O lúpus eritematoso sistêmico (LES) é uma doença inflamatória crônica autoimune que se caracteriza pela perturbação da homeostase imunológica. Envolve a indução e produção de autoanticorpos, bem como formação e deposição de complexos imunes, que conduzem a uma intensa resposta inflamatória e dano tecidual. Fatores imunológicos, ambientais, hormonais e genéticos podem estar implicados na patogênese da doença. A expressão alterada de genes e proteínas reguladores da apoptose em células T pode comprometer a tolerância imunológica e induzir autoantígenos responsáveis pelo desenvolvimento e perpetuação de condições autoimunes. Mas, pouco se sabe a respeito de como a expressão destas proteínas afeta o desenvolvimento e a função das células T. As células T regulatórias (Treg), uma subpopulação celular de importância para prevenir a autoimunidade, apresentam número e capacidade supressora alterados nos pacientes lúpicos. Buscando entender como anormalidades observadas nas vias apoptóticas contribuem para a autoimunidade, o presente estudo avaliou a frequência de apoptose inicial e morte celular de linfócitos nestes pacientes, a frequência de subtipos de células T expressando as proteínas envolvidas nas vias extrínseca e intrínseca da apoptose, como Fas, FasL, Bax, Bcl-2 e p53, além da expressão dos genes que as codificam. Analisou-se ainda a frequência de polimorfismos nos genes FAS, FASL, BCL-2 e BAX em pacientes, comparando estes dados com o de controles, buscando possíveis associações com a predisposição à doença e com os dados clínicos. Foram estudados 427 pacientes com LES do Hospital de Clínicas de Porto Alegre (HCPA) e 543 indivíduos saudáveis, como controles. Dados de 50 mulheres lúpicas mostraram maior frequência de células Treg, bem como maior densidade de expressão do fator de transcrição Foxp3 nestas células em relação a controles, embora não tenhamos observado diferenças estatísticas em relação à expressão de proteínas pró- e antiapoptóticas nesta subpopulação celular. Uma proporção aumentada de células T CD4+ expressando Fas e p53, e uma frequência reduzida expressando Bcl-2 foi encontrada nas pacientes, em relação aos controles. Esse último dado foi apoiado pela relação observada entre a menor frequência de T CD4+ expressando Bcl-2 e a atividade da doença, e pela expressão diminuída do gene BCL-2 em células mononucleares do sangue periférico (PBMCs) de mulheres lúpicas. Células T CD8+ expressando Bax e p53 também foram mais frequentes nas pacientes, ressaltando que células T CD8+p53+ foram mais numerosas nas pacientes com positividade para anticorpos anti-DNA. Maior frequência de morte de linfócitos foi observada em mulheres lúpicas. Com relação ao estudo dos polimorfimos, variantes alélicas dos genes FASL e BAX possivelmente se relacionam com o desenvolvimento e proteção da doença, respectivamente. Em conclusão, nossos achados apontam que a expressão modificada de genes e proteínas em células TCD4+ e TCD8+, relacionada a uma maior taxa de apoptose, podem conduzir à desregulação das vias apoptóticas e levar à perda da tolerância periférica, favoráveis ao desenvolvimento do LES. / Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease that is characterized by disruption of the immune homeostasis. It involves the induction and production of autoantibodies, as well as formation and deposition of immune complexes, leading to a severe inflammatory response and tissue damage. Immunological, environmental, hormonal and genetic factors may be implicated in the pathogenesis of the disease. The altered expression of genes and proteins regulating apoptosis on T cells may lead to breakdown of the immune tolerance and induce autoantigen responsible for the development and perpetuation of autoimmune conditions. Little information is known of how these proteins affect the development and function of T cells. Regulatory T cells (Treg), a cell subpopulation of importance to prevent the autoimmunity, exhibit changed number and suppressive capacity in SLE patients. Trying to understand how abnormalities observed in apoptotic pathways contribute to autoimmunity, the present study evaluated the frequency of initial apoptosis and cell death of lymphocytes in these patients, the frequency of T cell subsets expressing the proteins involved in the extrinsic and intrinsic pathways of apoptosis, such as Fas, FasL, Bax, Bcl-2 and p53, besides the expression of genes that encode them. We also examined the polymorphisms frequencies of Fas, FasL, Bcl-2 and Bax genes in patients, comparing these data with controls, looking for possible associations with the predisposition to disease and the clinical data. 427 SLE patients from Hospital de Clínicas de Porto Alegre (HCPA) and 543 healthy subjects, as controls were studied. Results from 50 SLE women showed a higher frequency of Treg cells as well as a higher density of Foxp3 expression in these cells compared to controls, although we have not observed statistical differences in relation to the expression of pro- and antiapoptotic proteins in this cell subpopulation. An increased proportion of CD4+ T cells expressing Fas and p53, and a reduced frequency expressing Bcl-2 were found in patients. The latter finding was supported by the negative relation found between the frequency of CD4+Bcl-2+ cells and the disease activity index and by the decreased expression of BCL-2 gene in PBMCs of lupus women. CD8+ T cells expressing Bax and p53 were also more frequent in patients, noting that CD8+ T cells expressing p53 were more frequent in patients positive for anti-DNA antibodies. A higher frequency of death of lymphocytes was observed in SLE women. Regarding to polymorphisms analysis, allelic variants of FasL and Bax are possibly relate to the development and protection of the disease, respectively. In conclusion, our findings indicate that the modified expression of genes and proteins in CD4+ and CD8+ T cells, related to an increased apoptosis, may lead to dysregulation of the apoptosis pathways and the loss of peripheral tolerance, favorable for SLE development.

A cohort of 306 patients was recruited. Questionnaire interview, review of medical records and clinical assessments were performed to obtain information regarding disease status, healthcare resources utilization and HRQoL. / Cost-of-illness studies measure the monetary burden that a disease imposes on society or individuals. The substantial financial burden of SLE has been demonstrated in a modest number of studies and a restricted number of countries. However, there is no study investigating the relationship between disease costs and NPSLE/flare. / In summary, this study has provided support for our hypotheses. The socioeconomic impact of SLE in Hong Kong is considerable. The presence of NPSLE and flare are significantly associated increase disease costs but not impaired HRQoL. These suggest that management, which can lead to early diagnosis and effectively control disease activity and prevent lupus flares, may reduce disease costs due to both healthcare consumption and loss of productivity. / Systemic lupus erythematosus (SLE) is a multi-factorial autoimmune disease that primarily affects young women, characterized by a chronic remitting-relapsing (flare) disease course. Central nervous system is one of the most common affect systems in SLE. Neuropsychiatrie SLE (NPSLE) is associated with impairment of quality of life, accumulated disease damage, disability and employment. Flare, an increase in disease activity over a defined period, is an important outcome in the assessment of SLE. Uncontrolled disease activity results in cumulative organ damage which is associated with increased mortality. / The main findings were as follows. 1. The average annual total costs were USD 13,307 (2006 US dollars) per patient. The direct costs dominated the total costs (62%), and the costs of inpatient care contributed 52% of the direct costs. Costs of SLE per subject are higher than those of other chronic diseases in Hong Kong. 2. Patients with NPSLE incurred significantly higher direct and indirect costs compared to those without NPSLE. The number of NPSLE event was an independent explanatory variable associated with both increased direct and indirect costs. 3. Annual direct costs and indirect costs were significantly higher in those with flares. The number of flare was an independent explanatory variable associated with increased direct costs. Patients with multi-organ flares or renal/neuropsychiatric flares incurred higher direct costs than those with single organ flare or those with minor organ flares. 4. Patients with SLE had significantly lower level of HRQoL compared with Hong Kong general population. The presence of NPSLE and flare only weakly associated with impairment of HRQoL. / The present thesis was a retrospective cost-of-illness study on Chinese patients in Hong Kong with SLE within working age, aiming to 1. estimate the direct and indirect costs of SLE from a societal perspective; 2. ascertain the relationship between NPSLE and direct and indirect costs; 3. ascertain the relationship between flare and direct and indirect costs; 4. investigate the relationship between HRQoL and NPSLE/flares. / We hypothesized that: I. SLE is associated with substantial socioeconomic burden as a result of NPSLE and flare; 2. patients with NPSLE or flares may experience more compromised health-related quality of life (HRQoL). / Zhu, Yaner. / Adviser: Edmund K. Li. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 190-214). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thèse d'exercice : Pharmacie : Nantes : 2003. / Thèse : 2003NANT034P. Bibliogr. f. 48-58 [91 réf.].

Thesis (Ph. D.)--University of Virginia, 2001. / Includes bibliographical references (leaves 129-144). Also available online through Digital Dissertations.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus, characterized by the production of autoantibodies and immune-mediated injury to the kidney. Hyaluronan (HA) is a major component of the extracellular matrix, which is involved in immune-mediated renal injury. We have previously demonstrated that HA expression is increased in the glomerulus of patients with severe lupus nephritis, attributed in part to anti-dsDNA antibody-mediated induction of low molecular weight (LMW) HA and high molecular weight (HMW) HA synthesis in mesangial cells. The causal role of HA and its fragments in the pathogenesis of lupus nephritis has not been explored. In this project, two separate in vivo studies were undertaken to delineate the role of HA in disease progression in NZBWF1/J mice. In the first study, we determined the effect of 4-methylumbelliferone (MU), a specific inhibitor of HA synthesis, on renal function and histology in NZBWF1/J mice with active disease, with particular emphasis on inflammatory and fibrotic processes. In the second study, we investigated whether exogenous HA could exacerbate disease manifestations in pre-disease NZBWF1/J mice. The mechanisms underlying the effects of MU and exogenous LMW HA and HMW HA were investigated in mesangial cells isolated from NZBWF1/J mice. Female NZBWF1/J mice with established nephritis were randomized into 3 groups and treated with (1) PBS, (2) Arabic gum (Gum) or (3) MU (3g/kg/day) for 2, 4, 8 and 12 weeks. Treatment of mice with MU for 12 weeks reduced serum HA levels and abrogated intra-renal expression of HA compared to PBS and Gum treated mice. Inhibition of HA synthesis in the kidney resulted in decreased IgG and C3 deposition, reduced B cell, T cell and macrophage infiltration, matrix accumulation and TNF-, IL-6 and MCP-1 expression, which was associated with improved renal functions. To confirm that HA influenced pathogenesis of lupus nephritis, pre-disease NZBWF1/J mice were randomized to receive (1) PBS, (2) LMW HA or (3) HMW HA for periods up to 24 weeks. Administration of LMW HA and HMW HA into NZBWF1/J mice by tail-vein injection induced intra-glomerular deposition of IgG and C3, B cell infiltration, glomerular hypercellularity and tubular atrophy, which was accompanied by induction of MAPK signaling pathways, enhanced MCP-1 expression, and increased matrix deposition in the glomerular and tubular basement membranes. In vitro studies showed that exogenous IL-6, IL-1, TGF-1 and TNF- induced HA synthesis in murine mesangial cells (MMC), with over 80% secreted into the conditioned medium. This was accompanied by an increase in pro-inflammatory cytokine secretion and synthesis of fibronectin and laminin. Inhibition of HA synthesis with MU significantly decreased cytokine secretion and fibronectin synthesis. The ability of HA to induce inflammatory and fibrotic processes in mesangial cells was confirmed in separate studies in which MMC were incubated with exogenous LMW HA and HMW HA. In summary, these original findings provide evidence of a direct effect of HA on intra-renal inflammation and fibrosis in lupus nephritis. Approaches to inhibit HA synthesis may offer novel therapeutic strategies to delay disease progression. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

SLE is a complicated autoimmune disease with a strong genetic basis. Owing to the successful application of genome-wide association study (GWAS) on complex traits, significant progresses have been made in identifying susceptible loci for SLE. So far, more than 30 loci have been shown to have robust association with the disease, significantly improving our understanding of this disease. On the other hand, these loci only confer relatively small increments in disease risk, leaving a large amount of disease heritability unexplained. Several limitations may lead to the issue of “missing heritability”, such as the insufficient statistical power of standard GWAS, the failure to explore independent signals with marginal effects, and other forms of genetic variations or interactions that can hardly be detected by the current GWAS approach. In the current study, efforts have been made on solving the issue of missing heritability. Firstly, in order to increase the statistical power of GWAS, we performed meta-analysis of two existing SLE GWASs on Chinese populations, and replicated the findings in additional samples from Hong Kong, Anhui, and Thailand. We identified ARID5B and CDKN1B as novel SLE susceptibility genes through this approach. Secondly, considering the limitation of previous GWASs that only focus on the most significant signal in an individual region, we revisited the established loci by in-depth analysis on the basis of meta-analysis and followed up the findings with large-scale replication efforts. We found three additional independent signals in 11q23.3 as being associated with SLE. Thirdly, given the importance of independent effects in predisposing disease susceptibility, we performed gene-based analysis to combine the marginal independent signals within a gene to identify novel susceptibility genes. Our results demonstrated the widespread existence of independent effects within known SLE susceptibility genes, and we also identified ANXA6 as a novel SLE susceptibility gene. Lastly, we also made some efforts in the characterization of the related genes, and we identified several regulatory SNPs (rSNPs) predisposing individuals to SLE via affecting expression of the corresponding genes. In addition, we also found that epistatic interaction of variants in a previously established susceptibility gene, ETS1,correlates with cytokine (e.g.IL-17) abnormality in SLE cases. The current work represents several practical approaches to improve the power of GWAS. The findings might enrich the list of SLE susceptibility genes as well as advance our knowledge on the etiology of this complicated disease. Importantly, the current study highlights the importance of independent effects and rSNPs in conferring disease susceptibility, which may shed light on further exploration on the genetic architecture of SLE. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Systemic Lupus Erythematosus (SLE) is an autoimmune disease which is characterized by high level of autoantibody detected in the body. This disease is female predominant with a male to female ratio 1: 9. SLE could cause damage to different organ systems and central nervous system is one of them. Patients diagnosed with SLE could suffer from psychiatric problems like cognitive dysfunction, depression and anxiety. Neurogenesis refers to the process by which new neurons are generated. Although it has been widely reported that neurogenesis could be enhanced under pathological conditions such as stroke, Huntington’s disease and epilepsy, study focusing on the relationship between neurogenesis and SLE remains limited. In the present study, by using NZB/W F1 mice as the animal model of SLE, we could demonstrate that there was dramatic increase of neuronal precursor cells at the corpus callosum after the onset of SLE symptoms. Meanwhile, as IBA-1 positive cells and GFAP positive cells also increased significantly there, this suggested inflammation has taken place. I hypothesized there were upregulation of immunological factors after the onset of SLE symptoms and those factors were responsible for the neurogenesis. In my in vitro study, cytokine- interferon gamma (IFN gamma) has been shown to promote neuronal progenitor cells (NPCs) to differentiate into neuronal linage but it did not obviously affect the cell proliferation and migration. For the other cytokine and chemokines, including interleukin-10 (IL-10), interleukin-8 (IL-8), macrophage-derived chemokine (MDC), stromal cell-derived factor 1 alpha (SDF-1alpha) and thymus and activation regulated chemokine (TARC), all of them had no effect on NPC proliferation and differentiation. As far as we know, this is the first study to report SLE could enhance neurogenesis. Concerning the role of inflammation and IFN gamma on the neurogenesis in our case, it should be worth for further investigation, which will benefit future development of novel treatment targeting psychiatric symptoms in SLE. / published_or_final_version / Anatomy / Master / Master of Philosophy

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against self-antigens. SLE has a complex multifactorial genetic basis. Genome wide association studies (GWAS) have implicated polymorphisms causing decreased expression in many genes in SLE etiology and will likely continue to implicate new genes. Therefore, the aim of my project was to create a modular system where the effect of knocking down a gene of interest can be studied in cell culture in a timely manner. This was accomplished by inserting shRNAmirs targeting our validation gene, A20, into a retroviral vector, creating viral particles using a packaging cell line, and infecting cultured cells. Two shRNAmirs were determined to be effective by qRT-PCR and Western blots on infected cells. Vectors carrying these shRNAmirs were used to infect ex vivo B cells and BMDCs, and results suggested that A20 knockdown may mediate functional changes in both cell types.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against self-antigens. SLE has a complex multifactorial genetic basis. Genome wide association studies (GWAS) have implicated polymorphisms causing decreased expression in many genes in SLE etiology and will likely continue to implicate new genes. Therefore, the aim of my project was to create a modular system where the effect of knocking down a gene of interest can be studied in cell culture in a timely manner. This was accomplished by inserting shRNAmirs targeting our validation gene, A20, into a retroviral vector, creating viral particles using a packaging cell line, and infecting cultured cells. Two shRNAmirs were determined to be effective by qRT-PCR and Western blots on infected cells. Vectors carrying these shRNAmirs were used to infect ex vivo B cells and BMDCs, and results suggested that A20 knockdown may mediate functional changes in both cell types.