be important for BDCA2 mediated IFN inhibition. Treatment of human pDCs with PI3kinase inhibitor followed by TLR9 stimulation confirmed that PI3K activity is critical for BDCA2 mediated IFN inhibition. This work describes a critical threshold of IRF5 expression in B cells necessary for the development of the lupus like disease in the FcγRIIB−/−Yaa mice and that systemic deletion of IRF5 after disease onset reduces the disease manifestations in the same mouse model. Additionally, this work demonstrated that BCAP and PI3K are important pathways to mediate the IFN inhibition observed with engagement of the BDCA2 pathway. This work supports IRF5 as a therapeutic target in SLE and furthermore, describes a molecular pathway important for BDCA2 mediated IFN-I inhibition from pDCs.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by a low platelet count (less than 100 x 10^9 platelets/L) and an increased risk of bleeding. ITP is difficult to diagnose and manage due to the deficiencies in our understanding of the pathophysiological mechanisms leading to thrombocytopenia. Anti-platelet autoantibodies are believed to be the primary mechanism of thrombocytopenia in ITP. In this thesis, I demonstrate that autoantibodies can only be detected in half of all ITP patients; therefore, other mechanisms should be investigated. CD8+ T cells have been implicated as a mechanism of disease in ITP, but platelet-specific CD8+ T cells have yet to be identified. I have characterized CD8+ T cells in ITP patients and found that platelet-specific CD8+ T cells can be detected in ITP patients. These platelet-specific CD8+ T cells can also be detected in healthy individuals, so they are not specific to ITP. However, regulatory defects were observed in ITP patients and CD8+ T cell activity was elevated in ITP patients relative to healthy individuals and thrombocytopenic non-ITP patients. Investigating whether platelet-specific CD8+ T cells can actively participate in platelet destruction and underproduction will be an essential step towards better understanding the role of CD8+ T cells as a disease mechanism in ITP, which will lead to improvements in the management of ITP. / Thesis / Doctor of Philosophy (PhD) / Platelets are small blood cells that are involved in minimizing blood loss at the site of a wound by forming a plug. In a disease called immune thrombocytopenia (ITP), patients have a low platelet count, which can result in bleeding. The bleeding symptoms of ITP decrease the quality of life for ITP patients and can be life-threatening in rare cases. It is believed that ITP is caused by proteins produced by the immune system called antibodies. I found that the antibodies that cause ITP can only be detected in half of all ITP patients. Therefore, there are probably additional causes of ITP. It is suspected that CD8+ T cells might cause ITP in some patients. CD8+ T cells are part of the immune system and they typically destroy other cells that are cancerous or infected by viruses. CD8+ T cells might also destroy healthy cells, like platelets. My goal was to characterize CD8+ T cells in order to determine their role in ITP. I found that CD8+ T cells from ITP patients can target platelets, and that healthy people have these CD8+ T cells as well. In regard to CD8+ T cells that target platelets, the difference between ITP patients and healthy people appears to be related to immune system regulation and CD8+ T cell activity. In the future, we should focus on understanding how platelet-specific CD8+ T cells can cause a low platelet count in order to improve the clinical management of ITP.

The development of autoimmune diseases is frequently linked to exposure to environmental factors such as chemicals, drugs or infections. In the experimental model of metal-induced autoimmunity, administration of subtoxic doses of mercury (a common environmental pollutant) to genetically susceptible mice induces an autoimmune syndrome with rapid anti-nucleolar antibody production and immune system activation. Regulatory components of the innate immune system such as the costimulatory molecules PD-1, CTLA-4 and their ligands PD-L1, PD-L2, B7-1 and B7-2 can also modulate the autoimmune process. We examined the interplay among environmental chemicals and these costimulatory molecules in the regulation of autoimmunity. Additionally, we studied PD-1, CTLA-4 and its ligands in a tolerance model where pre-administration of a low dose of mercury in the steady state renders animals tolerant to metal-induced autoimmunity. Overall, PD-1/CTLA-4 blockade by blocking antibodies enhanced the manifestations of metal-induced autoimmunity. Although we expected that the blockade of both PD-1 ligands would mimic blocking the receptor, blocking the ligands resulted in the opposite effect when co-injected with mercury, reducing the manifestations of metal-induced autoimmunity. Individual PD-1 ligands differed in their ability to enhance the mercury treatment, with PD-L1 being the major regulator in the model. Likewise, we showed that PD-L1 is essential to keep the recall response to mercury at check. Our data suggest that these effects could be mediated by the modification of cytokine profiles, B cells and T cell subpopulations numbers. / Microbiology and Immunology

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by loss of immune tolerance and the production of auto-antibodies which target various nuclear components. There is a 9:1 women to men ratio among lupus patients, indicating differing mechanisms of lupus pathogenesis between the sexes. Although lupus patients may develop many different manifestations, lupus nephritis (LN) remains to be one of the most devastating manifestations and an indicator of poor prognosis. Although both sexes develop LN, the nephritis in males often develops more rapidly and is more severe. Necrotic cell death is a characteristic of lupus nephritis and contributes to the exacerbation of the inflammatory immune response within the glomeruli. Previously our laboratory found that absence or pharmacological inhibition of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme involved in necrotic cell death, results in milder nephritis, reduced necrotic lesions, and higher survival rates only among males. Although RIPK3-mediated necrosis was a likely candidate for inducing necrosis during female LN, murine models of glomerulonephritis revealed that the development of LN occurs independently of RIPK3. In addition, during LN, there is no crosstalk between the RIPK3- and PARP-1 mediated pathways to induce necrotic cell death. The sex bias in SLE indicates sex hormones may play a role in pathogenesis. Interestingly, estrogen receptor alpha (ERα) in the renal tissue is highly expressed and the renal specific estrogen-induced gene activation is second only to that of reproductive organs. The absence of estrogen receptor alpha protects female mice from developing nephritis, despite the presence of immune complexes in the kidneys and production of pro-inflammatory cytokines. Analysis of gene expression changes during LN progression indicate the protection seen in ERKO females may be due to alterations in metabolic pathways, including PPAR and retinol metabolism. These results demonstrate the complexity of lupus nephritis. Despite the presence of necrosis in LN, this manifestation occurs in a RIPK3-independent manner, which leaves the pathway responsible for necrosis in female kidneys to still be investigated. In addition, lupus nephritis occurs in an ER-dependent manner in females, demonstrating the significant impacts sex-hormone environments play in the pathogenesis of immune-mediated nephropathies. / Biomedical Sciences